Molecularly Targeted Therapy for Childhood Cancer

Molecularly Targeted Therapy for Childhood Cancer w Peter J. Houghton â•‡ â•‡ Robert J. Arceci ● Editors Molecula...

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Molecularly Targeted Therapy for Childhood Cancer

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Peter J. Houghton â•‡ â•‡ Robert J. Arceci ●

Editors

Molecularly Targeted Therapy for Childhood Cancer

Editors Peter J. Houghton Elizabeth M. and Richard M. Ross Chair Director, Center for Childhood Cancer The Research Institute Nationwide Children’s Hospital 700 Children’s Drive Columbus, OH 43205 [email protected]

Robert J. Arceci King Fahd Professor of Pediatric Oncology Professor of Pediatrics Oncology and Cellular and Molecular Medicine Kimmel Comprehensive Cancer Center at Johns Hopkins The Bunting and Blaustein Cancer Research Building 1650 Orleans Street, Suite 207 Baltimore, MD 21231 USA [email protected]

ISBN 978-0-387-69060-5 e-ISBN 978-0-387-69062-9 DOI 10.1007/978-0-387-69062-9 Springer New York Dordrecht Heidelberg London Library of Congress Control Number: 2010930693 © Springer Science+Business Media, LLC 2010 All rights reserved. This work may not be translated or copied in whole or in part without the written permission of the publisher (Springer Science+Business Media, LLC, 233 Spring Street, New York, NY 10013, USA), except for brief excerpts in connection with reviews or scholarly analysis. Use in connection with any form of information storage and retrieval, electronic adaptation, computer software, or by similar or dissimilar methodology now known or hereafter developed is forbidden. The use in this publication of trade names, trademarks, service marks, and similar terms, even if they are not identified as such, is not to be taken as an expression of opinion as to whether or not they are subject to proprietary rights. Printed on acid-free paper Springer is part of Springer Science+Business Media (www.springer.com)

Acknowledgments

We would like to especially thank all the authors and investigators who contributed to the content of this book along with the publisher for believing in the importance of directly addressing important issues concerning children with cancer.

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Contents

Part Iâ•… Hematologic Malignancies The Emerging Era of Targeted Therapy in Childhood Acute Lymphoblastic Leukemia..................................................................... William L. Carroll and Rob Pieters

3

Molecular Targeted Therapies in T-CellAcute Lymphoblastic Leukemia................................................................................ Alejandro Gutierrez and A. Thomas Look

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Molecularly Targeted Therapy for Infant ALL............................................ Patrick A. Brown and Carolyn A. Felix

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Targeted Therapeutic Approaches for AML................................................. Robert J. Arceci and Donald Small

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Acute Promyelocytic Leukaemia.................................................................... Andrea Biondi, Anna Maria Testi, and Brenda E.S. Gibson

83

Down Syndrome and Acute Myeloid Leukemia: An Unique Genetic Sensitivity to Chemotherapy......................................... 109 Jeffrey W. Taub, Yubin Ge, and Yaddanapudi Ravindranath Targeting RAS Signaling Pathways in Juvenile Myelomonocytic Leukemia (JMML)........................................................................................... 123 Jennifer O’Hara Lauchle and Benjamin S. Braun Chronic Myeloid Leukemia: Pathophysiology and Therapeutics............... 139 Seth J. Corey and Jorge Cortes Molecularly Targeted Therapies in Pediatric Myelodysplastic Syndromes............................................................................ 155 Lia Gore vii

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Contents

New Therapeutic Frontiers for Childhood Non-Hodgkin Lymphoma............................................................................... 177 Megan S. Lim and Mitchell S. Cairo Molecular Targeting of Post-transplant Lymphoproliferative Disorders........................................................................................................... 215 Michael Wang and Thomas G. Gross Part IIâ•… Solid Tumors Molecularly Targeted Therapies for Astrocytomas...................................... 231 Ian F. Pollack Targeted Therapy in Medulloblastoma in Molecularly Targeted Therapy for Childhood Cancer...................................................... 267 Yoon-Jae Cho and Scott L. Pomeroy Future Treatments of Ependymoma.............................................................. 291 Richard J. Gilbertson Development of Targeted Therapies for Rhabdoid Tumors Based on the Functions of INI1/hSNF5 Tumor Suppressor........................ 305 Ganjam V. Kalpana and Melissa E. Smith Development of Targeted Therapies for Neurofibromatosis Type 1 (NF1) Related Tumors......................................................................... 331 Brigitte Widemann Molecular Therapy for Neuroblastoma......................................................... 351 Yaël P. Mossé and John M. Maris Ewing’s Sarcoma Family of Tumors: Molecular Targets Need Arrows..................................................................................................... 373 Jeffrey A. Toretsky and Aykut Üren Molecular Targeted Therapy for Wilms’ Tumor.......................................... 401 James I. Geller and Jeffrey S. Dome Molecular Therapy for Rhabdomyosarcoma................................................ 425 Raushan T. Kurmasheva, Hajime Hosoi, Ken Kikuchi, and Peter J. Houghton Molecularly Targeted Therapy for Osteosarcoma: Where Do We Go from Here?......................................................................... 459 Rosanna Ricafort and Richard Gorlick

Contents

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Nonrhabdomyosarcoma Soft Tissue Sarcoma in Children: Developing New Treatments Based on a Better Understanding of Disease Biology.................................................................. 499 Stephen X. Skapek Index.................................................................................................................. 521

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Introduction to Targeted Therapy Text Robert J. Arceci and Peter Houghton

The dream of “magic bullets” to treat patients with cancer, as defined by Paul Ehrlich in the early 1900s during his Harben Lectures, promised great hope for molecules that would specifically react with and eradicate tumors without harming the host. His lecture was entitled, “Experimental Researches on Specific Therapy. On Immunity with Special Reference to the Relationship between Distribution and Action of Antigens.”1 Many decades have come and gone since those concepts were first presented along with many lives prematurely lost to cancer. One might ask then “Where did we go wrong?” As if trying to push Ehrlich’s ideas more rapidly forward, Hollywood and Warner Brothers Studios premiered in 1940 the movie “Dr. Ehrlich’s Magic Bullets,” starring Emanuel Goldenberg, aka Edward G. Robinson. During that same period, under the auspices of the Department of Defense, Drs. Louis Goodman and Alfred Gilman were enlisted to investigate autopsy results of soldiers who had died following mustard gas exposures, leading to one of the earliest insights into the selective ablation of the hematopoietic elements and subsequently to early clinical trials in patients with primarily lymphoid malignancies.2 During the first half of the twentieth century, the Indian investigator, Dr. Yellapragada Subbarao, was successful in synthesizing a spectrum of antimetabolites, leading in turn to the landmark study by Farber etÂ€ al., “Temporary Remissions in Acute Leukemia in Children Produced by Folic Acid Antagonist, 4-aminopteroyl-glutamic acid (Aminopterin), in 1948.3 In 1956, Li, Hertz, and Spencer reported the effect of methotrexate on gestational related choriocarcinoma leading eventually to a curative therapy and demonstrating that indeed a small molecule could cure a widely metastatic cancer.4 These were of course all examples of targeted cancer therapy. Following the death of her husband, Albert Lasker, from cancer in the 1950s, Mrs. Mary Lasker and her Citizens Committee for the Conquest of Cancer, effectively promoted the conviction that the federal government had the ability and resources to develop a national crusade to cure cancer. A second United States National Cancer Act (The first was in 1937 and established the National Cancer institute.) was signed by President Richard Nixon in 1971. There was a tremendous amount of excitement during those decades for the day when Dr. Ehrlich’s magic bullets would cure all cancers. Comprehensive Cancer Centers arose, small molecule drug screening began on a wide scale leading to the development of new therapies, xi

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training in oncologic specialties expanded, the maturation of the clinical cooperative groups for adults and children occurred, the need for multidisciplinary treatment teams and supportive care became apparent, and the initiation of combination chemotherapy, adjuvant and neoadjuvant approaches to treatment along with sanctuary site prophylaxis were developed. Over the course of those decades, many cancer patients of all ages benefited. For children with cancer, cancer evolved from a disease that was nearly always fatal to a disease in which about 75% of patients can be cured. Yet, a large percentage of patients still could not expect to be cured. And for those who were cured, the adverse consequences of treatment were too often severe. Thus, as combinations of more intense doses and delivery schedules were tested, the very real plateau of balancing cure and toxicity became an increasingly difficult challenge. Had Dr. Ehrlich’s dream come to an end? Fortunately, throughout those decades, basic laboratory and translational investigators were pushing forward with novel tools and approaches to extend our knowledge of the molecular basis of different cancers and the hosts they afflict. This basic understanding of genetics and epigenetics has formed the substrate of a resurgence of hope in the form of the next generation of targeted therapies. The new therapeutic agents are based on the distinct genetic and epigenetic signatures that translate into unique protein expression patterns that can be exploited for the benefit of patients. From the dawn of this resurgence, stemming from the demonstration of targeting BCR-ABL with imatinib,5 a plethora of novel tumor specific or selective approaches are being applied. Targeting these tumor specific pathways is based on the development of small molecule signal transduction modulators to monoclonal antibodies to antitumor vaccines to nanoparticles. In addition, a growing recognition of the confluence of stem cell biology to the behavior of tumor initiating stem cells is provided for the first time in history the correct cellular targets. The treatment of children with cancer has been at center stage addressing the basic causes of cancer as well as developing and exploiting novel molecularly targeted therapies. This volume attempts to bring together in one place some of the successes, but mostly the hope of eradicating the morbidity and mortality of both hematopoietic malignancies and solid tumors in pediatric patients. The chapters, written by expert laboratory and clinical investigators, provide updated information on current and future approaches to targeted therapy in pediatric oncology. Further, there is a strong belief that the development of less toxic and more effective combinations of therapeutic approaches will also be important in improving the outcome of the 85% of children with cancer who live in developing countries and too often cannot be successfully treated. While the pace of progress has continued to accelerate, for too many patients the pace remains too slow. As Mario Andretti once said, “If things seem under control, you’re not going fast enough.” It is with that sense of urgency that the current state of targeted therapy should be catapulted to the next level based on an increasingly detailed molecular description of both tumors and hosts as we enter an era of increasing genetically directed therapies. There still remains an immense need for

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more financial support, more collaboration, more discovery, more innovation, and more translation, but there has never been, nor will there ever be a lack of passion and hard work. Given the opportunities, those dedicated in eradicating this ancient enemy of the young and old will most assuredly succeed and bring into reality the magic bullets of Paul Ehrlich.

References Witkop B. Paul Ehrlich and His magic Bullets, Revisited. Proceedings of the American Philosophical Society. Vol. 143. Philadelphia, PA: American Philosophical Society; 1999; 540–557. Goodman LS, Wintrobe MM, Dameshek W, Goodman MJ, Gilman A, McLennan MT. Nitrogen mustard therapy. Use of methyl-bis(beta-chloroethyl)amine hydrochloride and tris(beta-chloroethyl)amine hydrochloride for Hodgkin’s disease, lymphosarcoma, leukemia and certain allied and miscellaneous disorders. JAMA. 1946; 251:2255–2261. Farber S, Diamond LK, Mercer RD, Sylvester RF Jr, Wolff JA. Temporary Remissions in acute leukemia in children produced by folic acid antagnonist, 4-aminopteroyl-glutamic acid (aminopterin). NEJM. 1948; 238:787–793. Li M, Hertz R, Spencer DB. Effect of methotrexate therapy upon choriocarcinoma and chorioadenoma. Proc Soc Exp Biol Med. 1956; 93:361–366. Druker BJ, Talpaz M, Resta DJ, etÂ€al. Efficacy and safety of a specific inhibitor of the BCR-ABL tyrosine kinase in chronic myeloid leukemia. N Engl J Med. 2001; 344:1031–1037.

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Contributors

Robert J. Arceci King Fahd Professor of Pediatric Oncology, Professor of Pediatrics, Oncology and Cellular and Molecular Medicine, Kimmel Comprehensive Cancer Center at Johns Hopkins, The Bunting & Blaustein Cancer Research Building, 1650 Orleans Street, Suite 207, Baltimore, MD 21231, USA [email protected] Andrea Biondi Centro M. Tettamanti, Clinica Pediatrica Università di Milano-Bicocca, Ospedale San Gerardo, Via Pergolesi, 33, 20052 Monza, Italy [email protected] Patrick A. Brown Sidney Kimmel CCC at Johns Hopkins, Bunting-Blaustein Cancer Research Building, 1650 Orleans Street, CRB I-2M49, Baltimore, MD 21231, USA [email protected] Mitchell S. Cairo Division of Pediatric Blood and Marrow Transplantation, New York-Presbyterian Morgan Stanley Children’s Hospital, Columbia University, 3959 Broadway, CHN 10-03, New York, NY 10032, USA [email protected] William L. Carroll New York University Cancer Institute, 522 First Avenue, Smilow 1201, New York, NY 10016, USA [email protected] Seth J. Corey Departments of Pediatrics and Cell & Molecular Biology, Children’s Memorial Hospital and the Robert H. Lurie Comprehensive Cancer Center, Northwestern University, Chicago, IL [email protected]

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Contributors

Jeffrey S. Dome Division of Oncology, Center for Cancer and Blood Disorders, Children’s National Medical Center, 111 Michigan Avenue NW, Washington, DC 20010, USA [email protected] Carolyn A. Felix Division of Oncology, Department of Pediatrics, The Children’s Hospital of Philadelphia, University of Pennsylvania School of Medicine, Colket Translational Research Building, Room 4006, 3501 Civic Center Blvd, Philadelphia, PA [email protected] Lia Gore The University of Colorado Cancer Center and The Center for Cancer and Blood Disorders, The Children’s Hospital, University of Colorado Denver, Pediatrics Mail Stop 8302, P.O. BoxÂ€6511, Aurora, CO 80045, USA [email protected] Richard Gorlick Department of Pediatrics, Pediatric Hematology/Oncology, The Children’s Hospital at Montefiore and the Albert Einstein College of Medicine, 3415 Bainbridge Avenue, Rosenthal 3rd Floor, Bronx, NY 10467, USA [email protected] Richard J. Gilbertson Neurobiology and Brain Tumor Program, St. Jude Children’s Research Hospital, Memphis, TN, USA [email protected] Thomas G. Gross Division of Hematology/Oncology/BMT, The Ohio State University College of Medicine, 700 Children’s Drive, Columbus, OH 43205, USA [email protected] Peter J. Houghton Department of Director, Center for Childhood Cancer, The Research Institute Nationwide Children’s Hospital, 700 Children’s Drive, Columbus, OH 43205 [email protected] Ganjam V. Kalpana Department of Molecular Genetics and Albert Einstein College Cancer Center, Albert Einstein College of Medicine of Yeshiva University, 1300, Morris Park Ave., Ullman 821, Bronx, NY 10461, USA [email protected]

Contributors

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Jennifer O’Hara Lauchle University of California, Helen Diller Family Cancer Research Building (Optional), 1450 3rd Street, Room 264 (Lauchle) and 265 (Braun), San Francisco, CA 94158, USA [email protected] A. Thomas Look Department of Pediatric Oncology, Dana-Farber Cancer Institute and Children’s Hospital Boston, 44 Binney Street, Boston, MA 02115, USA and Harvard Medical School, Boston, MA, USA [email protected] Yaël P. Mossé Division of Oncology, Department of Pediatrics, Children’s Hospital of Philadelphia, University of Pennsylvania, 3615 Civic Center Blvd., ARC 907C, Philadelphia, PA, USA [email protected] Rob Pieters Department of Pediatric Oncology and Hematology, Erasmus MC, Sophia Children’s Hospital, Room Sp2456, Dr Molewaterplein 60, 3015 GJ Rotterdam, The Netherlands [email protected] Ian F. Pollack Department of Neurosurgery, Children’s Hospital of Pittsburgh, 3705 Fifth Avenue, Pittsburgh, PA, USA [email protected] Scott L. Pomeroy Department of Neurology, Children’s Hospital Boston, 300 Longwood Avenue, Enders 270 , 02115 Boston, MA, USA [email protected] Yaddanapudi Ravindranath Children’s Hospital of Michigan, 3901 Beaubien Boulevard, Detroit, MI 48201, USA [email protected] Donald Small Sidney Kimmel CCC at Johns Hopkins, Bunting-Blaustein Cancer Research Building, 1650 Orleans Street, CRB I-252, Baltimore, MD 21231, USA [email protected]

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Contributors

Stephen X. Skapek Pediatric Hematology/Oncology, The University of Chicago Comer Children’s Hospital, 900 E. 57th Street, Chicago, IL 60637, USA [email protected] Jeffrey W. Taub Division of Hematology/Oncology, Children’s Hospital of Michigan, 3901 Beaubien Boulevard, Detroit, MI 48201, USA and Department of Pediatrics, Wayne State University School of Medicine, Detroit, MI 48201, USA and Developmental and Therapeutics Program, Barbara Ann Karmanos Cancer Institute, 4100 John R Street, Detroit, MI 48201, USA e-mail: [email protected] Jeffrey A. Toretsky Departments of Oncology and Pediatrics, Lombardi Comprehensive Cancer Center, Georgetown University, 3970 Reservoir Rd. N.W., Washington, DC 20057-1469, USA [email protected] Brigitte Widemann Pharmacology and Experimental Therapeutics Section, National Cancer Institute, Pediatric Oncology Branch, 10 Center Drive, 10-CRC, Room 1-5750, MSC 1101, Bethesda, MD 20892, USA [email protected]

Part I

Hematologic Malignancies

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The Emerging Era of Targeted Therapy in Childhood Acute Lymphoblastic Leukemia William L. Carroll and Rob Pieters

One of the most fundamental goals of modern cancer research is to develop more effective therapies that specifically target the cancer cell while sparing normal cells from the collateral damage that is common to conventional therapies. The cornerstone of current cancer treatment depends on drugs associated with a very narrow therapeutic index in that the effective dose and the toxic dose frequently overlap. While progress in pediatric oncology, specifically, improved cure rates for the most common childhood malignancy, acute lymphoblastic leukemia (ALL), has outpaced improvements in other cancer subtypes, treatment for ALL still relies on conventional cytotoxic agents thereby exposing children to considerable short- and long-term side effects. Optimally, targeted therapy would converge on a specific lesion or pathway in the cancer cell, not shared by normal cells, that is essential to the maintenance of the population of leukemia cells. In order to be most effective, the agent must have efficient access to the biological target to maximize tumor kill. Fortunately, in the past few years, a spectrum of agents have been, or are currently being developed, that target cancer cells with increasing specificity. With the initial development of monoclonal antibodies to specific leukocyte antigens, it became clear that leukemia cells display a unique profile of surface proteins shared by a minority of normal cells. These antigens have proved to be useful targets for therapeutic antibodies. Likewise, breakthroughs in methods to analyze gross chromosome structure and the subsequent introduction of recombinant DNA technology led to the recognition that all human cancers were the result of disrupted biological pathways due to somatic, and sometimes germline, alterations in DNA copy number (e.g., amplifications, deletions) and/or structure (translocations, mutations, etc.). Thus, there are many examples of where altered biological pathways that drive cancer initiation and maintenance can be traced back to these sentinal genetic lesions.

W.L. Carrollâ•›(*) New York University Cancer Institute, 522 First Avenue, Smilow 1201, New York, NY 10016, USA e-mail: [email protected]

P.J. Houghton and R.J. Arceci (eds.), Molecularly Targeted Therapy for Childhood Cancer, DOI 10.1007/978-0-387-69062-9_1, © Springer Science+Business Media, LLC 2010

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W.L. Carroll and R. Pieters

The identification of the specific genes and pathways involved in transformation and their subsequent validation in various experimental models now provide the first bona fide molecular targets. These studies have also determined that the discovery of a somatically mutated gene in a cancer cell does not necessarily indicate that it will prove to be a useful target, and subsequent analysis to distinguish “driver” from “passenger” mutations will prove key to the prioritization potential targets (Frohling etÂ€ al. 2007). This complex process is now even more daunting with the massive amount of data coming from more recent advances in high throughput sequencing, proteomic approaches, epigenetic profiling, and analysis of non-coding RNAs. While many approaches to targeted therapy are being considered, this overview will focus on those targets and their corresponding targeted agents that are currently in clinical trials that best illustrate the challenges and opportunities for this, the next generation of clinical trials in ALL.

The BCR/ABL Tyrosine Kinase and Ph+ ALL The t(9;22)(q34;q11) or Philadelphia chromosome was the first nonrandom chromosomal abnormality detected in cancer and is identified in 2% of childhood ALL samples and in approximately 20% of adult cases (Moorman etÂ€al. 2007; Schultz etÂ€al. 2007). The translocation results in the well known BCR-ABL protein fusion that is a constitutively active tyrosine kinase (Chan etÂ€al. 1987; Clark etÂ€al. 1988). Historically, the presence of the Ph+ genotype correlates with an extremely poor outcome and has been used in risk stratification (Secker-Walker etÂ€al. 1997; Schultz etÂ€al. 2007). While age, WBC, and early response may select for patients with a better outcome, stem cell transplantation (SCT) strategies have been usually considered for this subgroup of patients (Ribeiro etÂ€ al. 1997; Schrappe etÂ€ al. 1998). However, even if stem cell transplantation is performed after successful induction, many patients will still succumb to the disease. In the largest analysis to date in children, the 5Â€ year disease-free survival is 25±4% with chemotherapy alone compared to 65±8% when SCT is performed (Arico etÂ€al. 2000). The discovery of the biological function of the fusion protein as well as recognition that BCR-ABL was essential for tumor maintenance led to efforts to screen for inhibitors. The ATP binding pocket proved to be an attractive target for selective inhibition. Imatinib mesylate was the first such compound to enter clinical trials. Considerable preclinical data showed that it bound to the inactive confirmation of the kinase domain, blocked kinase activity, and inhibited proliferation of leukemia cells harboring the t(9;22). The results from early trials in chronic myelogenous leukemia led to considerable enthusiasm for integration into treatment strategies for Ph+ ALL (Druker etÂ€al. 1996, 2001, 2006). In phase I studies performed in children and adults, the drug was well tolerated and the majority of patients showed a response including a complete response rate of approximately 20% (Ottmann etÂ€al. 2002). However, these responses were short-lived with most patients showing

The Emerging Era of Targeted Therapy in Childhood Acute Lymphoblastic Leukemia

5

disease progression within weeks of their initial response. Studies incorporating imatinib into a backbone of chemotherapy soon followed these initial observations. A number of protocols using a combination of imatinib and chemotherapy in adults showed that this approach improved the induction rate, led to a lower disease burden, increased the number of patients proceeding to SCT, and most importantly, showed an impact on EFS and overall survival. For example, a study by Thomas and colleagues from MD Anderson Cancer Center investigated the efficacy of imatinib given concurrently with the hyper-CVAD regimen (Thomas etÂ€al. 2004). The CR rate on this study was 93% and the 3Â€year relapse free and overall survival rate was 62% and 55%, respectively, far better than historical controls. The Children’s Oncology Group COG initiated a study for Ph+ ALL (AALL0031) in 2002. Children and adolescents 1 to 21Â€years of age with Ph+ ALL were eligible after initial induction using standard chemotherapy. Imatinib was introduced in a stepwise fashion over five cohorts of patients to assess potential toxicity when used with aggressive chemotherapy. Patients with an HLA identical sibling proceeded to stem cell transplantation after two consolidation courses. Other patients received three additional intensification and re-induction blocks followed by 12 8-week cycles of maintenance chemotherapy for a total protocol duration of just over 2Â€ years. Patients in cohort one received 42Â€ days of imatinib before maintenance whereas patients in cohort 5 received 280Â€days. All groups received 336Â€days of imatinib in maintenance and patients who received SCT had 6Â€months of imatinib post transplant. Imatinib was well tolerated in the context of aggressive chemotherapy on AALL0031. There was modest ALT elevation noted in maintenance that led to a shortening of the duration of imatinib from 21 to 14Â€days for each month of maintenance. In addition, there was an increase in grade III and IV neutropenia during re-induction block 2 and a lower total WBC in consolidation. The 3Â€year EFS of patients on cohort 5 who received continuous imatinib was 80.5±11.2%, well above the 35.0±4.4% EFS reported on previous cooperative group studies. Twenty one patients received matched sibling transplants, and there was no difference in outcome among those receiving chemotherapy versus SCT. Furthermore, end induction minimal residual disease was not prognostic in those patients exposed to continuous imatinib indicating that targeted therapy can overcome the adverse prognostic significance of slow early response to chemotherapy. These striking results, while early, are extremely encouraging and represent an example of the promise of targeted therapy. Has been well documented that imatinib resistance is most commonly due to the development of BCR-ABL mutants (usually in the P loop or ATP-binding site) that alter affinity for imatinib (Branford etÂ€al. 2003; O’Hare etÂ€al. 2007). Newer generation tyrosine kinase inhibitors such as dasatinib and nilotinib, among others, inhibit such mutants, show more potent suppression of BCR-ABL kinase activity, bind to both the inactive and active conformation of BCR-ABL (e.g., dasatinib), and inhibit important collateral pathways such as those involving Src that are known to play a role in Ph+ ALL (Shah etÂ€al. 2004; Weisberg etÂ€al. 2005; Talpaz etÂ€al. 2006). Preliminary data from an ongoing European phase I/II study with dasatinib show promising activity in Â�children with

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Ph+ ALL who are refractory or intolerant to imatinib (Zwaan etÂ€ al. 2008). Therefore, dasatinib is being used in the current COG study (AALL0622) and is integrated earlier in treatment during induction to maximize early tumor kill.

The FLT-3 Pathway in MLL Rearranged Infant ALL Infants under 1Â€year of age constitute an especially high risk subgroup of patients with ALL. The primary biological basis for this association is that leukemic blasts from such children harbor an 11q23 rearrangement involving the MLL gene, usually t(4;11), t(11;19) or t(9:11) (Silverman 2007). The 6Â€ year event free survival (EFS) for these infants ranges from 22% to 43% and aggressive attempts to improve outcome using augmented chemotherapy including stem cell rescue have largely failed (Pui etÂ€ al. 2002; Hilden etÂ€ al. 2006; Pieters etÂ€ al. 2007). Thus, novel approaches are clearly warranted. The FMS-like tyrosine kinase-3 (FLT-3) is a class III receptor tyrosine kinase, a family that includes the c-KIT and platelet derived growth factor (PDGF) receptors (Agnes etÂ€al. 1994) (Fig.Â€1). FLT-3 is expressed in almost all cases of AML and B-lineage ALL, and up to a third of T ALL (Carow etÂ€al. 1996; Rosnet etÂ€al. 1996).

FLT3L-Binding

Extracellular Domain

Transmembrane Domain Juxtamembrane Domain Kinase Domain

Internal Tandem Duplications Activation Loop Mutations P

P

Fig.Â€1â•… The FLT-3 Receptor. The FLT-3 receptor contains an extracellular domain consisting of five immunoglobulin like domains. The juxtamembrane portion is a target for tandem duplications. Two kinase domains are linked through a tyrosine kinase insert. The kinase domain is mutated in hematological malignancies and most mutations are single point substitutions with insertions and deletions occuring less frequently. Binding of FLT-3 ligand leads to receptor dimerization and phophorylation of the kinase domain that triggers the activation of downstream pathways. (Revised based on reference (Stirewalt and Radich 2003))

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Constitutive activation of FLT-3 is observed through somatic mutation and/or a through co-expression of the FLT-3 ligand as part of an autocrine loop (Brasel etÂ€al. 1995; Drexler 1996; Nakao etÂ€al. 1996). Activating mutations of FLT-3, either in the juxtamembrane region or kinase domain, are seen in 22% of childhood AML samples but are rare in childhood ALL cases (Schnittger etÂ€al. 2002; Thiede etÂ€al. 2002). Juxtamembrane mutations are characterized by internal tandem duplications (FLT/ITD) and insertion/deletion mutations. Importantly, FLT/ITD constitutes an adverse prognostic variable in AML (Meshinchi etÂ€al. 2001). High level mRNA and protein expression of FLT-3 is observed in infant and childhood ALL with rearrangements of MLL and cases associated with hyperdiploidy – two subtypes with distinctly different outcomes (Armstrong etÂ€ al. 2002; Yeoh etÂ€al. 2002). FLT-3 mutations are absent in most subtypes of childhood ALL except for MLL-rearranged and hyperdiploid cases where up to 18 and 25% of samples, respectively, show either kinase mutations (MLL and hyperdiploid) and/ or juxtamembrane mutations (hyperdiploid) (Armstrong etÂ€al. 2004; Taketani etÂ€al. 2004). Other studies showed a much lower incidence of FLT-3 mutations in these subtypes and that the level of expression may be related to outcome in MLL rearranged ALL (Stam etÂ€ al. 2007a, b). Finally, several studies have demonstrated constitutive phosphorylation of FLT-3 in MLL rearranged infant ALL and hyperdiploid cases even in the absence of mutation suggesting an autocrine loop (Brown etÂ€al. 2005; Stam etÂ€al. 2005). Given the importance of FLT-3 in childhood leukemia, there has been great interest in exploring the therapeutic potential of FLT-3 inhibitors. A number of such agents have been developed including CEP-701 (lestaurtinib), SU112248 (sunitinib malate), and PKC412 (midostaurin) among others (Stirewalt and Radich 2003). Lestuartinib is of particular interest because it is currently being evaluated in two Children’s Oncology Group (COG) trials for MLL rearranged infant ALL, as well as, relapsed FLT-3 mutant AML. Preclinical work has demonstrated the therapeutic potential of this agent (Brown etÂ€ al. 2005, 2006). Phase II trials in adults with refractory/relapsed AML demonstrate tolerability and clinical responses, especially in patients with FLT-3 mutant AML (Smith etÂ€al. 2004). A phase I trial in children with heavily pretreated neuroblastoma (lestuartinib also inhibits the Trk neurotropin receptors) has also demonstrated the feasibility of administration. It is noteworthy that preclincal studies with combination chemotherapy show that synergism is achieved when chemotherapy is delivered first followed by FLT-3 inhibition with lestuartinib while the reverse sequence is antagonistic (Brown etÂ€ al. 2006). This important observation underscores the need for careful preclinical evaluation when combining conventional chemotherapy with biologically targeted agents. COG AALL0631 will test whether FLT-3 inhibition with lestuartinib administered on a backbone of chemotherapy will improve outcome for children with MLLrearranged ALL. The trial has a limited institution safety phase where two cohorts of children (<90Â€days and ³90Â€days) will receive lestuartinib with chemotherapy, and after this portion of the study is completed, the full randomized study will commence group wide.

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A second interesting FLT3 inhibitor is midostaurin. This drug was identified in 2002 and was been shown to be active against MLL rearranged ALL cells in a mouse models (Weisberg etÂ€al. 2002; Armstrong etÂ€al. 2003). Also, midostaurin is differentially cytotoxic to primary ALL cells with MLL rearrangements derived from infants compared to cells from other ALL patients (Stam etÂ€al. 2005). In these studies, target validation was shown by dephosphorylation of constitutively active FLT3. Clinical studies in refractory/relapsed adult AML have demonstrated moderate clinical activity of midostaurin as a single drug (Stone etÂ€al. 2005). In combination, these data have led to the design of a phase I/II study with midostaurin in children with a relapse of MLL rearranged ALL in Europe.

The Notch Pathway in T-ALL The Notch family of proteins are transmembrane receptors for cell surface ligands of the Delta-Serrate-Lag2 (DSL) family (Demarest etÂ€ al. 2008). There are four members of the Notch family and structurally they exist as heterodimers (Fig.Â€2). The extracellular domain has an extended domain of epidermal growth factors repeats. Two regions at the C terminus of the extracellular domain, a cysteine rich

ICN MAML

EGF-like repeats

Lin12/Notch repeats

CSL

Myc

AKT PEST domain

mTOR

Heterodimerization domain Transmembrane domain

Notch intracellular domain

PTEN

HES1

SCF FBXW7

Proteosome Degradation

Fig.Â€2â•… The Notch Pathway. The Notch receptor contains an extracellular domain with a series of epidermal growth factor receptor (EGF) repeats followed by a negative regulatory region composed of three Lin12/Notch repeats and a heterodimerization domain. Upon ligand binding two cleavage events (first via ADAMS like protease and the second by g secretase) occur leading to release of the intracellular notch domain (ICN) that functions as a transcription factor. ICN degradation is mediated by phophorylation of the PEST domain followed by degradation in the proteosome. ICN translocates to the nucleus where it binds a transcriptional repressor CSL (CBF-1/ Suppressor of Hairless/Lag1). This complex then recruits MAML (Mastermind like) transcriptional activators leading to transcriptional activation of key target genes. (Figure based on (Nefedova and Gabrilovich 2008; Palomero and Ferrando 2008))

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region and the heterodimerization domain (HD), function in a negative fashion to prevent receptor activation (Gordon etÂ€al. 2007). The intracellular domain functions as a transcription factor and includes a region that binds to the DNA-binding CSL protein, as well as a PEST domain that mediates the protein half life of intracellular Notch (Fortini and Artavanis-Tsakonas 1994; Oberg etÂ€al. 2001). Upon ligand binding the negative regulatory region of the extracellular domain undergoes a conformational change and exposes a proteolytic site that is cleaved by an ADAM metalloproteinase. This is followed by a second cleavage mediated by a g-secretase complex, thus releasing intracellular Notch (ICN) from the plasma membrane (Schroeter etÂ€al. 1998). ICN translocates to the nucleus where it associates with the CSL protein and converts a transcriptional repressor complex into an activator, thus activating downstream biological pathways. Notch activation is terminated by phosphorylation in the PEST region and binding of the F-box protein Fbw7 that is part of an SCF-E3 ubiquitin ligase complex (SCFFBX7) (Oberg etÂ€al. 2001; Fryer etÂ€al. 2004). Notch was first implicated in T-ALL through the description of a t(7;9) (q34;q34.3) that is observed in <1% of all patients with the disease (Ellisen etÂ€al. 1991). By virtue of the translocation, the 3¢ region of Notch is juxtaposed to the TCR-b locus resulting in constitutive expression of intracellular Notch. However, more recently, it was discovered that over 50% of T-ALL cell lines and samples show point mutations in the HD domain (26%), the PEST domain (12.5%), or both (17.7%) (Weng etÂ€al. 2004; Malecki etÂ€al. 2006). Mutations in the HD domain result in ligand independent proteolytic cleavage of Notch, whereas the PEST mutations prevent Fbx7 interaction, thus prolonging half life of the protein. Furthermore, 16% of T-ALL samples show mutations in Fbw7 leading to loss of recognition of Notch, thus leading to prolonged half life of the protein (Thompson etÂ€ al. 2007). Many samples revealed mutations at relapse that were not present at initial diagnosis(Weng etÂ€ al. 2004; Thompson etÂ€ al. 2007). The importance of Notch activation in the T-ALL transformation pathway is underscored by the observation that forced expression of ICN in murine hematopoietic cells leads to T-ALL in 100% of recipient mice (Pear etÂ€ al. 1996). In summary, these data collectively indicate that the activation of Notch plays a dominant role in T-ALL transformation and maintenance of the malignant phenotype. The fact that the Notch pathway is involved in both hematological and solid neoplasms prompted many investigators to examine approaches to inhibit the pathway. g-secretase complex mediated activation of Notch has drawn the most attention. The complex is composed of many proteins including presenelin and it processes many important proteins including b-amyloid precursor protein (Lleo 2008). There was great interest in g-secretase inhibitors (GSIs) for the treatment of Alzheimer’s disease and GSIs were already in clinical development at the time of the initial discoveries of widespread Notch mutations in T-ALL. Early work showed that the GSI, compound E, was able to elicit cell cycle arrest in 5 of 30 T-ALL cell lines prompting an early clinical trial (Weng etÂ€al. 2004). While the full results of this clinical trial have yet to be published, it was marked by the lack of clinical responses and severe gastrointestinal toxicity. A recent article showed that GSI

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restored dexamethasone sensitivity of T-ALL blasts and importantly glucocorticoid treatment protected mice from GSI-induced gut toxicity. Thus, these results support future trials incorporating glucocorticoids and GSIs in the treatment of refractory T-cell disease (Real etÂ€al. 2009). The downstream pathways modulated by Notch are just beginning to be discovered and provide additional nodal points for inhibition. c-Myc is a direct target of Notch, and in turn, c-Myc activates the m-TOR pathway in at least a subset of T-ALLs (Palomero etÂ€al. 2006; Chan etÂ€al. 2007). In vitro data showed synergistic inhibition of T-ALL growth with simultaneous application of GSI (Compound E) and the m-TOR inhibitor rapamycin. Notch also positively regulates the NF-kB signaling pathway and inhibition of NF-kB with bortezomib results in apoptotic cell death in T-ALL cell lines and combined therapy with a GSI (LY411575) and bortezomib causes synergistic cytotoxicity. Finally, work comparing GSI sensitive and resistant cell lines showed equivalent Notch inhibition indicating corruption of downstream pathways in GSI resistant lines. Notch is known to downregulate the tumor suppressor PTEN (mediated by the direct Notch target genes MYC and HES1) that in turn inhibits AKT (Palomero etÂ€al. 2007). The PI3K-AKT pathway drives many cellular processes including cell growth, proliferation, and survival. GSI resistant cell lines showed mutational loss of PTEN and this finding was also observed in 17% of T-ALL samples. In this context, inhibitors of the PI3K-AKT signaling pathway already in development may be excellent candidates for therapy.

Monoclonal Antibodies in the Treatment of ALL Leukemia-associated antigens are promising targets for therapy for ALL and the impact of monoclonal antibodies (mAbs) is just being realized in spite of the fact that they have been in development for decades. While not truly tumor-specific, expression is restricted to narrow subsets of normal hematopoietic cells, and therefore, they are unlikely to be associated with the broad collateral damage to normal tissues so characteristic of conventional chemotherapy. Therapeutic Mabs, while of murine origin, are frequently engineered so that the bulk of the murine backbone is replaced with human immunoglobulin regions. mAbs can be delivered in an unconjugated format or linked to conventional agents, immunotoxins, or radioactive elements. In principle, unconjugated Mabs may be more safely integrated into chemotherapy regimens. Rituximab, directed against the CD20 antigen, was the first mAb approved by the Food and Drug Administration. It is a chimeric, unconjugated antibody whose mechanism of action depends on antibody and complement dependent cell-mediated cytotoxicity (Harris 2004). Rituximab also induces direct tumor cell apoptosis. The greatest experience with rituximab has been in adult indolent and aggressive B-cell non-Hodgkins lymphoma where it provides significant advantage over chemotherapy alone (Feugier etÂ€al. 2005; Pfreundschuh etÂ€al. 2006). In addition, it has

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been used in children with recurrent/refractory B-cell NHL and mature B-cell ALL with encouraging results (Griffin etÂ€al. 2009). Expression of CD-20 is associated with a poor prognosis in adult ALL but no such relationship is seen in pediatric ALL (Jeha etÂ€al. 2006; Thomas etÂ€al. 2008). CD-20 is expressed in only 50% of pediatric ALL but a recent study indicates that the antigen is upregulated during induction such that CD-20 positivity was 45% in initial samples but increased to 81% in residual blasts at end-induction (Dworzak etÂ€al. 2008). Moreover the relative level of expression is also increased. Thus, a clinical trial could be designed to incorporate rituximab for those with high levels of MRD at end induction. Given the prognostic significance of CD-20 expression in adult ALL, many trials have incorporated rituximab into chemotherapy protocols. Thomas etÂ€ al. reported their experience with chemoimmunotherapy with Hyper-CVAD and rituximab in adult Burkitt and Burkitt Type lymphoma and ALL(Thomas etÂ€ al. 2006). Overall 3Â€year survival (89%) and event free survival (80%) was superior to their experience with hyper-CVAD alone. Experience in children is limited to case reports and small series. Morris etÂ€al. described successful remission induction using rituximab alone in a patient with multiply relapsed ALL (Morris and Vora 2007). An additional three children with refractory ALL have been treated with rituximab-based chemoimmunotherapy with encouraging results (Claviez etÂ€al. 2006). CD-22 is another B-cell surface antigen that is expressed in over 90% of children with ALL (Raetz etÂ€al. 2008). Epratuzumab is a humanized mAb that has also shown encouraging results in adult indolent and aggressive NHL and has been used in combination with rituximab (Carnahan etÂ€ al. 2003; Leonard etÂ€ al. 2005). The Children’s Oncology Group has completed a pilot protocol using epratuzumab in combination with chemotherapy for children with ALL in first or later marrow relapse (Raetz etÂ€al. 2008). Therapy started with a 2Â€week window of epratuzumab alone administered twice weekly followed by four weekly doses combined with standard re-induction chemotherapy. Effective targeting of CD-22 was validated in 10 of 11 patients and epratuzumab was well-tolerated. Grade 1 and 2 infusion reactions were observed in 10 of 15 patients during the reduction phase but quickly resolved with temporary stopping of the infusions and administration of steroids and/or meperidine. All patients tolerated the rest of the initial as well as additional infusions. Nine patients achieved a complete remission in this highly refractory population and seven were MRD negative at end induction. A larger COG study using epratuzumab twice a week during induction for first early marrow relapsed ALL is underway. Alemtuzumab is another humanized mAb that recognizes CD52, which is expressed on most B and almost all T malignancies (Xia etÂ€al. 1993; Frampton and Wagstaff 2003). It has been approved for use in chronic lymphocytic leukemia and is also part of myelablative conditioning regimens. Limited experience with single agent alemtuzumab in adult ALL has been disappointing (Tibes etÂ€al. 2006). A COG phase II trial in children with ALL in second or greater relapse was suspended early due to low accrual, but only one of the 13 patients showed a significant response (Personal communication, Anne Angiollilo). Alemtuzumab is

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associated with a higher number of infectious complications that may limit its use in chemoimmunotherapy protocols. Gemtuzumab ozogamicin (GO) is a humanized anti-CD33 mAb that has significant activity in relapsed AML and is currently being evaluated as part of frontline therapy for that disease (Sievers etÂ€al. 2001). The CD33 antigen is expressed on 90% of AML samples but is restricted to 15 to 20% of ALL (Putti etÂ€al. 1998; Sievers etÂ€al. 2001). In vitro and inÂ€vivo mouse model activity has been observed for GO in CD-33+ ALL and case reports demonstrate that the agent has activity either alone or in combination with additional agents in relapsed ALL (Balduzzi etÂ€al. 2003; Cotter etÂ€al. 2003; Zwaan etÂ€al. 2003; Golay etÂ€al. 2005). GO is associated with hepatotoxicity and its use following BMT may be problematic. Moreover, its restriction to a small minority of ALL cases will limit its application to ALL in the future. CD-19 is a B lineage antigen that is expressed earlier in differentiation when compared to CD-22 and CD-20 and is expressed in almost all B-precursor ALLs. Many immunotherapeutic strategies have been developed including the use of unmodified antibodies, antibody-drug, and immunotoxin combinations as well as bi-specific antibodies (Grossbard etÂ€al. 1993; Rowland etÂ€al. 1993; Molhoj etÂ€al. 2007; Horton etÂ€al. 2008). Phase I studies have demonstrated safety but shortlived responses (Hekman etÂ€al. 1991). A particularly novel approach has focused on the development of bi-specific antibodies to promote the engagement of T-cells. A recent report documented the clinical utility of blinatumomab, a CD-19/CD-3 single chain bi-specific T cell engager, in patients with relapsed B NHL (Bargou etÂ€al. 2008). Eleven major responses were seen among 38 patients including four complete responses. The doses required to achieve these responses were far below those used for conventional mAb therapy highlighting the extraordinary lytic potential of cytotoxic T cells. This agent is currently being studied for use in ALL.

Cancer Stem Cells in ALL The existence of a small population of self-renewing cancer cells that are responsible for the proliferation and dissemination of the bulk population has now been documented for both hematological and solid tumors. Failure to eradicate such cancer stem cells may explain the emergence of resistant disease. Although recent studies indicate that the assay used to functionally characterize such cancer stem cells may be imperfect, they have been documented in CML, AML, central nervous system tumors, breast cancer, and colon cancer (Bonnet and Dick 1997; Wang etÂ€al. 1998; Al-Hajj etÂ€al. 2003; O’Brien etÂ€al. 2007). Work in many laboratories to identify leukemic stem cells LSCs, also known as leukemia initiating cells (LICs) in CML and AML has defined a phenotype associated with self renewal. However, similar work in ALL has led to conflicting results about the surface phenotype of

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13

LSCs (Cobaleda etÂ€al. 2000; Cox etÂ€al. 2004; Castor etÂ€al. 2005). However, some of these discordant results may be due to the precision of the technique used to isolate these cells (e.g., different monoclonal antibodies), the functional assay (e.g., mouse strain used to demonstrate self renewal), and the biologic subtype of the samples being analyzed (e.g., Ph+ vs. ETV6-RUNX1). Available data favor that a CD34+/CD38− ALL subpopulation is capable of reconstituting hematopoiesis in a xenograft mouse model and that this population can be further enriched with CD133, an antigen present on many cancer stem cells (Cox etÂ€ al. 2009). There is controversy, however, about whether LSCs express CD19 and this is crucial since this cell surface receptor is the target for monoclonal antibody therapy as described above (Castor etÂ€al. 2005; Hong etÂ€al. 2008). A recent study by Cox etÂ€al. demonstrates the critical issue of targeting the LSC (Cox etÂ€al. 2009). In a series of experiments it was noted that CD133+/CD19− cells were enriched during long-term inÂ€ vitro culture and that this subpopulation led to engraftment of NOD/SCID animals. Furthermore, the engrafted cells showed high expression of CD10 and CD19 as well as a karyotype similar to the original bulk leukemia. Further refinement of leukemia stem cells could be facilitated with three color flow cytometry since most investigators have shown that these stem cells lack CD38 expression (e.g., CD133+/CD38−/CD19−). Most importantly, CD133+/ CD19− leukemia initiating cells in the study by Cox etÂ€al. were resistant to glucorticoids and vincristine in contrast to the unsorted bulk leukemia. Thus new strategies to identify unique pathways in leukemia stem cells and assays to determine their sensitivity to pharmacological treatment will be essential to prevent re-emergence of the leukemia in selected cases.

Summary The development of truly tumor-specific therapy in childhood ALL is now a reality with the success of imatinib and other agents directed against the BCR-ABL fusion product. It is expected that other examples will soon follow and the challenge is how best to evaluate the scores of new agents entering the drug pipeline based on an understanding of the biology of childhood ALL. Such agents will undoubtedly make their initial impact when used with conventional chemotherapy. The relatively uncommon incidence of childhood ALL will limit the number of new agents that can be evaluated in a timely fashion. It is, therefore, incumbent on investigators to develop a rigorous prioritization scheme based on preclinical inÂ€vitro data and inÂ€vivo mouse models to promote the most promising compounds. In addition, the development of novel clinical trial designs based using a high bar for success with early stopping rules for those agents who fail to meet a meaningful standard is clearly needed. Nonetheless, it is clear that the gap between laboratory and clinical biologists is as narrow as it has ever been and the future holds great promise for major advances in the field.

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Acknowledgementsâ•… This work was supported by the Penelope London Foundation, the Friedman Fund for Childhood Leukemia, and the Walter Family Pediatric Leukemia Fund. The authors thank Drs. Teena Bhatla and Elizabeth Raetz for review of the manuscript.

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Molecular Targeted Therapies in T-Cell Acute Lymphoblastic Leukemia Alejandro Gutierrez and A. Thomas Look

Introduction T cell precursors arise from hematopoietic progenitors in the bone marrow and migrate to the thymus, where they undergo a series of proliferation and differentiation steps that include the somatic recombination of T cell receptor (TCR) gene loci (Market and Papavasiliou 2003). TCR gene rearrangements are followed by positive and negative selection steps that allow the survival of T cells only if their TCR functions appropriately within the context of an individual’s immune microenvironment. This highly regulated developmental process results in the generation of a population of T cells with a wide range of somatically acquired T cell receptor variation, which forms the foundation of a fully competent adaptive immune system that can respond to a countless variety of foreign antigens. Genetic alterations involving oncogenes or tumor suppressors can result in the aberrant proliferation, differentiation arrest, and clonal expansion of T cell precursors that is characteristic of T cell acute lymphoblastic leukemia (T-ALL). T cell lymphoblastic malignancies can be characterized based on the pattern of surface antigen expression in T cell lymphoblasts in relation to the pattern of surface antigen expression that occurs in normal thymocyte development (Reinherz and Schlossman 1980; Rothenberg and Taghon 2005; Staal et al. 2001). The earliest T cell precursors migrate from the bone marrow to the thymus, where they proceed through a series of so-called double-negative (CD4-, CD8-) differentiation stages, during which rearrangements of the T-cell receptor d, g and b genes occur. This is

A.T. Lookâ•›(*) Department of Pediatric Oncology, Dana-Farber Cancer Institute and Children’s Hospital Boston, 44 Binney Street, Boston, MA 02115, USA and Harvard Medical School, Boston, MA, USA e-mail: [email protected] P.J. Houghton and R.J. Arceci (eds.), Molecularly Targeted Therapy for Childhood Cancer, DOI 10.1007/978-0-387-69062-9_2, © Springer Science+Business Media, LLC 2010

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followed by the intermediate single-positive (CD4+, CD8-, surface CD3-), and the subsequent double-positive (CD4+, CD8+) stage, during which TCR a rearrangements occur. Intrathymic T cell development ends with the generation of singlepositive CD4+ or CD8+ T cells that migrate outside the thymus, although additional differentiation steps occur in T cells upon antigen recognition. T-ALL accounts for 10 to 15% of acute lymphoblastic leukemias in children, and it is most common in older children and adolescents. This disease typically presents with distinctive clinical features, including very high numbers of lymphoblasts in the peripheral blood, involvement of the central nervous system, and an anterior mediastinal mass. T cell lymphoblastic lymphoma is a closely related malignancy that typically presents with a thymic anterior mediastinal mass and is differentiated from T-ALL based on the degree of bone marrow involvement, with greater than 25% lymphoblasts in the bone marrow defining T-ALL. These diseases exhibit a high degree of similarity in morphology, immunophenotype, and response to therapy, and are generally thought to represent different clinical presentations of the same disease (Cairo etÂ€al. 2005). Most of the insights into the molecular pathogenesis of T-cell lymphoblastic malignancies have come through the study of T-ALL rather than T cell lymphoblastic lymphoma, probably due to the greater ease of obtaining primary tumor specimens. This chapter is focused on the key aspects of the molecular pathogenesis of T-ALL that are most relevant to the development of molecular targeted therapies for this disease.

Oncogenic Transcription Factors Many of the early insight into the pathobiology underlying T-ALL came through the study of genes affected by recurrent chromosomal translocations. In contrast to B-precursor ALL, where recurrent translocations often lead to the expression of a fusion gene product, T-ALL translocations typically lead to the aberrant overexpression of structurally intact transcription factors, due to the placement of these genes under the control of regulatory elements that are highly active in T cell precursors, such as the gene regulatory elements that normally drive the expression ofÂ€T-cell receptor (TCR) genes. Most often, the transcription factors involved play prominent roles in normal hematopoietic development. Oncogenic transcription factors dysregulated by this mechanism in T-ALL include basic region helix-loophelix (bHLH) genes such as TAL1, TAL2, LYL1, and MYC, cysteine-rich genes such as LMO1 and LMO2, and homeodomain genes such as HOX11 and HOX11L2. Furthermore, although chromosomal translocations involving these genes are present in only 25% of cases of T-ALL, gene expression profiling has identified overexpression of these genes in 80% of cases (Ferrando etÂ€al. 2004; Ferrando and Look 2003), strongly suggesting that the dysregulated expression of these proto-oncogenic transcription factors plays a fundamental role in the molecular pathogenesis of most cases of T-ALL.

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Although of great biologic interest due to their central role in the pathobiology of T-ALL, dysregulated transcription factor activity, which relies on intracellular protein–protein and protein–DNA interactions, has been difficult to target with currently available pharmacologic strategies. Small molecules with favorable pharmacologic properties can readily be designed to target intracellular proteins whose activity relies on small hydrophobic pockets, such as enzymes. However, it is estimated that only about 12% of the proteins encoded by the human genome are “druggable” by small molecules with current technology, due to structural and mechanistic considerations (Hopkins and Groom 2002; Hopkins and Groom 2003; Russ and Lampel 2005); most transcription factors are currently considered “undruggable.” On the other hand, antibodies can be used as therapeutic agents to inhibit protein–protein interactions and thus have a much broader range of potential targets; however, antibodies generally cannot cross the cell membrane, and are thus poorly suited as targeted modulators of intracellular proteins (Verdine and Walensky 2007). However, recent advances in peptide medicinal chemistry include the development of chemically stabilized alpha-helical peptides that can successfully target intracellular protein–protein interactions and that are taken up in vivo by intact cells (Gavathiotis etÂ€al. 2008; Walensky etÂ€ al. 2004). This strategy may allow the development of specific therapies targeting dysregulated oncogenic transcription factor activity in the human leukemias.

NOTCH1 The NOTCH1 Pathway Notch, which was initially identified by the characterization of a Drosophila melanogaster mutant strain with notched wings (Morgan 1917), plays central roles in the development of multicellular organisms, where it acts by regulating cell fate, proliferation, survival, migration, and differentiation (Lai 2004). The first human ortholog of Notch, NOTCH1, was initially identified as a partner gene in a t(7;9) translocation found in very rare cases of T-ALL, in which the expression of a truncated NOTCH1 protein is driven by T cell receptor b gene regulatory elements (Ellisen etÂ€ al. 1991). NOTCH1 plays central roles in T cell development, where it directs lymphoid progenitors toward a T cell fate at multiple stages of lymphoid development (Grabher etÂ€ al. 2006; Maillard etÂ€ al. 2006; Radtke etÂ€ al. 2004; Rothenberg and Taghon 2005). Additionally, constitutive NOTCH1 signaling transforms T cell precursors in experimental systems (ZweidlerMcKay and Pear 2004). Despite the very low frequency of translocations involving NOTCH1, activating mutations in the heterodimerization or PEST domains of the full-length NOTCH receptor have been found in greater than 50% of cases of T-ALL (Weng etÂ€al. 2004).

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NOTCH1 is a cell surface receptor with an unusual mechanism of action (reviewed in (Bray 2006; Grabher etÂ€al. 2006)). After its synthesis, the NOTCH1 protein undergoes cleavage by a furin-like protease into extracellular and transmembrane subunits that remain associated via noncovalent interactions at the cell membrane. The binding of ligand to the extracellular subunit of NOTCH1 triggers two additional cleavage events in the transmembrane subunit, the first catalyzed by ADAM-family metalloproteases and the second by the g-secretase enzyme complex, which consists of presenilin, nicastrin, PEN2, and APH1. This final g-secretase-mediated cleavage allows the release of the intracellular domain of NOTCH1, known as ICN, into the cytoplasm. The ICN then translocates to the nucleus, where it binds to the CSL/RBPJ transcription factor, leading to the displacement of transcriptional corepressors and the recruitment of coactivators, thus resulting in the transcriptional upregulation of target genes (Fig.Â€1). NOTCH1 activating mutations in T-ALL generally occur in two distinct regions of the protein, and result in the accumulation of nuclear ICN protein via distinct mechanisms. Missense mutations in the heterodimerization domain lead to constitutive proteolytic activation of ICN, whereas truncating mutations in the C-terminal PEST domain impair the degradation of nuclear ICN. Interestingly, mutations in

Fig.Â€1â•… Activation of NOTCH signaling via proteolytic cleavage and nuclear translocation of the intracellular NOTCH domain (ICN). Interaction with delta serrate ligand (DSL) stimulates proteolytic cleavage of NOTCH by metalloproteases and g-segretase. This leads to the release of the intracellular ICN domain, which translocates to the nucleus, displaces corepressors and recruits coactivators (MAM1), thereby converting CSL from a repressor to an activator of gene expression. Reprinted with permission from Armstrong and Look (2005)

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both regions of the protein frequently occur in cis in the same NOTCH1 allele, resulting in the synergistic activation of NOTCH1 signaling (Weng etÂ€ al. 2004). Prominent transcriptional targets of the NOTCH1 ICN include MYC, which has been shown to be critical for the growth-promoting effects of NOTCH1 in T-ALL cell lines (Palomero etÂ€ al. 2006; Sharma etÂ€ al. 2006; Weng etÂ€ al. 2006), while another NOTCH1 target, HES1, is a transcriptional repressor that is a critical mediator of the ICN-induced transcriptional repression of the PTEN tumor suppressor (Palomero etÂ€al. 2007), as discussed in more detail below.

Strategies for NOTCH1 Inhibition A number of strategies have been devised for the inhibition of NOTCH. Wild-type NOTCH1 signaling can be targeted at the receptor-ligand interaction, but such strategies are unlikely to be effective against T-ALL cells expressing mutant NOTCH1 because the mutant receptor undergoes ligand-independent receptor activation. Other strategies, including the development of monoclonal antibodies that lock NOTCH1 in an inactive conformation, are in preclinical development (reviewed in (Rizzo etÂ€al. 2008)). The identification of a requirement for enzymatic activity of the g-secretase complex for the proteolytic activation of ICN, both upon ligand binding and in the presence of activating NOTCH1 mutations, generated considerable excitement for the application of g-secretase inhibitors (GSI) in T-ALL. Small molecule inhibitors of g-secretase were developed prior to the discovery of NOTCH1 activation in T-ALL because the g-secretase complex also plays a central role in the proteolytic generation of the insoluble amyloid-b peptide that accumulates in the brain in Alzheimer’s disease (reviewed in (Gotz and Ittner 2008). Interestingly, toxicity to normal T cells was found to be a toxicity of GSI during their preclinical development for the treatment of Alzheimer’s disease (Wong etÂ€al. 2004). Clinical trials of GSIs have been undertaken in T-ALL; however, the clinical utility of these inhibitors has been hindered by the development of serious gastrointestinal toxicity (DeAngelo etÂ€ al. 2006; Rizzo etÂ€ al. 2008). GSI therapy induces apoptosis of intestinal epithelial cells and goblet cell metaplasia (Milano etÂ€al. 2004), an on-target toxicity resulting from altered intestinal cell differentiation toward a goblet cell fate in the absence of NOTCH signaling (van Es etÂ€al. 2005). Efforts to overcome this toxicity through alterations in drug schedules and doses are the focus of current investigations. It has been reported that patients with breast cancer treated with a combination of tamoxifen and GSI had greatly alleviated intestinal toxicity (Rizzo etÂ€al. 2008). Furthermore, another report has demonstrated that the treatment with a combination of dexamethasone and GSI dramatically ameliorates the GSI-mediated intestinal toxicity in an animal model, while GSI therapy could simultaneously reverse NOTCH-mediated glucocorticoid resistance in human T-ALL cell lines (Real etÂ€ al. 2009). This important finding suggests that combination therapy with dexamethasone and GSIs could

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lead to synergistic antileukemic activity while simultaneously abrogating the dose-limiting gastrointestinal toxicity of GSIs, a possibility that should be tested in T-ALL clinical trials in the near future.

Resistance to NOTCH1 Inhibitors and Therapeutic Strategies to Overcome Resistance Despite their initial promise, g-secretase inhibitors are not effective against all T-ALL cell lines harboring activating NOTCH1 mutations. The mechanism mediating the resistance of T-ALL cells to g-secretase inhibition has been the target of intense investigation, and at least two distinct mechanisms of resistance have been identified. The first of these involves the inactivation of the FBW7 tumor suppressor, which appears to mediate resistance to g-secretase inhibition by impairing the downregulation of NOTCH1 signaling. FBW7 is an E3 ubiquitin ligase that targets both the ICN and MYC for proteasomal degradation (O’Neil etÂ€al. 2007; Thompson etÂ€al. 2007). Despite the effective inhibition of NOTCH1 activation at the cell surface by g-secretase inhibitor therapy, cells harboring inactivation of FBW7 apparently fail to effectively downregulate NOTCH1 signaling because the proteasomal degradation of ICN, and of its prominent target MYC, is impaired by the inactivation of the FBW7 E3 ubiquitin ligase. The inactivation of PTEN, an important tumor suppressor that negatively regulates PI3K-AKT signaling, has been identified as a second prominent mechanism mediating resistance to g-secretase inhibitor therapy (Palomero etÂ€al. 2007). In contrast to FBW7 mutations, the inactivation of PTEN does not appear to exert its effect by promoting ICN transcriptional activity. Under physiologic conditions, active NOTCH1 signaling leads to the transcriptional upregulation of HES1, which then acts as a transcriptional repressor of PTEN. Thus, in the setting of active NOTCH1 signaling, active PI3K-AKT signaling is promoted due to the transcriptional repression of PTEN (Fig. 2a). Upon inhibition of g-secretase activity, NOTCH1 signaling is inhibited, the transcriptional repression of PTEN is released, and the resultant upregulation in PTEN expression leads to the inhibition of oncogenic PI3K-AKT signaling. Thus, g-secretase inhibitor therapy in a cell with intact PTEN results in the inhibition of both NOTCH1 and PI3K-AKT signaling (Fig.Â€2b). However, deletions and inactivating mutations of PTEN have been identified in 36% of primary T-ALL samples, as discussed in more detail below. PTEN-negative T-ALL cells with NOTCH1 activating mutations maintain active signaling through both NOTCH1 and PI3K-AKT pathways (Fig.Â€ 2c). Upon effective g-secretase inhibitor therapy, NOTCH1 signaling is inhibited in PTEN-negative cells. However, PTEN expression cannot be upregulated in these cells despite the downregulation of HES1, and PI3K-AKT signaling remains active in PTEN-negative cells. The failure to downregulate PI3K-AKT signaling thus leads to resistance to GSI treatment in PTEN-negative cells (Fig.Â€ 2d). Interestingly, Palomero etÂ€ al. have shown that, although PTEN loss induces resistance to NOTCH1 inhibition, PTEN-negative

Molecular Targeted Therapies in T-Cell Acute Lymphoblastic Leukemia

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Fig.Â€ 2â•… PTEN inactivation leads to resistance to NOTCH1 inhibition but dependence on AKT signaling. (a) T-ALL lymphoblasts with activating mutations of NOTCH1 constitutively generate intracellular NOTCH1 (ICN), whose transcriptional targets include MYC and HES1. MYC appears to be a transcriptional activator of PTEN, but HES1-mediated repression predominates under ICN signaling conditions. Low expression of PTEN leads to incomplete inhibition of the PI3K-AKT pathway. (b) Inhibition of proteolytic release of ICN from the NOTCH1 receptor by g-secretase inhibitors (GSI) blocks ICN-mediated proliferative and survival signals, leading to cell cycle arrest and apoptosis. Additionally, the HES1-mediated repression of PTEN expression is relieved, and PTEN can therefore inhibit prosurvival signaling mediated by the PI3K-AKT pathway. Thus, PTEN-positive T-ALL cells with activating NOTCH1 mutations depend on NOTCH1 activity for survival and proliferation. (c and d). In the absence of PTEN, uninhibited AKT activation leads to aberrant prosurvival and proliferative signaling independent of NOTCH1 pathway activity, thus leading to resistance to NOTCH1 inhibition. PTEN-null T-ALL cells that are resistant to NOTCH1 inhibition appear to be dependent on PI3K-AKT pathway signaling. Reprinted with permission from Gutierrez and Look (2007)

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NOTCH1-mutant T-ALL cells are instead dependent on AKT signaling (Palomero etÂ€al. 2007). A number of inhibitors of the PI3K-AKT pathway are currently undergoing clinical development, and it seems likely that these will be particularly effective in T-ALL when used in combination with NOTCH1 inhibitors.

PTEN, PI3K-AKT, and mTOR The role of the PTEN tumor suppressor in T-ALL has been suggested by murine studies demonstrating that T-ALL develops in mice after the conditional deletion of PTEN in hematopoietic stem cells (Guo etÂ€al. 2008; Yilmaz etÂ€al. 2006). The tumor suppressor activity of PTEN is mediated in large part due to its role as a negative regulator of PI3K-AKT signaling (Chow and Baker 2006), an oncogenic signal transduction pathway activated by growth factor signaling and by a variety of oncogenic mutations in human cancer (Brugge etÂ€ al. 2007). Deletions of PTEN have recently been described in approximately 8% of primary T-ALL patient samples (Maser etÂ€ al. 2007; Mullighan etÂ€ al. 2007). Additionally, we have recently completed an analysis of PTEN as well as PI3K and AKT genes in primary T-ALL samples, and have identified the presence of genetic lesions in the PTEN-PI3KAKT pathway in 48% of diagnostic specimens from a series of 44 children with T-ALL (Gutierrez et al. 2009). Activation of signaling through the PI3K-AKT pathway, as a result of PTEN loss or the introduction of a constitutively active AKT transgene, has been shown to mediate resistance to NOTCH1 inhibition, but this appears to induce sensitivity to PI3K-AKT pathway inhibitors, as discussed in the section “Resistance to NOTCH1 Inhibitors and Therapeutic Strategies to Overcome Resistance” (Palomero etÂ€al. 2007). Additionally, evidence suggests that the mammalian target of rapamycin (mTOR), which is activated by the PI3K-AKT pathway, mediates glucocorticoid resistance in at least some ALL cells, and the inhibition of mTOR effectively reversed glucocorticoid resistance in T-ALL and B-precursor ALL cell lines (Wei etÂ€ al. 2006). Furthermore, combination therapy with a glucocorticoid and a g-secretase inhibitor has been shown to both reverse glucocorticoid resistance and to mitigate the serious gastrointestinal toxicity of g-secretase inhibitors (Real etÂ€al. 2009), as discussed in the section “Strategies for NOTCH1 Inhibition.” Taken together, these findings suggest that combination therapy with an inhibitor of PI3KAKT-mTOR signaling, a g-secretase inhibitor, and a glucocorticoid may represent a particularly effective therapeutic combination in T-ALL, a possibility that will likely be tested by future clinical trials.

Tyrosine Kinase Genes The ABL tyrosine kinase normally plays physiologic roles in the regulation of proliferation, cell adhesion and migration, and apoptosis (Sirvent etÂ€al. 2008). Translocations resulting in the expression of a BCR-ABL fusion oncoprotein represent the characteristic

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genetic lesion in chronic myeloid leukemia (CML) and also occur in B-precursor ALL. Compared to the wild-type ABL tyrosine kinase, BCR-ABL exhibits aberrant subcellular localization and constitutive kinase activity. The ability of imatinib, a small molecule inhibitor of BCR-ABL kinase activity, to provide long-term disease control in CML has revolutionized therapy for this disease, and imatinib also has activity against BCR-ABL-positive acute lymphoblastic leukemia. Despite the rarity of BCR-ABL translocations in T-ALL, amplified episomes containing NUP214-ABL fusion genes have been described in approximately 6% of adults and children with T-ALL (Graux etÂ€al. 2004). The ABL breakpoint in these cases occurs in intron 1, the same location as in BCR-ABL translocations, and these fusions impart constitutive ABL tyrosine kinase activity to the fusion oncoprotein. Therapy with tyrosine kinase inhibitors designed to target BCR-ABL, including imatinib, dasatinib, and nilotinib, inhibits proliferation and induces apoptosis in T-ALL cell lines harboring NUP214-ABL fusions (Quintas-Cardama etÂ€al. 2008). Furthermore, ABL amplifications and EML1-ABL fusions have also been described in rare cases of T-ALL (Bernasconi etÂ€al. 2005; De Keersmaecker etÂ€al. 2005). Taken together, these findings suggest that ABL kinase inhibitors may have therapeutic utility in a subset of cases of T-ALL, and clinical trials should be initiated to test this possibility. Mutational activation of JAK1 has recently been reported in 18% of T-ALL cases, and these mutations were associated with poor response to therapy (Flex etÂ€al. 2008). The JAK1 gene encodes a nonreceptor tyrosine kinase that associates with cytokine receptors and mediates the activation of STAT proteins upon cytokine receptor activation. JAK1 has been shown to play critical roles in lymphocyte development, and JAK1 knockout mice show a severely hypocellular thymus. JAK1 mutations identified in T-ALL lead to the ligand-independent activation of STAT signaling. A number of inhibitors of JAK/STAT signaling are currently under clinical development, and the determination of their clinical activity against JAK1mutated T-ALL will be of great interest.

RAS The RAS proto-oncogenes encode proteins that play central roles in mediating growth factor signaling, and missense mutations at codons 12, 13, or 61 are frequent events in human cancer. Mutant RAS proteins are potently oncogenic and lead to the induction of signaling though the MAP kinase, PI3K-AKT, and RAL-GDS pathways (Schubbert etÂ€ al. 2007). Mutations activating NRAS or KRAS have been identified in approximately 15% of T-ALL and B-precursor ALL cases (Liang etÂ€al. 2006; Perentesis etÂ€ al. 2004). Due to their role in the pathogenesis of numerous cancers, a number of targeted RAS inhibitors are under development, many of which target the post-translational modifications required for the generation of functional RAS proteins, including farnesylation, palmitoylation and geranylgeranylation. Furthermore, recent studies in KRAS-dependent cell lines derived from diverse tumor types revealed inhibition of the STK33 kinase as effective therapy against KRAS-dependent tumors, indicating the need for preclinical studies of STK33

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inhibitors in RAS-dependent T-ALL (Scholl et al. 2009). Targeted inhibitors of RAS, or of signal transduction pathways activated by RAS, including the MAP kinase and the PI3K-AKT pathways, may prove to have a role in the therapy of RAS-mutated T-ALL.

Conclusions Recent advances in our understanding of the molecular pathogenesis of T-ALL have uncovered a number of therapeutic targets in this disease, including proteins in the NOTCH and PI3K-AKT pathways. Despite initial challenges, and the early difficulties with gastrointestinal toxicity with the use of NOTCH inhibitors, remarkable recent advances have suggested rational combination therapies that may lead to both synergistic antileukemic activity and reduced toxicity. Our understanding of T-ALL oncogenic pathways almost certainly remains incomplete, and new advances should lead to the successful clinical application of molecularly targeted agents in T-ALL.

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Molecularly Targeted Therapy for Infant ALL Patrick A. Brown and Carolyn A. Felix

Rational combinations of molecularly targeted agents with synergistic conventional cytotoxic chemotherapeutic agents or, ultimately, with one another are urgently needed for infants with acute leukemia. Leukemia is the commonest malignancy during infancy, comprises 2.5 to 5% of ALL and 6 to 14% of AML in pediatrics overall, (Gurney etÂ€ al. 1999; Smith etÂ€ al. 1999a; Pui etÂ€ al. 1995; SEER Cancer Statistics Review 1975–2006) and represents a special leukemia subtype characterized by MLL (Mixed Lineage Leukemia) gene translocations. MLL translocations with heterogeneous partner genes, of which there are >60, (Meyer etÂ€al. 2009) are the primary molecular aberrations in infant ALL and infant AML alike; approximately 75 to 80% of infant ALL cases and myelomonocytic/monoblastic AML feature MLL translocations (Pui etÂ€al. 1995; Rubnitz etÂ€al. 1994; Robinson et al. 2009). Within infant ALL cases with MLL translocations, 70 and 13% involve the AF4 (ALL-1 fused gene from chromosome 4) or ENL (Eleven-nineteen leukemia) partner genes, respectively, (Pui etÂ€al. 2003) whereas the partner genes in AML are more diverse. In one recent infant ALL treatment study, these more common partner genes were associated with only approximately 30% 5-year event free survival, with a poorer outcome associated with the CD10− immunophenotype and younger age at diagnosis (Hilden etÂ€al. 2006). The treatment options for acute leukemia in infants have been limited to intensive chemotherapy and hematopoietic stem cell transplantation. A deeper biological understanding of MLL disease in infants that becomes translatable to better treatment options would be of utmost impact because the prognosis is dismal due to profound vulnerabilities to treatment-related toxicities unique to this population, frequent relapses, and inherent chemotherapy resistance as a consequence of the translocations. While infant ALL and AML are orphan diseases with annual incidences of 19 and 10 per million, respectively, MLL translocations occur in 15 to 20%

C.A. Felixâ•›(*) Division of Oncology, Department of Pediatrics, The Children’s Hospital of Philadelphia, University of Pennsylvania School of Medicine, Colket Translational Research Building, Room 4006, 3501 Civic Center Blvd, Philadelphia, PA e-mail: [email protected]

P.J. Houghton and R.J. Arceci (eds.), Molecularly Targeted Therapy for Childhood Cancer, DOI 10.1007/978-0-387-69062-9_3, © Springer Science+Business Media, LLC 2010

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of pediatric AML (Balgobind etÂ€al. 2009) and comprise 5 to 10% of acquired chromosomal rearrangements in pediatric ALL and adult ALL and AML (Pui etÂ€al. 2003). Nearly all current-era anticancer regimens specify chemotherapy with one or more topoisomerase II poisons (i.e., agents that convert native topoisomerase II to a cellular toxin by altering the topoisomerase II cleavage–religation equilibrium, causing DNA breakage); examples are etoposide, anthracyclines, dactinomycin, and mitoxantrone (Felix etÂ€al. 2006). Various secondary leukemias with balanced translocations are a potential untoward complication of these agents, the most common of which involve MLL (Smith etÂ€al. 1999b). The complex circuitry of relevant target aberrations comprising resultant heterogeneous MLL fusion oncoproteins and proteins with which they are associated (Liedtke and Cleary 2009) has been gradually unraveling since the initial discovery of MLL as the gene disrupted by the translocations approximately 18Â€years ago (Ziemin-van der Poel etÂ€al. 1991; Djabali etÂ€al. 1992; Gu etÂ€al. 1992; Tkachuk etÂ€al. 1992). Such observations form the basis for the current development of targeted therapies for these highly aggressive and usually incurable diseases. Infant ALL and AML are embryonal diseases that originate in utero from DNA damage to MLL that results in the formation of the translocations (Ford etÂ€al. 1993; Megonigal etÂ€al. 1998). The native MLL protein is a complex transcriptional regulator that undergoes proteolytic cleavage into amino and carboxyl terminal fragments with transcriptional repression and activation properties, which reassemble in a multiprotein complex (Yokoyama etÂ€al. 2002) that controls epigenetic modifications of nucleosomes and histones and maintains HOX expression (Hess 2004). Amino terminal structural domains include AT hook motifs, SNL motifs that direct subnuclear localization, and a cysteine-rich CXXC region that recognizes CpG dinucleotides similar to the CXXC region in DNA methyltransferase 1 (Hess 2004). The adjacent more central PHD mediates homodimerization and protein interactions (Hess 2004). The carboxyl terminal SET domain interacts with the SWI/SNF chromatin remodeling complex and has histone H3K4 methyltransferase activity (Hess 2004). MLL is important in cell cycle regulation; MLL proteolytic cleavage by Taspase1 (Takeda etÂ€al. 2006) and degradation by specific E3 ligases are critical for proper cell cycle progression (Liu etÂ€al. 2007). MLL translocations disrupt a breakpoint cluster region and fuse 5¢ MLL with the 3¢ portion of the partner gene, producing diverse fusion oncoproteins comprising the amino-terminus of MLL and the carboxyl-terminus of the partner protein, which cause leukemia in mice (Hess 2004; Meyer etÂ€al. 2006). N-MLL-Partner-C fusion proteins are believed to mediate leukemogenesis by altering transcription, (Hess 2004) possibly through the gain of function mechanisms (e.g., binding to transcriptional coactivators, fusion protein dimerization, etc.) since HOX expression is increased even though the MLL histone H3K4 transactivation activity of the SET domain is missing (Hess 2004; Armstrong etÂ€ al. 2002a; Ayton and Cleary 2003; Martin etÂ€ al. 2003). However, in murine models, sustained HoxA gene expression is necessary but insufficient for altered differentiation and clonal expansion, (Ayton and Cleary 2003) and levels of overexpression of rate-limiting Meis1/ Pbx cofactors vary with the partner genes (Wong etÂ€al. 2007).

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Until 2008, no molecularly targeted agents had been used in infant ALL. The pathways and target genes that are critically affected by MLL fusion oncoprotein transcriptional deregulation have been challenging to resolve and remain incompletely understood, and there has been considerable controversy surrounding the nature of the transformed MLL leukemia stem cell (Somervaille and Cleary 2006; Krivtsov etÂ€al. 2006; Wang etÂ€al. 2005). Therefore, in the current landscape of infant leukemia therapy, the molecularly targeted agents that have begun advancing through the drug development pipeline (FDA 2004) are predominantly at the preclinical inÂ€vitro and inÂ€vivo stages of development. The initial focus in bringing molecularly targeted agents to the clinic for this disease has been on compounds with demonstrable activity in other leukemias and cancers that might also silence cooperating events in MLL leukemogenesis, rather than on agents directly targeting the fusion oncoproteins and interacting factors. A major milestone was achieved when comprehensive preclinical analyses formed the basis for the recently opened COG AALL0631 clinical trial, which is testing the combination of chemotherapy and the FLT3 tyrosine kinase inhibitor lestaurtinib for MLL-rearranged infant ALL; this is the first trial to incorporate a signal transduction pathway targeted agent into frontline therapy for infant ALL. In this chapter, the limited targeted agents that have advanced to the clinic, of which there are now two, will be reviewed in detail and promising targets and molecularly targeted agents progressing through the drug development pipeline will also be addressed.

FLT3 Tyrosine Kinase Inhibition The receptor tyrosine kinase FLT3 (FMS-Like Tyrosine Kinase 3) has been strongly implicated in the pathogenesis of infant leukemia (Brown and Small 2004a; Levis and Small 2003). With the development of FLT3-targeted agents, the hypothesis that FLT3-targeted therapy can improve the outcome for infant leukemia is now being tested. FLT3 is aberrantly expressed in greater than 90% of AML and nearly 100% of B-lineage ALL (Brown and Small 2004a; Carow etÂ€al. 1996; Rosnet etÂ€al. 1996; Birg etÂ€ al. 1992). Microarray studies have shown that in primary samples from children with leukemia, the highest levels of FLT3 expression occur in cases of infant and childhood ALL with MLL gene rearrangements (Armstrong etÂ€al. 2002b; Yeoh etÂ€ al. 2002; Ross etÂ€ al. 2003). Nearly all leukemia cells also express FLT3 ligand (FL), leading to constitutive FLT3 phosphorylation and activation through autocrine, paracrine, or intracrine mechanisms (Zheng etÂ€al. 2004; Drexler 1996; Meierhoff etÂ€al. 1995; Brasel etÂ€al. 1995). FLT3 can also be activated through two types of activating mutation: internal tandem duplications (ITD) in the juxtamembrane region and point mutations (PM) within the activation loop of the kinase domain (Yamamoto etÂ€al. 2001; Abu-Duhier etÂ€al. 2001; Nakao etÂ€al. 1996). Both mutations result in ligand-independent FLT3 autophosphorylation and subsequent activation of downstream targets, including

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proteins in the STAT, MAP kinase, and AKT pathways, which are involved in proliferation, differentiation, and survival (Lavagna-Sevenier etÂ€ al. 1998; Zhang etÂ€al. 1999, 2000 Zhang and Broxmeyer 2000; Kiyoi etÂ€al. 1998, 2002). FLT3 mutations have been found in many adult and pediatric leukemias, most notably in AML, where they are the most common somatic genetic alteration (Brown and Small 2004a; Levis and Small 2003; Meshinchi etÂ€al. 2001). FLT3/ITD is present in approximately 25% of adult AML and 15% of pediatric AML. FLT3/PM occurs in 7 to 9% of both adult and pediatric AML. In childhood ALL, FLT3/ITD is rare (1%), but FLT3/PM occurs commonly in ALL with rearrangements of the MLL gene in infants and children, where FLT3/PM are present in approximately 15% of cases (Armstrong etÂ€al. 2004a; Taketani etÂ€al. 2004a). Constitutively activated FLT3 signaling has been shown in many model systems to contribute to the process of leukemogenesis, but constitutive FLT3 activation alone is insufficient to fully transform primary hematopoietic cells (Kelly etÂ€ al. 2002a, b; Li etÂ€al. 2008a). Furthermore, a number of clinical studies in adult and pediatric AML have shown that activating mutations, particularly FLT3/ITD, are associated with inferior prognosis (Brown and Small 2004a; Levis and Small 2003; Meshinchi etÂ€al. 2001, 2006). The demonstrated importance of FLT3 signaling in the pathogenesis of human leukemia has led to the development of FLT3-targeted agents. CEP-701 (lestaurtinib) is a highly selective small molecule FLT3 tyrosine kinase inhibitor (TKI) with excellent oral bioavailability and an IC50 of 3Â€nM (Levis etÂ€al. 2002). Lestaurtinib is selectively cytotoxic to primary pediatric AML samples with FLT3/ITD mutations (Brown etÂ€ al. 2004). Further, in AML cells, lestaurtinib exhibits scheduledependent synergy with multiple chemotherapeutic cytotoxic agents (Levis etÂ€al. 2004). Prior adult phase II trials of lestaurtinib monotherapy for relapsed/refractory FLT3 mutant AML demonstrated clinical activity with clearance of peripheral blasts and, less frequently, bone marrow responses; now lestaurtinib is being tested in combination with standard chemotherapy (Smith etÂ€al. 2004; Stone etÂ€al. 2005). Based on recent evidence that lestaurtinib is selectively cytotoxic against MLL rearranged infant and childhood ALL with high levels of constitutively activated FLT3 expression, (Brown etÂ€ al. 2005a) the Children’s Oncology Group (COG) is currently studying lestaurtinib in combination with chemotherapy in FLT3-mutant AML and infant ALL. Brown etÂ€al. reported the effects of a FLT3 inhibitor (lestaurtinib, or CEP-701) on primary pediatric ALL (Brown etÂ€ al. 2005a) and AML (Brown etÂ€ al. 2004) blasts. The ALL study examined 36 childhood ALL samples, including seven infant samples, with known cytogenetics, quantitative FLT3 expression determined by microarray analysis and/or Western blotting, and FLT3 genotyping. MTT cytotoxicity and annexin V binding (AVB) apoptosis assays were done at 48Â€hours using increasing doses of CEP-701. CEP-701 was shown to be selectively cytotoxic to samples with high FLT3 expression; within this group, MLL-rearranged and high hyperdiploid samples were especially sensitive to FLT3 inhibition. A significant linear correlation between the MTT and AVB assays in the CEP-701-sensitive samples (Nâ•›=â•›18) indicated that the predominant mechanism of cytotoxicity involved

Molecularly Targeted Therapy for Infant ALL

35

Fig.Â€1â•… Sensitivity of infant and childhood ALL samples to FLT3 inhibition is based upon marked overexpression of constitutively activated FLT3

apoptosis. To investigate the molecular basis for the variability in CEP-701 cytotoxicity, FLT3 immunoprecipitation and immunoblotting (IP/IB) were performed on CEP-701 untreated and treated samples in cases where there were sufficient cells (Fig.Â€1); five of the six insensitive samples did not express appreciable FLT3 levels. The one sample that did express a high level of FLT3 had no constitutive FLT3 phosphorylation. By contrast, all seven sensitive samples expressed high levels of FLT3, and FLT3 was constitutively activated. Moreover, all but one of the sensitive samples expressed wild-type FLT3; suggesting the possibility of autocrine activation of FLT3 by FL coexpression, as has been demonstrated in some cases of AML expressing wild-type FLT3 (Zheng etÂ€al. 2004). In all seven cases, FLT3 phosphorylation was potently inhibited by 50Â€ nM CEP-701. Thus, there appears to be a strong correlation between the cytotoxic response to CEP-701 and high-level constitutively activated FLT3 expression, suggesting that analyses of FLT3 expression and activation status may prospectively identify patients likely to benefit from FLT3 TKI. In the AML study, 44 cryopreserved diagnostic infant (Nâ•›=â•›4) and childhood (Nâ•›=â•›40) AML samples from the COG leukemia bank were studied. The FLT3 mutation status had been determined by PCR on genomic DNA isolated from the blasts at diagnosis (Meshinchi etÂ€al. 2001, 2003). Cases were selected to evaluate approximately equal numbers of FLT3/ITD, FLT3/PM, and FLT3/WT samples; investigators were blinded to FLT3 mutation status until CEP-701 activity was assayed. MTT assays indicated selective cytotoxicity of CEP-701 for FLT3/ITD

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samples compared to FLT3/PM or FLT3/WT samples. These studies provide preliminary data supporting the potential for FLT3 inhibitors for infant leukemia, although more samples will need to be studied. Since any molecularly targeted agent is unlikely to be curative as monotherapy in acute leukemia, how FLT3 inhibitors might be incorporated most effectively into standard chemotherapy regimens was also investigated. These studies have shown that lestaurtinib and chemotherapy agents consistently result in synergistic killing of FLT3/ITD+ AML and MLL-rearranged ALL cell lines and primary patient samples when used in the specific sequence of exposure to chemotherapy followed by lestaurtinib (Levis etÂ€al. 2004; Brown etÂ€al. 2006a). Other sequences of exposure (simultaneous and lestaurtinib followed by chemotherapy) are less synergistic and sometimes antagonistic due to the cell-cycle inhibition by lestaurtinib. The results of these experiments have informed the design of the ongoing adult clinical trials of the combination of chemotherapy and lestaurtinib, as well as the ongoing COG trials for pediatric patients (discussed below), illustrating the potential for well-designed preclinical laboratory studies to guide the clinical development of novel agents. Anti-FLT3 monoclonal antibodies represent another potential approach for targeting FLT3, including a fully humanized anti-FLT3 monoclonal antibody (IMC-EB10) that avidly binds FLT3 and blocks FL-mediated receptor activation (Piloto etÂ€al. 2005, 2006). Because the primary mechanism of FLT3 activation in infant leukemia is FL activation of the overexpressed wild type receptor, EB10 may be particularly effective in infant leukemia. Other potential advantages include inhibiting FLT3 more specifically than is possible with small molecule inhibitors, and eradicating FLT3-expressing leukemia cells through antibody-dependent cellular cytotoxicity (ADCC) and complement fixation independently of effects on FLT3 signaling. Thus, antibodies may prove efficacious in leukemias where FLT3 is expressed, regardless of the degree to which leukemia cell survival is FLT3 dependent. A NOD/SCID xenograft model has been used to explore the effects of FLT3targeted agents on the leukemic stem cell (LSC) in infant leukemia inÂ€vivo (Piloto etÂ€al. 2006). Leukemic blasts from a 3-month-old infant with MLL-rearranged ALL (MLL-AF4 fusion) and high level constitutively phosphorylated FLT3 expression, which also exhibited pronounced inÂ€ vitro cytotoxicity with lestaurtinib, were injected by tail vein (1â•›×â•›106 cells/mouse) into sublethally-irradiated 6-week-old NOD/SCID mice. The mice then were treated with subcutaneous injections of vehicle control vs. CEP-701 twice daily, or intraperitoneal injections of C225 (antiEGFR control antibody) vs. EB10 (anti-FLT3 antibody) three times weekly. After 14Â€ weeks, the mice were sacrificed and assessed by flow cytometry for human leukemia cell engraftment in the bone marrow. CEP-701 and EB10 significantly reduced the marrow engraftment of primary infant ALL cells in NOD/SCID mice. To further examine EB10 activity, a luciferase-GFP construct was packaged in a lentiviral vector and transfected into the SEM-K2 cell line, which was derived from a case of childhood MLL-AF4 ALL at relapse and has high level expression of activated wild-type FLT3. After sorting for GFP+ cells, 1â•›×â•›106 cells were injected into the tail vein of sublethally irradiated 6-week-old NOD/SCID mice. On day 11,

Molecularly Targeted Therapy for Infant ALL

37

inÂ€vivo imaging showed early bone marrow engraftment and a single IP EB10 injection was administered, resulting in marked reversal of established engraftment and prevention of further engraftment as demonstrated 3Â€days later by reimaging. Thus, FLT3 targeted therapy demonstrates activity not only against bulk leukemia cells, but also against the self-renewing leukemia stem cells responsible for long-term engraftment in xenograft models. An important consideration for the ensuing clinical development of FLT3 inhibitors was whether sustained FLT3 inhibition could be achieved at doses that are tolerable in patients. Small molecules such as lestaurtinib are often highly (>99%) plasma protein bound, such that total drug levels measured by standard pharmacokinetic assays give little information regarding drug activity. Furthermore, direct assessment of FLT3 inhibition in leukemic blasts in patients is particularly unfeasible in clinical trials in which the chemotherapy administered beforehand results in clearance of blasts from the blood. Therefore, a plasma inhibitory activity (PIA) was developed as a surrogate for direct assessment of inÂ€ vivo FLT3 inhibition (Levis etÂ€al. 2006). Plasma is collected from patients prior to and at various trough time points after CEP-701 is administered. Aliquots of TF-1/ITD cells (TF-1 human leukemia cell line engineered to express a FLT3/ITD mutation) are exposed to the plasma, then lysed and subjected to FLT3 IP and P-FLT3 Western blotting. The percent inhibition of P-FLT3 compared to cells exposed to the patient’s pretreatment plasma (untreated control) is calculated to give an estimate of FLT3 inhibitory activity present in the patient’s plasma (PIA) at each time point. Validation of this approach has been done by comparing results in cases where FLT3 inhibition could simultaneously be measured directly in leukemic blasts and in plasma for the same patient at the same time point. The PIA is considered adequate if it is >80% at trough time points, corresponding to the degree of prolonged FLT3 inhibition required for cytotoxicity. The adult AML clinical trials with lestaurtinib have successfully correlated clinical responses with FLT3 PIA levels (Smith etÂ€al. 2004; Levis etÂ€al. 2005). PIA analysis is being performed in the ongoing COG clinical trials of lestaurtinib in childhood AML and infant ALL, and excellent levels of inhibitory activity at tolerable dose levels have been observed in some patients, suggesting that an adequate PIA can be achieved in infants and children also. Lestaurtinib is now being tested in the COG for children with relapsed/refractory FLT3-mutant AML. Lestaurtinib is given in sequential combination with reinduction chemotherapy (HiDAC and idarubicin), where the sequence of exposure (chemotherapy followed by FLT3 inhibitor) is based upon preclinical studies demonstrating maximal synergy between FLT3 inhibition with this sequence, compared to only additive effects with simultaneous exposure and antagonism with FLT3 inhibitor followed by chemotherapy (Levis etÂ€al. 2004; Brown etÂ€al. 2006a). The antagonism seen with the sequence of FLT3 inhibition followed by chemotherapy is due to the cell cycle arrest from FLT3 inhibitors, which protects the cells from the cytotoxic effects of chemotherapeutic agents that are cell-cycle dependent. Plans to incorporate FLT3 inhibitors into COG phase III clinical trials for patients with deÂ€novo FLT3/ITD positive AML are in progress.

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Enroll

Induction

A

Post-induction Arm A (SR chemo)

B

Post-induction Arm B (IR/HR chemo)

C

Post-induction Arm C (IR/HR chemo + lestaurtinib)

MLL-G

MLL-R

A

Randomize

B

C

Fig.Â€2â•… Design of COG study AALL0631. MLL-G, germline (wt) MLL; MLL-R, rearranged MLL

Lestaurtinib is also being tested in the COG for infants with newly diagnosed MLL-rearranged ALL. Lestaurtinib is being added in a randomized fashion to the multicourse chemotherapy regimen used in the previous clinical trial for infant ALL (COG P9407). The design of this study (Fig.Â€2) takes into account the preclinical data regarding combinations of lestaurtinib and chemotherapy, as lestaurtinib will be given immediately following the exposure to standard cytotoxic chemotherapy in an effort to maximize potential synergy, and will not be given for at least 24Â€hours prior to chemotherapy to avoid potential for antagonism.

Targeting Anti-apoptotic BCL-2 Family Members Deregulated apoptosis due to imbalanced expression of BCL-2 family proteins is a general avenue to chemotherapy resistance because the cytotoxicity of most anticancer chemotherapy occurs through this pathway. The focus on silencing antiapoptotic BCL2 family members in MLL leukemia in infants grew primarily from protein and gene expression level data on abundant expression of BCL-2 and other pro-survival BCL-2 family members in MLL leukemia in infants (Robinson etÂ€al. 2008; Zhang etÂ€ al. 2008a) coupled with preclinical and clinical adult forerunner data in different cancer cell types suggesting the utility of apoptosis pathway modulation, with selection of compounds to silence anti-apoptotic BCL-2 family members that were already in the clinical phases of development and showed minimal toxicities in adults.

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BCL-2 family members fall into three classes: multidomain anti-apoptotic proteins homologous in all four BCL-2 homology (BH) domains; multidomain pro-apoptotic proteins homologous in BH1, BH2, and BH3 domains; and BH3only proteins, which all are pro-apoptotic. BCL-2 family protein interactions occur via docking of the BH3 domain with a deep binding groove on the surface of the binding partner (Green 2005; Reed 2002, 2003). Pro-apoptotic multidomain BAX and BAK exist as inactive monomers, but they homo-oligomerize upon BH3-only BIM or BID activation, leading to mitochondrial outer membrane permeabilization and release of apoptotic cofactors cytochrome c and Smac/DIABLO to the cytosol (Danial and Korsmeyer 2004; Nguyen etÂ€al. 2007). Cytochrome c is then bound by Apaf-1, which forms Apaf-1 oligomers and recruits pro-caspase 9. Caspase-9 activation activates effector caspases that execute apoptosis (Danial and Korsmeyer 2004). Many targeted strategies, including antisense therapies, siRNAs, peptides, and small molecule inhibitors have emerged for silencing anti-apoptotic BCL-2 family proteins at the transcript and the protein levels (Reed 2003; Gewirtz etÂ€al. 1998; Letai etÂ€al. 2002; Garber 2005). Since quantitative real-time PCR and Western blot analysis demonstrated that BCL-2 mRNA and protein are abundant and suggested that BCL-2 provides a cell survival mechanism in cases of pediatric/infant ALL and AML with the t(4;11) or other MLL translocations, (Robinson etÂ€al. 2008) the first foray to modulate apoptosis in infant leukemias involved preclinical inÂ€ vitro studies on Genasense™ (Oblimersen, G3139; Genta, Inc.), an 18-mer phosphorothioate antisense oligodeoxynucleotide (ODN), which forms a DNA–RNA hybrid with the first six codons of the BCL-2 mRNA (Nicholson 2000). Among the forerunner adult studies on this agent was an encouraging Phase I trial of Genasense™ with chemotherapy for refractory/relapsed adult acute leukemia, in which 45% of patients had disease response and BCL-2 mRNA decreased in 75% of cases; (Marcucci etÂ€al. 2003) in addition, the toxicities (low-grade fever, transient liver function abnormalities, fatigue, thrombocytopenia) were limited (Marcucci etÂ€al. 2003). Its clinical activity in refractory and relapsed adult B-cell malignancies (O’Brien etÂ€al. 2005; Waters etÂ€al. 2000) also was encouraging. Abundant BCL-2 expression was detected in the cell lines RS4:11, MV4-11, and SEM-K2 with t(4;11), similar to primary MLL leukemia cases, enabling drug activity and mechanistic assays on the cell line models (Robinson etÂ€ al. 2008). Genasense™ abrogated BCL-2 mRNA expression in RS4:11 cells and significantly reduced the protein, though to recognize the changes these molecular analyses were performed at high concentrations (Robinson etÂ€ al. 2008). However, studies of cytotoxicity and effects on apoptosis and cell cycle suggested that lower concentrations were sufficient to change the apoptosis threshold (Robinson etÂ€al. 2008). In MTT assays of cytotoxicity, the most sensitive of the three cell lines to single-agent Genasense™ was RS4:11, and SEM-K2 was the least sensitive (Robinson etÂ€al. 2008). Applying pharmacostatistical response surface modeling of drug interactions, Genasense™ at low concentrations was also found to sensitize RS4:11 and MV4-11 cells to select anti-leukemia cytotoxic drugs (ADR, VP16, ARAC, 6-TG in RS4:11; VP16, 6-TG in MV4-11), indicating synergistic

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interactions (Robinson etÂ€al. 2008). In further flow cytometric mechanistic studies, combining Genasense™ with doxorubicin in RS4:11 cells increased active caspase-3 and TUNEL staining compared to doxorubicin alone, consistent with greater apoptosis (Robinson etÂ€ al. 2008). In addition, Genasense™ increased S-phase progression, (Robinson etÂ€al. 2008) which is consistent with an effect on the BCL-2 target transcript since the anti-apoptotic effects of BCL-2 are linked to retardation of the G1/S transition. (Deng etÂ€al. 2003) This evaluation of the effects of the pro-apoptotic BCL-2 antisense compound Genasense™ designed to decrease BCL-2 protein levels via selective BCL-2 mRNA degradation as a prototypic strategy to modulate cell death, indicated that abundant BCL-2 affords a molecular target in leukemias with t(4;11). Although the cardinal anti-apoptotic regulator in the intrinsic cell death pathway, BCL-2, has a general role in chemotherapy resistance (Danial and Korsmeyer 2004), the formation of homo- and heterotypic dimers by many different BCL-2 family proteins with opposing anti- or pro- apoptotic actions together determines the apoptosis threshold and regulates the intrinsic (mitochondrial) cell death pathway. Therefore, an increasing number of small molecule inhibitors also have emerged to restore balance in this pathway. Obatoclax (GX15-070; GeminX, Inc.) is one that antagonizes a broad spectrum of anti-apoptotic BCL-2 family proteins (BCL-2, BCL-XL, A1, BCL-w, MCL-1) (Nguyen etÂ€al. 2007). Removal of the mitochondrial block by obatoclax also may enhance TRAIL-mediated extrinsic apoptosis (Huang etÂ€ al. 2009). Thus, there has been significant impetus for preclinical and clinical obatoclax development to potentiate chemotherapy by activating apoptosis mechanisms in malignancies with inherent or acquired chemotherapy resistance. Obatoclax represents the first molecularly targeted agent for silencing anti-apoptotic BCL-2 family members that will be tested in children. The recently approved COG trial ADVL0816, a Phase I study of obatoclax in combination with vincristine/ doxorubicin/dexrazoxane in children with relapsed/refractory solid tumors or leukemia, incorporates a separate stratum for patients with MLL disease on the basis of preclinical activity data elaborated on below. The pan-anti-apoptotic BCL-2 family small molecule inhibitor obatoclax has the ability to bind to the BH3 binding pocket of the anti-apoptotic BCL-2 family members and block their anti-apoptotic function (Garber 2005). A synthetic derivative of the bacterial prodiginines family of red-pigmented tripyrrolic compounds developed on the basis of structure activity relationships; (Williamson etÂ€al. 2007) obatoclax has been reported to cause apoptosis inÂ€vitro in a wide range of human cancer cells derived from adult leukemias, lymphomas, and solid tumors, (Campas etÂ€al. 2006; Galan etÂ€al. 2005; Perez-Galan etÂ€al. 2007; Li etÂ€al. 2007; Li etÂ€al. 2008b; Martinez-Paniagua etÂ€al. 2007; Trudel etÂ€al. 2007a; Witters etÂ€al. 2007) and single agent responses of several human solid tumor xenografts occurred. In solid tumor models, obatoclax showed synergy with cisplatin in nonsmall cell lung cancer cell lines (Li etÂ€al. 2008b). Synergy with cytosine arabinosine was reported in AML cell lines (Konopleva etÂ€ al. 2008). In mantle cell lymphoma lines, obatoclax induced apoptosis and enhanced the cytotoxicity of vincristine, (Bebb etÂ€al. 2006) proteasome inhibitors and doxorubicin (Yazbeck etÂ€ al. 2006). In addition to inducing

Molecularly Targeted Therapy for Infant ALL

41

synergistic cytotoxicity when combined with cisplatin or doxorubicin (Martinez-Paniagua etÂ€ al. 2007; Hernandez-Ilizaliturri etÂ€ al. 2006a), obatoclax increased rituximabmediated antibody dependent cellular cytotoxicity and complement mediated cytotoxicity in non-Hodgkins lymphoma cell lines (Hernandez-Ilizaliturri etÂ€al. 2006b). Obatoclax also exhibited synergy in combination with dexamethasone in a dexamethasone sensitive multiple myeloma cell line and additive activity in several other multiple myeloma lines (Trudel etÂ€al. 2007a). Therefore, obatoclax rapidly advanced into adult Phase I and II solid tumor and leukemia clinical trials (Borthakur etÂ€ al. 2006; Goy etÂ€ al. 2007a; O’Brien etÂ€ al. 2009; Schimmer etÂ€al. 2007a; Verstovsek etÂ€al. 2007). The salient observations of adult Phase I trials evaluating various infusion durations and schedules have been that obatoclax is well tolerated with minimal toxicities, and that its primary toxicities, which affect the central nervous system (i.e., somnolence, euphoria, confusional state, ataxia), are reversible, infusion-related, and self-limited once the infusion stops. Importantly, modest obatoclax activity was suggested against various relapsed/refractory adult leukemias and solid tumors with minimal toxicity (O’Brien etÂ€ al. 2009; Goy etÂ€ al. 2007b). Remarkably, single agent obatoclax resulted in a complete cytogenetic remission, resolution of cytopenias, and transfusion independence in a patient with secondary AML with the t(9;11) translocation (Schimmer etÂ€al. 2007b). Thus, the attractive toxicity profile and the activity in MLL disease in an adult, made this agent especially suitable for preclinical pediatric studies, especially studies on MLL rearranged leukemia in infants as a potential pediatric cancer where obatoclax might have eventual utility in the clinic and substantial impact. In addition, HOXA9 repression of pro-apoptotic BIM (BCL2L11) was shown in a cell line with t(9;11), (Stubbs etÂ€al. 2007) and silencing of the MLL-AF4 fusion oncoprotein in cell lines with t(4;11) using siRNAs not only decreased BCL-XL protein, but also increased activated caspase 3 and increased apoptosis (Thomas etÂ€al. 2005). The rationale to test obatoclax also extended more generally to pediatric cancers and leukemia. In neuroblastoma, the inherent cancer cell survival mechanism derives from anti-apoptotic MCL-1 (Goldsmith etÂ€al. 2006). In contrast, BCL-XL is believed to be the direct transcriptional target of the PAX3-FKHR translocation, the primary molecular aberration in alveolar rhabdomyosarcoma (Xia etÂ€al. 2002). In childhood ALL, pro-apoptotic BIM expression predicted early response to treatment in the high-risk patients, (Bhojwani etÂ€ al. 2008) and decreased expression of the extrinsic apoptosis effector caspase CASP8 predicted MRD during and following induction (Flotho etÂ€al. 2007). Studies of apoptosis genes in relation to inÂ€vitro drug responses in childhood ALL uncovered the importance of MCL-1 in prednisolone sensitivity and of the new BCL-2 family members BCL2L13 and HRK in L-asparaginase resistance (Holleman etÂ€ al. 2006). Importantly, preclinical inÂ€ vitro studies suggested that obatoclax restored the glucocorticoid response in steroid resistant childhood precursor B-cell and T-cell ALL (Bonapace etÂ€ al. 2007). That not all of the emerging BH3 mimetics have high MCL-1 affinity, (van Delft etÂ€ al. 2006) also made obatoclax an attractive agent to advance in pediatrics.

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The principal pediatric preclinical studies leading to the ADVL0816 obatoclax Phase I trial in relapsed/refractory solid tumors and leukemia were performed on MLL rearranged leukemia in infants. Quantitative real-time PCR suggested that not only anti- but also several pro-apoptotic BCL-2 family mRNAs, apoptosis execution, and various mitochondrial maintenance mRNAs were abundant in MLL rearranged infant ALL (Zhang etÂ€al. 2008a). Since basal expression levels of cell death and cell survival factors are relevant to finding novel therapeutic targets, these studies formed the basis to evaluate the potential of obatoclax to silence the activity of anti-apoptotic BCL-2 family protein targets in primary MLL leukemia cases (Zhang etÂ€al. 2008b). MTT assays of MLL rearranged infant ALL and bilineal acute leukemias showed variable single-agent sensitivities, many within clinically achievable range (Zhang etÂ€ al. 2008b). In a primary ALL with the MLL-AF4 rearrangement, pharmacostatistical response surface modeling indicated synergy between obatoclax and many of the common antileukemia cytotoxic chemotherapeutic drugs (cytosine arabinoside, doxorubicin, etoposide, dexamethasone, L-asparaginase, vincristine) (Zhang etÂ€ al. 2008b). Interestingly, apoptosis and the alternative cell death pathway of autophagy, in which cellular components are degraded through the lysosomal machinery, are partially overlapping in downstream cell death mediators (Bonapace etÂ€ al. 2007; Klionsky etÂ€ al. 2008; Maiuri etÂ€ al. 2007). Accumulating evidence from standard studies of autophagy (Klionsky etÂ€al. 2008) and autophagy factors is suggesting that the obatoclax cell death mechanism in MLL leukemia involves the autophagy cell death pathway (Zhang etÂ€al. 2008b). Biomarkers in the autophagy pathway will be further studied as the Phase I clinical trial of obatoclax ADVL0816 advances. Beyond the demonstration of preclinical inÂ€vitro activity, obatoclax also is attractive to advance for leukemia in infants because pharmacometric modeling strategies and a pharmacokinetic study carried out in a diseased NOD-scid-IL-2Rgnull (NOG) immunodeficient xenograft model that recapitulates the hyperleukocytosis and extramedullary involvement of MLL leukemias, (Zhang etÂ€al. 2008c) have demonstrated that obatoclax has excellent biodistribution to sites of involvement by MLL leukemia in infants, as indicated by tissue:plasma concentration ratios, including excellent CNS penetration (Zhang etÂ€al. 2008c, 2007). The ADVL0816 Phase I clinical study of obatoclax summarized in Fig.Â€3 was approved by Clinical Trials Evaluation Program (CTEP) and opened for enrollment in COG Phase I Consortium Institutions in 2009. After an initial single agent dose, obatoclax will be administered in combination with vincristine, doxorubicin, and dexrazoxane. Patients will be enrolled in three separate strata. Patients with relapsed/refractory solid tumors (Stratum 1) will be enrolled in a dose escalation trial with a starting obatoclax dose of approximately 70% of the adult MTD. As a result of the striking preclinical obatoclax activity, patients with relapsed/refractory MLL-rearranged leukemia (Stratum 2) will be eligible for enrollment concurrently with the dose finding phase in solid tumor stratum. Once the dose finding phase has been completed, a patient cohort with relapsed/refractory non-MLL leukemia will be enrolled in Stratum 3.

Molecularly Targeted Therapy for Infant ALL

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Fig.Â€3â•… COG study ADVL0816 treatment schema

The foregoing studies have built a foundation for overcoming drug resistance from abnormal cell death regulation in leukemia in infants where current treatments offer little hope due to the inherent drug resistance and unique vulnerabilities of infants to toxicities from intensive treatments. However, that MLL leukemias exhibit variable preclinical obatoclax sensitivities, (Zhang etÂ€ al. 2008b) although promising, also underscores that no one agent targeting this pathway is expected to be active in all cases. The small molecule inhibitor ABT737, which targets anti-apoptotic BCL-2, BCL-XL, and BCL-w, (van Delft etÂ€al. 2006) is another candidate to evaluate for cell death pathway modulation in leukemia in infants. ABT-737 disrupts BCL-2 family protein interactions by targeting the BH3 binding pocket of select anti-apoptotic BCL-2 family members (van Delft etÂ€ al. 2006). Inhibition of interactions of anti-apoptotic BCL-2 family proteins with proapoptotic BCL-2 family proteins by ABT-737 and activity of ABT-737 alone and in combination with various cytotoxic drugs has been demonstrated in several preclinical studies of leukemia (Kang etÂ€ al. 2007; Trudel etÂ€ al. 2007b; Chauhan etÂ€al. 2007; Konopleva etÂ€al. 2006; Kline etÂ€al. 2007; Kojima etÂ€al. 2006) and ABT263, which has identical pharmacologic properties, is advancing to adult clinic trials. This compound demonstrates mechanism-based single agent activity in lymphomas (Oltersdorf etÂ€al. 2005). Of particular interest, inÂ€vitro activity of ABT737 was recently studied in seven different ALL cell lines [T-cell ALL: COG-LL-317, CEM, MOLT-3, MOLT-4; pre-B ALL: COG-LL-317, Nalm-6, RS4:11], (Kang etÂ€ al. 2007) among which the RS4:11 cell line was established from a case of adult ALL with t(4;11); however, no cases of infant leukemia have been tested. Strongly synergistic or synergistic cytotoxicity as defined by combination indices £0.3 or £0.7, respectively, of ABT-737 combined with cytotoxics, especially L-asparaginase or vincristine, was shown in all of the cell lines, even when there was no single agent sensitivity; the strongest synergy was with Lasparaginase. Further study of the pro-Â�apoptotic effects of the ABT-737/L- asparaginase combination in RS4:11 and COG-LL-317 cells showed evidence of

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mitochondrial apoptosis pathway Â�activation including an increase in the truncated form of BID, tBID, increased hypophosphorylated BAD, an increase in the activated form of BAX, BAX(s), an increase in the cleaved form of CASP8, which cleaves BID to tBID, and cleavage of CASP9 and CASP3. The ABT-737/L- asparaginase combination in COG-LL-317 cells resulted in increased Annexin V/FITC staining and increased JC-1 probe staining, and Western blot analysis showed increased mitochondrial cytochrome c release. Importantly, ABT-737 did not increase apoptosis in normal PBMCs. ABT-737 showed synergistic cytotoxicity when combined with vincristine, L- asparaginase and dexamethasone in 5 of the 7 cell lines tested, including RS4:11. ABT-737 combined with vincristine, L- asparaginase, and dexamethasone delayed the progression of established xenografts in two NOD/SCID models of relapsed primary ALL and increased EFS compared to chemotherapy alone or ABT-737 alone in one of the two xenografts. As described in detail above, infant acute leukemia exhibits deregulated FLT3 receptor tyrosine kinase signaling as a separate resistance mechanism (Armstrong etÂ€ al. 2003, 2004b; Brown etÂ€ al. 2006b; Taketani etÂ€ al. 2004b; Brown and Small 2004b; Brown etÂ€al. 2005b; Stam etÂ€al. 2005; Stubbs and Armstrong 2007) in addition to the imbalanced expression of cell death regulatory factors (Zhang etÂ€ al. 2008a). Therefore, it may be of substantial interest that Abbott Pharmaceuticals has developed small molecule inhibitors to both pathways. ABT-869 is an ATPcompetitive small molecule inhibitor with specificity for the VEGF and PDGF families of receptor tyrosine kinases (RTKs), thereby targeting multiple resistance mechanisms (Rodila etÂ€al. 2006). There is rationale to test ABT-869 (Carlson etÂ€al. 2005; Dai etÂ€al. 2007; Shankar etÂ€al. 2007; Albert etÂ€al. 2006) alone and combined with the pro-apoptotic agent ABT-737 in leukemias in this population, since FLT3 is a member of the PDGF family of RTKs (Dai etÂ€al. 2007). The activity of ABT-869 is dependent on mutant kinases (Albert etÂ€al. 2006). In the AML cell lines MV4-11 and Molm13 with MLL translocations and FLT3ITD mutations, ABT-869 already has exhibited inÂ€vitro activity in cell proliferation/ viability assays, and downregulation of FLT3, STAT5, and ERK phosphorylation and the expression of the STAT5 target gene Pim-1 (Shankar etÂ€al. 2007). ABT-869 prevented the formation of subcutaneous MV4-11 xenografts, caused the regression of established subcutaneous MV4-11 xenografts, slowed the progression of subcutaneous Molm13 xenografts and prolonged the survival of mice with marrow engraftment of MV4-11 cells (Shankar etÂ€al. 2007). Even though FLT3 tyrosine inhibitors may effectively decrease FLT3 phosphorylation, persistent phosphorylation of downstream signaling proteins (e.g., AKT, MAPK), activating mutations in parallel signaling pathways (e.g., RAS) or overexpression of other RTKs may be avenues to resistance, as has been shown in cell lines with MLL translocations (Piloto etÂ€al. 2007). Importantly, however, the ABT737 pro-apoptotic drug can neutralize resistance to FLT3 tyrosine kinase inhibition (Kohl etÂ€ al. 2007). Studies of ABT-737 and ABT-869 together in leukemia in infants would enable more detailed characterization of additional pathways to deregulated cell death mechanisms and RTK signaling and how these pathways can be targeted.

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Targeting MLL Fusion Transcripts The plethora of MLL partner genes and heterogeneity in genomic breakpoint locations, which increases transcript heterogeneity even further, would make targeting the fusion transcripts difficult. Nonetheless, it has been possible to silence several of the more common MLL fusion transcripts experimentally using nucleic acid therapeutics (Gewirtz etÂ€al. 1998). Antisense oligodeoxynucleotides have resulted in successful downregulation of translation of MLL fusion proteins inÂ€vitro in cell lines (Kawagoe etÂ€al. 2001; Niitsu etÂ€al. 2001a; Akao etÂ€al. 1998). The MLL-AF9 downregulation in the THP-1 cell line resulted in reduced HOXA7 and HOXA10 gene expression and increased apoptosis (Kawagoe etÂ€ al. 2001). MLL-CBP downregulation using antisense rendered the AML cell line SN-1 sensitive to all-trans retinoic acid or 1a,25-dihydroxyvitamin D3 induced differentiation (Niitsu etÂ€al. 2001a). Importantly, pro-apoptotic effects of targeting the fusion transcript with an antisense oligodeoxynucleotide also were observed in the KOCL33 cell line derived from a case of infant ALL with the MLL-ENL translocation (Akao etÂ€al. 1998). More recently, fusion transcript specific siRNAs were used to deplete the respective fusion oncoproteins resulting from two different MLL-AF4 genomic rearrangements in the SEM-K2 and RS4:11 cell lines (Thomas etÂ€ al. 2005). The inÂ€ vitro effects of the respective siRNAs included decreased colony formation on methylcellulose, decreased proliferation in MTT assays, an increase in G0/G1 phase cells and a decrease in cells in S phase, increased activated caspase 3 and decreased antiapoptotic BCL-XL protein, decreased HOXA7, HOXA9, and MEIS1 gene expression, and decreased expression of the stem cell marker CD133 (Thomas etÂ€ al. 2005). Furthermore, injection of SEM-K2 cells that had been pretreated MLL-AF4 specific siRNA into SCID mice completely suppressed engraftment in a xenotransplantation assay (Thomas etÂ€al. 2005).

PFWT Targeting of MLL Partner Protein Interactions The MLL partner proteins AF4, ENL, AF9, and AF10 interact in a multiprotein complex, which is involved in the regulation of transcriptional elongation and recruitment of the histone H3K79 methyltransferase Dot1 to elongating RNA Pol II (Bitoun etÂ€al. 2007). The PFWT peptide mimetic of the AF9 binding domain of AF4 with a conjugated Penetratin transduction sequence at the amino terminus is in preclinical development for disruption of the AF4-AF9 interaction (Srinivasan etÂ€ al. 2004). The AF-9 interaction domain of AF4, which binds at the AF9 carboxyl terminus, is highly conserved in all LAF4 family proteins (AF4, LAF4, AF5, FMR2) and is retained in the MLL fusion oncoproteins (Srinivasan etÂ€al. 2004; Erfurth etÂ€al. 2004). PFWT disruption of AF4-AF9 complexes was validated in GST pull-down assays and in NIH 3T3 cells, and PFWT decreased cell proliferation of RS4:11 cells, KP-L-RY (MLL-AF5) cells and, to a lesser degree, THP-1 cells with the MLL-AF9

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rearrangement (Srinivasan etÂ€ al. 2004). In the MV4-11 and Molm13 (MLL-AF9) myeloid cell lines, PFWT rapidly induces ultrastructural changes of necrosis; abrogation of PFWT cytotoxicity by the serine protease inhibitor TLCK implicated serine proteases in the cell death mechanism (Palermo etÂ€al. 2008). In MV4-11 cells, promising synergistic interactions were suggested between PFWT and cytosine arabinoside, etoposide, the HSP-90 inhibitor 17-allylamino17-demethoxygeldanamycin (17-AAG), or the FLT3 inhibitor 3,4-dimethoxybenzoylamide, in Molm13 cells, between PFWT and cytosine arabinoside or etoposide and, in KOPN-8 (MLL-ENL) cells, though only at half the IC50 concentration, between PFWT and cytosine arabinoside (Bennett etÂ€ al. 2009). Further study of MV4-11 cells showed that, with exception of 17-AAG, the penetratin sequence did not itself augment cytotoxicity (Bennett etÂ€al. 2009). Interestingly, in MV4-11 cells in which synergy was sequence-dependent (cytosine arabinoside or etoposide before PFWT), PFWT alone caused an increase in cells in G1 and a decrease in cells in S phase (Bennett etÂ€al. 2009). Treatment with etoposide and PFWT resulted in EM features of both apoptosis and necrosis and an increase activated caspase 3, indicating that the two cell death mechanisms are not mutually exclusive (Bennett etÂ€al. 2009).

Targeting Glycogen Synthase Kinase 3 The recent basic research discovery demonstrating that the serine/threonine kinase GSK3 is a required, cell type specific MLL leukemia maintenance factor, suggested GSK3 as a potential therapeutic target. The role of GSK3 in MLL leukemia is paradoxical because GSK3 is involved in tumor suppression in other cancer types, and also because GSK3 normally phosphorylates and inactivates b -catenin, MYCN, and JUN, and inhibits pathways involved in self renewal and proliferation, whereas GSK3 inhibition increases stem cell pluripotency and HSC repopulation (Wang etÂ€al. 2008). Targeting GSK3 using small molecule inhibitors (GSK3-IX, SB216763, alsterpaullone) in MLL-AF4 (SEMK2, RS4:11, MV4-11) and MLL-AF5 (KP-L-RY) cell lines inhibited proliferation and increased b-catenin expression, decreased G1–S phase cell cycle progression, and increased sub-G0-G1 DNA consistent with cell death. Culture of murine primary myeloid progenitors expressing transduced MLL-ENL, MLL-LAF4, MLL-AF6, or MLL-GAS7 in the presence of SB216763, decreased clonogenic potential and proliferation; myeloid differentiation and decreased c-Kit expression were suggested with prolonged exposure (Wang etÂ€al. 2008). Transduction of murine Gsk3b−/− cells with MLL fusion genes, or shRNAmediated Gsk3b knockdown in murine myeloid progenitors transduced with MLL fusion genes, increased sensitivity to pharmacological GSK3 inhibition, even though Gsk3b was not required for transformation, whereas Gsk3a depletion alone

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did not have these effects. Still cooperativity was suggested since the sensitivity to pharmacological inhibition, decreased proliferation and clonogenicity, and increased myeloid differentiation were more profound in a Gsk3b−/− Gsk3aKD MLL fusion gene model in which both Gsk3 isoforms were depleted, and Gsk3b−/− Gsk3aKD MLL-ENL transduced cells did not induce leukemia in mice (Wang etÂ€al. 2008). Additionally, the treatment of MLL-AF4 leukemia bearing mice with the GSK3 inhibitor lithium carbonate prolonged their survival (Wang etÂ€al. 2008). With GSK3 inhibition, the CDKI protein p27Kip1 significantly increased in the MLL-AF5 leukemia cell line KP-L-RY, and in MLL-ENL transduced murine myeloid progenitors, indicating that the effects of GSK3 in MLL leukemias involve p27Kip1 suppression (Wang etÂ€al. 2008). None of these effects were observed in non-MLL leukemia cell lines (Wang etÂ€al. 2008).

HSP90 as a Potential Therapeutic Target The heat shock protein HSP90 is an ATP-dependent chaperone protein that functions in a multi chaperone protein complex with roles in the regulation of conformational maturation and refolding of client proteins, (Wiech etÂ€al. 1992; Stebbins etÂ€al. 1997; Terasawa etÂ€al. 2005) tertiary structure of the proteasome, (Imai etÂ€al. 2003) and steroid receptor functions and interactions (Terasawa etÂ€ al. 2005). Of particular interest to MLL leukemia, FLT3 is another HSP90 client protein (Yao etÂ€ al. 2003). Interestingly, in cancer cells HSP90 stabilizes PI3K (Belova etÂ€ al. 2008; Fujita etÂ€al. 2002) and AKT (Zhang etÂ€al. 2005) and has an extracellular role in invasion and metastasis via regulation of MMP2 protein maturation (Picard 2004; Eustace and Jay 2004; Eustace etÂ€al. 2004). HSP90 is of interest as a potential therapeutic target in infant leukemia. Proteomics-based 2D-DIGE analysis of whole cell proteins identified HSP-90 as a differentially expressed protein in the MLL-AF4 cell lines RS4:11 and MV4-11 compared to normal CD34+ cells (Yocum etÂ€al. 2006). In studies testing HSP90 inhibition, MLL leukemia cell lines exhibited differential 17-AAG sensitivity depending on FLT3 status; RS4:11 cells in which FLT3 is wild type were less sensitive than MV4-11 or Molm13 with the FLT3 ITD (Yao etÂ€al. 2003). In all three of these cell lines, HSP90 inhibition was associated with decreased FLT3, RAF, and AKT expression (Yao etÂ€ al. 2003). In MV4-11 cells, not only did quantitative proteomics indicate that 17-AAG effectively depleted the HSP90a target, but also 17-AAG resulted in decreased viability and proliferation and increased apoptosis; a decrease in nucleoside diphosphate kinase nm23 suggested nm23 as a novel biomarker of HSP90 inhibition (Yocum etÂ€ al. 2006). More recently, when synergy between 17-AAG and etoposide was tested, greater synergy was observed in Molm13 and MV4;11 with the FLT3 ITD than in RS4:11 cells with wild-type FLT3 (Yao etÂ€al. 2007). Synergistic effects of combining

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17-AAG with the FLT3 inhibitor GTP14564 in cell lines with FLT3-ITD (MV411) or amplified wild-type FLT3 (SEM-K2) have also been suggested, with reductions in phospho-FLT3 and phospho-STAT5, G0–G1 arrest and increased apoptosis (Yao etÂ€al. 2005).

Epigenetic Strategies In cell lines with the MLL-AF9 translocation (THP-1, Mono-Mac-6, and Molm13), histone deacetylase inhibition with valproic acid has been associated with decreased cell viability, cell cycle arrest in G1, increased apoptosis and induction of p21, and the G cyclin CG2, which is involved in G1 cell cycle arrest (Tonelli etÂ€al. 2006). In the SN-1 AML cell line with the MLL-CBP translocation, various HDAC inhibitors (sodium butyrate, trichostatin A, polyloxyl bytyrate) exhibited synergy with all-trans retinoic acid in inducing differentiation as measured by NBT-reduction (Niitsu etÂ€ al. 2001a). In the SN-1 cell line, and in the KOCL33, KOCL51, and KOCL44 cell lines, all with the MLL-ENL translocation, the demethylating agent 5-aza-2¢-deoxycytidine resulted in p16/INK4A upregulation and sensitivity to differentiation therapy with all-trans retinoic acid and 1a,25-dihydroxyvitamin D3 (Niitsu etÂ€al. 2001b). Taken together these studies may suggest that combinations of epigenetic therapies with differentiation therapies may have utility in MLL disease.

mTOR Inhibition The serine/threonine kinase mTOR (mammalian Target of Rapamycin) is an integrator of several signal transduction pathways, and is involved in the regulation of cell cycle, apoptosis and angiogenesis. The mTOR pathway is aberrantly activated in many tumor types, and the inhibition of mTOR signaling (using sirolimus or one of its analogs, such as temsirolimus or everolimus) has shown antitumor activity in several model systems as well as early phase clinical trials. In fact, temsirolimus was recently FDA-approved for the treatment of advanced renal cell carcinoma (Hudes etÂ€al. 2007). There is some preclinical evidence that inhibition of the serine/ threonine kinase mTOR (mammalian Target of Rapamycin) may be a useful therapeutic strategy in infant leukemia. Sirolimus (and the related mTOR inhibitor temsirolimus) inhibit the growth of precursor B cell ALL cell lines inÂ€vitro and are also active in ALL transgenic and xenograft murine models (Brown etÂ€ al. 2003). In further studies, mTOR inhibitors showed synergistic cytotoxicity with methotrexate against human ALL xenografts (Teachey etÂ€al. 2008). Moreover, sirolimus has been shown to overcome glucocorticoid resistance in ALL cells via anti-apoptotic MCL1 downregulation (Wei etÂ€al. 2006). Although none of these studies were performed on MLL-rearranged leukemias specifically, several groups have suggested that the

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glucocorticoid resistance in MLL-rearranged infant ALL may be related to the poor prognosis (Pieters etÂ€al. 1998; Palle etÂ€al. 2005). Since mTOR inhibition can reverse glucocorticoid resistance, these studies may provide rationale to consider clinical mTOR inhibition in infant ALL.

Targeting CD33 Cell Surface Antigen Gemtuzumab ozogamicin is a humanized anti-CD33 antibody linked to the antitumor antibiotic calicheamicin. Not only is CD33 positivity observed in a preponderance of the subtypes of pediatric AML including cases with MLL translocations but also this agent may have a role in MLL rearranged ALL because of the frequent coexpression of myeloid cell surface markers (Zwaan etÂ€al. 2003). The results of clinical testing of gemtuzumab ozogamicin for remission induction in conjunction with intensive post induction therapy for relapsed/refractory CD33+ AML have been encouraging (Zwaan etÂ€al. 2003; Arceci etÂ€al. 2005).

Conclusions Several examples of molecularly targeted agents at various stages of development for infant leukemia have been discussed. As the molecular pathobiology of infant leukemia continues to unravel, the characterization of MLL fusion oncoproteins and factors that interact with MLL fusion oncoproteins in multiprotein complexes or cooperate with them in downstream or parallel signaling pathways have led to identification of a number of promising targets and experiments on potentially efficacious molecularly targeted agents that might benefit this patient population. The agents are at various stages of testing along the drug development pipeline from basic to preclinical inÂ€vitro and inÂ€vivo testing, and more recently to testing in clinical trials. In 2010, most still are at the basic and preclinical stages of development. Identification of efficacious agents with a low toxicity profile holds promise to change the outcome of this subset of pediatric cancer that has heretofore been so refractory to more conventional treatments because the mechanisms of action of targeting specific signaling cascades are very different from the more general mechanisms of action of conventional chemotherapy, which result in such profound toxicities in infants in particular. Appropriate choices of molecularly targeted treatments for infant leukemia in the clinic are also expected to be reliant on molecular testing for the relevant target aberrations because of the heterogeneity in MLL partner proteins and the cooperating events in disease pathogenesis within this patient subset. Furthermore, molecular characterization will be critically important to understand mechanisms of response and resistance. As well exemplified by FLT3 tyrosine kinase inhibition, (Piloto etÂ€al. 2007) the future therapeutic opportunities and challenges lie not only in identifying

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primary mutations and coexisting mutations, but also exploiting acquired resistance mechanisms as avenues to new treatment in instances of treatment failure. Moreover, it has also been suggested that the eradication of the leukemia stem cell will likely prove essential to cure MLL disease (Somervaille and Cleary 2006). Acknowledgmentâ•… The work is supported by Leukemia & Lymphoma Society SCOR 7372-07.

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Srinivasan RS, Nesbit JB, Marrero L, Erfurth F, LaRussa VF, Hemenway CS. The synthetic peptide PFWT disrupts AF4-AF9 protein complexes and induces apoptosis in t(4;11) leukemia cells. Leukemia. 2004;18:1364–1372. Erfurth F, Hemenway CS, de Erkenez AC, Domer PH. MLL fusion partners AF4 and AF9 interact at subnuclear foci. Leukemia. 2004;18:92–102. Palermo CM, Bennett CA, Winters AC, Hemenway CS. The AF4-mimetic peptide, PFWT, induces necrotic cell death in MV4-11 leukemia cells. Leuk Res. 2008;32:633–642. Bennett CA, Winters AC, Barretto NN, Hemenway CS. Molecular targeting of MLL-rearranged leukemia cell lines with the synthetic peptide PFWT synergistically enhances the cytotoxic effect of established chemotherapeutic agents. Leuk Res. 2009;33:937–947. Wang Z, Smith KS, Murphy M, Piloto O, Somervaille TC, Cleary ML. Glycogen synthase kinase 3 in MLL leukaemia maintenance and targeted therapy. Nature. 2008;455:1205–1209. Wiech H, Buchner J, Zimmermann R, Jakob U. Hsp90 chaperones protein folding inÂ€vitro. Nature. 1992;358:169–170. Stebbins CE, Russo AA, Schneider C, Rosen N, Hartl FU, Pavletich NP. Crystal structure of an Hsp90-geldanamycin complex: targeting of a protein chaperone by an antitumor agent. Cell. 1997;89:239–250. Terasawa K, Minami M, Minami Y. Constantly updated knowledge of Hsp90. J Biochem. 2005;137:443–447. Imai J, Maruya M, Yashiroda H, Yahara I, Tanaka K. The molecular chaperone Hsp90 plays a role in the assembly and maintenance of the 26S proteasome. EMBO J. 2003;22:3557–3567. Yao Q, Nishiuchi R, Li Q, Kumar AR, Hudson WA, Kersey JH. FLT3 expressing leukemias are selectively sensitive to inhibitors of the molecular chaperone heat shock protein 90 through destabilization of signal transduction-associated kinases. Clin Cancer Res. 2003;9:4483–4493. Belova L, Brickley DR, Ky B, Sharma SK, Conzen SD. Hsp90 regulates the phosphorylation and activity of serum- and glucocorticoid-regulated kinase-1. J Biol Chem. 2008;283:18821–18831. Fujita N, Sato S, Ishida A, Tsuruo T. Involvement of Hsp90 in signaling and stability of 3-phosphoinositide-dependent kinase-1. J Biol Chem. 2002;277:10346–10353. Zhang R, Luo D, Miao R, etÂ€al. Hsp90-Akt phosphorylates ASK1 and inhibits ASK1-mediated apoptosis. Oncogene. 2005;24:3954–3963. Picard D. Hsp90 invades the outside. Nat Cell Biol. 2004;6:479–480. Eustace BK, Jay DG. Extracellular roles for the molecular chaperone, hsp90. Cell Cycle. 2004;3:1098–1100. Eustace BK, Sakurai T, Stewart JK, etÂ€al. Functional proteomic screens reveal an essential extracellular role for hsp90 alpha in cancer cell invasiveness. Nat Cell Biol. 2004;6:507–514. Yocum AK, Busch CM, Felix CA, Blair IA. Proteomics-based strategy to identify biomarkers and pharmacological targets in leukemias with t(4;11) translocations. J Proteome Res. 2006;5:2743–2753. Yao Q, Weigel B, Kersey J. Synergism between etoposide and 17-AAG in leukemia cells: critical roles for Hsp90, FLT3, topoisomerase II, Chk1, and Rad51. Clin Cancer Res. 2007;13:1591–1600. Yao Q, Nishiuchi R, Kitamura T, Kersey JH. Human leukemias with mutated FLT3 kinase are synergistically sensitive to FLT3 and Hsp90 inhibitors: the key role of the STAT5 signal transduction pathway. Leukemia. 2005;19:1605–1612. Tonelli R, Sartini R, Fronza R, etÂ€ al. G1 cell-cycle arrest and apoptosis by histone deacetylase inhibition in MLL-AF9 acute myeloid leukemia cells is p21 dependent and MLL-AF9 independent. Leukemia. 2006;20:1307–1310. Niitsu N, Hayashi Y, Sugita K, Honma Y. Sensitization by 5-aza-2¢-deoxycytidine of leukaemia cells with MLL abnormalities to induction of differentiation by all-trans retinoic acid and 1alpha,25-dihydroxyvitamin D3. Br J Haematol. 2001;112:315–326. Hudes G, Carducci M, Tomczak P, etÂ€al. Temsirolimus, interferon alfa, or both for advanced renalcell carcinoma. N Engl J Med. 2007;356:2271–2281. Brown VI, Fang J, Alcorn K, etÂ€al. Rapamycin is active against B-precursor leukemia inÂ€vitro and inÂ€vivo, an effect that is modulated by IL-7-mediated signaling. Proc Natl Acad Sci U S A. 2003;100:15113–15118. Teachey DT, Sheen C, Hall J, etÂ€al. mTOR inhibitors are synergistic with methotrexate: an effective combination to treat acute lymphoblastic leukemia. Blood. 2008;112:2020–2023.

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Targeted Therapeutic Approaches for AML Robert J. Arceci and Donald Small

Introduction Acute myeloid leukemia comprises about 20% of the acute leukemias in children, but it is responsible for more than half of leukemic deaths due to leukemia. Compared to the tremendous success in the treatment of acute lymphocytic leukemia in the last three decades, resulting in more than 80% cure rate, improvements in AML therapy have been more limited with only about half of patients with AML being cured. Risk-adapted therapy has been the cornerstone of ALL therapy. One of the reasons for the success of this approach in ALL is that standard ALL induction and consolidation have been able to be intensified without causing significant morbidity and mortality. In contrast, the leukemic stem cell in most AML subtypes is inherently more drug resistant requiring significantly intensified courses of near myeloablative combinations of chemotherapeutic agents. This has resulted in a plateau in survival at approximately 50% along with significant morbidity and mortality. Current AML therapy is based on the use of multi-agent combinations of Â�noncross-resistant chemotherapeutic agents, dose intensification, risk-adapted use of allogeneic HSCT as well as aggressive, pre-emptive use of supportive care interventions. Such therapeutic approaches are also relatively nonselective and associatedÂ� with significant treatment-related toxicities. Advances in the molecular basis for AML have provided improvements in subtype classification, a better understanding of risk stratification, and the introduction of molecularly targeted therapies. This increased the understanding of the molecular basis of AML in combination with a growing list of host genetic risk factors provides for the possibility of truly individualized therapy.

R.J. Arceci (*) The Sidney Kimmel Comprehensive Cancer Center at Johns Hopkins, Pediatric Oncology, Johns Hopkins University, Baltimore, MD 21231, USA e-mail: [email protected] P.J. Houghton and R.J. Arceci (eds.), Molecularly Targeted Therapy for Childhood Cancer, DOI 10.1007/978-0-387-69062-9_4, © Springer Science+Business Media, LLC 2010

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Mechanisms of Leukemogenesis: Implications for Targeted Therapy Development The explosion of new discoveries relating to our understanding of the cellular and molecular basis of AML in adults and children has significant implications for the development of less toxic and more effective therapies but also is associated with challenges in terms of validating potential therapeutic targets both in preclinical models and in clinical trials. The genetic changes that lead to AML can be both inherited as well as somatically acquired. Many of these molecular changes have been largely derived from abnormal chromosomal rearrangements that in turn have led to the identification of gene translocations and resulting fusion products that play roles in the development of AML. The genes targeted by these abnormal chromosome changes often include transcription factors that are important in regulating myeloid differentiation as well as partner genes that may relocate the DNA binding or regulatory region of the transcription factor to altered sites within the cell or along DNA. For example, although the cytogenetic abnormalities observed in AML are quite heterogeneous, they may result in the altered targeting of the same molecular pathways. For example, all the t(8;21), t(3;21), t(16;16), t(16;21), and inv(16) chromosomal changes observed in AML alter the function of core binding factor complexes that regulate chromatin structure and function. Another example includes translocations that involve PML and RAR alpha in APL. Such genetic abnormalities result in a change in the normal function of these core binding factors leading to transcriptional repression of target genes regulating proliferation, cell survival, and differentiation. Furthermore, the convergence of these altered differentiation regulatory pathways has important implications for therapeutic targeting. For example, instead of developing therapeutic inhibitors directed toward each fusion product, it may be possible to target the critical and shared transcriptional pathways that contribute to this step in AML development (Ichikawa etÂ€al. 2006). However, preclinical murine models designed to test the contribution of important translocations in AML have determined that the presence of only the translocation fusion product is usually insufficient for leukemic transformation. This observation led to a search for cooperating genetic pathways, which, in turn, led to the recognition of the importance of activating mutations of tyrosine kinase receptors such as FLT3 and c-KIT as regulators of leukemia cell survival and proliferation. The presence of activating mutations of specific tyrosine kinase receptors has also been shown to contribute to chemotherapeutic resistance through their activation of downstream resistance pathways as well as increased proliferation and survival of leukemic blasts. Thus, the pathways affected by these mutated receptors and signal transduction molecules also represent additional therapeutic targets.

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Refining the Definition and Relevance of Targeted Therapy The considerable overlap in the phenotypes of AML and normal hematopoietic precursor cells has presented significant challenges in terms of distinguishing them for targeted therapies. The development of fluorescence activated cell sorting in conjunction with monoclonal antibodies directed to lineage specific markers has provided an approach to isolate primitive self-repopulating leukemic stem cells apart from normal progenitors. The AML, self-repopulating primitive cells are quite rare within the population of leukemic blasts, with estimates of their Â�frequency being in the 0.2 to 200 per 106 mononuclear cells range. These data have led to the conclusion that in many instances, the subtype of AML observed clinically depends on the types of initiating and subsequent molecular events that occur in an early stem cell population of hematopoietic precursors rather than from transformation occurring in a later stage, lineage-determined, precursor cell (Jordan 2007; Barabe etÂ€al. 2007; Hope etÂ€al. 2004). The complex hierarchy of the self-initiating AML stem cell creates important challenges for targeted therapy development. Thus, for a pathway to represent a potentially curative target and not simply a cytoreductive one, the altered pathway should have a specific, or at least a highly selective, effect on the leukemia selfinitiating stem cell. Thus, a potentially curative target should (1) be expressed in the relevant cell populationÂ�, (2) survival of the relevant cell type should be dependent on the expression of the target, and (3) modulation of the targeted pathways should result in the demise or permanent inactivity of the relevant cell type. As noted above, the relevant cell type in AML should include the self-replicating leukemic stem cell. Clinical trials to assess the relevance of agents directed toward enumerating and eradicating this Â�self-repopulating AML in order to test the clinical relevance of these concepts are needed.

Development of Targeted Therapies for AML Although many therapies are particularly effective at cytoreducing leukemic cell burden in patients with AML, very few have been selectively developed to target the self-repopulating leukemic stem cell population. In order to make this type of targeting a reality, a significant amount of investigation needs to be done into what distinguishes AML stem cells from their normal counterparts. Little information has thus far been reported on this issue, although some reports are starting to reveal potentially important differences between leukemic and the normal stem cell counterparts (Andersson etÂ€al. 2005a,b; Ross etÂ€al. 2004; Oshima etÂ€al. 2003). In addition, while most cytoreductive agents produce rapid antileukemic responses, agents targeted at the relatively rare but self-repopulating leukemic stem cell population would be expected to result in slow clinical responses. Most clinical trials do not Â�usually provide the opportunity to observe such slow responses, especially in

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Cancer Self-Renewing Stem Cell

Non-Self-Replicating Cancer Cell

Replication, Proliferation and Differentiation

Lineage Specific Antigens Drug Transporter Inhibitors Cytokine Receptors Signal Transduction Pathways Chromatin/Transcription

Replication or Dormancy Immunotherapy

Proliferation and Differentiation Microenvironment

Fig.Â€1â•… Schematic of possible cellular and molecular targets in AML (adapted from Arceci and Cripe (2002))

patients with acute leukemia. Thus, if a targeted agent is not an effective cytoreductive agent, it may still provide important antileukemic effects, particularly when used in Â�vcombination with more traditional cytoreductive chemotherapies. Quantitation of the self-repopulating AML stem cells in such combination trials may be an approach to evaluate the effect of such stem-cell directed treatments. Several pathways and targets are being currently tested in pediatric AML that are directed alone or in combination toward reversing the abnormalities in leukemia cell survival/drug resistance, proliferation, and differentiation. Promising new approaches to more selective treatments include inhibition of proliferation and survival pathways such as FLT3-ITD and c-KIT receptors and their downstream targets, such as RAS, as well as transcriptional or chromatin-based strategies and immunotargeted therapies with monoclonal antibodies and vaccines (Fig.Â€1).

Specific Examples of Targeting AML Targeting Pathways that Alter Leukemia Cell Proliferation and Survival Tyrosine Kinases Laboratory studies demonstrating the importance of cytokines (survival and differentiation factors) and their receptors along with the induced downstream Â�signaling

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pathways that direct normal hematopoiesis were fundamental in elucidating the role of these same pathways in the etiology and pathophysiology of AML. Several of these cytokine receptors, such as FLT3, c-KIT, c-FMS, PDGF-R, and VEGF-R, have been shown to be expressed at high levels or have activating mutations that lead to increased proliferative, survival, and drug resistance in AML (Kiyoi etÂ€al. 1997, 1998; Meshinchi etÂ€al. 2001, 2003a; Yamamoto etÂ€al. 2001). These changes in leukemic cell behavior have furthermore been shown in several instances to changes in clinical outcome of patients. For example, pediatric and adult patients with FLT3/ITD mutations, particularly in the group of patients with normal karyotypes, have a poor outcome. Such observations have provided the rationale to target these mutant receptors and their downstream signaling pathways. Mutations effecting c-KIT occur more frequently in the subset of AML characterized by alterations in core binding factors and have been reported to portend a poor outcome in an otherwise favorable group of patients (Schnittger etÂ€al. 2006, 2007; Shimada etÂ€al. 2006). In both the examples of FLT3/ITD and c-KIT, the mutant receptors result in distinct changes in signal transduction compared to activation of the respective wild type receptors, leading to cytokine independent survival, increased proliferation, and greater resistance to chemotherapeutic drugs (Tse etÂ€ al. 2000; Ning etÂ€ al. 2001a,b,c). Another important observation regarding FLT3/ITD is that at least some cases of AML appear to have the mutation in a leukemia repopulating subset of cells as well as their progeny, thus in part addressing the issues raised above concerning the targeting of the leukemia initiating stem cell as well as the right molecular target (Levis etÂ€al. 2005a; Pollard etÂ€ al. 2006). However, in some patients with FLT3/ITD positive AML, relapsed samples lacked the FLT3/ITD, suggesting that in these patients, FLT3/ITD is more likely to be a secondary mutational event rather than arising in a leukemia selfrepopulating cell; an alternative explanation would be that more than one leukemia initiating cell was initially present and differentially selected for during treatment (Kottaridis etÂ€al. 2002; Cloos etÂ€al. 2006). Several inhibitors of FLT3/ITD, such as CEP-701 (Cephalon) (Brown etÂ€al. 2006; Levis etÂ€al. 2002, 2004; Smith etÂ€al. 2004), MLN518 (Millenium) (DeAngelo etÂ€al. 2006) and PKC412 (Novartis) (Cools etÂ€ al. 2003; Stone etÂ€ al. 2005), have been developed and tested in preclinical models and early clinical trials. One of these inhibitors, CEP-701 (lestaurtinib), showed excellent activity in a preclinical model (Levis etÂ€al. 2002) followed by clinical trials as a single agent (Smith etÂ€al. 2004; Levis etÂ€ al. 2006). In these clinical trials, decreases in peripheral blood leukemic blasts were observed but no significant marrow responses leading to complete remissions. Such results have also been observed with other inhibitors of FLT3 (DeAngelo etÂ€al. 2006; Stone etÂ€al. 2005; Knapper etÂ€al. 2006). However, an important aspect of the single agent clinical trials with CEP-701 was that methods were developed and validated which showed that sufficiently high plasma levels of the inhibitor could be obtained to inhibit the activated, mutant receptor (Levis etÂ€al. 2006). Additional preclinical work demonstrated increased efficacy of combining CEP701 with cytosine arabinoside (Levis etÂ€al. 2004) which subsequently led to encouraging early results in a randomized phase II trial in adults with relapsed AML expressing FLT3/ITD receptors (Levis etÂ€al. 2005b). In the preliminary data reported

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from this trial, 76% (13 of 17) of patients showed plasma levels of drug that inhibited FLT3 activity by at least 85%. Further, true clinical bone marrow responses were observed primarily in the group of patients whose AML was sensitive and who had sufficient, inhibitory plasma levels of CEP-701 (Levis etÂ€al. 2005b). COG AAML06P1 in combination with chemotherapy (idarubicin and cytosine arabinoside) for pediatric patients with relapsed AML harboring FLT3/ITD mutations. In addition, the increased expression of wild type FLT3 in AML, particularly in infant leukemia, has led to a COG clinical trial combining CEP-701 with chemotherapy in this very high risk leukemia in infants (Brown etÂ€al. 2006; Armstrong etÂ€al. 2002). There are plans to test the addition of FLT3 inhibitors into the COG phase III trial for patients with newly diagnosed AML expressing the FLT3/ITD mutant receptor. The c-KIT tyrosine kinase receptor has also been associated with poor outcome in the subtype of otherwise good prognostic core binding factor AML (Schnittger etÂ€al. 2006; Shimada etÂ€al. 2006) although a recent report from COG was not able to confirm a worse prognosis associated with c-KIT mutations (Poland et al. 2009). Mutations of c-KIT involve single base pair changes leading to amino acid changes in the kinase domain and resulting in an auto-activating receptor. Less common in AML, some c-KIT mutations involve the cytoplasmic, juxtamembrane portion of the receptor; such mutations have different kinase inhibitor characteristics than the kinase domain mutations (Roberts etÂ€al. 2007). c-KIT mutations are observed in only about 3 to 5% of AML, but in 20 to 30% of AML characterized by changes in core binding factors (Schnittger etÂ€al. 2006; Shimada etÂ€al. 2006; Meshinchi etÂ€al. 2003b). Thus, kinase inhibitors with activity against such c-KIT mutations would be expected to be potentially useful in this subset of AML. Imatinib, first developed for the inhibition of the BCR-ABL fusion kinase and the treatment of CML, also has significant activity against c-KIT as well as the platelet derived growth factor receptor (PDGFR) (Heinrich etÂ€al. 2000; Fernandez etÂ€al. 2007). In vitro results have demonstrated that imatinib selectively inhibits wild type and mutant c-KIT activity as well as its activation of downstream signaling molecules, such as MAPK and AKT (Heinrich etÂ€al. 2000). An initial phase II study of imatinib in patients with refractory or recurrent AML showed disappointing results with no significant clinical responses (Kindler etÂ€al. 2004). However, a subsequent clinical trial using imatinib in 21 patients with refractory AML expressing c-KIT demonstrated 5 out of 21 patients with hematologic responses, two of which were complete hematologic remissions in patients who had relatively low blast counts in the bone marrow or in peripheral blood (Ito etÂ€al. 2005; Jentsch-Ullrich etÂ€al. 2004; Pompetti etÂ€al. 2007). Imatinib and low dose cytosine arabinoside has been tested in older adults in a phase II trial based on inÂ€vitro data that showed synergistic cytotoxicity with the combination compared to either agent used alone (Heidel etÂ€al. 2007). However, the clinical response rate was only 11% and did not appear significantly better than AraC alone based on comparison with historical controls (Heidel etÂ€al. 2007). There are several potential problems with such studies. First is that imatinib may not be a particularly potent or specific inhibitor of mutant c-KIT receptors; second, the inhibition of the c-KIT receptor

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kinase activity and/or downstream signaling pathways were not assessed in leukemic blasts from patients, thus raising the possibility that the kinase was not sufficiently inhibited. Although c-KIT remains a potentially important therapeutic target in a subset of patients with AML, more selective and potent inhibitors need to develop along with synergistic combinations conventional chemotherapy or other targeting agents. Although their name suggests the unlikely possibility that the IGF-I and VEGF receptors would play a role in AML, there are data to the contrary. IGF-IR expression has been demonstrated on AML cells although activating mutations have not been reported (Hizuka etÂ€al. 1987). In addition, IGF-I has been shown to contribute along with other cytokines to the survival and proliferation of AML cells as well as their response to AraC (Abe etÂ€al. 2006; Frostad and Bruserud 1999). In vitro studies have further shown that blockade of the IGF-IR was able to induce apoptosis in AraC resistant AML cells (Abe etÂ€al. 2006). As IGF-IR inhibitors, whether small molecules or monoclonal antibodies, are further studied in the laboratory and in clinical trials (Cosaceanu etÂ€al. 2007; Jerome etÂ€al. 2006), a potential role of these approaches alone or in combination with other targeted therapies, may prove worthwhile in AML. Several lines of evidence have established a role for VEGF and its receptor in hematopoiesis and in AML. Laboratory studies have demonstrated that a high percentage of AML cells express VEGF as well as VEGFR (Perl and Carroll 2007). Furthermore, the secretion of VEGF by AML blasts can stimulate bone marrow stromal fibroblasts to secrete hematopoietic cytokines such as G-CSF and GM-CSF, which, in turn, may contribute to further myeloid progenitor expansion (Aguayo 2004; Aguayo etÂ€al. 1999, 2003; Hussong etÂ€al. 2000; Kessler etÂ€al. 2007; Schuch etÂ€ al. 2002). In addition, increased bone marrow angiogenesis, observed in both MDS and AML, may be in part a result of VEGF secretion. Thus, VEGF may play both an autocrine and paracrine role in MDS and AML. Leukemic cell proliferation, survival, and resistance to chemotherapy have also been directly linked to signaling through VEGF-C and its activation of VEGFR-3 (Dias etÂ€ al. 2002; Liersch etÂ€ al. 2007). Inhibitory monoclonal antibodies that bind and inactivate VEGF (e.g., Avastin or Bevacizumab) (Karp etÂ€al. 2004), or small molecule inhibitors of VEGFR (e.g., SU5416 or AZD2171) (Fiedler etÂ€al. 2003; Maris etÂ€al. 2008), have been developed for therapeutic anti-angiogenic treatment approaches. Karp etÂ€al. (2004) tested Bevacizumab post chemotherapy treatment in adults with AML and showed clearance of blasts and decreased bone marrow microvasculature. Bevacizumab is also being tested in adults with newly diagnosed AML in combination with idarubicin and AraC. Initial clinical studies with SU5416 reported a complete remission in a patient with AML, although subsequent studies have not shown significant response rates to this agent when used alone (Perl and Carroll 2007; Fiedler etÂ€al. 2003). It is possible that the selective preference of SU5416 may be too narrow or mechanisms of resistance are activated to prevent significant leukemic cell killing. Current attempts to combine such agents with chemotherapy or to use tyrosine kinase inhibitors with a broader spectrum of targets are being developed (Perl and Carroll 2007).

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The above examples demonstrate several key principles for targeted therapy in AML using TK inhibitors. First, unlike in CML, such approaches using single agents are unlikely to be highly effective in AML. However, these TK inhibitors appear to share the characteristic of sensitizing AML cells to conventional chemotherapeutic drugs, without necessarily resulting in significant overlapping toxicities. Nevertheless, because some of the activated TKs signal through common, downstream pathways, investigators are attempting to determine whether inhibition of these shared, convergence points can be therapeutically exploited (Fig.Â€2). However, similar to imatinib, resistance to TK inhibitors has been demonstrated, although the pathways leading to resistance may be different (Piloto etÂ€al. 2007).

Inhibition of Signaling Pathways Downstream of TK Receptors Not long after its initial identification as an important dominant oncogene in malignant transformation, RAS mutations were documented in about 25% of AML/MDS samples (Farr etÂ€al. 1991). Further, in the absence of RAS mutations, RAS activation through its conversion from GDP to GTP and farnesylated tethering to the inner cell membrane is known to play a critical role in the development of AML as well as the myeloproliferative syndrome, juvenile myelomonocytic leukemia (JMML) (Loh etÂ€al. 2004). While specific inhibitors of GTP activation of RAS have not yet been successfully reported, farnesyl transferase inhibitors (FTI) have been tested in patients with AML (Karp 2001, 2005; Emanuel etÂ€al. 2000; Gotlib 2005). Phase I studies in adults with AML reported significant clinical responses that did not appear to be dose dependent (Karp 2001). A subsequent multi-institutional phase II study reported a 5% complete remission rate using tipifarnib as a single agent in adult patients with relapsed and/or refractory disease (Harousseau etÂ€ al. 2007). Tipifarnib as a single agent in patients aged 65 or older with high risk AML showed a response rate of 10 to 20% (Lancet etÂ€al. 2007). Based on such results, a randomized phase III trial comparing tipifarnib to supportive care in patients aged 70 years or more with AML is being conducted in Europe. A pediatric randomized trial of tipifarnib versus no drug in the postallogeneic HSCT setting by the COG in patients with relapsed AML. While tipifarnib has shown a low, but real level of activity in AML as a single agent, future use and testing will likely be in combination with other targeted agents and/or conventional chemotherapy. Of note, however, is that the mechanism of action of such FTI in AML and whether they are mediating their effect through the RAS pathway remain unclear. Downstream of RAS are the RAF, MEK, ERK, and STAT pathways, all of which are being considered as potential therapeutic targets in AML (Fig.Â€2). For example, small molecule inhibitors of the MAPK pathway, such as PD98059 and PD184352, show significant and selective induction of cell cycle arrest and apoptosis in AML cells (Milella etÂ€al. 2001, 2002, 2003). MAPK inhibition can also result in increased sensitization to chemotherapy of AML cells, particularly from AML expressing the

Fig.Â€2â•… Schematic of FLT3 signaling pathways as an example potential downstream molecular targets as a result of activating mutations of tyrosine kinases. From Doepfner etÂ€al. (2007)

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stem cell marker, CD34 (James etÂ€ al. 2003). RAF-1 kinase inhibitors, such as sorafenib, are also being clinically developed. Of note, sorafenib also has significant activity against c-KIT (Tong etÂ€al. 2006). The mTOR (mammalian Target Of Rapamycin) serine/threonine kinase plays a primary role as a regulator of cell cycle progression, proliferation, apoptosis, and angiogenesis in both normal and neoplastic cells. mTOR functions as a type of Â�utility player of signal transduction and in that it is a central regulator of a variety of signaling pathways originating from TK receptor activation, including FLT3, c-KIT, PDGFR, c-FMS, and VEGF, as well as through RAS, various phosphatases, such as PTEN and SHP-1/2 (Weisberg etÂ€al. 2008). Coactivation of the PI3K/AKT pathway is also usually observed with mTOR activation (Tamburini etÂ€al. 2008; Xu etÂ€al. 2003, 2005). In each of these examples, a role of mTOR has been demonstrated in the proliferation, survival, and drug resistant phenotype of approximately 70% of AML cases examined (Xu etÂ€al. 2003; Recher etÂ€al. 2005; Min etÂ€al. 2003). Several highly selective inhibitors of mTOR, including rapamycin, CCI-779, RAD001, AP23573, and MK-8669 have been developed. A clinical trial in adults with refractory/relapsed or secondary AML reported that four of nine patients had partial responses to rapamycin when used as a single agent (Recher etÂ€al. 2005). A phase I/II study using RAD001 (everolimus) as a single agent in adults with refractory and/or relapsed hematologic malignancies showed that a 10Â€mg daily dose was well tolerated and two of five patients with MDS had a major and minor platelet responses. No patients with AML were included on this trial (Yee etÂ€ al. 2006). Because of its central role and activation in a majority of patients with AML, mTOR inhibition as a therapeutic approach, is being planned for clinical trial testing in pediatric patients with relapsed/ refractory AML. Combining mTOR inhibitors with conventional chemotherapy, particularly ARAC containing regimens, or with other signal transduction inhibitors will likely be the best approach for definitive clinical testing.

Drug Resistance Mechanisms and Leukemic Cell Survival As discussed above, in addition to conveying a proliferative advantage, mutant or highly expressed tyrosine kinase receptors and their downstream signaling pathways can also lead to increased drug resistance on the part of AML blasts. Other molecular pathways may also lead to deÂ€novo or acquired drug resistance as well. The identification of the drug efflux transporter P-glycoprotein (Pgp 170 or MDR1 P-glycoprotein) as an important mechanism of multidrug resistance in cancer cells provided a potential therapeutic target. To this end, a variety of competitive inhibitors, including cyclosporine A and its nonimmunosuppressive D isomer, PSC833, as well as a number of other small molecules, were developed to block the drug efflux capacity of this ATP-dependent transporter. Inhibition of the MDR1 transporter results in decreased efflux of multiple chemotherapeutic, natural product drugs, such as anthracyclines and vinca alkaloids, leading to their increased intracellular concentration and subsequent cell death. The observations that many tumor types that are resistant to

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such chemotherapeutic agents also express high levels of the MDR1 P-glycoprotein and could be resensitized to chemotherapy in the presence of an MDR1 inhibitor provided the experimental foundation to clinically test this approach. Of note, the expression of MDR1 P-glycoprotein was found to be high in adult AML and, furthermore, showed even higher expression at the time of relapse (Baer etÂ€al. 2002; Leith etÂ€al. 1995, 1999; Willman 1996). Such high levels of MDR1 P-glycoprotein were correlated with poor outcome (Willman 1997). Expression in childhood AML was reported to be considerably lower (Sievers etÂ€al. 1995). After the completion of phase I/II trials with MDR1 P-glycoprotein inhibitors, it became clear that significant dose adjustments of the chemotherapeutic drugs needed to be made to correct for decreased drug elimination secondary to inhibition of cellular efflux in normal cells of the liver, kidney, and intestine (List etÂ€al. 1993; Dahl etÂ€ al. 2000). Most randomized phase III trials in adults with AML tested the impact on outcome of inclusion of an MDR1 inhibitor, e.g., PSC833, in combination with chemotherapy, did not show an improvement compared to chemotherapy along (Greenberg etÂ€al. 2004). One exception was a randomized comparison of cyclosporine A and no cyclosporine in combination with daunorubicin and ARAC during consolidation for adults with poor-risk AML (List etÂ€al. 2001). Although the rate of complete remission was not different in the two groups of patients (39 vs. 33%, pâ•›=â•›0.14), relapse free survival and overall survival were significantly improved in the group receiving cyclosporine (RFS 34 vs. 9% at 2 years, pâ•›=â•›0.031 and OS 22 vs. 12%, pâ•›=â•›0.46, respectively). Currently, the Eastern Cooperative Oncology group is testing the nonpharmacologically active inhibitor, zosuquidar and the Southwest Oncology Group is testing cyclosporine A in randomized, phase III trials. Phase I and II studies in pediatric patients with relapsed/refractory AML established the dose of cyclosporine A and chemotherapy when used in combination (Dahl etÂ€al. 2000). Based on these results, the POG 9421 study randomized patients to receive in consolidation cyclosporine or no cyclosporine in combination with mitoxantrone and ARAC during the consolidation phase of treatment (Becton etÂ€al. 2006). In addition, this study randomized high dose to standard dose ARAC during induction therapy. The results showed that the best outcome was associated receiving high dose ARAC during induction and cyclosporine plus mitoxantrone and ARAC during consolidation. However, no statistically significant differences in outcome were observed for either the high dose ARAC or the addition of cyclosporine when considered as independent variables (Becton etÂ€al. 2006). The lack of a definitive benefit for MDR1 P-glycoprotein inhibition may be related to the emergence of other mechanisms of resistance in the presence of MDR1 inhibition, including the possible role of one or more of the dozens of other transport pumps expressed in cells, increased expression of the MDR1 P-glycoprotein as well as ineffective blockade and increased toxicity. While further MDR transporter inhibition trials in pediatrics are unlikely in the near future, one exception might be in conjunction with anti-CD33-calicheamicin immunotargeted therapy because a known mechanism of resistance to calicheamicin is MDR1 P-glycoprotein efflux (Arceci etÂ€al. 2005; Linenberger etÂ€al. 2001).

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A second pathway to chemotherapy-induced resistance is the upregulation of bcl-2. Extensive work on the role of bcl-2 in drug resistance has established an important role in AML. The ability of inhibitors of bcl-2 to sensitize AML cells to chemotherapeutic agents has led to the development of clinical trials using bcl-2 inhibitors (e.g., antisense oligonucleotide oblimersen or Genasense) (Marcucci etÂ€ al. 2003, 2005). While this approach is being tested in adults with AML, no immediate clinical trials are being planned in pediatric AML (Moore etÂ€al. 2006). An exciting new approach of therapeutically targeting protein degradation mechanisms in cancer cells as a means to sensitize them to chemotherapy has arisen out of experimental studies demonstrating the extreme dependency of tumor cells on such pathways (Zavrski etÂ€al. 2007). In part because of the increased metabolic rate, the increased concentration of reactive oxygen species and the expression of multiple, mutated or aberrant proteins, malignant cells heavily depend on proteasome degradation. By blocking this system, proteasome inhibitors have been shown to increase the sensitivity of tumor cells to chemotherapeutic agents as well as induce apoptosis. Thus, this “proteasome addiction” of tumor cells, similar to their “kinase addiction” may make them more susceptible to inhibition of these pathways than normal cells. The observations that AML stem cells express high levels of the transcription and survival promoting factor, NF-KB, along with the expression of the NF-KB inhibitor, IKB, provides a potentially important link between survival factors and proteasome inhibition (Guzman etÂ€al. 2001, 2002, 2007). For example, the use of the proteasome inhibitor, bortezomib (PS-341 or Velcade), has been shown to decrease the degradation of IKB and thereby reducing the amount of translocation of NF-KB to the nucleus, resulting in increased apoptosis of AML self-repopulating or stem cells (Guzman etÂ€al. 2001, 2002). Further, the apoptotic effects of an anthracycline, for example, idarubicin, were greatly enhanced in AML stem cells in the presence of proteasome inhibition (Guzman etÂ€al. 2001, 2002). The induction of the proapoptotic factors, BIM and Bax, have also been observed to be increased in AML cells exposed to idarubicin and proteasome inhibition (Pigneux etÂ€al. 2007). Based on such data, the COG is planning to conduct a clinical trial with bortezemib in combination with chemotherapy in children with relapsed and/or refractory AML. This type of approach should help resolve the issue of the ability to selectively target AML stem cells in a clinical setting.

Epigenetic and Chromatin Remodeling-Directed Targets The modifications of DNA that help regulate its expression, whether cytosine methylation, histone type and/or modification or changes in other DNA-associated proteins, are considered epigenetic events (Holliday 2006). Epigenetic modifications of the genome have been causally linked to altered gene expression, often involving gene silencing of tumor suppressor genes and the development of various types of cancer. Several of the chromosomal translocations that characterize

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various subtypes of AML are also known to generate fusion proteins of gene products that profoundly influence DNA and/or chromatin structure and function. For instance, several of these fusion proteins, including the t(15;17)/PML/RAR alpha in APL, those involving core binding factors such as AML1/ETO resulting from t(8;21), or those involving the MLL gene product, have been demonstrated to recruit repression-associated chromatin remodeling protein complexes to specific genomic regions (Wood etÂ€al. 2005; Nie etÂ€al. 2003). In a significant percentage of AML, silencing of expression coincides with promoter hypermethylation of the tumor suppressor gene, p15INK4b or the mismatch repair gene, hMLH1, is observed (Preisler etÂ€al. 2001; Herman etÂ€al. 1997; Seedhouse etÂ€al. 2003). Other chromatin marks of the repression of gene expression are decreased histone H3 acetylation as well as trimethylation of lysine 27 of histone H3 (Bernstein etÂ€al. 2007; Ting etÂ€al. 2006). Rearrangements of the MLL gene along with its fusion to other partner genes results in both activation and repression of a subset of genes involved in AML, particularly the upregulation of HOX gene expression (Nie etÂ€ al. 2003; So etÂ€ al. 2003, 2004). Although the epigenetic changes that characterize different subtypes of AML are complex, many of them may converge on several critical changes, similar to the convergence of some downstream signaling pathways following activation of different TK receptors. Thus, strategies to reprogram the aberrant epigenetic changes observed in cancer and, in particular, MDS and AML are being pursued. Small molecule inhibitors or modulators of both DNA methylation and histone deacetylation have been developed and are being tested alone and in combination with the intent of inducing the reactivation of expression of genes which may promote leukemia cell maturation, senescence, or apoptosis (Ghoshal and Bai 2007; Santini etÂ€al. 2007). Studies on MDS in adults led to the approval by the US Food and Drug Administration for the DNA methylation inhibitor 5¢-azacytidine (Garcia-Manero etÂ€ al. 2007; Gattermann etÂ€ al. 2007; Plimack etÂ€ al. 2007; Kaminskas etÂ€ al. 2005; Silverman 2001). However, inÂ€ vitro and subsequently inÂ€vivo data have demonstrated synergy in terms of the induction of gene expression with the use of combinations of inhibitors of DNA methylation and HDACs (Griffiths and Gore 2008; Qin etÂ€al. 2007). Clinical trials testing the DNA methyltransferase inhibitors, 5¢-azacytidine or the deoxy-form, decitabine, alone and in combination with HDAC inhibitors, such as suberoylanilide hydroxamic acid (SAHA, Vorinostat or Zolinza), the benzamide derivative, MS275, depsipeptide, and valproic acid, are being intensely pursued in adults with MDS and high risk AML (Griffiths and Gore 2008; Gore etÂ€al. 2006). Some of the results from these trials have demonstrated clinical responses that are comparable to conventional chemotherapy in older adults with high risk MDS/AML (Garcia-Manero etÂ€ al. 2006; Issa etÂ€ al. 2004; Soriano etÂ€ al. 2007; Kantarjian etÂ€ al. 2007). Trials using DNA methyltransferase and HDAC inhibitors alone and in combination are either ongoing or being planned in pediatric patients. Several challenges exist for this type of therapeutic targeting. The first is how specific or global the effects of the inhibitors should be in terms of effecting epigenetic change. An unwanted result would be if DNA demethylation, for example,

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induced genes that would prevent apoptosis or induce drug resistance; there is some precedent for this occurring (Li etÂ€al. 1999). Another challenge is how best to use epigenetically directed therapies in combination with each other, with other targeting agents or with conventional chemotherapy. For example, DNA replication is required for the effects of DNA methyltransferase inhibitors to be most effective. HDAC inhibitors have been known to cause cell cycle arrest. Thus, the sequencing of agents with these characteristics will be a significant issue to solve. A third challenge is to determine how such epigenetic therapies will impact the AML stem cell population. Increasing evidence suggests that the cancer stem cells share many epigenetic marks with embryonic stem cells. How such complex genomic signatures can be therapeutically manipulated remains an immense challenge.

Targeted Immunotherapy and Immunostimulatory Therapy Immense potential of the discovery of how to generate antigen specific, monoclonal antibodies from B cell/myeloma hybridomas by Kohler and Milstein has only started to be realized in the last decade. An outcome of such work in target AML has been thus far most using monoclonal antibodies directed against the differentiation antigen, CD33, a sialic acid-dependent cell adhesion molecule that is differentially expressed during myeloid differentiation. Gemtuzumab ozogamicin (GO; Mylotarg™) is a humanized, IgG4 subtype, monoclonal antibody conjugated to calicheamicin and directed against CD33; GO has demonstrated significant activity in AML and was the first toxin/monoclonal antibody fusion approved by the US FDA (Larson etÂ€al. 2005). Initial clinical trials in adults and children with relapsed or primary refractory disease AML showed response rates of approximately 30 to 35% when GO is used as a single agent (Arceci etÂ€ al. 2005; Larson etÂ€ al. 2005). Of particular interest, the response in children with primary refractory disease was the same as that for patients with relapsed disease who had achieved a prior remission, suggesting that some conventional resistance mechanisms can be circumvented with this agent (Arceci etÂ€al. 2005). The maximal tolerated dose (MTD) for children was determined to be 6Â€mg/m2 on the pediatric phase I study. The main toxicity was myelosuppression. However, there was a 24% (7 of 29 patients) overall incidence of veno-occlusive disease (VOD) with 6 of 13 (40%) developing VOD during a subsequent HSCT. VOD was most frequently observed in patients undergoing allogeneic SCT in less than 3.5 months from receiving GO. A COG phase I trial using GO in combination with either mitoxantrone plus high dose ARAC (regimen A) or with Capizzi II chemotherapy (regimen B) showed a maximum tolerated dose of GO to be 3 and 2Â€ mg/m2, respectively (Aplenc etÂ€ al. 2008). Significant toxicities included those typically observed in patients with AML. DLTs for the Capizzi II included grade III transaminitis, pancreatitis and hyperbilirubinemia requiring a dose de-escalation to 2Â€mg/m2. The starting dose of 3Â€mg/m2 of GO in combination with mitoxantrone plus high dose ARAC was well tolerated. Responses included 52 and 40% CR

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or CRp for regimens A and B, respectively (Aplenc etÂ€ al. 2008). The MRC has performed a randomized trial of GO plus induction chemotherapy with ARAC, daunomycin, and etoposide (ADE) as well as with the second intensification course of mitoxantrone plus ARAC (Kell etÂ€al. 2003). Results from this trial have been reported in abstract form and an advantage of EFS and OS for good risk patients who received GO (Burnett etÂ€al. 2006). The current COG trial is test in a randomized fashion whether the addition of GO to induction and intensification courses of treatment results in an improved outcome for pediatric patients with newly diagnosed AML. In addition to the potential role of GO in improving the outcome of patients with AML, several additional questions remain. The question of why many patients are resistant to GO therapy has been partially addressed by the findings that calicheamicin is a substrate for the MDR1 P-glycoprotein drug transporter (Arceci etÂ€al. 2005; Linenberger etÂ€al. 2001). Thus, AML cells that express high levels of this transporter are more likely to be resistant to GO. These observations have led to the consideration of a clinical trial in pediatric patients with relapsed or refractory AML that combines GO with an inhibitor of the MDR1 transporter. A second question is whether GO will augment killing of AML repopulating stem cells. There is now definitive evidence of expression of CD33 on the surface of some AML stem cells, although the expression does not appear to be uniform across all AML (Pollard etÂ€al. 2006; Taussig etÂ€al. 2005). Phase III randomized trials will help answer the question of the utility of adding GO to conventional chemotherapy, but trials developed to look more specifically at the effects of such combination on AML stem cells should also be performed. The potential for targeting AML with monoclonal antibodies remains an important avenue for future research. Defining antigens that are more selectively or specifically expressed on AML stem cells remains an important challenge. CLL-1 has been reported to be such a specific AML stem cell antigen although its utility as a therapeutic target will need to be definitively tested in clinical trials (van Rhenen etÂ€al. 2007). The hope that the inherent ability of the immune system to recognized foreign antigens and cells could be directed toward antitumor therapy has been a challenge for many decades. Several preclinical models for vaccine-mediated treatment of AML have shown excellent antileukemic effects, including cures (Arceci 1998; DunussiJoannopoulos etÂ€al. 1996, 1997, 1998). Most of these preclinical models have involved upon the transduction of costimulatory receptors into AML cells or the use of cytokines to induce immune costimulation (Cheuk and Guinn 2008; Chan etÂ€al. 2005). Clinical trials in adults with AML have involved similar approaches but remain in very early stages of development (Houtenbos etÂ€al. 2006). Another approach has been to expose patients with potentially immunogenic antigens that are preferentially expressed on AML blasts (Greiner etÂ€ al. 2004, 2005). For example, WT1 peptides are being tested in an attempt to augment anti-AML responses (Greiner etÂ€al. 2005, 2006; Gaiger etÂ€al. 2000). Attempts to stimulate anti-AML immune responses in pediatric patients have been tested in the randomized CCG-2961 trial, which used a limited course of IL-2 in the postremission period (Lange etÂ€al. 2008). No significant difference in out-

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come was observed between the patients who received IL-2 and those who did not (Lange etÂ€al. 2008). This result could certainly have been due to the relatively short course or dosing of IL-2. Based in part on the empiric evidence that significant graft versus host disease is correlated with a lower relapse rate for patients with AML undergoing allogeneic HSCT, the use of donor lymphocytes and attempts to modulate graft versus tumor responses with cytokines and or early withdrawal of immunosuppressive drugs have been tested but with limited success. Another possibility, supported by preclinical models, is that this approach simply may not be sufficiently robust to elicit significant antitumor responses. Attempts to augment anti-AML immune responses in the allogeneic HSCT setting are also being tested in a COG trial attempting to exploit HLA mismatching and blockade of natural killer cell inhibition.

Future Challenges A major hope for improving the outcome of patients with AML is that more molecularly targeted therapies that will increase antileukemic activity without adding significant toxicity will be developed. Some of these agents, such as antibody-directed therapies and tyrosine kinase inhibitors, are currently in phase II and III clinical trials. The results of such trials should determine whether these approaches will be integrated into future regimens, establishing a new standard backbone for AML therapy. Although the thought of eliminating the need to use currently available cytotoxic chemotherapeutic agents with their short- and long-term associated adverse sequelae may seem improbable at this time, this should be our goal. Subtypes of AML with distinct genomic, gene expression or proteomic signatures or epigenetic patterns as well as with mutations of genes encoding critical molecules affecting signaling pathways should be prime targets for this type of therapy. In addition, essential to future strategies will be the important consideration of targeting leukemic self-repopulating progenitors in clinical trials. There is also a significant need for more predictive inÂ€vitro and animal models for the many subtypes of AML. Such truly predictive inÂ€ vitro or inÂ€ vivo models would help greatly in the prioritization of new agents. However, at the current time and in the conceivable future, the definitive proof that a drug will improve outcomes in patients is to demonstrate this in the context of a well-designed and statistically powered clinical trial. The increasing emphasis on subgroup analysis, prognostic factor stratification and patient characteristics should lead to more individualized therapeutic approaches. The historical approach of using one or two hammers to hit a heterogeneous group of nails will be challenged by the biologically based delineation of increasingly smaller subgroups of patients for testing targeted therapies. Thus, the accrual to phase III trials will also continue to be a challenge with diminishing numbers of patients characterized by specific subgroups. The use of smaller, albeit less statistically robust, trials, such as randomized phase II or trials comparing an experimental treatment with a comparable historical

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control group, may provide approaches to allow for both the testing of novel agents and decision-making concerning how to incorporate them into future regimens. We should also not forget that about 85% of children with cancer reside in Â�developing countries. And although significant obstacles may need to be overcome, it is becoming increasingly clear that approaches for effectively performing international trials need to be established. In addition, the predicted reduction in toxicity to be realized with molecularly targeted therapies is likely to be of significant benefit to patients in developing parts of the world where the complications associated with intensive chemotherapy regimens are often difficult to manage.

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Acute Promyelocytic Leukaemia Andrea Biondi, Anna Maria Testi, and Brenda E.S. Gibson

Introduction Since the time of its first description (Degos 2003), Acute Promyelocytic Leukaemia (APL) has drawn the attention of clinicians and scientists for its clinical and biological features (Degos 2003). APL is notable for distinctive clinical and biological characteristics, including its particular morphological features, the presence of potentially devastating hemorrhagic syndrome that is related to disseminated intravascular coagulopathy and abnormal fibrinolysis, and finally, the sensitivity to anthracyclinecontaining chemotherapy (Bernard etÂ€ al. 1973). The availability of differentation therapy with All-Trans Retinoic Acid (ATRA), and more recently, the discovery of the beneficial effect of arsenic trioxide (ATO), together with the molecular characterization of the t(15;17) specific translocation (Grignani etÂ€al. 1994) have produced a remarkable improvement in patient outcome in the last decade. Most patients with APL are now cured (Sanz 2006). APL is a paradigm of a malignant disease that can be treated by cell modulation, using agents that act specifically on oncogenic, molecular events. This chapter reviews its impact in children APL.

Demographic Features APL represents approximately 4 to 8% of paediatric AML (Kaspers and Creutzig 2005). The median age at presentation is probably similar to that of other AML subtypes (7 to 9Â€years), but APL has rarely been reported in the first year of life. A remarkable epidemiologic feature of APL is its high incidence in certain ethnic groups (Ribeiro and Rego 2006). Douer described the increased incidence of APL

A. Biondiâ•›(*) Centro M. Tettamanti, Clinica Pediatrica Università Milano-Bicocca, Ospedale San Gerardo,Via Pergolesi, 33, 20052 Monza, Italy e-mail: [email protected]

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among patients of Latin American descent (Douer 2003). Several series from hospitalbased registries in Italy (Biondi etÂ€al. 1994), Spain (Tomas and Fernandez-Ranada 1996), Mexico (Ruiz-Arguelles 1997), Perù (Otero etÂ€ al. 1996), and Nicaragua (Malta Corea etÂ€al. 1993) have noted a higher-than expected frequency of APL. It is still unknown whether such differences may suggest a genetic predisposition to APL and/or exposure to distinct environmental factor(s).

Pathogenesis of APL Acute promyelocytic leukaemia (APL) is characterized by rearrangements of the retinoic acid receptor-a(RARA) gene on chromosome 17q21 reviewed by Melnick and Licht (1999). To date, six different partner genes have been identified, with the vast majority of cases having the majority of the RAR gene fused to the PML gene on chromosome 15q21 reviewed by Grignani etÂ€al. (1994). Determination of the underlying molecular lesion is critical for appropriate management of APL, with the presence of an underlying PML-RARA fusion gene predicting a favorable response to molecularly targeted therapies in the form of ATRA and ATO. Two rare subtypes of retinoid-sensitive APL have been reported in the paediatric population, that is, the NPM1-RARA and NuMA-RARA fusions resulting from t(5;17)(q34-35;q21) and t(11;17)(q13;q21), respectively (Redner etÂ€al. 1996; Wells etÂ€al. 1997; Grimwade etÂ€al. 2000). The response of these rare subtypes of APL to ATO has not been established in patients. PLZF-RARA fusion, associated with t(11;17)(q23;q21) has only been reported in adults with the disease but is clinically important since it is resistant to ATRA and ATO reviewed by Mistry etÂ€al. (2003). The STAT5b-RARA fusion resulting from an interstitial deletion on chromosome 17 is also considered to be retinoid-resistant (Arnould etÂ€al. 1999). Fusion of the PRKAR1A to RARA was described as a variant APL. (Catalano etÂ€al. 2007). The presence of the PML-RARA fusion gene is responsible for the peculiar response to ATRA and ATO. RARA (there are three homologous RAR proteins, called a, b and g) is a ligand-dependent transcriptional activator that binds through its zinc (Zn) finger domain, as heterodimer with members of the RXR family of nuclear receptors (Grignani etÂ€al. 1994), to specific DNA sequences (called retinoic acid responsive elements, RARE) found in the promoters of retinoic acid-responsive genes (Umesono etÂ€al. 1991). This process is accomplished in concert with a coactivator complex that includes proteins such as p300 and pCAF. As shown schematically in Fig.1, in the absence of retinoic acid, a co-repressor complex is recruited comprising either Nuclear Receptor Co-Repressor (N-CoR) or Silencing Mediator of Retinoid and Thyroid Receptors (SMRT), which binds to another protein called Sin3 and then to HDAC1 (Umesono etÂ€ al. 1991; Alland etÂ€ al. 1997; Laherty etÂ€al. 1997; Heinzel etÂ€al. 1997; Redner etÂ€al. 1999). HDAC1 is a histone deacetylase protein, which epigenetically alters histones to keep DNA in an untranscribable form. The expression of retinoic-acid responsive genes is essential for

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Differentiation RAR (wild-type) PML-RARa

Cofactors

in3

N-CoR

mS

RAR

N-CoR

3

in mS

HDAC

HDAC

RAR

PML

Myelocyte

All-transretinoic acid RAR

X Transcription arrest

APL blast cell

PML

PML (dispersed)

Metamyelocyte PML-RARα degradation

PML in PODs

Granulocyte

Fig.Â€1â•… A schematic representation of the effects of PML-RAR and ATRA in the blast cells of acute promyelocytic leukaemia. DNA-bound PML-RARA interacts with N-CoR (or SMRT) and recruits the m-Sin3-HD complex, decreasing hystone acetylation and producing repressive chromatin organisation and transcriptional repression. Retinoic acid interaction with PML/RAR leads to rapid degradation of this fusion protein and assembly of the wild-type PML protein into normal nuclear structures called PODs (PML oncogenic domains). Moreover, it induces dissociation of the N-CoR-mSin3-HD complex, recruitment of coactivators with acetyltransferase activity, increased levels of hystone acetylation, chromatin remodelling, and transcriptional activation

normal myeloid development (Tsai and Collins 1993). In APL, the presence of the PML-RARA fusion gene generates a chimeric protein that is capable of DNA binding, but it recruits the N- CoR/Sin3/HDAC1 co-repressor complex and prevents transcription of RAR target genes (Collins 1998) and myeloid cell differentiation (Tsai and Collins 1993) under normal conditions. Additionally, the PML-RARA forms a heterodimer with the wild type PML protein and disrupts the PML nuclear body/PML oncogenic domain (Koken etÂ€al. 1994). ATRA functions by binding to the RAR and causing degradation of the PML-RAR protein through both the ubiquitin-proteosome and caspase systems, thus allowing for the terminal differentiation of leukemic promyelocytes (Nervi etÂ€ al. 1998; Zhu etÂ€ al. 1999; Liu etÂ€ al. 2000). This binding also results in N-CoR being dissociated from the PML-RAR fusion protein, and subsequently, the nuclear co-activator complex is recruited to reverse histone deacetylase-mediated repression Fig.Â€1. Finally, PML nuclear bodies are restored to their normal structure.

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Diagnosis of APL Compared with diagnosis of other AML subtypes, the identification of APL conveys unique therapeutic and prognostic implications. In fact, (1) this leukaemia is a medical emergency, and up to 10% of early hemorrhagic deaths are currently recorded even in patients receiving modern state-of-the-art treatments; (2) the optimised front-line approach (ATRA plus chemotherapy) is different from that used in other AMLs and is effective also in controlling the life-threatening coagulopathy. Figureâ•›2 summarises the different tools available for the proper diagnosis of APL. According to FAB classification, the term M3-AML was assigned to hypergranular promyelocytic leukaemia characterised by blast cells with heavy azurophilic granules, bundles of Auer rods, and a reniform or bilobed nucleus (Fig.Â€2 panel A). Although the vast majority of M3 cases fit the description of hypergranular or classical M3, a cytological hypogranular or microgranular variant form, M3v, has been identified (Fig.Â€2 panel B). It is commonly associated with hyperleukocytosis, accounts for 15 to 20% of APL cases, and shares the same t(15;17). Albeit less frequently observed, M3v occurs even in paediatric patients (Rovelli etÂ€al. 1992). As compared with control cases with the classical t(15;17) translocation, in the cases with PLZF/RARA rearrangements, the majority of blasts had (1) a regular nucleus, and, (2) an abundant cytoplasm with either coarse granules or, less frequently, with fine or no granules and Chediak-like granules are rarely detectable in such cases Sainty etÂ€al. (2000). The presence of a regular nuclear outline is a key feature of t(11;17)-associated APL. The few cases so far described of APL expressing NPM/RARA (Redner etÂ€al. 1996) could be classified as M3 and presented with hyperleukocytosis the remaining case had an M3v morphology. As for morphology and molecular genetics, the immunophenotype of acute promyelocytic leukaemia is also very distinctive. Both in adults and children, APL blasts show a typical surface marker expression characterised by positivity for CD33, CD13, CD9, absence of HLA-DR and rare expression of CD10, CD7, and CD11b (Guglielmi etÂ€al. 1998). The t(15;17) is the diagnostic hallmark of APL and initially had been considered to be present in all patients with this condition reviewed by Grimwade etÂ€al. (2000). Conventional cytogenetics or Fluorescence in situ hybridization (FISH) enable the identification of both derivatives chromosome 15q and 17q (Fig.Â€ 2, panel C). In addition to the classic form of the t(15;17) translocation, the existence of cryptyic translocations or microinsertion have been reported (Grimwade etÂ€al. 2000). The study of the PML distribution pattern in leukemic cells provides a rapid, specific, low-cost, and relatively simple diagnostic approach (Dyck etÂ€ al. 1995; Falini etÂ€al. 1997). Different from the wild-type (speckled) staining, which corresponds to the localization of PML into 5 to 20 discrete nuclear particles (so-called “nuclear bodies”), APL cells show a characteristic and easily distinguishable nuclear PML positivity known as “microspeckled,” resulting from the disruption of the nuclear bodies and redistribution of the protein into greater than 50 small

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Fig.Â€2â•… Diagnosis of acute promyelocytic leukaemia. Representative morphology of M3-hypergranular (Panel a) and M3v (Panel b). Bone marrow smears were stained with May-Grumwald-Giemsa. ×1,000-fold magnification. Panel c: FISH analysis of an APL case. The result shown has been obtained by using the Vysis probe set, which is designed to detect only the PML-RARA fusion gene. It comprises a mixture of directly labelled probes: a PML probe, which begins in intron 7 and extends toward the centromere for 180Â€kb, and a RARA probe, which begins in intron 4 and extends toward the telomere for 400Â€kb, Panel d: Immunolabeling APL blast with thePG-M3MoAb shows a microgranular distribution of the PML/RARa protein within the nucleus. Panel e: Results of the RT-PCR analysis of the PML-RARA fusion gene. Ethidium bromide-stained agarose gel showing bcr1+ and bcr3+ nested PCR products according to the procedures in van Dongen et al. (1999). Pt.pos: positive samples, Pt:neg: negative samples; Ctrl neg: negative controls, MK: markers

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granules/per cell. Either immunocytochemistry or immunofluorescence have been successfully used as detection systems. The use of Reverse-Transcription PCR (RT-PCR) for the detection of the PMLRARA and RARA/PML fusion genes is the only technique that defines the PML breakpoint type and that allows the definition of a correct strategy for subsequent MRD monitoring. The advantage of routinely using this assay at diagnosis to better address treatment (Miller etÂ€ al. 1992; Biondi etÂ€ al. 1992) has been subsequently validated in prospective multicenter reviewed by Grimwade (2002). According to most investigators, high-quality RNA and efficient RT are the crucial determinants for successful RT-PCR of PML-RARA (van Dongen etÂ€ al. 1999). As shown in Fig.Â€2, panel E, a proper set of primers has been identified that allow the detection of all the different PML-RARA junctions, generated by the existence of different PML breakpoint regions in the PML locus and the presence of alternative PML splicing of PML transcripts. Moreover, the alternative usage of two alternative usage of RARA sites generates extra PML-RARA transcripts of different size (van Dongen etÂ€al. 1999).

Treatment of APL Despite areas of controversy in the management of APL, there is reasonable consensus in both adults and children for the approach of induction therapy with simultaneous ATRA and anthracycline, two or three blocks of anthracycline-based consolidation with simultaneous ATRA, with or without cytarabine or nonintercalating agents, and 1 or 2Â€ years of maintenance therapy with intermittent ATRA, with or without 6-mercaptopurine and methotrexate. Consolidation may be risk adapted and usually based on the presenting white cell count (WCC). Many frontline protocols incorporate serial molecular monitoring during treatment and at three month intervals for 2Â€years following completion of therapy to detect persistent or recurrent molecular disease, which would be an indication for further treatment. There is no role for hematopoietic stem cell transplantation (HSCT) in frontline therapy, except for the small number of patients with persistent minimal residual disease (MRD) at the end of consolidation who may proceed to either allogenic or autologous transplantation based on molecular status after further treatment. While the incorporation of ATO in frontline studies appears encouraging, there are limited data regarding children and none based on a randomized comparison of ATO-based therapy against ATRA and anthracycline based therapy. Pediatric APL trials, which include ATRA with anthracycline-based chemotherapy, report a 5Â€year overall survival (OS) of 87 to 90%, disease-free survival (DFS) of 78 to 82% and event-free survival (EFS) of 71 to 77% (Mann etÂ€al. 2001; de Botton etÂ€ al. 2004; Ortega etÂ€ al. 2005; Testi etÂ€ al. 2005) (Table 1). However, ATRA and anthracycline-based chemotherapy is only appropriate for children confirmed positive for a fusion gene associated with ATRA sensitivity and should not be used for children with APL who lack these fusion genes, and who should receive standard intensive AML type therapy with anthracyclines and cytarabine.

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TableÂ€1â•… Four studies of children with newly diagnosed APL treated with combinations of ATRA and anthracycline-based chemotherapya Study results German-Austrian- European Trial Swiss BFM APL 93 GIMEMA PETHEMA No. of children 22 31 124 66 Study period 1994–2000 1993–1998 1993–2000 1996–2004 Treatment: ATRA dose (mg/m2/d) 25(15) 45(7) 45 25 25 Anthracycline dose DNR 180 or IDR DNR 495 IDR 80â•›+â•›MTZ IDR 80/100â•›+â•›MTZ (mg/m2) 36â•›+â•›ADR 120 50 50 ATRA toxicity (%): APL DS 14 13 7.5 19.5 Definite 2 4.5 Indeterminate 6 15 Pseudotumour cerebri 5 16 9 6 Headache 27 39 13 30 Results: CR Rate % 95 97 96 92 Deaths in CR 0 0 0 0 NA NA 3 3 Molecular persistence at the end of consolidation 5-year EFS, % 76 71 76 77 5-year DFS, % NA 78 82 5-year OS, % 87 90 89 87 GIMEMA Gruppo Italiano Malattie Ematologiche Maligne dell’Adulto group; PETHEMA Programa de Estudio y Tratamiento de las Hemopatias Malignas Group; NA not available; DNR daunorubicin; IDR idarubicin; ADR adriamycin; MTZ mitoxantrone; CR complete remission; APL DS APL differentiation syndrome; EFS event-free survival; OS overall survival; DFS disease free survival a Adapted from Ortega etÂ€al. (2005)

Induction Therapy The simultaneous use of ATRA and anthracycline monotherapy is considered optimal induction chemotherapy (Fenaux etÂ€al. 1999; Sanz etÂ€al. 2003). Primary resistance to ATRA and idarubicin is extremely rare and there may be no advantage for the addition of cytarabine or other chemotherapeutic agents to ATRA and anthracyclines in induction. A study, which did not include ATRA, found no advantage in CR or EFS for dual therapy with idarubicin and cytarabine compared to monotherapy with idarubicin (Avvisati etÂ€al. 2002), although a larger anthracycline dose may have conferred an advantage in the monotherapy arm. However, an increase in the relapse rate, but no difference in CR, was reported when cytarabine was omitted from a schedule employing daunorubicin as the anthracycline (Ades etÂ€al. 2006). Daunorubicin and idarubicin have not been randomly compared in APL although idarubicin is often favored because of its relatively long half-life and CNS penetration.

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In two separate trials employing simultaneous anthracycline (idarubicin) and ATRA as induction therapy, the CR and EFS for children were reported at 92 and 96% and 77 and 76%, respectively (Ortega etÂ€al. 2005; Testi etÂ€al. 2005). While ATRA followed sequentially by chemotherapy is superior in terms of CR and RR to single agent ATRA or chemotherapy alone (Ortega etÂ€al. 2005; Fenaux etÂ€ al. 2000; Tallman etÂ€ al. 1997; Kanamaru etÂ€ al. 1995), the simultaneous use of chemotherapy in combination with ATRA is superior to the sequential use (de Botton etÂ€ al. 2004; Fenaux etÂ€ al. 1999; Burnett etÂ€ al. 1999; Mandelli etÂ€ al. 1997; Tallman etÂ€al. 2002). The European APL 93 study (Fenaux etÂ€al. 1999) randomized patients to simultaneous induction treatment (ATRA, with chemotherapy added on day 3) or sequential treatment (ATRA followed by chemotherapy after CR to a maximum of 90 days) in patients with a WCCâ•›<â•›5â•›×â•›109/L and allocated simultaneous treatment with ATRA and chemotherapy from day 1 to patients with a WCCâ•›>â•›5â•›×â•›109/L. Fifty-five percent of patients randomized to sequential treatment had chemotherapy added before CR achievement because of a rising WCC. The CR rate was similar for simultaneous and sequential treatment at 94 and 95% (pâ•›=â•›0.79) respectively, but the estimated RR at 2Â€ years was in favor of simultaneous treatment (pâ•›=â•›0.04). This implies that the advantage of simultaneous over sequential ATRA is due to a reduction in relapse risk rather than a significant increase in CR or reduction in early deaths. The number of children less than 18Â€years of age and treated on this trial was too small (31; 5%) to compare simultaneous with sequential treatment, but overall, there was no difference in outcome between adults and children, except for a significantly better survival in children after adjustment for WCC (Ortega etÂ€al. 2005). Within the pediatric age group, there are limited data on the advantage of ATRA and anthracycline monotherapy over ATRA and polychemotherapy and on simultaneous over sequential ATRA with strategies extrapolated from adult studies. AML- BFM 93 reported a CR rate of 95% with a single induction death (4.5%) in a cohort of 22 children who received 3 days of ATRA followed by simultaneous multi-agent chemotherapy, compared to a CR rate of 64% with seven induction deaths (32%) in a historical cohort of 22 children who were treated with similar chemotherapy alone (Mann etÂ€al. 2001) and no ATRA (pâ•›<â•›0.04). The GIMEMA-AIEOP (Testi etÂ€al. 2005) and PETHEMA (Ortega etÂ€ al. 2005) groups both delivered simultaneous chemotherapy with idarubicin and ATRA and reported CR rates of 96 and 92% and induction death rates of 4 and 7.5%, respectively. European APL 93 study using a combination of sequential and simultaneous treatment reported a CR rate of 97% with a single induction death (3%) (de Botton etÂ€al. 2004). There are therefore clear advantages for the combined use of ATRA and chemotherapy with excellent CR rates being achieved and low induction death rates.

Complications of Induction Therapy Primary resistance of APL to simultaneous ATRA and anthracycline-based chemotherapy is exceptionally rare and the most common cause of failure to achieve CR is death during induction therapy from hemorrhage. A bleeding diathesis, particularly

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intracranial hemorrhage is more common in APL than in other AML subtypes, and most commonly involves children with a high presenting WCC (>10â•›×â•›109/L) and in those with M3 variant. The severe coagulopathy initiated or exacerbated by chemotherapy, can result in both hemorrhagic and thrombotic complications. While ATRA significantly reduces the incidence of intracranial hemorrhage, this complication remains a cause of early death in 3 to 4% of children despite increased awareness, preemptive therapy, and the use of ATRA (Mann etÂ€al. 2001; de Botton etÂ€al. 2004; Ortega etÂ€al. 2005; Testi etÂ€al. 2005). The risk of early hemorrhagic death, often prior to, or in the first week of treatment, classifies APL as a medical emergency, and ATRA, which improves the coagulopathy and may reduce the risk period, should be started immediately on morphological suspicion (Sanz etÂ€ al. 1999; Falanga 2003; Huang etÂ€al. 1988; Chomienne etÂ€al. 1990; Tallman etÂ€al. 2004a) and not delayed until the diagnosis is molecularly confirmed. The rapid initiation of combined ATRA and anthracycline-based chemotherapy is particularly important in patients with a high WCC, who should not undergo leucopheresis as this can exacerbate the coagulopathy (Tallman etÂ€al. 2004b). Coagulation parameters remain abnormal (low fibrinogen, elevated d-dimers) until hypocellularity of the bone marrow is achieved; a median of about 9 days. The coagulation screen and platelet count should be checked twice daily during induction therapy. Fibrinogen levels should be maintained above 1.5 to 2Â€g/L with fresh frozen plasma (FFP) or cryoprecipitate and the platelet count above 50â•›×â•›109/L until the coagulopathy resolves (Sanz etÂ€al. 1999; Chomienne etÂ€al. 1990; Tallman etÂ€al. 2004a, b; Milligan etÂ€al. 2006). There is no proven benefit for the use of heparin, Tranexamic acid, or antifibrinolytic drugs and these drugs should not be used routinely. Indeed, anti-fibrinolytic agents, when combined with ATRA, may increase the risk of thrombosis. Anti-fibrinolytic agents and NOVOSEVEN may be considered in life-threatening hemorrhage unresponsive to platelets, FFP, and cryoprecipitate. The APL differentation syndrome (APL DS) is characterized by fluid retention and capillary leak and is associated with a high mortality if not promptly treated (Sanz etÂ€al. 2005a; Vahdat etÂ€al. 1994; Frankel etÂ€al. 1992; De Botton etÂ€al. 1998). It is thought to be related to surface adhesion molecule modulation and cytokine release following differentiation of APL cells and can occur at any time from day 1 to day 35 after the start of induction therapy with a mean of day 7 (Frankel etÂ€al. 1992). Patients with a high WCC (>10â•›×â•›109/L) or a rising WCC are at greatest risk and should receive simultaneous chemotherapy and ATRA, which is reported to reduce the incidente (Frankel etÂ€al. 1992; De Botton etÂ€al. 1998). Early recognition and prompt treatment with dexamethasone reduces the associated mortalità (Milligan etÂ€al. 2006; De Botton etÂ€al. 1998). At the first sign or symptom suggesting APL differentation syndrome, dexamethasone should be promptly initiated at a dose of 5Â€mg/m2â•›bd (max single dose 10Â€mg) iv, until disappearance of symptoms and signs, and for a minimum of 3 days. ATRA should be stopped if symptoms are severe or unresponsive to steroid (Milligan etÂ€ al. 2006; de Botton etÂ€ al. 2003). ATRA can be cautiously reintroduced because the occurrence of APL differentiation syndrome during induction, linked to the differentiation of APL blasts, is not a contraindication to its subsequent use. Although the prophylactic use of steroids

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with induction therapy is practised, its benefit is uncertain (Firkin etÂ€ al. 1999; Wiley and Firkin 1995). APL differentiation syndrome appears to occur with the same incidence in children as in adults. In adults receiving combined ATRA and chemotherapy and the early use of dexamethasone, the incidence is reported at 15%, but with a low associated mortality of 1.2% (De Botton etÂ€ al. 1998). The incidence in similarly treated children of definite APL differentiation syndrome is reported at 3 to 5% (de Botton etÂ€al. 2004; Ortega etÂ€al. 2005; Testi etÂ€al. 2005) with an additional 5 to 15% having indeterminate APL differentiation syndrome and a mortality rate of less than 1% (Mann etÂ€ al. 2001; de Botton etÂ€ al. 2004; Ortega etÂ€al. 2005; Testi etÂ€al. 2005). Children have a higher incidence of ATRA-associated headache and pseudotumor cerebri than adults (Mahmoud etÂ€ al. 1993). Symptoms are reversible with discontinuation of the drug, which may be tolerated when reinstituted at a lower dose. The incidence of headaches may be dose-dependent and age-dependent (<10Â€years). Doses as low as 25Â€mg/m2 may be effective in children (Mann etÂ€al. 2001; Ortega etÂ€al. 2005; Testi etÂ€al. 2005; Castaigne etÂ€al. 1993). The incidence of headache and pseudotumor cerebri has been reported between 13 to 30 and 5 to 9% respectively, for children receiving ATRA at a dose of 25Â€mg/m2 (Ortega etÂ€al. 2005; Testi etÂ€al. 2005) and 39 and 16%, respectively, for those receiving 45Â€mg/m2. There are no randomized studies comparing 25Â€mg/m2 with 45Â€mg/m2 in terms of relapse reduction but similar outcomes have been reported (Castaigne etÂ€al. 1993). Three studies using an ATRA dose of 25Â€mg/m2 report EFS of 76 and 77% and OS of 87 and 89%, suggesting efficacy. Failure of ATRA may reflect a reduction in plasma drug levels due to induction of cytochrome p450 or due to cellular retinoic acid binding proteins (CRABPs) (Warrell 1993), which cannot be overcome by dose escalation. Care should be taken with the concomitant use of azoles and ATRA because ATRA is metabolized by cytochrome P450, which is inhibited by azoles. Resistance to retinoids may be due to mutations in the PML-RAR alpha fusion gene (Ding etÂ€al. 1998). ATRA capsules can be difficult for small children to swallow. The pharmokinetics of formulations prepared from capsules and of such preparations given by nasogastric tube are poorly defined. Liposomal ATRA has the advantage of being an intravenous preparation.

Consolidation Therapy The controversial issues in consolidation therapy are the benefits of simultaneous ATRA with chemotherapy, of cytarabine or any chemotherapy additional to anthracyclines and of a risk-stratified approach. Morphological assessment at the end of induction therapy is uninformative. About 50% of patients have molecular evidence of residual disease at the end of induction, while more than 95% (Testi etÂ€ al. 2005; Mandelli etÂ€ al. 1997) are in molecular remission at the end of consolidation following two or three intensive anthracycline-based courses. There is no correlation between molecular positivity

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at the end of induction, but molecular positivity at the end of consolidation is highly predictive of relapse. ATRA may have a synergistic effect when given simultaneously with chemotherapy in consolidation, but this has not been demonstrated in a randomized trial. The use of ATRA for 28 days during induction and with two cycles of daunorubicin and cytarabine consolidation without subsequent maintenance reported comparable outcomes (5Â€year OS and LFS of 82 and 78%, respectively) to studies employing prolonged maintenance with ATRA with or without low dose chemotherapy, suggesting that ATRA in consolidation may allow shortening of the overall duration of APL treatment (Gupta etÂ€ al. 2005). The PETHEMA group, employing anthracycline monotherapy in consolidation, reported a reduction in the relapse rate in high and intermediate risk patients from 20.1% to 8.7% (pâ•›=â•›0.004) when ATRA was added to consolidation. The relapse rate fell from 14 to 2.5% (pâ•›=â•›0.006) in intermediate risk patients (Sanz etÂ€al. 2004). The role of cytarabine in consolidation therapy is questioned comparable results reported for anthracycline monotherapy and combination chemotherapy, commonly an anthracycline combined with cytarabine. However, some groups report benefit for high dose cytarabine in consolidation (Schlenk etÂ€al. 2005), while others report encouraging results for anthracycline monotherapy (Tallman etÂ€ al. 2002; Sanz etÂ€al. 2004). Fifty-one percent of patients receiving anthracycline monotherapy in induction and consolidation were PCR negative after induction, and 93%, after consolidation. The OS, DFS, and cumulative RR at 5Â€years were 87, 82 and 17%, respectively (Ortega etÂ€al. 2005) for the 66 children who received anthracycline monotherapy in consolidation with which ATRA was subsequently added for intermediate and high-risk patients defined by their presenting WCC and platelet counts. Combined analysis of the GIMEMA and PETHEMA APL trials, which differ only in the inclusion or exclusion of drugs other than anthracyclines during consolidation showed that the omission of nonanthracycline drugs was not associated with an inferior antileukaemia effect (Sanz etÂ€al. 2000). The view that cytarabine plays a minor role in consolidation therapy has been challenged by the European APL Group who found benefit for cytarabine in combination with ATRA and daunorubicin in newly diagnosed patients with APL (Avvisati etÂ€al. 2002). Any advantage for cytarabine may be protocol-dependent, and particularly, may relate to the anthracycline, its cumulative dose, and the content of consolidation and maintenance courses. Cytarabine or polytherapy consolidation may play a particular role in patients with a high WCC, a more common situation in childhood APL (Lo-Coco etÂ€al. 2004a; Diverio etÂ€al. 1998). Heterogeneous therapeutic regimens appear to produce similar results, but all deliver high cumulative doses of anthracycline, which may be very important in APL. While anthracycline monotherapy in consolidation has the advantage of reduced morbidity and mortality, there were no remission deaths in four reported pediatric studies (Mann etÂ€al. 2001; de Botton etÂ€al. 2004; Ortega etÂ€al. 2005; Testi etÂ€al. 2005) using variable consolidation courses. The optimal number of consolidation courses has not been identified by randomized trial. WCC at presentation is a strong predictive indicator of outcome

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and the number and intensity of the blocks of consolidation may be tailored to the individual patient. Children with a low presenting WCC (<10â•›×â•›109/L) may require less treatment than those with a high presenting WCC (>10â•›×â•›109/L) and treatment may be further modified by response as assessed by MRD monitoring. In the GIMEMA-AIEOP study, the EFS was 83% versus 59% at 10Â€years for children with a low versus high WCC, respectively (Testi etÂ€al. 2005). The WCC is not as predictive as MRD.

Maintenance Treatment A number of groups have reported that maintenance therapy, given for 2Â€ years, reduces the relapse risk, with the best results being obtained with intermittent ATRA (15 days every 3Â€months) in combination with 6-mercaptopurine and methotrexate (Testi etÂ€al. 2005; Fenaux etÂ€al. 1999; Tallman etÂ€al. 1997). The intermittent administration of ATRA recognizes its pharmacological properties; particularly that continuous use leads to a fall in plasma levels secondary to hypercatabolism of the drug. The European APL study (Fenaux etÂ€al. 1999) reported improved OS in patients who received maintenance chemotherapy (pâ•›=â•›0.01) and a trend in favor of ATRA (pâ•›=â•›0.22). The best results were obtained in patients who received both ATRA and low-dose maintenance chemotherapy, particularly patients with a high WCC, and therefore, the combined use of ATRA, 6MP, and MTX may have particular benefit for this group (Fenaux etÂ€al. 1999). However, the GIMEMA study found no benefit for maintenance in patients who are in molecular remission at the end of consolidation (Avvisati etÂ€ al. 2003), questioning its value for this cohort. Similarly, it is unclear if maintenance retinoid confers a survival advantage in patients who receive prolonged ATRA as a component of induction and consolidation chemotherapy. The benefit of maintenance may depend on the intensity of previous induction and consolidation therapy.

Molecular Monitoring in APL MRD Detection Is an Independent Predictor of Outcome in APL Presenting leucocyte count has been shown to be a key prognostic factor in clinical trials involving pediatric and adult patients with APL, with WCCâ•›>â•›10â•›×â•›109/L predicting a significantly increased risk of induction death and relapse risk reviewed by Grimwade (2002). Relapse risk among patients treated with ATRA and anthracycline-based chemotherapy with WBCâ•›<â•›10 is typically approximately 10%, as opposed to about 30% in those who are being overtreated with current protocols leading to unnecessary morbidity, while patients at higher risk of relapse could benefit from additional therapy. There is evidence to suggest that more precise tailoring of therapy may be achieved through

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MRD monitoring, which has been shown to provide an independent risk factor for relapse reviewed by Lo-Coco and Ammatuna (2006). Using conventional end-point PCR assays, which achieve a sensitivity of approximately 1 in 104, Italian studies undertaken in children and adults in conjunction with the AIDA protocol have established that patients with PML-RARA transcripts still detectable at the end of consolidation (who account for less than 10% of cases overall) or those subject to a later recurrence of PCR positivity (molecular relapse) are destined to undergo subsequent hematological relapse, which may however in both instances, be averted by additional therapy (Diverio etÂ€al. 1998). Indeed, preliminary data from the Italian trials suggest that preemptive treatment at the time of molecular relapse may lead to improved survival in comparison to patients who are re-treated in frank hematological relapse (Lo Coco etÂ€al. 1999). In addition, analysis of children and adults treated in the UK Medical Research Council (MRC) ATRA trial showed that MRD monitoring can distinguish subgroups of patients at differing risk of subsequent relapse relatively early during the treatment course. Patients with detectable fusion transcripts following three courses of treatment who accounted for 10% of patients had a significantly increased risk of subsequent relapse (57% vs. 26% at 5Â€years, pâ•›=â•›0.008) associated with poorer overall survival (57% vs. 82%, pâ•›=â•›0.03) compared to those testing PCR negative at the same stage (Burnett etÂ€al. 1999). These findings have recently been supported by a study from the German AML Cooperative Group using real-time quantitative RT-PCR (RQ-PCR), in which patients who failed to achieve a 3-log reduction in PML-RARA transcript level within the first 3 to 4Â€ months of therapy, had an increased risk of early relapse (Schnittger etÂ€al. 2003).

Real-Time Quantitative RT-PCR Assays Enhance MRD Detection in APL While MRD monitoring using conventional nested RT-PCR has provided valuable prognostic information, its clinical utility has been somewhat compromised by failure to detect residual disease in a significant proportion of APL patients who ultimately relapse reviewed by Grimwade (2002). This may be a reflection of the relatively limited sensitivity of conventional assays and/or variation in RNA quality/ quantity and efficiency of the reverse transcription (RT) step. Quantitative PCR approaches using hydrolysis (Taqman) or hybridization probe technology afford a number of advantages in comparison to conventional end-point assays. In particular, quantitation of fusion gene and endogenous control gene transcripts enables more reliable determination of kinetics of molecular remission achievement or relapse and readily identifies poor quality samples that could potentially give rise to falsenegative results. Moreover, RQ-PCR assays are rapid, facilitate high throughput sample analysis, are highly reproducible, and readily standardized, thereby lending themselves to MRD assessment in multicenter clinical trials. Optimized RQ-PCR protocols for detection of the PML-RARA fusion gene and ubiquitously expressed control genes have been established by the Europe Against Cancer (EAC) Group

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(Gabert etÂ€al. 2003). These assays have subsequently been validated in relation to conventional nested RT-PCR (Gallagher etÂ€al. 2003). However, despite the higher sensitivity of RQ-PCR, a significant number of patients who ultimately relapsed tested PCR negative at the end of consolidation, underlining the importance of serial monitoring to increase the predictive value of this approach.

Anthracycline Cumulative Dose and Cardiotoxicity Idarubicin and daunorubicin have not been randomly compared in APL. Idarubicin is more often used as monotherapy and daunorubicin in combination therapy. The optimum cumulative dose of anthracycline in children is unknown. A high cumulative dosage of anthracyclines (650Â€ mg/m2) was delivered to children in the GIMEMAAIEOP AIDA protocol with no serious cardio-toxicity observed in the 101 long-term survivors, but late cardiotoxicity may occur (Testi etÂ€al. 2005). While anthracyclines are very effective in APL, the favorable outcome increases the importance of minimizing late effects and clearly the lowest effective dose should be identified. The role of liposomal anthracycline and cardioprotectants have not been evaluated in a clinical trial.

Stem Cell Transplantation Because of the favorable results achieved with chemotherapy and ATRA, Hematopoietic stem cell transplantation (HSCT), allogenic or autologous, are not recommended in first remission (Burnett etÂ€al. 1998, 2002). Patients with persistent molecular disease at the end of consolidation may be candidates for HSCT after further chemotherapy.

Arsenic Trioxide ATO, like ATRA, is a differentiating agent, which offers the potential for a non or low chemotherapy approach to the treatment of APL. While the data on ATO use in children are limited, it may be the most effective single agent in APL. Preliminary data from adult studies suggest at least comparability between ATO and single agent ATRA in terms of the achievement of CR (>90%) and that combined ATO and ATRA is superior to ATO or ATRA alone (Niu etÂ€al. 1999; Soignet etÂ€al. 2001; Shen etÂ€al. 2004). ATRA and ATO may act in part synergistically in triggering down regulation of telomerase causing telomerase shortening and subsequent cell death (Tarkanyi etÂ€al. 2005). A small single center pediatric study using monotherapy with ATO reported a haematological and molecular remission rate of 91% with a RFS and OS of 81 and 91%, respectively at 30Â€months (George etÂ€al. 2004). The addition of ATO to consolidation following remission induction with ATRA and chemotherapy has been reported to significantly improve the EFS and OS in adults with newly

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diagnosed APL (Powell 2007). While ATO appears to be excellent at inducing remission, its long-term benefit and comparability with ATRA-based chemotherapy is less clearly defined in frontline therapy, especially in children, where there are many unanswered questions. ATO may have a role in both induction and consolidation, combined with either ATRA or chemotherapy and used with the aim of minimizing or eliminating chemotherapy. ATO is usually well tolerated, although its use is associated with serious adverse events including hyperleukocytosis, the APL differentiation syndrome, and prolongation of QT interval. Although severe neuropathy has been rarely observed, mild peripheral neuropathy, which usually resolves after ATO discontinuation, has been reported in approximately 40% of patients reviewed by Sanz etÂ€al. (2005b). Little is known of late ATO cardio and neurological long-term toxicity. There is a need to define toxicity in children and to assess the role and best strategy for the use of ATO within a pediatric study.

What to Do When Treatment Fails? Failure to treatment currently constitutes an infrequent event in APL, involving only one-fourth of patients, owing to the high efficacy of frontline therapy. Failure is represented by disease recurrence at hematological and/or molecular level (relapse rate 20 to 25%) and by the persistence of molecular disease at recovery from 2 to 3 consolidation cycles (about 3 to 5%). Relapse occurs more frequently in those patients with a WCC countâ•›>â•›10â•›×â•›109/L at presentation (Tallman etÂ€al. 2002; Sanz etÂ€al. 2004; Tallman 2007). Approximately 3 to 5% of APL patients develop extramedullary (EM) sites of relapse, predominating in the central nervous system (CNS) and the skin, followed by other sites (testes, sites of vascular access, external ear, and auditory canal). It has been suggested that EM relapse also may be associated with initial adverse prognostic features such as high WCC count, and bcr3 PML-RARA isoform, but the risk factors for this kind of relapse have not been precisely defined (Tallman 2007; de Botton etÂ€al. 2006; Specchia etÂ€al. 2001; Breccia etÂ€al. 2003). Nonetheless, the adequate management of relapsed APL patients is still unclear, and few studies have analyzed the outcome of these patients in a prospective fashion. In particular, very limited information is available in paediatric age, with only few sporadic cases, mainly included in adult series, described in literature. In any case, the prognosis following hematological relapse is usually dismal; on the contrary, institution of therapy at the first evidence of molecular disease results in more favorable outcome, reinforcing the rationale of PCR-triggered therapy in APL (Lo Coco etÂ€al. 2003). Reinduction with ATRA alone in patients did not provide long-term benefit, since second complete remission (CR) achieved (approximately 50%) in this manner, lacked durability and required consolidation by further intensive chemotherapy (Castagnola etÂ€al. 1998). Better results have been achieved in relapsed APL with salvage therapy consisting of ATRA and chemotherapy for induction, generally containing high-dose cytarabine, followed by further chemotherapy and/or hematopoietic stem cell transplant (HSCT) (CR rate 87%; disease-free-survival 50 to 60

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(99, 100). In these studies, the choice of transplant modality was mainly based on PCR status achieved with chemotherapy: autologous HSCT was the preferred option in patients without detectable MRD, while allogeneic HSCT was chosen for patients failing to achieve a second molecular remission. However, the outcome of patients treated in molecular relapse compared favorably to those who received the treatment for hematological relapse, with longer survival and lower relapse risk (Specchia etÂ€ al. 2001). Additionally, molecular response to salvage therapy predicted longer leukaemia-free survival (LFS), independently by the therapeutic consolidation choice (Castagnola etÂ€al. 1998; Esteve etÂ€al. 2007). Currently, given the high antileukemic efficacy observed with ATO in APL patients relapsing after ATRA-containing regimen, this agent is regarded as the best option in this context. ATO has emerged as the single most active agent in patients with relapsed APL (Tallman 2007; Lo Coco etÂ€al. 2007; Ghavamzadeh etÂ€al. 2006). In the several published trials with relatively small numbers of patients, the CR rate ranges from approximately 80 to 90%, accompanied by molecular remission after two cycles in the majority of cases. ATO is administered at the dosage of 0.15Â€mg/ kg/day by 3Â€hours intravenous infusion, until CR achievement, and for a maximum of 50 days. Combining ATO with other agents may also be useful: in a report from China, the combination of ATO and anthracycline resulted in a superior outcome compared to ATO alone. However, the addition of ATRA with ATO, providing high CR rate in newly diagnosed patients, does not appear to be beneficial in the setting of relapse disease. In the randomised French study, ATO alone was compared to ATO plus ATRA in 20 patients with relapsed APL. Haematological and molecular response (8/10 patients in each treatment group), time necessary to reach CR (42 days in the two groups) and outcome (2-year overall survival 59% in the two groups) were comparable in both treatment groups (Tallman 2007; Lo-Coco etÂ€al. 2004b, 2007; Ghavamzadeh etÂ€al. 2006; Raffoux etÂ€al. 2003). Also in these experiences, best results are achieved in patients treated in molecular relapse; in these cases molecular and durable remission can be obtained after ATO monotherapy with less risk of ATO-related toxicity. The quality of CR obtained after ATO salvage treatment remains an open question. However, the best consolidation strategy after ATO-induced second remission is unknown. Several options are available, including repeated cycles of ATO (0.15Â€ mg/kg/day, ×5 days ×5Â€ weeks; 1 to 2 courses), combination with standard chemotherapy, HSCT, and recently, antiCD33. Gemtuzumab ozogamicin combines an anti-CD33 antibody with calicheamicin a cytotoxic agent with similarities to anthracyclines, and therefore, this targeted therapy might be expected to have activity in APL. CD33 is expressed in virtually 100% of APL cases with a highly homogenous expression pattern and calicheamicin belongs to the anthracycline family. While it has been used mainly in relapsed disease, it has shown efficacy in adult frontline studies (Lo-Coco etÂ€al. 2004b; Takeshita etÂ€al. 2005). CR rates comparable to ATRA and anthracycline have been reported for ATRA and Gemtuzumab (Takeshita etÂ€al. 2005). Nonetheless, the precise role of this agent in the management of relapsed APL remains unsettled. In a limited series, molecular response was obtained in 9/11 (91%) patients tested by RT-PCR after two doses and in all 13 (100%) patients tested after the third dose. GO resulted in mild

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myelosuppression and little extrahematologic toxicity, even in heavily pretreated patients (Zheng etÂ€al. 2007). Furthermore, it is presumable that combining GO with simultaneous ATRA or other agents, such as ATO, may result in improved outcome also in more advanced APL (Takeshita etÂ€al. 2005; Zheng etÂ€al. 2007). The selection of one of the above-mentioned options for consolidation therapy, as well as the modality of HSCT, should take into account various variables that may influence the outcome. Recent data published from the EBMT Group indicate that HSCT has continued to be part of treatment strategy for patients with second CR (CR2) (Sanz etÂ€al. 2007). These results show that a high proportion of patients in CR2 achieve a long-term overall survival (OS) after auto- and allo-HSCT, and both procedures present valid therapeutic options in this setting. The choice of one or other procedures will depend on the molecular status, age, the availability of an HLA identical donor and the time from diagnosis to transplant. For the 195 autografted patients in CR2, the 5-year cumulative incidence of LFS was 51% and the transplant-related mortality (TRM) 16%; for the 137 patients allografted in CR2, the 5-year LFS and TRM were 59% and 24%, respectively. The use of peripheral blood stem cells was associated with decreased TRM as compared to bone marrow (12 and 31%). In the near future the results of all consolidation procedures in similar series of CR2 patients need to be compared for better understanding how to improve the prognosis in this group of patients.

Extramedullary Relapse Because of the rarity of this event in APL, the outcome of these patients remains undetermined and only few studies have been reported in literature. However, since the introduction of ATRA in the treatment of this disease, cases of EM relapse have been increasingly described in the last years (3-year cumulative incidence from 1 to 5 to 12% of the first relapse) (Tallman 2007; de Botton etÂ€al. 2006; Specchia etÂ€al. 2001; Breccia etÂ€ al. 2003). The reason for the potential increase in EM relapse observed with ATRA treatment are unknown. One possibility is that the longer survival of patients may increase the number of patients at risk of relapse. The role of ATRA in mediating increased expression of adhesion molecules has been implicated as possible mechanism in the infiltration of EM sites. EM relapses largely predominate in the CNS and are frequently associated with overt or molecular relapse (about 80% of cases). Some reports found, in patients with EM relapse, a high incidence of presenting features such as microgranular M3 variant, the bcr3 PML-RARalpha isoform, both features being correlated to high WBC counts and younger age. Finally, it has been suggested that occurrence of APL differentation syndrome during induction, might be associated with an increased risk of EM relapse (Tallman 2007; de Botton etÂ€al. 2006; Specchia etÂ€al. 2001; Breccia etÂ€al. 2003; Knipp etÂ€al. 2007). The available data suggest that EM relapse in patients with APL should be regarded as systemic disease. CNS-directed therapy with intrathecal methotrexate and cytarabine and cranial radiotherapy, has to be associated to systemic treatment

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including drugs administered at high doses and HSCT. Heterogeneous protocols have been used for the treatment of EM relapse; second CR is achieved in almost 90% of the patients. Despite intensive systemic therapy followed by autologous or allogeneic HSCT in most cases, patients with EM relapse have a poor outcome and the overall survival is comparable to those patients who relapsed at hematological level only. The new drugs, such as ATO, alone or in combination with other chemotherapeutic agents, were recently employed in the treatment of EM relapse; ATO crosses the blood-brain-barrier but the concentrations in Cerebral Spinal Fluid (CSF) achieved by intravenous infusion, are probably insufficient for the treatment of meningeal leukemia (Knipp etÂ€ al. 2007). Also, the described ATO neurological toxicity might be increased by the previous or subsequent intrathecal and radiotherapy, necessary for the whole treatment of CNS relapse. For these reasons and because of the small number of patients, it is difficult to give more exact indications about the treatment of CNS relapse in APL. In response to the above considerations, many institutions have adopted CNS intrathecal prophylaxis in the frontline protocols (starting from consolidation phase), only for those patients with unfavorable presenting features, at higher risk of CNS relapse.

New Drugs Recent novel therapeutic agents potentially effective in APL are currently under investigation. In particular, there is great promise for the FLT-3 inhibitors in APL. FLT-3 aberrations, in the form of an internal tandem duplication (ITD) or mutation at the activation loop position 835, D835, have been reported in 20 to 45% of APL cases (Arrigoni etÂ€al. 2003; Au etÂ€al. 2004; Sohal etÂ€al. 2003). The FLT-3 ITD has also frequently associated with high WBC, microgranular morphology, and PML breakpoint at bcr3. The FLT-3 inhibitors have been shown to be effective with ATRA in a APL mouse model (Fazi etÂ€al. 2005). These raise the enticing possibility that APL may be more effectively managed in the future using orally bioavailable drugs that molecular target both classes of mutation that contribute to the pathogenesis of APL: FLT-3 inhibitors for FLT-3 ITD and ATRA that targets the PML/ RARalpha fusion. The upfront combination of ATRA and FLT-3 antagonists could be highly effective for the treating of APL patients possessing these adverse factors. On the contrary, FLT-3 aberrations might only exist in subclones or represent secondary changes in relapse, and finally might be lost at relapse. The use of FLT-3 antagonists in APL patients in relapse might not be warranted. These data should be addressed in future large prospective trials. A potential role of histone deacetylase (HDAC) inhibitors has been also suggested in the setting of APL. The addition of HDAC inhibitor to ATRA, may restore the sensitivity to retinoids in ATRA-resistant relapse. The effects of a novel mixed retinoic/butyric hyaluronan ester (HBR) were explored inÂ€vitro and inÂ€ vivo on a retinoic acid (RA)-sensitive human myeloid cell line and on its RA-resistant subclone. The results showed that HBR blocks cell growth in both RA-sensitive and RA-resistant cell lines and induces terminal granulocytic

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differentiation in RA-sensitive cells and apoptosis in RA-resistant cells. The antiproliferative effect observed inÂ€vitro is confirmed by inÂ€vivo significant prolongation of the lifetime expectancy in treated immunodeficient mice. Also, HBR increased the host life spam similarly to maximum tolerated doses of RA alone. HBR, due to its strong affinity for the CD44 membrane receptors could be a promising antineoplastic agent for APL patients regardless of their possible resistance to RA. Other retinoid/HDCA inhibitor associations are currently under investigation with promising results. Finally, a possible role for alemtuzumab, an anti-CD52 monoclonal antibody, has also been suggested in APL. ATRA treatment of a patient’s APL cells inÂ€vitro, induced high level of CD52 expression on the leukemic cells. This antigen may become a new target for antibody therapy in patients with APL.

Conclusions Much of the data on which strategies for the treatment of children with APL are based have been extrapolated from adult trials. Questions which require to be addressed exclusively in children include both the optimal ATRA and cumulative anthracycline dose, the role of lipsomal anthracyclines and cardioprotectants, the optimal risk-adapted therapy (as children often have a higher WCC at presentation compared to adults), optimal maintenance and its duration, the role of GO and ATO in combination with traditional first line therapies, the ideal frequency of molecular monitoring and best preemptive therapy, and finally, the role that novel therapies will play in this disease.

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ed Oncologia Pediatrica Cooperative groups Molecular remission in PML/RARAa positive acute promyelocytic leukemia by combined all-trans retinoic acid and idarubicin (AIDA) therapy. Blood 1997; 90:1014–1021. Tallman MS, Nabhan CH, Feusner JH, Rowe JM. Acute promyelocytic leukemia: evolving therapeutic strategies. Blood 2002; 99:759–767. Sanz MA, Martin G, Rayon C, Esteve J, Gonzalez M, Diaz-Mediavilla J, Bolufer P, Barragan E, Terol MJ, Gonzalez JD, Colomer D, Chillon C, Rivas C, Gomez T, Ribera JM, Bornstein R, Roman J, Calasanz MJ, Arias J, Alvarez C, Ramos F, and Deben G for the PETHEMA Group. A modified AIDA protocol with anthracycline-based consolidation results in high anti leukemic efficacy and reduced toxicity in newly diagnosed PML/RARa -positive acute promyelocytic leukemia. Blood 1999; 94:3015–3021. Falanga A, & Rickles FR Pathogenesis and management of the bleeding diathesis in acute promyelocytic leukemia. Best Practice & Research Clinical Hematology 2003; 16: 463–482. Huang ME, Ye YC, Chen SR, Chai JR, Lu JX, Zhoa L, Gu LJ & Wang ZY. Use of all-trans retinoic acid in the treatment of acute promyelocytic leukemia. Blood 1988; 72: 567–572. Chomienne C, Ballerini P, Balitrans N, Daniel MT, Fenaux P, Castaigne S, Degos L. All-trans retinoic acid in acute promyelocytic leukemias. II. In vitro studies: structure-function relationship. Blood 1990; 76: 1710–1717. Tallman MS, Lefebvre P, Baine RM, Shoji M, Cohen I, Green D, Kwaan HC, Peitta E & Rickles FR Effects of all-trans retinoic acid or chemotherapy on the molecular regulation of systemic blood coagulation and fibrinolysis in patients with acute promyelocytic leukemia. Journal of Thrombosis and Haemostatis 2004, 2, 1341–1350. Tallman MS, Brenner B, Serna Jde L, Dombret H, Falanga A, Kwaan HC, Liebman H, Raffoux E, Rickles FR. promyelocytic leukemia-associated coagulopathy, 21 January 2004, London, United Kingdom. Leuk Res 2005; 29: 347–351. Milligan DW, Grimwade D, Cullis J O, Bond L, Swirsky D, Craddock C, Kell J, Homewood J, Campbell K, McGinley S, Wheatley K, Jackson G. Guidelines on the management of acute myeloid leukemia in adults. British Committee for Standards in Haematology (BCSH). Br J Haematol 2006; 135(4):450–474. Sanz MA, Tallman MS & Lo Coco F. The tricks of the trade for the appropriate management of newly diagnosed acute promyelocytic leukemia. Blood 2005; 105: 3019–3025. Vahdat L, Maslak P, Miller WH, Eardkey A, Heller G, Scheinberg DA, Warrell RP. Early mortality and the retinoic acid syndrome in acute promyelocytic leukaemia: impact of leucocytosis, low – dose chemotherapy, PML-RAR(alpha) isoform, and CD 13 expression in patients treated with all-trans retinoic acid. Blood 1994; 84: 3843–3849. Frankel S.R, Eardley A, Lauwers G, Weiss M, Warrell R.P. The “retinoic acid syndrome” in acute promyelocytic leukaemia. Annals of Internal Medicine 1992; 117: 292–296. De Botton S, Dombret H, Sanz M, San Miguel J, Caillot D, Zittoun R, Gardembas M, Stamatoulas A, Conde E, Guerci A, Gardin C, Gelser K, Cony Makhoul D, Reman O, de la Serna J, Lefrere F, Chomienne C, Chastang C, Degos L, Fenaux P and the European APL Group. Incidence, clinical features, and outcome of all trans-retinoic acid syndrome in 413 cases of newly diagnosed acute promyelocytic leukemia. The European APL Group. Blood 1998; 92: 2712–2718. de Botton S, Chevret S, Coiteux V, Dombret H, Sanz M, San Miguel J, Calliot D, Vekhoff A, Gardembas M, Stamatoulas A, Conde E, Guerci A, Gardin C, Fey M, Cony Makhoul D, Reman O, de la Serna J, Lefrere F, Chomienne C, Degos L, Fenaux P, European APL group. Early onset of chemotherapy can reduce the incidence of ATRA syndrome in newly diagnosed acute promyelocytic leukaemia (APL) with low white blood cell counts: results from APL 93 trial. Leukemia 2003; 17 (2): 339–342. Firkin F, Matthews J, Bradstock K & Wiley JS A phase II study of all-trans retinoic acid (ATRA) with Prednisolone prophylaxis in the treatment of acute promyelocytic leukemia (APL). Blood 1999; 94 (Suppl. 2): 228B (abstract). Wiley JS & Firkin FC. Reduction of pulmonary toxicity by Prednisolone prophylaxis during alltrans retinoic acid treatment of acute promyelocytic leukemia. Leukemia 1995; 9: 774–778.

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González M, Viehmann S, Malec M, Saglio G, van Dongen JJ. Standardization and quality control studies of ‘real-time’ quantitative reverse transcriptase polymerase chain reaction of fusion gene transcripts for residual disease detection in leukemia - a Europe Against Cancer program. Leukemia. 2003;17(12):2318–2357. Review. Gallagher RE, Yeap BY, Bi W, etÂ€al. Quantitative real-time RT- PCR analysis of PMLRAR alpha mRNA in acute promyelocytic leukemia: assessment of prognostic significance in adult patients from intergroup protocol 0129. Blood. 2003;101:2521–2528. Burnett AK, Wheatley K, Goldstone AH, Stevens RF, Hann IM, Rees JH, Harrison G. The value of allogeneic bone marrow transplant on patients with acute myeloid leukaemia at differing risk of relapse; results of the UK MRC AML 10 trial. British Journal of Haematology 2002: 118: 385-400. Burnett A.K, Goldstone A.H., Stevens R.M.F., Hann I.M., Rees J.K.H. Gray R.G.,Wheatley K. Randomised comparison of addition of autologous bone-marrow transplantation to intensive chemotherapy for acute promyelocytic leukaemia in first remission – results of MRC AML 10-trial. Lancet 1998; 351: 700–708. Niu C, Yan H, Yu T, Sun HP, Liu JX, Li XS, Wu W, Zhang FQ, Chen Y, Zhou L, Li JM, Zeng XY, Yang RR, Yuan MM, Ren MY, Gu FY, Cao Q, Gu BW, Su XY, Chen GQ, Xiong SM, Zhang TD, Waxman S, Wang ZY, Chen Z, Hu J, Shen ZX, Chen SJ. Studies on treatment of acute promyelocytic leukemia with arsenic trioxide: remission induction, follow-up, and molecular monitoring in 11 newly diagnosed and 47 relapsed acute promyelocytic leukemia patients. Blood 1999; 94: 3315–3324. Soignet SL, Frankel SR, Douer D, Tallman MS, Kantarjian H, Calleja E, Stone RM, Kalaycio M, Scheinberg DA, Steinherz P, Sievers EL, Coutré S, Dahlberg S, Ellison R, Warrell RP Jr. United States Multicenter Study of arsenic trioxide in relapsed acute promyelocytic leukemia. J Clin Oncol 2001; 19: 3852–3860. Shen ZX, Shi ZZ, Fang J, Gu BW, Li JM, Zhu YM. All-trans retinoic acid/As2O3 combination yields a high quality remission and survival in newly diagnosed acute promyelocytic leukaemia. Proc Natl Acad Sci USA 2004; 101: 5328–5335. Tarkanyi I, Dudognon C, Hillion J, Pendino F, Lanotte M, Aradi J, Sigal L, Bendirdjian E. Retinoid/arsenic combination therapy of promyelocytic leukaemia: induction of telomerasedependent cell death. Leukemia 2005; 19(10):1806–1811. George B,Mathews V, Poonkuzhali B, Shaji RV, Srivastava A,Chandy M. Treatment of children with newly diagnosed acute promyelocytic leukemia with arsenic trioxide: a single centre experience. Leukemia 2004; 18(10): 1587–1590. Powell BL. Effect of consolidation with arsenic troxide (As2O3) on event-free survival (EFS) and overall survival (OS) among patients with newly diagnosed acute promyelocytic leukaemia (APL): North American Intergroup Protocol C9710. Journal of Clinical Oncology 2007; 25(18S): 2 Sanz MA, Fenaux P, Lo Coco F. Arsenic trioxide in the treatment of acute promyelocytic leukemia. A review of current evidence. Haematologica 2005; 90: 1231–1235. Tallman MS. Treatment of relapsed refractory acute promyelocytic leukemia. Best Pract res Clin Haematol 2007; 20: 57–65. de Botton S, Sanz MA, Chevret S, Dombret H, Martin G, Thomas X, Mediavilla JD, Recher C, Ades L, Quesnel B, Brault P, Fey M, Wandt H, Machover D, Guerci A, Maloisel F, Stoppa AM, Rayon C, Ribera JM, Chomienne C, Degos L, Fenaux P; European APL Group; PETHEMA Group. Extramedullary relapse in acute Promyelocytic leukemia treated with alltrans retinoic acid and chemotherapy. Leukemia 2006; 20: 35–41. Specchia G, Lo Coco F, Vignetti M, Avvisati G, Fazi P, Albano F, Di Raimondo F, Martino B, Ferrara F, Selleri C, Liso V, Mandelli F. Extramedullary involvement at relapse in acute promyelocytic leukemia patients treated or not with all-trans retinoic acid: a report by the Gruppo Italiano Malattie Ematologiche dell’Adulto. J Clin Oncol 2001; 19: 4023–4028. Breccia M, Carmosino I, Diverio D, De Santis S, De Propris MS, Romano A, Petti MC, Mandelli F, Lo-Coco F. Early detection of meningeal localization in acute promyelocytic leukemia patients with high presenting leucocyte count. British J Haematol 2003; 120: 266–270.

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Down Syndrome and Acute Myeloid Leukemia: An Unique Genetic Sensitivity to Chemotherapy Jeffrey W. Taub, Yubin Ge, and Yaddanapudi Ravindranath

Introduction Following the first description of leukemia in a Down syndrome (DS) child in 1930 (Brewster and Cannon 1930), a national survey in 1957 confirmed that DS individuals had an increased risk of developing leukemia (Krivit and Good 1957). It has been estimated that DS children have a 10 to 20-fold increased risk of developing both acute lymphoblastic leukemia (ALL) and acute myeloid leukemia (AML) compared to nonDS children (Taub 2001). A Danish population-based study reported that the cumulative risk of developing leukemia in DS children by the age of 5 was 2.1%, and there was an approximately fourfold higher standardized incidence ratio of AML compared to ALL (Hasle etÂ€al. 2000). The Nordic Society of Pediatric Hematology and Oncology (NOPHO) cooperative group reported that DS children with ALL and AML comprised 2.1% and 14%, respectively, of total childhood leukemia cases (Zeller etÂ€al. 2005). In the Children’s Cancer Group (CCG) 2891 study, 15% of the AML patients had DS, indicating that DS children comprise one of the largest subgroup of AML patients. Acute megakaryocytic leukemia (AMkL; M7) is the most common FrenchAmerican-British (FAB) subtype of DS AML patients, as reported by the Pediatric Oncology Group (POG), CCG, Berlin-Frankfort-Münster (BFM)-AML, NOPHO, Medical Research Council (MRC), Toronto and Japanese Childhood AML

J.W. Taub (*) Division of Hematology/Oncology, Children’s Hospital of Michigan, 3901 Beaubien Boulevard, Detroit, MI 48201, USA and Department of Pediatrics, Wayne State University School of Medicine, Detroit, MI 48201, USA and Developmental and Therapeutics Program, Barbara Ann Karmanos Cancer Institute, 4100 John R Street, Detroit, MI 48201, USA e-mail: [email protected]

P.J. Houghton and R.J. Arceci (eds.), Molecularly Targeted Therapy for Childhood Cancer, DOI 10.1007/978-0-387-69062-9_6, © Springer Science+Business Media, LLC 2010

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Â�cooperative groups (Zeller etÂ€al. 2005; Ravindranath etÂ€al. 1992, 1996; Gamis etÂ€al. 2003; Creutzig etÂ€ al. 2005; Al-Ahmari etÂ€ al. 2006; Kudo etÂ€ al. 2005; Rao etÂ€ al. 2006) (TableÂ€1). In contrast, AMkL is estimated to represent approximately 10% of pediatric AML cases and 1 to 2% of adult AML cases. The reported frequencies of the AMkL phenotype in DS AML cases have ranged from 39% to 100% and the discrepancy among the reported frequencies is likely due to variations in identifyingÂ� megakaryoblasts by either morphology or expression of platelet-associated membrane antigens (glycoprotein IIb/IIIa) using CD41/61 antibodies. Zipursky has estimated that DS children have a 500-fold increased risk of developing AMkL compared to nonDS children (Zipursky etÂ€al. 1994). Prior to the early 1990s, the prognosis for DS AML patients was considered to be extremely poor. In 1992, 12 DS AML patients treated on the POG 8498 study, had a 100% event-free survival (EFS) rate compared to 28% for nonDS AML patients treated with the identical therapies (Ravindranath etÂ€ al. 1992). Subsequent studies from multiple pediatric oncology cooperative groups have confirmed this observation (TableÂ€1). The improved survival rates highlighted by studies from these groups coincided with the utilization of high-dose cytosine arabinoside (ara-C)-based therapy for DS AML patients (Ravindranath etÂ€ al. 1992, 1996; Lange etÂ€ al. 1998; Gamis etÂ€ al. 2003). The high EFS rates of DS AML patients (particularly patients with AMkL) contrasts with the extremely poor complete remission (CR) rates and EFS rates for nonDS pediatric and adult patients with AMkL (Athale etÂ€ al. 2001; Barnard etÂ€ al. 2007; Reinhardt etÂ€ al. 2005; Ravindranath etÂ€ al. 2005; Lie etÂ€ al. 2005; Ruiz-Arguelles etÂ€ al. 1992; Duchayne etÂ€al. 2003; Tallman etÂ€al. 2000; Oki etÂ€al. 2006; Pagano etÂ€al. 2002) (TableÂ€2). The EFS rates of nonDS AMkL patients are also markedly lower compared

TableÂ€1â•… Treatment outcome of Down syndrome children with AML/MDS Induction failure/ Rx relapse deaths Protocol N M6/7 EFS References POG 8498 12 58% 100% 0% 0% Ravindranath etÂ€al. (1992) POG 8821 34 47% 68% 15% 18% Ravindranath etÂ€al. (1996) POG 9421 62 91% 80% 11% 11% CCG-2891 161 55% 77% 13% 4% Gamis etÂ€al. (2003) NOPHO 38 66% 83% 11% 0% Zeller etÂ€al. (2005) BFM AML98 58 97% 89% 6% 6% Creutzig etÂ€al. (2005) Japan-AML72 90% 83% 9% 9% Kudo etÂ€al. (2005) Down MRC AML10/12 46 39% 74% 3% 27% Rao etÂ€al. (2006) Toronto (low 18 100% 72% 28% 0% Al-Ahmari etÂ€al. dose ara-C) (2006) Toronto (standard 16 100% 75% 25% 0% Al-Ahmari etÂ€al. dose ara-C) (2006)

Down Syndrome and Acute Myeloid Leukemia: An Unique Genetic Sensitivity TableÂ€2â•… Treatment results of non-Down syndrome AMkL cases Group N CR rate EFS St. Jude 35 (12.7%) 60.5% 14% De novo 20% Secondary CCG 2891 52 (5.9%) 66% 22.5% BFM 89 (7.1%) 75% 34%

111

References Athale etÂ€al. (2001) Barnard etÂ€al. (2007) Reinhardt etÂ€al. (2005) Ravindranath etÂ€al. (2005) Lie etÂ€al. (2005) Ruiz-Argüelles etÂ€al. (1992) Duchayne etÂ€al. (2003)

POG 8821

32 (6.5%)

Not reported

25%

NOPHO-AML93 Mexico

16 (6.6 %) 10 children 35 adults 23 children

Not reported 90% 74% 77%

35% 30% (OS) 9% (OS) 26%

23 adult 20 (1.2%) 37 (2%)

33% 50% 43%

0% 10% 0%

Tallman etÂ€al. (2000) Oki etÂ€al. (2006)

24 (0.6%)

50%

10% (OS)

Pagano etÂ€al. (2002)

Groupe Francais De Cytogenetique Hematologique ECOG (adult) MD Anderson (adult) Gimema (adult)

to current EFS rates for nonDS pediatric AML patients, overall. This indicates that the AMkL group is one of the highest risk AML subgroups, despite the use of intensive chemotherapy protocols.

Chemotherapy Sensitivity and Down Syndrome What is the basis for the significantly higher EFS rates of DS AML patients compared to other subtypes of AML? In the 3-(4,5-Dimethylthiazol-2-yl)-2,5diphenyltetrazolium bromide (MTT) inÂ€vitro drug sensitivity assays, DS megakaryoblasts (nâ•›=â•›22) were 4.5-fold more sensitive to ara-C (median IC50: 77.5Â€ nm) and 12-fold more sensitive to daunorubicin (median IC50: 5.8Â€nm) compared to a large sample (nâ•›=â•›362) of nonDS AML blasts (median IC50’s: 350.9Â€nm and 71.2Â€nm, respectively) (Taub and Ge 2005). Further, DS megakaryoblasts generated significantly higher levels (4.4-fold) of the active intracellular metabolite, ara-CTP, following inÂ€vitro incubations with 3H-ara-C, indicating that the metabolism of ara-C in DS cells is altered (Taub etÂ€al. 1999). Increased inÂ€vitro sensitivities of DS AML blasts to ara-C and anthracyclines have been confirmed by other groups (Frost etÂ€al. 2000; Zwaan etÂ€al. 2002). These studies suggest that chromosome 21-localized genes, which may be overexpressed in trisomy 21 containing cells, contribute to enhanced drug sensitivities of DS AML blasts. Do the increased chemotherapy sensitivities of DS megakaryoblasts translate into increased treatment related toxicity of DS children with AML therapy? For the CCG-2891 AML study (standard timed induction), there were no significant Â�differences in Grade III/IV toxicity at any site between DS and nonDS patients

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treated with the high-dose ara-C (Capizzi II) cycle (Gamis etÂ€ al. 2003). NonDS patients experienced significantly greater hepatic toxicity (7.4% vs. 2%; Pâ•›=â•›0.036) compared to DS patients. In contrast, DS patients experienced greater mucositis (9.6% vs. 3.7%; Pâ•›=â•›0.023) and skin toxicity (3.7% vs. 0.4%; 0.024) compared to nonDS patients. However, other studies have reported a high frequency of treatment-related toxicity and mortality of DS patients. The intensive-timed DCTER regimen used in the CCG 2861/2891 AML protocol was associated with a 32% mortality rate for DS patients (Lange etÂ€ al. 1998). Similarly, the MRC AML 10/12 studies reported a 27% treatment related mortality rate for DS patients, which used no modification in chemotherapy dosing for DS compared to nonDS AML patients (Rao etÂ€ al. 2006). Infectious complications have also had a negative impact on the outcome of DS AML patients in the BFM studies (Lehrnbecher etÂ€ al. 2004). A study from Argentina reported seven treatment-related deaths among 11 DS AML patients (Zubizarreta etÂ€al. 1998). Studies from the POG have suggested that DS children have an increased risk of anthracycline-induced cardiac toxicity. A multivariate analysis of 6,493 children treated on POG protocols from 1974 to 1990 with anthracycline chemotherapy found that DS children had a relative risk of 3.4 to develop cardiac toxicity (Krischer etÂ€al. 1997). On the POG 9421 AML study (total cumulative anthracycline dose: daunorubicin: 135Â€ mg/m2; mitoxantrone: 80Â€ mg/m2), symptomatic Â�cardiomyopathy developed in 10 (17.5%) DS patients during or soon after completion of treatment, with three patients dying of congestive heart failure (O’Brien etÂ€al. 2006). The treatment outcome of DS ALL patients have been distinctly different Â�compared to DS AML patients. Prior studies have reported inferior survival rates of DS ALL patients compared to nonDS ALL patients (Whitlock 2006) and Â�frequently DS patients have experienced treatment-related toxicity, particularly with methotrexate (Peeters and Poon 1987). In vitro drug sensitivity studies have also demonstrated that DS lymphoblasts do not have the same patterns of increased inÂ€vitro chemotherapy sensitivities as in DS AML cases (Frost etÂ€al. 2000; Zwaan etÂ€ al. 2002). This suggests that DS ALL and DS AML cells differ biologically, Â�possibly reflecting the lack of GATA1 mutations (discussed below) in DS ALL and potential differences in expression of chromosome 21-localized genes.

The Role of Chromosome 21-Localized Genes and Chemotherapy Sensitivity in Down Syndrome AMkL Several chromosome 21-localized genes may be involved in the increased Â�sensitivity of DS megakaryoblasts to chemotherapy agents. The transsulfuration pathway enzyme, cystathionine-ß-synthase (CBS; localized to chromosome 21q22.3), Â�catalyzes the condensation of serine and homocysteine to form Â�cystathionine, an intermediate step in the synthesis of cysteine. Increased CBS

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activity is associated with significantly lower homocysteine, methionine, and S-adenosylmethionine (AdoMet) levels in DS individuals, which may have downstream effects on reduced folate metabolism. Increased CBS activity may also indirectly impact metabolism of nucleotides (including ara-C) and their antileukemic activity, analogus to the established synergism of sequential methotrexate and ara-C therapy (Newman etÂ€al. 1990). We previously hypothesized that increased CBS expression in DS leukemia cells leads to decreased allosteric regulation of 5,10-methylenetetrahydrofolate reductase (MTHFR) by AdoMet, which diverts increased levels of 5,10-methylenetetrahydrofolate to 5-methyltetrahydrofolate. The lower 5,10-methylenetetrahydrofolate pools could result in decreased dTMP and dTTP, which would relieve inhibition of deoxycytidylate deaminase (DCD) and result in lower dCTP pools. The decreased dCTP pools, in turn, would result in increased deoxycytidine kinase (dCK) catalytic activity and greater activation of ara-C to the active intracellular metabolite, araCTP, and decreased competition with ara-CTP for incorporation into DNA (Taub etÂ€al. 1999, 1996) (Fig.Â€1). Portions of this hypothesis have been validated experimentally in both clinical leukemia samples and leukemia cell line models. (1) The CBS gene was expressed at significantly higher levels in DS leukemia cells (median 12-fold higher) Â�compared to nonDS leukemia cells and correlated with both inÂ€ vitro ara-CTP Â�generation and ara-C sensitivities (Taub etÂ€al. 1999). Interestingly, CBS transcripts were not detected in DS and nonDS ALL blasts. (2) Transfection of the CBS-null CCRF-CEM leukemia cell line with the CBS coding cDNA resulted in significantly increased inÂ€vitro and inÂ€vivo ara-C sensitivities compared to wild-type CEM cells (Taub etÂ€al. 2000). (3) In AMkL cell lines with gene expression patterns similar to clinical AML samples, high CBS transcripts in DS CMK cells were accompanied by tenfold greater ara-C sensitivities and 2.4-fold higher levels of ara-CTP Â�generation compared to the CBS-null, nonDS AMkL cell line, CMS (Ge etÂ€al. 2003). A relationship may also exist between the presence of the CBS 844ins68 polymorphism and inÂ€vitro ara-C sensitivities of DS and nonDS AML blast cells (Ge etÂ€al. 2002). Copper/zinc superoxide dismutase (SOD1, gene localized to 21q22) catalyzes the reaction from superoxide anions to hydrogen peroxide, resulting in the generation of hydroxyl free radicals. Imbalances of oxygen radicals secondary to increased SOD activity are known to be involved in the pathophysiology of DS, reflecting increased generation of oxygen free radicals and increased susceptibility of cells to undergo apoptosis (Busciglio and Yankner 1995; de Haann etÂ€ al. 1997). The increased susceptibility of DS cells to undergo apoptosis via oxygen radicals, may contribute to enhanced chemotherapy sensitivity, particularly following daunorubicin therapy, which mediates its cytotoxicity via the generation of oxygen radicals (Chien etÂ€al. 2004). Consistent with this notion, SOD transcripts were significantly higher (median fourfold) in DS megakaryoblasts compared to nonDS AML cells with a trend toward a correlation with inÂ€vitro daunorubicin sensitivities (Taub etÂ€al. 1999). Altered oxygen radical metabolism in DS may also contribute to the increased risk of anthracycline-induced cardiotoxicity.

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a

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E C

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Fig.Â€1â•… The Role of GATA1 in cytidine deaminase expression and cytarabine metabolism in AML cells. After facilitated intracellular transport via the nucleoside transporter, HENT1 (a), cytarabine (ara-C) undergoes three sequential phosphorylation steps to form the active metabolite ara-CTP. The first phosphorylation step is rate limiting and is catalyzed by deoxycytidine kinase (dCK) (b), which is negatively regulated by dCTP. Cytidine deaminase (CDA) deaminates ara-C to the inactive metabolite, uridine arabinoside (ara-U) (c). Ara-CTP competes with dCTP for incorporation into DNA and inhibits DNA polymerase (d). Down syndrome (DS) acute megakaryocytic leukemia (AMkL) blasts are characterized by the presence of somatic mutations in exon 2 of the X-linked transcription factor gene, GATA1, resulting in the sythesis of a truncated GATA1 protein, GATA1s (40-kDa), which has reduced transactivation activity compared to the wild-type 50-kDa GATA1 protein (e). The CDA gene consists of a CDAsf (short-form) intronic promoter that acts as an enhancer to the CDAlf (long-form) promoter, which may enhance the transcription of the CDA gene. Therefore, lower CDA expression in DS megakaryoblasts compared to nonDS AMkL, may be due to the synthesis of the GATA1s protein, secondary to GATA1 mutations (f). Decreased CDA in DS megakaryoblasts results in greater ara-C phosphorylation and higher ara-CTP levels compared to nonDS megakaryoblast cells (g). Increased CBS expression in DS AMkL blasts results in altered intracellular reduced folate pools and decreased dCTP pools. This leads to: (1) reduced feedback inhibition of dCK resulting in greater phosphorylation of ara-C to form ara-CTP and (2) greater ara-CTP incorporation into DNA due to reduced competition from the lower dCTP pools (h). Bone marrow stromal cell antigen 2 (BST2), is a cell surface membrane protein expressed in normal tissues and malignant cells and supports the stromal cell-dependent growth of other cells. The transcription of BST2 gene is regulated by GATA1. Stimulation of BST2 promoter activity by the GATA1s protein is substantially reduced compared to GATA1, resulting in lower BST2 expression in DS megakaryoblasts. This results in reduced leukemia cell protection from ara-C-induced cytotoxicity modulated by bone marrow stromal cells (i). Other potential genes regulated by GATA1, may modulate the cytotoxicity of ara-C and other chemotherapy drugs via apoptosis pathways (e.g., HSP70, bcl-2) (j). In nonDS megakaryoblasts, higher CDA expression results in greater ara-C deamination to ara-U (k), decreased generation of ara-CTP and reduced ara-C cytotoxicity. Increased BST2 expression leads to greater leukemic blast protection from ara-C-induced apoptosis in the presence of bone marrow stromal cells (l)

Down Syndrome and Acute Myeloid Leukemia: An Unique Genetic Sensitivity

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Non-Down Syndrome Megakaryoblast

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Fig.Â€1â•… (continued)

Relationship Between GATA1 and Chemotherapy Drug Sensitivities A seminal discovery in understanding the biology of AML in DS children was initially reported in 2002, (Wechsler etÂ€ al. 2002) and subsequently confirmed by other groups (Hitzler etÂ€al. 2003; Rainis etÂ€al. 2003; Ahmed etÂ€al. 2004). Acquired somatic mutations of the transcription factor gene, GATA1 (localized to Xp11.23), have been detected with nearly 100% penetrance in DS AMkL and transient myeloproliferative disorder (TMD) cases, while mutations have not been detected in nonDS AML and nonAMkL DS leukemia cases. Hence, GATA1 mutations appear to be synonymous with the DS AMkL phenotype and are not detected in remission bone marrows. GATA1 encodes a zinc finger transcription factor that binds to the WGATAR motif and is essential for normal erythroid and megakaryocytic differentiation. Reported sequence alterations in the region encoding the N-terminal activation domain of GATA1 include insertion, deletion, missense, nonsense, and splice site mutations at the exon 2/intron boundary (Muntean etÂ€al. 2006). The net effect of GATA1 mutations is the introduction of stop codons and synthesis of a shorter GATA1 (designated GATA1s) protein (40-kDa), initiated from a downstream initiation site and distinguishable from the wild-type GATA1 protein (50-kDa) (Wechsler etÂ€al. 2002). Both GATA1s and the wild-type GATA1 proteins show similar DNA binding abilities and interact with FOG1, though the GATA1s protein exhibits altered transactivation capacity due to the loss of the N-terminal activation domain.

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Thus, mutations at the GATA1 locus in DS are believed to result in the accumulationÂ� of poorly differentiated megakaryocytic precursors and represent initiating or early genetic hits in a multistep process of leukemogenesis. The uniform detection of somatic mutations in the GATA1 gene in DS AMkL cases suggested the possibility that the mutant GATA1 protein in DS AMkL may contribute to the high EFS rates. A relationship between GATA1 and AML outcome has been suggested by a Japanese study in which nonDS AML patients with lower GATA1 expression had the highest complete remission rates (Shimamoto etÂ€al. 1995). It is conceivable that GATA1 may regulate the expression of several differentially expressed genes (based on the localization of GATA1 binding sites in their promoters), leading to altered activity of chemotherapy drugs. One likely target is cytidine deaminase (CDA; gene localized to chromosome 1p), which deaminates ara-C to the inactive metabolite, uridine arabinoside (ara-U). CDA transcripts were a median 5.1-fold lower in DS megakaryoblasts compared to nonDS AML blast cells (Ge etÂ€al. 2005). The CDA transcript is transcribed from a CDA “long form” promoter (CDAlf) while a “short form” promoter (CDAsf) acts as an enhancer for the CDAlf promoter (Ge etÂ€ al. 2004). Thus, mutations of the GATA1 gene in DS AMkL blasts that generate a functionally altered GATA1 protein, CDA enhancer activity and decreased overall CDA expression, decreased net conversion of ara-C to ara-U (Fig.Â€1). To assess the relationship between ara-C and GATA1, the DS AMkL cell line, CMK, was stably transfected with the full-length GATA1 cDNA (Ge etÂ€al. 2005). The CMK transfectants expressing the full-length GATA1 protein were approximately 8 to 17-fold less sensitive to ara-C compared to wild-type and mock-transfected CMK cells. The GATA1-transfected cells showed slightly decreased daunorubicin (approximately 2-fold) sensitivity compared with the wild-type and mock-Â�transfected CMK cells, though the relationship between GATA1 and genes involved in daunorubicin metabolism/activity is unknown.

Differential Gene Expression Studies and Down Syndrome AMkL Two studies have used microarrays to identify differentially expressed genes between DS and nonDS AMkL cases, (Ge etÂ€al. 2006; Bourquin etÂ€al. 2006), which may account for the biological differences between the two groups of AMkL and identify genes linked to the increased chemotherapy responses of DS AMkL cases and/or chemotherapy resistance of nonDS AMkL cases. In the study by Ge etÂ€al. (2006), 551 differentially expressed genes (105 overexpressed in the DS group) with a minimum twofold change were identified between DS and nonDS AMkL cases. Among the genes overexpressed in the nonDS AMkL group was bone Â�marrow stromal cell antigen 2 (BST2; localized to 19p13.2), a surface membrane protein expressed in normal tissues, malignant cells (e.g., nonDS AMkL cell line, Mo7, multiple myeloma cells). BST2 has been reported to support the stromal

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Â� cell-dependent growth of the DW34 cell line (Ishikawa etÂ€al. 1995; Ohtomo etÂ€al. 1999). Coincubation of leukemia cells with bone marrow stromal cells protects the former from chemotherapy-induced apoptosis, while megakaryocytic differentiation of progenitor cells is inhibited by direct contact with bone marrow stromal cells (Garrido etÂ€al. 2001; Konopleva etÂ€al. 2002; Zweegman etÂ€al. 1999). The BST2 promoter region contains numerous putative cis elements including GATA1. Using gel shift and chromatin immunoprecipitation (ChIP) assays, BST2 was confirmed to be a GATA1 target gene with transcript levels significantly higher in nonDS AMkL blasts compared to DS megakaryoblasts (Ge etÂ€ al. 2006). Interestingly, stable transfection of the BST2 cDNA into the DS AMkL cell line, CMK, resulted in reduced ara-C-induced apoptosis of the CMK transfectant cells in the presence of bone marrow stromal cells compared to a mock transfectant (Ge etÂ€ al. 2006). These results suggest that bone marrow stromal cells protect leukemia cells through cell interaction mediated by cell surface antigens (e.g., BST2) and receptors. In addition to bone marrow stromal cells, bone marrow derived mesenchymal cells have also been found to protect leukemia cells from asparaginase cytotoxicity (Iwamoto etÂ€al. 2007). Additional genes overexpressed in the nonDS AMkL group including bcl-2 and HSP70, (Ge etÂ€al. 2006) have known antiapoptosis activity, suggesting a potential common mechanism of resistance to chemotherapy drugs in nonDS megakaryoblasts. Interestingly, the chromosome 21-localized gene, AML1 (also known as RUNX1) is underexpressed in the DS AMkL group compared to the nonDS AMkL group despite the gene dosage effect of trisomy 21 (Bourquin etÂ€al. 2006). Knockdown expression of AML1 in a cell line model was associated with increased ara-C sensitivities (Edwards et al. 2009). Savasan etÂ€al. (2006) used CD 36 expression by flow cytometry as a marker for megakaryocytic blast maturation in DS and nonDS AMkL blasts. Interestingly, in a small sample of nonDS megakaryoblasts expressing CD36 at similar levels as DS AMkL blasts (high CD 36 expression is universal in DS AMkL), exhibited the same level of inÂ€vitro sensitivities as DS megakaryoblasts and the corresponding patients had markedly superior outcome compared to nonDS AMkL patients with low CD36 expression (Savasan etÂ€al. 2006).

Future Challenges The optimal therapy for DS AML patients has not been clearly defined, balancing curative therapy against the potential risk of significant morbidity and treatmentrelated mortality. The current COG AAML041 clinical trial “Treatment of Down Syndrome Children with AML and MDS Under the Age of 4Â€Years” is designed to determine whether intensification of ara-C therapy during the second cycle of induction therapy, a reduction of the total cumulative anthracycline dose by 25% and a reduction in the number of intrathecal chemotherapy treatments, can maintain the high EFS rates of DS patients. Correlative pharmacologic and genetic studies may also

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identify DS patients who could be treated with further reduced intensive therapies, such as low dose ara-C containing regimens, as pioneered by Zipursky (Al-Ahmari etÂ€ al. 2006) and/or identify “high risk” DS AML patients. In an analysis of DS patients treated on the POG 9421 and CCG-2891 (standard timing arm) AML studies, 11% (24/218) of the patients relapsed with an overall survival of only 12%, despite intensive salvage therapies including bone marrow transplantation (Loew etÂ€al. 2004). In addition, DS AML patients >4Â€years of age (which comprise 5% of total DS AML cases), have EFS rates of <35%, (Gamis etÂ€al. 2003) Â�indicating potential biological differences in AML blasts of DS children <4Â€years compared to >4Â€years of age. This suggests that a small subset of DS AML cases have an inherent resistance to chemotherapy, which may require intensification of therapy at diagnosis. No studies to date have analyzed GATA1 mutations in the group of DS AML patients >4Â€years of age. It is conceivable that these cases may represent true deÂ€novo AMLs rather than the classic DS AMkL phenotype associated with GATA1 mutations.

Conclusion Can the mechanisms of enhanced chemotherapy sensitivities in DS AMkL be translated to the treatment of nonDS children with AMkL, and possibly AML overall? From the studies to date, it appears that the high cure rates of DS AML patients, and in particular, patients with the AMkL phenotype, are multifactorial based upon the trisomy 21 phenotype (overexpression of the chromosome 21 localized genes, CBS and potentially SOD1) and the unique defining genetic abnormality in DS megakaryoblasts (mutations in the GATA1 gene leading to the synthesis of the 40-kDa GATA1s protein). Using gene targeting methods to knock down GATA1 expression in nonDS AMkL may be one potential strategy, though difficulties in targeting genes inÂ€ vivo exist. Alternative approaches include identifying downstream GATA1 target genes (e.g., genes in the apoptotic pathways), which mediate chemotherapy sensitivity/resistance. Ultimately, a universal mechanism linking the generation of GATA1 mutations, leukemogenesis, and enhanced chemotherapy sensitivities in DS AMkL may be identified. Acknowledgmentsâ•… Supported by grants RO1 CA92308 and CA120772 from the National Cancer Institute, the Leukemia and Lymphoma Society, The Elana Fund, The Ring Screw Textron Chair in Pediatric Cancer Research and The Georgie Ginopolis Chair for Pediatric Cancer and Hematology.

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Targeting RAS Signaling Pathways in Juvenile Myelomonocytic Leukemia (JMML) Jennifer O’Hara Lauchle and Benjamin S. Braun

Juvenile myelomonocytic leukemia (JMML) is an aggressive, clonal Â�myeloproliferative disorder (MPD) of childhood characterized by the overproduction of myelomonocytic cells that infiltrate the spleen, lung, and gastrointestinal tract (Arico etÂ€al. 1997; Emanuel etÂ€al. 1996). Children frequently present with anemia, thrombocytopenia, splenomegaly, and failure to thrive. The median age of diagnosis is 2Â€years of age. Without definitive treatment, the median survival of JMML patients is less than 1Â€year. Allogeneic hematopoietic stem cell transplantation (HSCT) is the only curative therapy with a probability of event-free survival at 5Â€ years of 50% (Liu etÂ€ al. 2004). The main cause of treatment failure continues to be leukemia relapse with death due to organ infiltration, infection, or transformation to acute myeloid leukemia. Stem cell transplantation is also associated with significant acute and chronic morbidity in young children. Therefore, new approaches to therapy are needed for children with newly diagnosed and relapsed JMML. Hematopoietic progenitors isolated from the peripheral blood and bone marrow of JMML patients are hypersensitive to multiple cytokines, particularly granulocytemacrophage colony-stimulating factor (GM-CSF) (Emanuel etÂ€ al. 1991). This is demonstrated by increased growth of granulocyte-macrophage colony forming units (CFU-GM) at low doses of GM-CSF compared to progenitor cells from healthy children. The molecular genetics of JMML implicates hyperactive RAS as an essential initiating event, as 85% of patients harbor mutations in either NRAS, KRAS, NF1, PTPN11 or CBL (reviewed in Lauchle etÂ€al. 2006; Loh et al. 2009). Due to deregulation of the RAS pathway in many malignancies, a tremendous effort has been expended developing novel inhibitors targeted at RAS signaling for cancer therapies. We will summarize recent experience with preclinical and clinical evaluation of targeted therapeutics for JMML and discuss opportunities to study agents currently in Â�development. With molecular insights into pathogenesis, new assays to evaluate molecular response and preclinical testing systems in murine

J. O’Hara Lauchle (*) University of California, Helen Diller Family Cancer Research Building (Optional), 1450 3rd Street, Room 264 (Lauchle) and 265 (Braun), San Francisco, CA 94158, USA e-mail: [email protected]

P.J. Houghton and R.J. Arceci (eds.), Molecularly Targeted Therapy for Childhood Cancer, DOI 10.1007/978-0-387-69062-9_7, © Springer Science+Business Media, LLC 2010

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models and human samples, JMML researchers are uniquely poised to evaluate novel mechanism-based therapeutics for RAS pathway malignancies.

GM-CSF and Deregulated RAS Signaling in Pathogenesis of JMML RAS Signaling is a critical determinant of proliferative cell output in response to extracellular stimuli including GM-CSF and other hematopoietic growth factors. Upon ligand binding, the GM-CSF receptor activates a receptor associated tyrosine kinase, JAK2. JAK2 phosphorylates the b-chain of the GM-CSF receptor and other substrates including Signal Transducer and Activator of Transcription (STAT) proteins. The phosphorylated GM-CSF receptor also recruits adaptor and signaling molecules such as SHP-2 and results in activation of RAS signaling pathways. RAS Proteins are molecular switches that cycle between an inactive GDP-bound state and an active GTP-bound state after receptor activation. Guanine nucleotide exchange factors (GEFs), such as son of sevenless homolog (SOS1), increase the proportion of RAS bound to GTP by disassociating guanine nucleotides from RAS and favoring passive binding to GTP, which is abundant in the cytosol. GTP bound RAS interacts with effector molecules including phosphatidylinositol 3-kinase (PI3K), RAF, and Ral-GDS to activate downstream kinase cascades. Fig.Â€1 shows a simplified schematic of RAS pathway effector cascades, although both cell context specific interactions between effectors and higher order network regulation are likely to be relevant. RAS signaling is terminated by hydrolysis of bound GTP to GDP by a slow intrinsic GTPase, which is markedly accelerated by the binding of GTPase activating proteins (GAPs), including p120GAP and neurofibromin. RAS Proteins are encoded by the closely related KRAS, NRAS, and HRAS genes, which undergo somatic point mutations in approximately one-third of human cancers. NRAS and KRAS mutations have been documented in 25% of JMML cases, though HRAS mutations are rare (Flotho etÂ€al. 1999; Miles etÂ€al. 1996). These mutations introduce amino acid substitutions that lead to accumulation of RAS in the GTP-bound conformation due to defective intrinsic GTPase activity and insensitivity to GAPs. Children with neurofibromatosis type 1 (NF1) are predisposed to developing several distinct malignancies, especially JMML, brain tumors, and malignant peripheral nerve sheath tumors. The NF1 gene encodes neurofibromin, which is a GAP that negatively regulates RAS signaling by accelerating the conversion of RAS-GTP to RAS-GDP (reviewed in refs. Cichowski and Jacks 2001; Donovan etÂ€al. 2002). Genetically, NF1 functions as a classic tumor suppressor gene. Patients with NF1 demonstrate germline heterozygosity for NF1 and develop malignancies that have undergone biallelic inactivation of NF1 (Schubbert etÂ€al. 2005; Shannon etÂ€al. 1994). In JMML, homozygous inactivation of NF1 has been shown to result in reduction in neurofibromin-specific GAP activity and elevated levels of RAS-GTP (Bollag etÂ€al. 1996). Recent studies have demonstrated that there is acquired isodisomy of the mutant NF1 allele in these patients, a commonly observed phenomenon in human cancer (Flotho etÂ€al. 2007; Michaelson etÂ€al. 2005).

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GF

JAK2 XL019 lestaurtinib INCB018424

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Shc

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RAS GTP

tipifarnib salirasib

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IC87114 GDC0941 PX866 XL147 BGT226

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PD0325901 AZD6244 XL518

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Fig.Â€1â•… RAS Pathway signaling and inhibitors in clinical development. A simplified schematic of the RAS signal transduction network is depicted. RAS cycles between active (GDP-bound) and inactive (GTP-bound) states upon activation of growth factor (GF) receptors, and in turn stimulates multiple cytoplasmic signal transduction cascades (see text). JMML-associated mutations in KRAS, NRAS, NF1, CBL, or PTPN11 (SHP-2) activate this system. Selected agents in clinical development that might be used to treat JMML are shown with their most relevant targets. Note that, in some cases, “off-target” proteins not shown in this figure are inhibited to an equal or greater degree. As reviewed in the text, initial efforts were focused on inhibition of RAS. Current efforts are more focused on targeting downstream RAS effectors, singly or in combination. An alternative approach is to inhibit upstream biochemical events, such as JAK2 activation

Germline PTPN11 mutations are present in 40 to 50% cases of Noonan syndrome, which shares clinical features with NF1 (Sun etÂ€al. 2003). Similar to NF1, the occurrence of JMML and transient myeloproliferative disorders in children with Noonan syndrome led investigators to screen nonsyndromic patients with deÂ€novo JMML for somatic mutations in PTPN11. Somatic mutations in PTPN11 are found in 35% of nonsyndromic JMML cases (Locatelli etÂ€ al. 2005; Tartaglia etÂ€ al. 2001). PTPN11 encodes the nonreceptor protein tyrosine phosphatase (PTP) SHP-2. SHP-2 contains two src homology 2 (SH2) domains and a PTP domain. In its basal state, the PTP activity is blocked by an interaction with the N-SH2 domain. Ligand binding disrupts this auto-inhibitory interaction resulting in PTP activation and enhanced RAS/ERK pathway activation. Most PTPN11 mutations in JMML alter residues in the interface between the N-SH2 and PTP domains thereby preventing basal auto-inhibition (Keilhack etÂ€al. 2005). Leukemia associated alleles encode SHP-2 variants, such as D61Y and E76K, that tend to have higher PTP activity than those found in germline mutations found in NS (Nakamura etÂ€al. 2005; Rotblat etÂ€al. 2008).

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More recently, mutations in CBL have been found in approximately 10% of JMML cases (Loh et al. 2009). The Cbl protein has multiple roles in signal transduction. A ubiquitin ligase function targets various growth factor receptors for degradation to terminate signaling, whereas other domains promote signal transduction by formation of signaling complexes. Leukemia associated point mutations appear to disrupt ubiquitin transfer activity but leave adapter functions intact, leading to a net activation of signal transduction by aberrant CBL proteins (Sanada et al. 2009). Complete loss of Cbl ubiquitin ligase activity may be important, because the normal allele is usually lost (specifically, by acquired uniparental isodisomy). In one child with JMML, the mutant allele was found to be heterozygous in buccal DNA, suggesting possible germline inheritance. In this scenario, patients that inherit a heterozygous point mutation would be at risk for developing JMML upon acquired somatic homozygosity, in a manner similar to patients heterozygous for NF1. Mutations in PTPN11, RAS and NF1 are rarely found in the same patient, supporting the hypothesis that hyperactive RAS signaling is essential to the pathogenesis of JMML. The molecular events in 25% of JMML cases are unknown, yet they are likely to also dysregulate the RAS pathway. Analysis of JMML samples has not yet uncovered mutations in GM-CSF, SOS1, SHC1, GRB2, GAB1, BRAF, MEK1, or MEK2 (de Vries etÂ€al. 2007a; Kratz etÂ€al. 2007). In addition, FLT3 and JAK2 mutations, relatively common in other myeloid leukemias and myeloproliferative disorders, are rarely detected in JMML (de Vries etÂ€al. 2007b; Winter-Vann and Casey 2005). In addition to providing insights into pathogenesis, detection of mutations in NF1, RAS, PTPN11, or CBL will be incorporated into revised diagnostic criteria for JMML, and may have prognostic implications. For therapeutic studies, mutation detection provides a way to evaluate molecular response to treatment. Specific quantitative assays for JMML-associated mutant alleles have been developed and used to detect relapse following stem cell transplant with high sensitivity (Archambeault etÂ€al. 2008). Importantly, this assay could yield objective response criteria for therapies given prior to transplantation or in the context of minimal residual disease. Because the morphology of neoplastic cells in JMML is nearly normal, quantitative assessment of the mutant clone by DNA analysis provides the only realistic way to measure disease burden in these settings. More recently, a population of cells with increased STAT5 phosphorylation in response to GM-CSF was identified in diagnostic JMML samples with sensitivity of 91% and a specificity of 95% (Kotecha etÂ€al. 2008). Presence of this population correlated with clinical disease status in several patients for whom diagnostic, remission, and relapse samples were evaluated (Kotecha etÂ€al. 2008). Application of this assay to evaluate therapeutic response and disease status may be especially important in cases for which PCR assays are not available such as those patients without a known mutation or with a diagnosis of NF1. Beyond its potential use as a diagnostic tool, intracellular phosphoprotein analysis of JMML samples may provide opportunities to identify new disease targets, facilitate preclinical drug testing, and evaluate clinical responses. For example, inÂ€ vitro exposure of JMML samples to a JAK2 inhibitor abrogated both the phospho-STAT5 and phospho-ERK response to GM-CSF (Kotecha etÂ€al. 2008). This work follows prior efforts to develop assays using patient samples for drug Â�development but will

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need to be linked to leukemia growth inÂ€ vitro, and most importantly, clinical responses. The GM-CSF hypersensitive myeloid progenitor colony growth that aids in diagnosis has also been applied as an inÂ€vitro assay to screen new therapeutic compounds (Emanuel etÂ€ al. 2000; Loh etÂ€ al. 2004). Inhibition of spontaneous versus GM-CSF stimulated colonies may be assessed, and therapeutic thresholds can be defined when colony growth from normal bone marrow progenitors is evaluated concurrently. Engraftment of JMML samples into NOD/SCID mice provides another method to test new agents on patient-derived samples (Mohi etÂ€al. 2005).

Mouse Models Genetically engineered mouse models have substantial potential to address several important issues in drug development. Clinical research in patients is limited by high cost, long duration, and the need to protect the safety of subjects. The rarity of JMML is also a major barrier, as even cooperative groups can expect to enroll fewer than 50 patients each year. Hopefully, mouse models that recapitulate the genetic, biochemical, and cell biologic features of JMML will have useful positive and negative predictive value for screening candidate therapeutics. Studies in these animal models can be performed at far lower cost, complexity, and time in comparison to conventional human trials. Several strategies have been used to model JMML in the mouse. The first used gene targeting to disrupt Nf1, and heterozygous mice indeed developed a JMML-like disease associated with somatic loss of the wild type allele (Bollag etÂ€al. 1996; Jacks etÂ€al. 1994). This occurred only in old mice, however, so homozygous loss of Nf1 was accomplished by breeding Nf1−/− mice. Because of late-term embryonic lethality, hematopoietic cells were harvested from Nf1−/− fetal livers and transplanted into irradiated recipients (Largaespada etÂ€al. 1996). This method reliably produced mice with MPD but also required significant effort and introduced potentially confounding effects of radiation. To create a more tractable and physiologic model, a conditional Nf1flox allele was constructed by placing loxP recombination sites around exons 31 and 32 (Zhu etÂ€al. 2001). When the Cre recombinase protein is expressed, it performs a splicing event between the loxP sites that removes the intervening DNA. This results in the deletion of these two exons and inactivation of Nf1. Importantly, this lesion is similar to mutations found in NF1 patients, who frequently have deletion, nonsense, or frameshift mutations in this region. The inducible Mx1-Cre transgene allows Nf1 disruption to be induced in hematopoietic stem cells after birth by treating mice with interferon or substances that elicit interferon production such as polyinosinic-polycytidilic acid (pIpC). Nf1flox/flox, Mx1-Cre mice that are treated with pIpC at 3–5Â€days of age uniformly develop a monocytic MPD that closely resembles JMML (Le etÂ€al. 2004). This represents a major technical advance over the earlier systems, and the conditional system is much more amenable to preclinical studies. Similar conditional alleles have also been used to model effects of dominant oncogenes such as Kras and Ptpn11 (Braun etÂ€al. 2004; Chan etÂ€al. 2004, 2009). In this case, an oncogenic mutation is introduced in the germ line, but it is initially

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silenced by a repressor cassette (“LoxP-Stop-LoxP” or LSL). The LSL is then excised by Cre in juvenile mice. In contrast to the indolent MPD seen in Nf1flox/flox and LSL-Ptpn11D61Y mice, which survive for 6–12Â€months, the LSL-KrasG12D model is characterized by aggressive disease leading to death in less than 3Â€months. The LSL-KrasG12D model is also the most readily transplanted into naïve recipients. Interestingly, despite broad phenotypic similarities among all these models, there remain distinctions between them with respect to erythroid maturation, progenitor distribution, signaling biochemistry, and stem cell function. It is yet unknown as to which of these features will reflect genotype-specific findings in JMML patients and which will be unique to mice. Nonetheless, it is a testament to the efficiency of modern genetic engineering that three of the 5 known JMML oncogenes have now been modeled using conditional targeted alleles in mice. Such systems have significant advantages, most notably (a) ease of use, (b) preservation of normal cis-acting regulatory control of the targeted locus, including normal copy number, and (c) the ability to study the effects of somatic mutation in animals that have undergone normal development. In principle, these mouse cancer models will allow researchers to conduct well-controlled experiments aimed at both discovering fundamental mechanisms of leukemogenesis and evaluating potential therapies prior to human testing. However, it is prudent to note that no inÂ€vitro or inÂ€vivo model system has yet been used to identify a clinically important therapy for JMML.

Targeted Therapeutics Considering its role in many cancers, inhibition of activated RAS is a logical therapeutic strategy. However, there are several obstacles to this approach. The importance of RAS signaling in diverse physiologic processes in normal cells raises the concern of systemic toxicities from any therapies in which normal RAS proteins or effectors are inhibited. In addition, oncogenic mutations in RAS lead to decreased GTPase activity, and developing agents that help restore rather than inhibit enzyme function has not been achieved to date. Competitive GTP inhibitors would need to confront the very high affinity of RAS for GTP, as well as the high intracellular concentration of GTP. With this in mind, researchers turned their focus to inhibiting posttranslational RAS processing at the C-terminal CAAX sequence. The multistep process of CAAX protein modification renders proteins hydrophobic at their C-termini, allowing for membrane association. The rate-limiting step in normal and mutant RAS protein modification is prenylation, which covalently attaches an isoprenoid lipid at the CAAX motif at the carboxyl terminus. Based on the terminal amino acid (X) of the CAAX motif, proteins are prenlyated by farnesyltransferase (FTase), which transfers a 15 carbon farnesyl isoprenoid, or geranylgeranyltransferase (GGtase), which in turn, transfers a 20 carbon geranylgeranyl isoprenoid. As prenylation of H-RAS, N-RAS, and K-RAS is catalyzed by FTase, farnesyltransferase inhibitors (FTIs) were developed to prevent RAS localization to the plasma membrane and inhibit RAS signaling. Colony formation of JMML cells was reduced by 25 to 50% at Â�concentrations of the FTI L739,749 that only modestly reduced colony growth

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of control hematopoietic progenitors (Emanuel etÂ€al. 2000). Treatment of Nf1 deficient murine models of JMML with a similar FTI, L744,832, did not inhibit Nf1−/− hematopoietic cell growth or splenic infiltration with myeloid progenitors (Mahgoub etÂ€ al. 1999). FTI treatment was associated with a decrease in farnesylation of H-RAS but not N-RAS (Mahgoub etÂ€al. 1999). Response rate and acute toxicities of a nonpeptidic CAAX-competitive selective inhibitor of FTase tipifarnib ( R115777 ) was studied in a Children’s Oncology Group (COG) Phase II treatment window in newly diagnosed JMML (Castleberry etÂ€ al. 2005). The majority of JMML patients treated had partial clinical responses defined by decreased white blood count and organomegaly prior to transplantation; however, event-free survival was not impacted by FTI treatment. Similar to other clinical trials with tipifarnib, clinical response did not correlate with the degree of inhibition of FTase activity as measured by farnesylation of the heat shock molecule, HDJ2 (Castleberry etÂ€al. 2005). Residual FTase activity at clinical doses, alternative prenylation of N-RAS and K-RAS by GGTase, as well as a lack of correlation between RAS mutation status and response in other tumor models underscore that the authentic target(s) of FTIs are unknown. The cellular effects of FTIs may require inhibition of any of the many cellular proteins that require farnesylation such as the Rho family GTPases. As N-RAS and K-RAS have been shown to be prenylated by GGTase during FTase inhibition, GGTase inhibitors have been developed and their clinical applications are under investigation (Park etÂ€al. 2008; Side etÂ€al. 1997). Prenylation is one step in a larger metabolic pathway that targets RAS to plasma membranes in both physiologic signaling and transformation. After prenyl transfer by FTase or GGTase, cleavage of the AAX residues occurs by RAS converting enzyme 1 (Rce1). This is followed by methylation catalyzed by isoprenylcystine carboxyl methyltransferase (ICMT). These steps increase efficiency of membrane association of the RAS proteins. A few small molecule inhibitors of Rce1 protease and ICMT have recently been described and remain interesting for preclinical evaluation (reviewed in Wilhelm etÂ€al. 2004). Analysis of Rce1 and Icmt null cells suggests that farnesylated RAS and other farnesylated proteins are mislocalized to a greater degree than geranylated proteins (Mahgoub etÂ€ al. 1999). Disruption of Icmt or Rce1 results in embryonic lethality, suggesting an important physiologic role for this pathway and potential for systemic toxicities from its inhibition (Bergo etÂ€al. 2001; Kim etÂ€al. 1999). In the Kras MPD model, conditional deletion of Icmt in the malignant populations reduced severity of the disease but did not correct anemia or prolong survival (Wahlstrom etÂ€ al. 2007). Paradoxically, conditional deletion of Rce1 caused acceleration of MPD, perhaps due to effects of Rce1 on CAAX-containing proteins that inhibit RAS signaling (Tartaglia etÂ€al. 2003). Overall, investigation of agents targeting RAS processing has revealed complex and poorly predicted effects on growth of normal and malignant cells. These problems can be attributed both to ineffective targeting of mutant RAS, in the case of FTIs that spare GGTase modification of N-RAS and K-RAS, and to a large class of “off-target” substrates other than RAS with both positive and negative effects on cell growth. This experience underscores the need to investigate the therapeutic targets, mechanism, and selectivity of these drugs if we are to understand which diseases and patients might benefit from their clinical application.

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Direct targeting of RAS by strategies that do not inhibit prenylation is also being evaluated. Farnesylthiosalicylic acid (FTS, Salirasib) mimics the carboxy-terminal farnesyl cysteine carboxymethyl ester of RAS and competes with RAS-GTP for binding sites on cellular membrane proteins, galectin 1 (Gal-1) and galectin 3 (Gal-3) (Ashery etÂ€al. 2006; Rinehart etÂ€al. 2004). It is postulated that disruption of galectin binding displaces all isoforms of RAS-GTP from the plasma membrane interfering with effector interactions and increasing the rate of inactivation to GDP (Ashery etÂ€al. 2006; Rinehart etÂ€al. 2004). FTS was recently shown to inhibit growth of NF1 mutant MPNST cell lines in a dose-dependent fashion that correlated with basal RAS-GTP levels (Barkan etÂ€al. 2006). FTS treatment shortened duration of RAS-GTP induction following serum stimulation and was associated with lower levels of phosphorylated ERK, AKT, and Ral-GTP (Barkan etÂ€al. 2006). Phase I and II trials of Salirasib in pancreatic and nonsmall cell lung cancer are currently enrolling patients and will be important in evaluating the pharmacodynamics and toxicity of this compound in malignancies associated with activated RAS signaling. Finally, N-RAS needs to be both palmitoylated and prenylated for stable membrane association, and inihibition of the palmitoylation step has been proposed as a therapy for N-RAS mutant leukemias (Cuiffo and Ren 2010). However, the K-RAS 4B isoform is not palmitoylated and therefore such a strategy would not apply to cases with K-RAS mutations. As targeting mutated RAS, SHP-2, and neurofibromin is difficult, inhibiting effectors of RAS signaling has emerged as an alternative therapeutic strategy. Nonmutated effector proteins would be inhibited to the same degree in neoplastic and normal cells, and thus, a therapeutic response would arise only if cancer cells respond differently to pathway inhibition. Cellular adaptation to oncogenic signals can alter the dependence on specific signals and determine the outcome of pathway inhibition, a process referred to as “oncogene addiction” (Hingorani and Tuveson 2003; Watanabe etÂ€ al. 1996). One potential advantage of this strategy is that inhibiting a critical cellular signal that is altered by diverse molecular mechanisms has broader clinical applicability. Given the complexity of normal and malignant signaling networks, it is hard to predict a priori whether inhibition of one or more signaling proteins will be selectively toxic to cancer cells. The two RAS effector pathways most highly associated with cancer are the Raf/MEK/ERK and the PI3K/AKT/mTOR networks, though their individual contributions to malignant phenotypes are not clear. A growing number of compounds targeting various components of these systems are now available for preclinical and clinical investigations. Studying cellular responses and signaling in the presence of specific inhibitors will inform the pathobiology of cancer and hopefully yield therapeutic discoveries.

RAF/MEK/ERK RAS-GTP associates with and activates the Raf kinases (A-Raf, B-Raf, and c-Raf /Raf-1). Raf phosphorylates and activates mitogen-activated protein kinase kinase (MEK1 and MEK2). MEK, in turn, activates ERK, which phosphorylates diverse

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nuclear and cytosolic substrates. RAF gene mutations that encode constitutively active proteins are found in several cancers, and therefore, it is reasonable to hypothesize that inhibiting the Raf/MEK/ERK cascade has therapeutic potential in cancers associated with elevated RAS-GTP. Degradation of Raf-1 with a DNA enzyme resulted in a reduction of spontaneous colony formation and GM-CSF hypersensitivity in JMML samples without effect on colony formation in normal bone marrow cells (Iversen etÂ€ al. 2002). Reduced levels of GM-CSF mRNA and protein were observed in a panel of treated JMML samples (Iversen etÂ€al. 2002). Immunodeficient mice transplanted with primary JMML cells and treated for 4Â€weeks with an Raf-1 degrading enzyme experienced a greater than 50% reduction in JMML bone marrow cell mass compared to control mice (Iversen etÂ€al. 2002). Several Raf inhibitors have been developed that vary in their specificity for Raf isoforms and mutant (V600E) B-Raf commonly found in melanoma (reviewed in Caraglia etÂ€al. 2006). To date, the largest clinical experience with Raf inhibitors is with Sorafenib (BAY 43-9006), which is approved by the FDA for treatment of advanced renal cell carcinoma and hepatocellular carcinoma. Sorafenib is a novel bi-aryl urea that has been shown to inhibit Raf-1 (c-Raf) as well as B-Raf, and reduces tumor proliferation in several human tumor xenograft models (Weinstein 2002). In preclinical testing, antitumor activity was noted to correlate with a reduction in MEK and ERK phosphorylation in the presence of Sorafenib in diverse tumor cell lines and murine models (Weinstein 2002). However, Sorafenib is also active against a variety of other kinases, including VEGFR-2 and 3, PDGFR-b, FGFR-1, FLT3 and c-KIT (Weinstein 2002). RAS and BRAF mutation status failed to correlate with growth inhibition in xenograft models suggesting the importance of inhibiting angiogenesis in these responses. Early phase testing is currently being conducted in pediatric solid tumor patients. A future study of this FDA-approved multikinase inhibitor in relapsed JMML is feasible, yet the preclinical activity in JMML cells is unknown. The broad kinase inhibition of Sorafenib may have more disadvantages than benefits in hematopoietic malignancies, as inhibition of angiogenesis may cause toxicities and is unlikely to have therapeutic benefit. These potential “off-target” toxicities may be avoided with more specific inhibitors of Raf or MEK. Although not frequently mutated in cancers, MEK activation is implicated in tumor development based on the frequency of RAS and RAF mutations in tumors. MEK is an attractive target because the only known substrate is ERK. Highly selective inhibition of MEK can be achieved with non competitive inhibitors that bind a novel region and lock the kinase in a catalytically inactive state (Niihori etÂ€ al. 2005). The addition of U0126, a MEK inhibitor, to myeloid colony assays selectively inhibited the growth of Ptpn11E76K expressing cells when compared with wild-type cells (Miyauchi etÂ€ al. 1994). In contrast, the MEK inhibitor CI-1040 inhibited myeloid colony growth in Nf1 mutant bone marrow only at high concentrations that equivalently inhibited myeloid colonies in wild-type bone marrow (Lauchle etÂ€al. 2007). Treatment with CI-1040 twice a day at the maximal tolerated dose (MTD) is known to inhibit phosphorylation of ERK for 6 to 8Â€ hours

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(Peterson etÂ€al. 2006). Four weeks of treatment with this regimen failed to decrease WBC or Â�splenomegaly in the Nf1flox/flox MPD model (Lauchle etÂ€al. 2007). There may be a role for MEK inhibition for patients with refractory JMML, especially in those patients that transform to acute myeloid leukemia. This is suggested by the inhibition of Nf1 mutant acute myeloid leukemic colony growth at low doses of CI-1040 and prolonged survival of mice transplanted with Nf1 mutant AMLs when treated with CI-1040 (Lauchle etÂ€al. 2007). Recently developed MEK inhibitors (PD0325901 and AZD 6244) that have more favorable pharmacodynamic profiles are currently in clinical trials, including a Phase II trial of AZD 6244 in adults with acute myeloid leukemia (Adjei etÂ€al. 2008).

PI3K/AKT/mTOR The PI3K/AKT/mTOR pathway is activated in human cancers by diverse genetic events including RAS mutations, activating PI3K mutations, and inactivation of PTEN, a negative regulator of PI3K. The largest clinical experience is with PI3K/ AKT/mTOR pathway inhibitors that target the more distal component of the pathway, mTOR. Rapamycin and its analogs, temsirolimus (CCI-779) and everolimus (RAD001), bind to the FK506 binding protein, FKBP-12, which then binds to and inhibits mTOR. Initially discovered in 1975, rapamycin is FDA approved as an immunosuppressant and is frequently used after HSCT to prevent graft versus host disease. In JMML, antileukemic activity of low dose rapamycin (1 to 10Â€nM) has been observed in Ptpn11E76K mutant transformed cells and in JMML patient samples (Le etÂ€al. 2004; Miyauchi etÂ€al. 1994). In Ptpn11 mutant cells, the addition of a MEK inhibitor to rapamycin resulted in a synergistic decrease in colony formation (Miyauchi etÂ€al. 1994). Inhibition of mTOR by rapamycin results in AKT activation in some cell systems. This, combined with the potential of other effectors downstream of PI3K/ AKT activation to contribute to the malignant phenotype, has increased interest in chemical inhibitors of more proximal pathway components. The first generation PI3K inhibitors, wortmannin and LY294002, both target the p110 catalytic subunit of PI3K, yet they also inhibit other kinases. They are associated with broad cellular toxicities that limit therapeutic evaluation of PI3K pathway inhibition. Recently, development of more specific inhibitors of PI3K (PX-866, GDC-0941, IC87114, XL147, BGT226) has revitalized the study of the PI3K pathway in malignancies, and several agents are under clinical investigation. In addition, several dual inhibitors of PI3K and mTOR (NVP-BEZ235, XL765 and PI-103) are under active preclinical and clinical study. Antileukemic activity in AML has recently been reported for PI-103, a dual p110 and mTOR inhibitor, which was not induced either by selective PI3K inhibition with a p110 inhibitor, IC87714, or by mTOR inhibition with everolimus (Pardanani 2008). In AML samples, these authors noted both AKT activation during mTOR inhibition as well as mTOR activation during PI3K inhibition, supporting a rationale for combined inhibition of pathway components

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(Pardanani 2008). AKT inhibitors with diverse mechanisms including ATP Â�competition, prevention of membrane localization, and allosteric inhibition provide another approach to inhibit this complex pathway.

PTPN11/SHP-2 As SHP-2 is the most commonly mutated protein in sporadic JMML and promotes signal transduction through RAS pathway, inhibiting the nonreceptor protein tyrosine phosphatase (PTP) is a rational therapeutic strategy for JMML. By screening the NCI diversity set chemical library, NSC-87877 was identified as a specific inhibitor of SHP-2 and SHP-1 (Chen etÂ€ al. 2006). Site directed mutagenesis and modeling suggested that NSC-87877 inhibits SHP-2 PTP activity by binding to the catalytic cleft (Chen etÂ€al. 2006). NCS-87877 may preferentially inhibit activated SHP-2, as 50% inhibition of PTP activity in fetal liver cells transduced with fulllength wild-type SHP-2 occurred at a NSC-87877 dose 4.5-fold higher than the dose required to achieve the same inhibition of the E76K mutant SHP-2 or the wildtype PTP domain lacking the autoinhibitory N-SH2 domain (Chen etÂ€ al. 2006). Cells exposed to inhibitor had lower RAS-GTP and phospho-ERK levels following cytokine stimulation (Chen etÂ€al. 2006). The cytotoxic effects of NSC-87877 on a human breast cancer cell line were not significantly different than those seen with a MEK inhibitor or a PI3K inhibitor (LY294002) alone; however, greater cell death was noted with the combination of NSC-878777 and LY294002 (Chen etÂ€al. 2006). A second compound, phenylhydrazonopyrazolone (PHSP1) was identified in a screen modeling binding interactions with the SHP-2 catalytic site (Hellmuth etÂ€al. 2008). PHSP1 decreased growth factor stimulated SHP-2 PTPase activity, phosphorylation of ERK, and SHP-2 dependent morphologic changes in epithelial cells (Hellmuth etÂ€al. 2008). PHSP1 inhibited growth and colony formation in a panel of human tumors in a SHP-2 dependent manner, with high expression of mutant SHP-2 predicting strong inhibition (Hellmuth etÂ€al. 2008). A reasonable next step is to study these compounds in clinical and murine JMML samples to evaluate therapeutic potential of SHP-2 inhibitors as they are currently unavailable for clinical administration.

JAK2-STAT5 Cellular responses to GM-CSF are primarily regulated by the JAK2 kinase (Wahlstrom etÂ€al. 2008). With the recent discovery of STAT5 phosphorylation in response to low dose GM-CSF in JMML cells and a prominent role of activating JAK2 mutations in myeloproliferative disorders, it is logical to evaluate the functional consequences of inhibiting JAK signaling (Kotecha etÂ€al. 2008). Based on the gain of function mutation JAK2 V617F, identified in the majority of patients with

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polycythemia vera, essential thrombocythemia, and primary myelofibrosis, a number of inhibitors (XL019, INCB018424, and TG101348) that inhibit wild-type and mutant JAK2 are in various stages of preclinical and clinical trial testing (Ohren etÂ€al. 2004). In addition, several agents designed to inhibit Aurora kinases (VX680, and AT9283) or FLT3 (CEP-701, lestaurtinib) have significant JAK2 kinase inhibition and Phase I and II clinical trials in MPDs are being conducted (Hexner etÂ€al. 2008; Ohren etÂ€ al. 2004). Therefore, these agents may be among the first signal transduction inhibitors used in clinical trials for JMML. In a preclinical setting, the orally available JAK inhibitor (XL019) abrogated the phosphorylation of both STAT5 and ERK in response to GM-CSF (Kotecha etÂ€al. 2008). Further correlating an inhibitor’s effect on RAS signaling proteins and colony formation may be used to evaluate the role of JAK/STAT5 signaling in JMML disease pathogenesis.

Conclusion Therapy with selective small molecule inhibitors has revolutionized cancer treatment for several hematopoietic malignancies, most notably CML. Advances in the molecular understanding of JMML have arrived concurrently with development of many inhibitors directed at RAS signaling. This provides an unprecedented opportunity to evaluate the impact of targeted inhibitors alone and in combination in JMML treatment. At a minimum, the drugs reviewed in this chapter will serve as chemical probes to explore the critical biochemical alterations in JMML cells and inform future drug development. Ultimately, the successful identification of a new therapy for JMML will require collaborative research to generate and analyze preclinical data necessary to prioritize the growing number of drugs available for clinical trial. Trial design must give careful consideration to incorporation of novel agents and biologic correlates into current clinical trial design to evaluate each target thoroughly. The options for JMML therapy are expanding rapidly and hold promise for improved clinical outcomes.

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Chronic Myeloid Leukemia: Pathophysiology and Therapeutics Seth J. Corey and Jorge Cortes

Chronic Myeloid Leukemia (CML) is a clonal myeloproliferative disorder Â�characterized by a translocation between the long arms of chromosomes 9 and 22. Often referred to as the Philadelphia (Ph) chromosome, this genetic rearrangement results in an oncogenic tyrosine kinase, Bcr-Abl. The disease may smolder for years before terminating in a blast crisis. Once curable, mostly with bone marrow transplantation and only seldom with interferon alpha-based therapy, its management has been radically changed with the introduction of imatinib mesylate, an orally available Abl kinase inhibitor, in 1998. More common in adults, only an estimated 50 pediatric cases occur annually in North America. The treatment of CML will remain in flux as second-generation Bcr-Abl kinase inhibitors are studied clinically. Because of its rarity in pediatrics, clinical insights must come from Â�experience with adult CML. CML serves as the paradigm of how molecular understanding of a cancer’s pathophysiology can produce a revolutionary form of Â�targeted therapy, providing hope for those pediatric cancers with a well-defined genetic lesion.

Pathophysiology of Bcr-Abl in CML More than 95% of all adult and pediatric CML possess the Ph chromosome (designated as Ph+ or Ph1) (Melo and Barnes 2007; Sherbenou and Druker 2007). This results from a reciprocal and balanced translocation in hematopoietic stem cells, which may be more precisely described t(9;22)(q34;q11.2) (Nowell and Hungerford 1960; Rowley 1973). Consequently, the Abl gene on chromosome 9 fuses to the Bcr

S.J. Corey (*) Departments of Pediatrics and Cell & Molecular Biology, Children’s Memorial Hospital and the Robert H. Lurie Comprehensive Cancer Center, Northwestern University, Chicago, IL e-mail: [email protected]

P.J. Houghton and R.J. Arceci (eds.), Molecularly Targeted Therapy for Childhood Cancer, DOI 10.1007/978-0-387-69062-9_8, © Springer Science+Business Media, LLC 2010

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gene on chromosome 22 (Ben-Neriah etÂ€al. 1986; Shtivelman etÂ€al. 1985). The fusion mRNA transcript encodes a protein, Bcr-Abl, with constitutive tyrosine kinase activity. Bcr stands for breakpoint cluster region, because there are variable breakpoints at which the translocation arises. The two most common breakpoints produce transcripts designated as e13a2 and e14a2 (also known as b2a2 and b3a2) typically found in CML. Mapping specific sites of chromosomal translocations aids in the design of quantitative PCR, which is commercially available, to monitor disease activity. The fusion of the partial genes for Bcr and Abl produce transcripts encoding either a 210 or approximately 190Â€kDa protein (Shtivelman etÂ€al. 1985). The larger (p210 Bcr-Abl) is found predominantly in CML, while the smaller form (p190 Bcr-Abl) occurs most frequently in Ph+ acute lymphoblastic leukemia (ALL) (Chan etÂ€al. 1987). Discussion of therapeutically targeting the p190 Bcr-Abl in ALL is discussed in Chap. 20. Bcr itself is a serine/threonine kinase and contains regulatory domains that affect the activities of RAS-related proteins Rac and Cdc42 (Diekmann etÂ€al. 1991; Maru and Witte 1991). Named after a viral transforming gene (v-Abl, Abelson murine leukemia virus) that Herb Abelson discovered as pediatric hematology fellow (Abelson and Rabstein 1970; Wang etÂ€al. 1984), cellular Abl (c-Abl) is a tyrosine kinase (Groffen etÂ€ al. 1983). Weakly active in its proto-oncogenic form, Abl becomes a much more potent intracellular tyrosine kinase that is constitutively activated upon fusion with Bcr. (Fig.Â€1). Bcr-Abl kinase drives cellular proliferation and inhibits apoptosis of myeloid progenitor and precursor cells. Ultimately, differentiation to neutrophils is impaired and blasts accumulate, leading to an acute leukemia-like presentation known as blast phase (BP). As the disease evolves, additional chromosomal abnormalities and genetic mutations may arise. Corresponding to the progression are three clinically defined phases: chronic, accelerated, and blast (discussed below). Murine retroviral transplantation experiments and the clinical success of imatinib confirm that Bcr-Abl is necessary and sufficient for CML. Bone marrow mononuclear cells harvested from healthy mice can be infected with retrovirus containing the Bcr-Abl gene (Daley etÂ€al. 1990). These modified cells, now expressing Bcr-Abl kinase, can be transplanted into irradiated, genetically related mice. Within several weeks, the transplant engrafts and a fatal myeloproliferative disease develops. At the same time that the mouse model for CML was being developed, organic chemists were synthesizing an array of tyrosine kinase inhibitors (Lydon and Druker 2004). Tyrosine kinases may be found in one of about a dozen families of receptors, such as those for epidermal growth factor, insulin, and Â�platelet-derived growth factor. At least eight families of non-receptor (or cytosolic) tyrosine kinases exist: Csk, Src, Btk/Itk, Syk/Zap-70, Fes, FAK/Pyk2, Jak, and Abl/Arg. Structural similarities among the several hundred receptor and cytosolic tyrosine kinases exist, which result in only relative specificity for most inhibitors. One lead compound (CGP57148B, later renamed STI571, and now known as imatinib mesylate) was discovered by scientists at Ciba-Geigy and showed potency against Abl and PDGF Receptors (Buchdunger etÂ€ al. 1996). Administration of that Abl tyrosine kinase inhibitor, eventually known as imatinib mesylate Â�(commercially known as

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Fig.Â€1â•… Structure and signaling function of Bcr-Abl. Upper panel, structure of Bcr and Abl. Arrows point to sites of where the two proteins fuse forming either a p190 or, more commonly, p210 fusion. For Bcr, portions of the N-terminal Ser/Thr kinase is preserved. For Abl, most of the domains, except for SH3, are retained. Lower panel, the Bcr-Abl fusion kinase recruits multiple signaling pathways. A few of the more important ones are shown here. When phosphorylated, Grb2 binds to Tyr177. This recruits two pathways, Sos-RAS-Raf-MAP Kinase and Gab2-PI 3¢kinase-Akt. Additional signaling come through activation of the Src kinase Lyn and the transcription factor STAT5. Mitochondrial associated apoptosis is deregulated through effects on Bad and Bcl-XL by Akt and STAT5, respectively. Altogether, these signaling pathways affect proliferation, survival, altered adhesiveness, and loss of differentiation

Gleevec™ or Glivec™), to these retrovirally transduced cell lines (Druker etÂ€ al. 1996) or mice (le Coutre etÂ€ al. 1999) or patients (Druker etÂ€ al. 2001) prevented development of the fatal disease. Additional genetic lesions contribute to or drive the disease through its stages. However, Bcr-Abl remains a critical target in disease progression (Shah etÂ€al. 2007). The structure and signaling function of Bcr-Abl has been thoroughly investigated, and still, new insights into how this fusion tyrosine kinase causes CML arise (Melo and Barnes 2007). Bcr-Abl tyrosine kinase’s activity is absolutely required for leukemogenesis. Increased genomic instability arising from the abnormal kinase likely contributes to disease progression with accumulation of additional genetic mutations. The constitutive tyrosine kinase activity of Bcr-Abl Â�phosphorylates a number of substrates, which activate a variety of signaling pathways (Fig.Â€ 1). Abl’s kinase activity autophosphorylates tyrosine 177 of Bcr-Abl, which serves as

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a docking site for the SH2 domain of Grb2 (Pendergast etÂ€al. 1993). In turn, Grb2 recruits SOS, a GTP exchange factor for RAS. RAS activates both PI 3¢kinase and Raf (and subsequently MAP Kinases). Grb2 also binds another scaffolding protein, Gab2, which also activates PI 3¢kinase (Sattler etÂ€al. 2002). PI 3¢kinase phosphorylates phosphatidylinositol at the 3¢ position of inositol. This leads to several 3¢OH-phosphoinositides. One major effect is to activate the serine/threonine kinase Akt (Skorski etÂ€al. 1997). Akt phosphorylates a variety of substrates involved in the regulation of metabolism and apoptosis control. One physiologically important substrate is Bad, which when phosphorylated, can no longer promote mitochondrial-associated apoptosis. Through less understood mechanisms, Bcr-Abl activates Src family kinases, such as Lyn and Hck (Danhauser-Riedl etÂ€al. 1996). These may serve to recruit additional or salvage growth-promoting pathways. Bcr-Abl also leads to activation of the transcription factor STAT5 (Carlesso etÂ€al. 1996; Ilaria and Van Etten 1996). STAT5 promotes the expression of the anti-apoptotic factor, Bcl-XL. Another group of Bcr-Abl substrates are cytoskeletal proteins, such as CrkL, paxillin, talin, and focal adhesion kinase, which affect cellular adhesiveness (Salgia etÂ€al. 1997). As a result of fusion of Bcr with Abl, normal cellular functions of c-Abl (and Bcr) are lost. Since c-Abl binds to DNA and is involved in mediating genotoxic repair, genetic instability results. Altogether, these signaling events promote cell cycle division, survival, altered adhesiveness, and genetic instability (Skorski 2002). Ultimately, a loss of differentiation occurs (Schuster etÂ€al. 2003).

Clinical Phases Pediatric CML is a rare disease with an annual incidence of 1 case per million children (the incidence in adults is approximately ten times greater) (Linet etÂ€ al. 1999). The clinical characteristics do not differ from those seen in the adult population, with the exception of age at presentation, and perhaps, white blood cell count (Millot etÂ€al. 2005). There is a slight preponderance of boys affected (ratio of 1.5:1) with a median age at diagnosis of 12.5 years. A third of the children will be diagnosed within the first decade of life. Almost all children with CML (approximately 95%) are diagnosed during the chronic phase (CP). Common presenting symptoms are: weakness, abdominal discomfort or fullness, weight loss, bleeding, fever, and bone pain. More than 2/3 of the children will have a palpable spleen on examination; most will not have a palpable liver or enlarged lymph nodes. Hematologic findings consist of elevated white blood cell count, with almost two-thirds having a count greater than 100â•›×â•›109/L. Two-thirds will also have an anemia (hemoglobin less than 12Â€g/dL) and thrombocytosis (greater than 450â•›×â•›109/L). The CP of CML presents as a myeloproliferative disease, with an increasing number of normal appearing and functioning granulocytes and their precursors (metamyelocytes and promyelocytes). Blasts count remains below 15% in both the peripheral circulation and the bone marrow. On examination, the spleen becomes increasingly enlarged. The CP typically lasts between 3 and 5 years. Before the disease terminates in a BP, which may be either myeloid (predominantly) or

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Â� lymphoid, there is frequently an intermediary accelerated phase (AP). Almost all of the children never go through AP, or it goes unrecognized. There are several criteria the different stages of CML (Cortes etÂ€al. 2006). As defined by the World Health Organization, criteria for AP include any of the following: 10 to 19% blasts in either the periphery or bone marrow, greater than 20% basophils in blood or marrow, platelet count either less than 100â•›×â•›109â•›L or greater than 1,000â•›×â•›109â•›L, new cytogenetic abnormalities, or increasing splenomegaly or white blood count unresponsive to therapy. However, these criteria have never been used prospectively, and several characteristics do not appear to remain as predictive of outcome in patients treated with tyrosine kinase inhibitors. The duration of AP is short, lasting 1 to 2 years, and usually terminates with a BP. Though there may not be an antecedent AP, BP occurs when there are at least 30% blasts (20% according to the WHO classification) in either the periphery or bone marrow or evidence of a chloroma or large clusters of blasts in the bone marrow. Blasts most frequently display myeloid markers, but a few will be lymphoid, and some undifferentiated.

Targeted Therapies Imatinib is a small molecule tyrosine kinase inhibitor, a 2-phenylaminopyrimidine compound originally identified in a drug screen for activity against the PDGF Receptor (Lydon and Druker 2004; Buchdunger etÂ€al. 1996). Pre-clinical evaluation of the compound (known then as CGP57148B and later as STI571) demonstrated its exquisite sensitivity against Bcr-Abl-transfected cell lines, cells derived from patients with CML, and mouse retroviral transduction models (Druker etÂ€al. 2001). Active also against stem cell factor receptor (c-Kit) and Abl, imatinib was tested first in patients with interferon-resistant CML, and subsequently, in patients with gastrointestinal stromal cell tumor (GIST) where activating mutations of c-Kit are commonly found (Demetri etÂ€al. 2002). Of note, imatinib interferes with the ATP binding domain of the inactive form of Bcr-Abl. For both diseases, responses were swift and dramatic. Complete cytogenetic remission can now be achieved in more than 80% of patients with CML in CP. The initial phase I trial of imatinib in patients with CP CML resistant to interferon-a was conducted in 1998 (Druker etÂ€al. 2001). Though a maximally tolerated dose was not estAblished, even at doses up to 1,000Â€ mg daily, an oral dose of 400Â€mg daily for adults was selected for phase II trials. Responses in CML may be defined as hematologic, cytogenetic, or molecular (TableÂ€1). A phase III International Randomized Study of Interferon and STI571 (IRIS) cross-over study compared imatinib 400Â€mg/day with combination of interferon-a and cytarabine in untreated CML patients in CP (O’Brien etÂ€al. 2003). As reported in 2003, complete hematologic response was seen in 96% of patients treated with imatinib (76% with Â�complete cytogenetic responses). With 5 years of additional follow-up, projected rates of complete hematologic responses were 98% and complete cytogenetic responses were 87%. Responses are mostly durable, with 5-year event-free survival rates of 83%, with 93% alive and free from transformation to accelerated or BP.

144 TableÂ€1â•… Response criteria Hematologic â•… Complete

â•… Partial

Cytogenetic â•… Complete â•… Major â•… Minor Molecular â•… Complete

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Complete normalization of peripheral blood counts Leukocyte count <10â•›×â•›109/L Platelet count <450â•›×â•›109/L No immature cells in peripheral blood Disappearance of palpable spleen Presence of immature cells (myelocytes, promyelocytes, blasts) Platelet count <50% of pre-treatment count, but >450â•›×â•›109/L Persistent splenomegaly No Ph+ metaphases 1–35% Ph+ metaphases >35% Ph+ metaphases

Bcr-Abl undetectable by qPCR 5-Log reduction in Bcr-Abl transcript levels â•… Major Bcr-Abl/Abl <0.05% 3-Log reduction in Bcr-Abl transcript levels Cytogenetic analysis requires at least 20 metaphases qPCR quantitative PCR

Five-year survival was 90% (92% when censoring patients at the time of transplant). Importantly, the response achieved at 12 months correlated with the longterm outcome. Patients who achieved a complete cytogenetic response and at least a 3-log reduction in Bcr-Abl transcript levels (i.e., major molecular response) after 12 months of therapy had a significantly better probability of being alive and free from transformation to accelerated or CP at 5 years compared to those with complete cytogenetic response but less than a 3-log reduction in transcript levels, and those without a complete cytogenetic response (100% vs. 95% vs. 88%, respectively; pâ•›<â•›0.001) (Druker etÂ€al. 2006). With these results, achieving a major molecular response has become the main goal of therapy in CML, and adequate molecular monitoring has become mandatory. Clinical experience based on treatment of over 60,000 patients has shown that imatinib is well-tolerated with once-daily administration (Baccarani etÂ€ al. 2006). The drug is cleared through the liver, and thus, the ingestion of grapefruit juice or other known cytochrome P450 inhibitors is to be discouraged (Peng etÂ€ al. 2005). These drugs include dexamethasone, phenobarbital, carbamezipine, and St. John’s Wort. Administering the drug with meals reduces gastrointestinal distress; however, an occasional patient may need anti-emetics such as prochlorperazine or ondanestron (Deininger etÂ€al. 2003). Diarrhea may also occur but it is frequently mild, not needing therapy; when it does, imodium adequately controls it in most instances. Approximately 60% will show signs of peripheral edema (e.g., peripheral edema, periorbital swelling) (Cohen etÂ€al. 2002). This tends to subside, and can be treated with diuretics (Esmaeli etÂ€al. 2002). Dyspnea can occur in approximately 12%, usually Â�representing fluid retention; it may be treated with diuretics and/or brief pulse

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of prednisone. Myelosuppression occurs frequently, particularly in the first 2 to 3 months of therapy (Cohen etÂ€al. 2002). If grade 3 to 4 neutropenia (<109/L) and/or thrombocytopenia (<50â•›×â•›109/L) occur, imatinib may be discontinued until the neutrophils and/or platelets return to above these levels, and then resumed. The dose may be reduced if the recovery takes longer than 2 weeks. A recent report of severe congestive heart failure in ten patients treated with imatinib and without known prior history of cardiovascular disease has not been confirmed by subsequent monitoring (Kerkela etÂ€al. 2006; Verweij etÂ€al. 2007; Atallah etÂ€al. 2007a) The incidence of heart failure among patients treated with imatinib appears not to be different than the risk in the general population. The incidence of congestive heart failure is rare and is associated with advanced age with pre-existing heart disease (Atallah etÂ€al. 2007b). Very little is known about long-term effects of imatinib in children (Bond 2007; Millot etÂ€al. 2006a; Millot etÂ€al. 2006b; Champagne etÂ€al. 2004). Although imatinib uncommonly affects bone metabolism through hypophosphatemia (Berman etÂ€ al. 2006), stunting of growth or osteoporosis has not been noted in pediatrics.

Imatinib in Children A Children’s Oncology Group Phase I study reported that imatinib was well tolerated (Champagne etÂ€al. 2004). The study group of 31 patients included 14 patients with CP, 6 with myeloid BP, and 9 with Ph+ ALL. CML patients were resistant to interferon-a. All 14 patients with CP had a hematologic response, with 10 having a major cytogenetic response. There was a 55% partial response rate among those with advanced disease. No maximally tolerated dose was defined, with 570Â€mg/m2 as the largest dose studied (comparable to 800Â€mg adult high-dose). Pharmacokinetic data showed steady state levels and area under the curve concentrations similar to those found in adults given 400Â€mg/m2/day. Also, as seen in adult patients, there was interpatient variability, perhaps due to CYP3A activity (Peng etÂ€al. 2005; Peng etÂ€al. 2004). A multi-center phase II study of imatinib conducted in children with AP (either after transplantation or with interferon resistance) showed a 75 to 80% complete hematologic response (Millot etÂ€ al. 2006a). One-year survival in this high-risk group was 95% for those with CP and 75% with AP. Since imatinib is the only currently FDA-approved drug for pediatric CML, we propose in Fig.Â€2, the following imatinib-based management of a child or adolescent with CML.

Monitoring With improved results with imatinib, adequate monitoring has become more important than ever. In addition to performing clinical evaluations and monitoring of peripheral blood counts, response assessment requires measurement of disease by the presence of the Philadelphia chromosome. Routine cytogenetics by G-banding are required at baseline to determine the presence of the Philadelphia chromosome as

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Fig.Â€2â•… Guidelines for managing a pediatric patient with CML. To prevent leukostasis, hydroxyurea (500 to 1,000Â€ mg/m2/d) may be given for several days. Imatinib dose should be started at 250Â€mg/m2/d. Dosage may be doubled if no response occurs. Therapy may be temporarily discontinued for neutropenia (<1â•›×â•›109/L) or thrombocytopenia (<50â•›×â•›109/L). For side effects, ondanestron may be given with imatinib. Upon attainment of cytogenetic response, qPCR of peripheral blood should be performed every 3 months. Bone marrow examination with cytogenetics should be performed yearly. Treatment options for imatinib/dasatinib resistance include cytarabine/interferon (IFN)a and allogeneic stem cell transplantations (SCT)

well as other chromosomal abnormalities that may have prognostic implications. During follow-up, cytogenetic studies of the bone marrow may be needed at least once per year to investigate the possible emergence of clonal evolution and the occurrence of chromosomal abnormalities in Ph-negative metaphases. This phenomenon is found in approximately 15% of patients and has rarely been associated with development of myelodysplastic syndromes and acute leukemias (Kovitz etÂ€al. 2006). Patients must also be followed with real-time (quantitative) PCR to measure the transcript levels of Bcr-Abl. This is particularly important after a patient has achieved a complete cytogenetic remission since achievement of a major molecular remission correlates with an improved outcome. Unfortunately, the methodology used for PCR testing is not universal and there is considerable variability between the results obtained in different laboratories. Recently, a group of experts in the field proposed a harmonization that would convert the results obtained in different laboratories to an international standard (Hughes etÂ€al. 2006). This process is still being developed.

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Resistance to Kinase Inhibitors and New Therapeutic Strategies The optimal duration of imatinib therapy is not known (Goldman 2007). Despite anecdotal reports of patients maintaining remission off imatinib, most patients who discontinue therapy, despite having achieved undetectable disease by PCR, relapse. Thus, the current recommendation is that patients should remain on imatinib indefinitely. Resistance to imatinib therapy has been defined based on the response achieved at different times and can be either primary (refractory) or secondary. After 5 years of follow-up, it appears that the rate of resistance is approximately 3 to 4% per year and may decrease as patients remain on therapy longer (Druker etÂ€al. 2006; Hughes etÂ€al. 2003). Resistance occurs by several mechanisms (Michor etÂ€al. 2005). The most common of these is the development of mutations in the Bcr-Abl kinase domain (Gorre etÂ€ al. 2001; Shah etÂ€ al. 2002). Mutations have been identified in 40 to 60% of patients affecting over 50 different residues. Mutations may confer different levels of resistance to imatinib, with some conferring only partial resistance (i.e., resistance can be overcome by higher concentrations of imatinib) and others conferring high Â�levels of resistance (i.e., T315I). Additional mechanisms for resistance include amplification or overexpression of Bcr-Abl. Non-Bcr-Abl related mechanisms of resistance have also been described. These include overexpression of multi-drug resistance phenotype that may expel imatinib out of the cell and decreased activity of the hOCT1 transporter that is required for importing imatinib into the cell (Thomas etÂ€ al. 2004). The emergence of salvage pathways (in particular, the increased expression of the Src kinase Lyn) has been reported in some patients after imatinib failure, although the actual incidence of this mechanism among patients who fail imatinib therapy is not known (Dai etÂ€ al. 2004; Donato etÂ€ al. 2003). In addition, it has been suggested that the CML leukemic stem cell remains in a Â�dormant state and is insensitive to imatinib and could thus be responsible for the Â�persistence of disease (Elrick etÂ€al. 2005). A second generation of tyrosine kinase inhibitors has been developed to Â�overcome resistance to imatinib. The Food and Drug Administration approved dasatinib (Sprycel™) in 2006 and nilotinib (Tisigna™) in 2008 for the treatment of patients with CML who are resistant or intolerant to imatinib. Whereas dasatinib targets both Src and Abl kinases, nilotinib does not inhibit Src kinases (Hazarika etÂ€al. 2008). Other inhibitors still in clinical trials are: Bosutinib (SKI606) and INNO-406. All of these small molecule inhibitors are more potent than imatinib in blocking Â� Bcr-Abl tyrosine kinase activity and are active against a wide variety of Bcr-Abl kinase domain mutations associated with resistance to imatinib, except for the T315I mutation. The inÂ€ vitro IC50 for imatinib against Bcr-Abl is 260Â€nM, whereas that for dasatinib it is 0.8Â€nM, for nilotinib 13Â€nM. In addition, dasatinib and Â�bosutinib inhibit several members of the Src family of kinases, and INNO-406 inhibits Lyn. Lyn has been involved in mediating Bcr-Abl salvage pathways or contributing to B-cell ALL. The role of Src kinases in CML is otherwise unknown.

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Dastinib, nilotinib, and bosutinib have shown significant clinical activity in patients with CML who have failed prior therapy with imatinib (Talpaz etÂ€al. 2006; Kantarjian etÂ€al. 2006; Cortes etÂ€al. 2007). The results achieved with those agents already in phase II trials are summarized in TableÂ€ 2. Complete cytogenetic responses have been achieved in 49% of patients with dasatinib, 40% with nilotinib, and 32% with bosutinib, although the follow-up time is different for each study, which may affect the overall response rate as responses continue improving over time. Still, these studies demonstrate that responses can be achieved in a significant number of patients after failure of response to imatinib therapy. Responses occur in patients with a wide range of different mutations or without mutations, with the one common exception being patients with the T315I mutation. Albeit with short follow-up, responses have been mostly durable and have translated into a survival benefit compared to historical controls treated with other modalities of therapy after imatinib failure. These agents have become the preferred therapy for patients who fail imatinib therapy. A COG phase I study of dasatinib was recently completed (Aplenc et al. 2007). To date, the drug has been well tolerated at 75Â€mg orally daily. Generally, dasatinib therapy has similar toxicity profile to that of imatinib. Management of drug interactions, myelosuppression, and edema follows the same guidelines (see above). Since the T315I mutation is still insensitive to all these new inhibitors, there are several agents that are being developed to counter this mutation (Quintas-Cardama etÂ€ al. 2007a). Some of them are tyrosine kinase inhibitors whose binding to the kinase domain is not affected by the bulky residue of isoleucine in T315I. Among them, MK-0457 (formerly known as VX-680) has provided the most clinical data. This agent inhibits Aurora and Jak2 kinases in addition to Bcr-Abl. VX-680 is highly potent inÂ€ vitro against Bcr-Abl with or without the T315I, inhibiting the proliferation of cell lines with either isoform of the kinase. Early results of an ongoing TableÂ€2â•… Response to second generation tyrosine kinase inhibitors in patients with CML in chronic phase after imatinib failure Percentage Response Dasatinib Nilotinib Bosutinib Response â•… CHR 91 74 84 â•… MCyR 59 56 42 â•… CCyR 49 40 32 Toxicity (grade 3–4) â•… Thrombocytopenia 22–49 33 9 â•… Neutropenia 34–49 31 6 â•… Pleural effusion ~6 <1 <1 â•… Ý transaminases 2 1–4 <5 â•… RASh 0.5 2 6 â•… Ý lipase 11 15 <5 â•… Ý bilirubin 0 9 <5 Median FU (mo) 15 12 3

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trial have shown clinical efficacy in patients with CML who failed imatinib and carry the T315I. Other similar multi-kinase inhibitors are being investigated both preclinically and in the clinic. Other agents, such as homoharringtonine (HHT), decrease protein synthesis with some selectivity for Bcr-Abl in CML cells. HHT is being investigated in patients who failed at least two prior tyrosine kinase inhibitors or who have the T315I mutation with some early suggestion of favorable clinical activity (Quintas-Cardama etÂ€al. 2007b).

Indications for Transplantation Because of the lower incidence of treatment-related mortality in pediatric bone marrow transplantation, allogeneic stem cell transplants had been widely preferred for children with an HLA-matched sibling donor (Pulsipher 2004). According to the European BMT study on childhood CML, those who received sibling-matched transplants had a 76% 5 year survival for pediatric patients in CP and 46% for AP/BP (Cwynarski etÂ€al. 2003). Much of the survival rested on prevention of graftversus-host disease. Those with grade 0 to 1 had 91% survival, but survival dropped to 61% for those with grades 2 to 4. Children who received a matched, unrelated donor transplant during CP had 65% 5 year survival. Transplant recipients have fared less well in other studies (Unal etÂ€al. 2007; Creutzig etÂ€al. 1996). Regardless of the availability of a sibling donor, there remains significant morbidity associated with severe immunosuppression, short- and long-term complications associated with graft-versus-host disease, and costs of frequent clinic visits, laboratory monitoring for transplant-associated complications, and hospitalization. Considering the 5-year probability of survival with imatinib is currently 90% (at least in adults), one should no longer recommend transplantation for pediatric patients with CML in CP as first line of therapy since the 5-year probability of survival is inferior. This is particularly true for patients receiving match unrelated donor transplants. Instead, evaluation for transplantation should be considered if (1) there is failure to reach response landmarks, (2) identification of the T315I mutation, and (3) emergence of a blast crisis. Patients who fail imatinib should be offered a trial with a second generation tyrosine kinase inhibitor (Guilhot etÂ€al. 2007). Those pediatric patients with an HLA-matched sibling donor should be offered the option of transplant after salvage tyrosine kinase inhibitor fails. Adequate monitoring is critical to ensure the best outcome, as patients who are not responding optimally could be considered for alternative therapies. Landmark responses consist of cytogenetic response by 6 months, major cytogenetic responses by 12 months, and complete cytogenetic response by 18 months. Because there is no approved inhibitor active against T315I mutation, identification of this mutation would prompt the search for a stem cell transplant donor, either related or unrelated. Until a donor is identified, recruitment into clinical trials of investigational agents, for example, VX-680, that block T315I kinase activity should be encouraged. There is very limited experienced of the outcome of patients with T315I receiving an allogeneic transplant.

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Future Therapies The clinical success of imatinib and related tyrosine kinase inhibitors has led to the emergence of a number of critical biological and therapeutic questions. Must a CML stem cell be eradicated to achieve cure? How can one attack the quiescent CML stem cell? Because of their greater potency, are second generation Bcr-Abl kinase inhibitors better than imatinib for patients with untreated CML? How long should therapy be continued with either imatinib or second generation Bcr-Abl kinase inhibitor? Which clinical parameters dictate when to switch from one BcrAbl kinase inhibitor to another or undergo a stem cell transplant? What therapies can be developed to target the CML stem cell? How does one best manage relapse after transplantation? Continued research will give us the answer to these questions. If the recent past is predictive of the future, we might be able to get the answers to these important questions from our clinical specialists in adult leukemia.

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Elrick LJ, Jorgensen HG, Mountford JC, Holyoake TL. Punish the parent not the progeny. Blood 2005;105:1862-6. Hazarika M, Jiang X, Liu Q, etÂ€al. Tasigna for chronic and accelerated phase Philadelphia chromosome–positive chronic myelogenous leukemia resistant to or intolerant of imatinib. Clin Cancer Res 2008;14:5325–31. Talpaz M, Shah NP, Kantarjian H, etÂ€al. Dasatinib in imatinib-resistant Philadelphia chromosomepositive leukemias. N Engl J Med 2006;354:2531–41. Kantarjian H, Giles F, Wunderle L, etÂ€ al. Nilotinib in imatinib-resistant CML and Philadelphia chromosome-positive ALL. N Engl J Med 2006;354:2542–51. Cortes J, Kantarjian H, Baccarani M, etÂ€al. A phase I/II study of SKI-606, a dual inhibitor of Src and Abl kinases, in adult patients with Philadelphia chromosome positive (Ph+) chronic meylogenous leukemia (CML) or acute lymphoblastic leukemia relapsed, refractory or intolerant of imatinib. Blood 2007;108:54a, (abstract 168) Aplenc R, Strauss LC, Shusterman S, et al. Pediatric phase I trial and pharmacokinetic (PK) study of dasatinib: a report from the Children’s Oncology Group. J Clin Oncol 2007;25 (Suppl):14094. Quintas-Cardama A, Kantarjian H, Cortes J. Flying under the radar: the new wave of Bcr-Abl inhibitors. Nat Rev Drug Discov 2007;6:834–48. Quintas-Cardama A, Kantarjian H, Garcia-Manero G, etÂ€ al. Phase I/II study of subcutaneous homoharringtonine in patients with chronic myeloid leukemia who have failed prior therapy. Cancer 2007;109:248–55 Pulsipher MA. Treatment of CML in pediatric patients: should imatinib mesylate (STI-571, Gleevec) or allogeneic hematopoietic cell transplant be front-line therapy? Pediatr Blood Cancer 2004;43:523–33. Cwynarski K, Roberts IA, Iacobelli S, etÂ€al. Stem cell transplantation for chronic myeloid leukemia in children. Blood 2003;102:1224–31. Unal S, Fidan G, Tavil B, Cetin M, Cetinkaya DU. Allogeneic hematopoietic stem cell transplantation in pediatric chronic myelogenous leukemia cases: Hacettepe experience. Pediatr Transplant 2007;11:645–9. Creutzig U, Ritter J, Zimmermann M, Klingebiel T. [Prognosis of children with chronic myeloid leukemia: a retrospective analysis of 75 patients]. Klin Padiatr 1996;208:236–41. Guilhot F, Apperley J, Kim DW, etÂ€al. Dasatinib induces significant hematologic and cytogenetic responses in patients with imatinib-resistant or -intolerant chronic myeloid leukemia in accelerated phase. Blood 2007;109:4143–50.

Molecularly Targeted Therapies in Pediatric Myelodysplastic Syndromes Lia Gore

Introduction New technologies have improved our understanding of hematopoietic and leukemia biology, and expanding knowledge of underlying molecular pathways and genomic aberrations is facilitating the development of new therapies for relapsed and refractory disease (Bhojwani etÂ€al. 2006; Carroll etÂ€al. 2006). Myelodysplasia and associated syndromes constitute a broad range of bone marrow dysfunction, characterized by variable clinical features; cytopenias due to bone marrow production and/or dysfunction and insufficiencies are typical. Clinical symptoms are frequently due to anemia, neutropenia, and/or thrombocytopenia, and transfusion dependence is common although not universal. The progression from myelodysplastic syndromes (MDS) to leukemia, particularly in children, is unequivocal, although the time course over which this occurs is highly variable. Risk factors for progression and lability of disease have been identified, and great effort is being made to identify the molecular triggers in MDS that are both causative and clinically relevant. This chapter presents a brief review of selected targets and new agents under investigation for MDS, the majority of which are in early phase clinical trials, and some of which remain in preclinical exploration. The novelty of such agents stems from their more selective nature in capitalizing on a characteristic that is unique or more predominant in cells with the aberrant MDS phenotype, compared to their normal counterparts. The current rate of drug development and the availability of new agents far outpace the realistic ability for any review to be comprehensive in nature. However, an attempt is made to focus on a number or promising molecularly and/or mechanistically targeted agents, with the hope of characterizing pathways that may be utilized more effectively to treat patients while minimizing short- and long-term toxicities. The therapy of choice for MDS in children has traditionally been

L. Gore () The University of Colorado Cancer Center and The Center for Cancer and Blood Disorders, The Children’s Hospital, University of Colorado Denver, Pediatrics Mail Stop 8302, P.O. BoxÂ€6511, Aurora, CO 80045, USA e-mail: [email protected]

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hematopoietic stem cell transplantation as soon as feasible. As our understanding of the disease takes on a more molecular basis, analysis of the targets for therapy is increasingly likely to take on a more cross platform nature and the timing to transplantation may change.

Myelodysplastic Syndromes Incidence Reporting of MDS to the Surveillance, Epidemiology, and End Results (SEER) program in the US began in 2001; prior to this time, statistical information about the incidence and case rate were more difficult to extract from databases and relied on literature reviews and meta-analyses. MDS occurs in adults with a substantially higher frequency than in children, the incidence increases with each decade of life, and men have a higher incidence than women, particularly over the age of 60. Based on SEER data, there are estimated to be at least 10,000 cases of MDS per year in the US, with an incidence of up to 54.7 per 100,000 patients per year in the older age cohorts (http:// seer.cancer.gov/registries). However, there are recent suggestions that the incidence reported by SEER may be significantly underestimated (Rollison etÂ€al. 2008). Based on an analysis of 1.7 million Medicare patients in the Medicare Standard Analytic file for 2003, an age-adjusted incidence of 181 per 100,000 patients, or approximately 76,000 patients in the Medicare system at the time of the analysis (Finn 2009) is postulated. SEER data for 2001 to 2003 in persons under the age of 20 indicate that there is an approximately equal distribution between males and females, and that the rate remains steady over the first two decades of life at 0 to 0.3 per 100,000 patients per year (Ma etÂ€al. 2007). Overall, it is estimated that MDS represents less than 5% of hematologic malignancies in children. However, 15 to 20% of pediatric Acute Myelogenous Leukemias (AML) may have a preceding MDS (Locatelli etÂ€al. 1995).

Classification Classification of MDS is sometimes separated from that of myelofibrosis or the myeloproliferative syndromes (Cervantes etÂ€al. 2009) where the French–American– British (FAB) and World Health Organization (WHO) (Bennett etÂ€al. 1982; Jaffe etÂ€al. 2001) have developed stringent uniform criteria based on laboratory findings and clinical correlates. In addition, the International Prognosis Scoring System (IPSS) is frequently used to identify risk groups based on variables such as percent bone marrow blasts, karyotype, and number of cytopenias (Greenberg etÂ€al. 1997; Kaushansky 1998). Patients are categorized as having low, intermediate-1, intermediate-2, or high risk disease based on IPSS score and prognostic information is available to

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guide clinical practice and patient education on the basis of the scores. Classification of MDS in children has met with more challenges, largely because the FAB and WHO criteria do not account for some of the features unique to pediatric patients with these syndromes, as parallel diseases in adults do not exist. As a result, the pediatric WHO criteria were developed in 2003 to include myeloproliferative and myelodysplastic disease (Hasle etÂ€al. 2003). The Category, Cytology, Cytogenetics (CCC) system uses descriptive terminology and etiology to group diseases, but does not include the myeloproliferative syndromes, including juvenile myelomonocytic leukemia (JMML) (Mandel etÂ€al. 2002). TableÂ€1 attempts to collate several of the diagnostic criteria categories from these different systems. One challenge has been to determine which scoring system is most appropriate to help determine both diagnostic and prognostic information for children with MDS. Lorand-Metze and colleagues evaluated the FAB and WHO classifications, together with IPSS score, and found that in 150 patients, ages 12 to 90, the younger patients had a relatively higher proportion of WHO “unclassified” disease, with cellular findings most similar to childhood MDS (Lorand-Metze etÂ€ al. 2004). Similarly, Occhipinti and colleagues compared the CCC and pediatric WHO criteria in a retrospective chart review that outlines a number of advantages and disadvantages noted when applying each staging system to a series of pediatric cases. They concluded that the CCC and pediatric WHO criteria were superior in being able to classify and predict outcomes in pediatric patients with MDS, than the FAB and adult WHO criteria overall, however, felt that both the CCC and pediatric WHO had limitations in individual cases (Occhipinti etÂ€ al. 2005). There remain discrepancies in several areas: (1) how to classify disorders with cytopenias other than refractory anemia; (2) whether 20 or 30% blasts should be the cut-off between MDS and AML, and the role of cytogenetics in diagnosis and prognostic systems; (3) the lack of prognostic information in the FAB classification system; (4) the broad use of the WHO “unclassifiable” category for unusual cases, and how to best accommodate those patients; and (5) that the pediatric IPSS system currently excludes patients with Down syndrome, other congenital marrow failure syndromes, or therapy-related disease, and provides no prognostic information for patients with JMML.

Etiology In children, MDS typically arises in one of three settings: Children who have genetic or metabolic syndromes, which predispose them to bone marrow dysfunction and failure, and eventual leukemic transformation; children with deÂ€novo MDS who have no known predisposition or triggering exposure; and children who have had prior exposure to agents known to induce marrow dysplasia and/or aplasia. Regardless of the cause of the development of MDS, the overwhelming majority of children who develop myelodysplasia will eventually progress to leukemic transformation of their disease, which is typically resistant to therapeutic intervention.

Leukocytosis, thrombocytopenia frequent Leukocytosis, thrombocytopenia frequent

Refractory anemia with excess blasts (RAEB) Refractory anemia with excess blasts in transformation (RAEB-T)

BCR-ABL negative chronic myeloid leukemia (Ph-CML)

20–29% blasts

2–19% blasts

10–19% blasts ³20% basophils No BCR-ABL fusion or Ph chromosome Myelodysplastic syndromes (pediatric and adult WHO) Pediatric Refractory cytopenia (RC) – includes <2% blasts RARS, if meets adult criteria Anemia not always present

Myelodysplastic/myeloproliferative disease Monocytosis >1e9/L Juvenile myelomonocytic leukemia (JMML) <20% blasts No BCR-ABL fusion or Ph chromosome Monocytosis >1e9/L Chronic myelomonocytic leukemia <20% blasts (CMML) (*must be only in patients No BCR-ABL fusion or Ph chromosome with prior chemotherapy)

Down syndrome disease Transient abnormal myelopoiesis (TAM) Myeloid leukemia of down syndrome

<5% blasts Immature erythropoiesis Decreased megakaryocytes or micromegakaryocytes 5–19% blasts May or may not have Auer rods 20–29% blasts

<20% blasts No BCR-ABL fusion or Ph chromosome May or may not have monosomy 7 <20% blasts No BCR-ABL fusion or Ph chromosome Dyplasia in ³1 myeloid lineages or clonal cytogenetic abnormality detected 10–19% blasts No BCR-ABL fusion or Ph chromosome detected

³30% blasts Higher incidence of M7 morphology

TableÂ€1â•… Combined adult and pediatric WHO classifications with IPSS prognostic scoring of myelodysplastic syndromes Clinical/peripheral blood Disease appearance Bone marrow appearance Myeloproliferative disease <10% blasts Transient myeloproliferative syndrome Usually normal maturation Cellularity usually increased and morphology ³1 myeloid cell lines increased Acute leukemia Dysplasia in ³1 myeloid lines ³30% blasts common ³20% blasts Cellularity usually increased, but may be decreased

IPSS 1.5–2

IPSS 0.5–1.5

IPSS 0-0.5

N/A

N/A

N/A

N/A N/A

N/A

N/A

IPSS prognostic scoring

Two or more cytopenias Few or no blasts No Auer rods <1e9/L monocytes Two or more cytopenias Few or no blasts No Auer rods <1e9/L monocytes Cytopenias <5% blasts No Auer rods <1e9/L monocytes Cytopenias <5% blasts Auer rods with or without <1e9/L monocytes Cytopenias No or rare blasts No Auer rods Anemia Normal or increased platelet count <5% blasts

Refractory cytopenia with multilineage dysplasia (RCMD)

N/A not applicable

MDS associated with isolated deletion of 5q

Myelodysplastic syndrome, unclassified (MDS-U)

Refractory anemia with excess blasts 2 (RAEB-2)

Refractory anemia with excess blasts (RAEB-1)

Refractory cytopenia with multilineage dysplasia and ringed sideroblasts (RCMD-RS)

Anemia No blasts

Anemia Few or no blasts

Refractory anemia with ringed sideroblasts (RARS)

Adult Refractory anemia (RA)

IPSS 0.5 One myeloid cell line dysplasia <5% blasts No Auer rods Normal or increased megakaryocytes with hypolobulated nuclei <5% blasts Isolated del(5q) cytogenetics No Auer rods

IPSS 0

IPSS 2.0

IPSS 1.0

IPSS 0.5

IPSS 0.5

IPSS 0

IPSS 0

Uni- or multilineage dysplasia 10–19% blasts Auer rods may or may not be present

<5% blasts <15% ringed sideroblasts Erythroid dysplasia only <5% blasts ³15% ringed sideroblasts Erythroid dysplasia only ³10% cells with dysplasia of two or more myeloid lines <5% blasts No Auer rods <15% ringed sideroblasts ³10% cells with dysplasia of two or more myeloid lines <5% blasts No Auer rods ³15% ringed sideroblasts Uni- or multilineage dysplasia 5–9% blasts No Auer rods

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Predisposing genetic syndromes associated with the development of MDS include entities such as Fanconi anemia, Shwachman-Diamond syndrome, Diamond-Blackfan anemia, Bloom syndrome, Ataxia-telangiectasia, Kostmann syndrome, Nonan syndrome, neurofibromatosis type I, and Down syndrome (Alter 1996; Alter 2002; Homans etÂ€ al. 1993; Creutzig etÂ€ al. 1996; Rischewski etÂ€ al. 2000). Other conditions associated with acquired predisposition to MDS include paroxysmal nocturnal hemoglobinuria, aplastic anemia, dysmegakaryocytopoiesis, selected congenital metabolic acidemias and acidurias, Griscelli syndrome, and Langerhans cell histiocytosis (Surico etÂ€ al. 2000; Cetin etÂ€ al. 1998; Ohara etÂ€ al. 1997), among others. The development of MDS following chemotherapy or toxin exposure is typically thought to be less than 1 to 2% of leukemia cases overall. Inciting agents often implicated are the epipodophyllotoxins, especially etoposide (Winick etÂ€al. 1993; Pui etÂ€al. 1991), or alkylating agents such as cyclophosphamide, chlorambucil, melphalan, busulfan, and BCNU (carmustine) (Tew etÂ€al. 2001; Smith etÂ€al. 1996). Antimetabolite therapy may be implicated as well, as has been observed in patients who have altered thiopurine methyltransferase (TPMT) drug metabolism and elevated erythrocyte 6-thioguanine nucleotide levels (Bo etÂ€al. 1999). In addition, exposure to toxins that are known to induce marrow aplasia, such as benzene and similar organic solvents, has been implicated. Patients with prior exposures as the purported etiology of MDS will often have rearrangements of the MLL gene or deletions of (or portions of) chromosomes 5 or 7 (Felix etÂ€al. 1995; Privitera etÂ€al. 1992; Yesilipek etÂ€al. 1994; Maris etÂ€al. 1997; Harrison etÂ€al. 1995; Chantrain etÂ€ al. 2000). Therapy-related MDS/AML (t-MDS/t-AML) is noted to have a particularly poor overall survival. Recently, Chakraborty and colleagues have reported that accelerated telomere loss precedes and is associated with t-MDS/AML following autologous stem cell transplantation for lymphomas (Chakraborty etÂ€al. 2009). The authors suggest that this may reflect clonal expansion or altered telomere regulation in the preleukemic condition. As such, therapies directed toward normalization of telomere structure and function would be a potential intervention to reverse this process. Genetic alterations in MDS are distinctive, and involve a wide array of cytogenetic structural and gene dysregulation abnormalities (Maris etÂ€al. 1997; Taketani etÂ€al. 2003; Tartaglia etÂ€al. 2003; Lo Coco etÂ€al. 1997; Perkins etÂ€al. 1997; Block etÂ€al. 2002). As mentioned above, the most commonly identified abnormalities are of chromosomes 5 and 7, with monosomy and partial deletions being most frequent. Selected other genetic abnormalities, including mutations in critical target genes such as AML1/RUNX1, JAK2, PTPN11, and other members of the RAS/ Raf/Mek pathway are presented in TableÂ€2. MDS is essentially ineffective hematopoiesis, typically with high proliferative rates and increased apoptosis. A variety of rare hematopoietic myelodysplastic and myeloproliferative disorders have been identified, including the chronic myelomonocytic leukemias, atypical chronic myeloid leukemia, and JMML; only JMML is found with any frequency among the already infrequent pediatric MDS disorders. The 4th edition of the World Health Organization classification of

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TableÂ€2â•… Selected genetic abnormalities identified in pediatric myelodysplastic syndrome patients Cytogenetic abnormality noted Gene(s) associated Clinical subgroup correlates Reference 1q Morerio etÂ€al. (2006), Mogul etÂ€al. (1997) De novo and t-MDS t(1;3)(p36;q21) MEL1-PRDM16 t-MDS, t-AML Block etÂ€al. (2002), Lahortiga etÂ€al. (2004) t(1;7)(p11;p11) MDS with preceeding Horsman etÂ€al. (1988) marrow hypoplasia t(1;17)(p36;q21) t-MDS, t-AML Block etÂ€al. (2002) t(1;22) Perkins etÂ€al. (1997) t(2;8)(p23;p11.2) MOZ t-MDS Imamura etÂ€al. (2003) t(3;5)(q21;q31) t-MDS, t-AML Block etÂ€al. (2002) 3q21q26 t-MDS, t-AML Stark etÂ€al. (1994), Block etÂ€al. (2002), Perkins etÂ€al. (1997) t(3;21)(q26;q22) MDS1-EVI1 t-MDS, t-AML, CML Rubin etÂ€al. (1990), Lo Coco etÂ€al. (1997), Lahortiga etÂ€al. (2004) del(4q)â•›+â•›t(9;22)(q34;q11) KIT Dalla Torre etÂ€al. (2002), Galili etÂ€al. (2007) t(5;12)(q31;p12-p13) KIT MDS with eosinophilia Galili etÂ€al. (2007), Pellier etÂ€al. (1996) Monosomy 5 or 5qPerkins etÂ€al. (1997) t(6;9)(p23;q34) t-MDS, t-AML Block etÂ€al. (2002), Perkins etÂ€al. (1997) Monosomy 7 or 7qPerkins etÂ€al. (1997) t(8;14)(q24;q32) t-MDS, t-AML Block etÂ€al. (2002) t(8;16)(p11;p13) MOZ-CBP t-MDS, t-AML Block etÂ€al. (2002), Imamura etÂ€al. (2003) t(8;21)(q22;q22) AML1/RUNX1-ETO Lo Coco etÂ€al. (1997), Perkins etÂ€al. (1997) Trisomy 8 Perkins etÂ€al. (1997) Pentasomy 8 Wong and Wu (2000) inv(8)(p11;q13) AML t(9;22)(q34;q11) t-MDS, t-AML Block etÂ€al. (2002), Perkins etÂ€al. (1997) Block etÂ€al. (2002), Taki etÂ€al. (1997), Perkins etÂ€al. (1997) MLLâ•… MLL-CBP Epipodophyllotoxin 11q23 abnormalities exposure â•… t(9;11) (q13;q13) â•… t(10;11)(p13;q13) â•… t(11;16)(q23;p13) â•… t(11;19)(q13;q13) (continued)

Gene(s) associated

Clinical subgroup correlates

Reference

Trisomy 11 Perkins etÂ€al. (1997) 11p15 t-MDS, t-AML Stark etÂ€al. (1994), Block etÂ€al. (2002) 12p13 and 12q13 ETV6 t-MDS, t-AML Block etÂ€al. (2002), Perkins etÂ€al. (1997), Tosi etÂ€al. (1998) 13q12 Adjacent to BRCA2 MDS Coignet etÂ€al. (1999) Inversion 16 Perkins etÂ€al. (1997) 20q- and 20q11.2-12 NNAT MDS, AML Perkins etÂ€al. (1997), Kuerbitz etÂ€al. (2002) 21q22 AML1 Lo Coco etÂ€al. (1997), Perkins etÂ€al. (1997), Taketani etÂ€al. (2003) Trisomy 21 Perkins etÂ€al. (1997) Selected gene mutations relevant in pediatric myelodysplastic syndromes Gene Fusion partners Clinical correlates Reference ETV6 Tosi etÂ€al. (1998) FLT3 Co-duplication with MLL AML Jamal etÂ€al. (2001) JAK2 AML, JMML Tartaglia etÂ€al. (2003), Hellstrom-Lindberg and Cazzola (2008) Aberrant histone Imamura etÂ€al. (2003) MOZ CBP acetylation regulation TIF2 p300 Side etÂ€al. (1998) NF1 JMML not associated with clinical neurofibromatosis NNAT MDS, AML Kuerbitz etÂ€al. (2002) NPM1 Zhang etÂ€al. (2007) PPARgamma loss Silveira etÂ€al. (2009) RAS May be associated with JMML Mahgoub etÂ€al. (1998); Side etÂ€al. (1998) MLL translocations P53, TP53 deletion Gafanovich etÂ€al. (1999), Silveira etÂ€al. (2009) PTPN11 JMML Kratz etÂ€al. (2005), Bentires-Alj etÂ€al. (2004), Loh etÂ€al. (2004) WT1 Rosenfeld etÂ€al. (2003) AML acute myeloid leukemia; JMML juvenile myelomonocytic leukemia; MDS myelodysplastic syndrome; t-AML therapy-related acute myeloid leukemia; t-MDS therapy-related myelodysplastic syndrome

TableÂ€2â•… (continued) Cytogenetic abnormality noted

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hematopoietic tumors, adopted in 2009, will replace the use of the term “myeloproliferative disorder” with “myeloproliferative neoplasm” to better describe the myriad molecular abnormalities associated with the malignant nature of these diseases. Regardless of the cause of the development of MDS, there is general agreement that the treatment in children should be the same. One particular population has separated itself somewhat: adults with 5q31 deletions appear to have a better outcome when treated with the immunomodulatory agent lenalidomide than patients with other cytogenetic findings. This will be discussed later in this chapter, and the significance in pediatrics is not yet known due to the rarity of 5q31 in pediatric patients, and the current lack of any clinical studies evaluating the use of lenalidomide in children with MDS.

The Relationship Between Acute Myeloid Leukemia and MDS The AML are addressed elsewhere in this text. However, AML relates significantly to this discussion. If not treated, MDS will invariably progress to a leukemic state, most often AML. AML comprises only 15 to 20% of childhood leukemia cases, but accounts for one-third of childhood leukemia deaths (Woods etÂ€al. 2001). Current therapies are intensive and carry with them a significant risk for acute toxicity and late effects. It is doubtful that significant gains will be achieved by continued intensification of conventional chemotherapy regimens without paying a significant price of morbidity to patients. In addition, patients whose AML arises from a preceding MDS typically have more aggressive and refractory disease than those patients in whom AML arises deÂ€novo. This makes the development of targeted therapies with greater efficacy and less toxicity of paramount importance. Fortunately, much has been learned about the biology and genetics of MDS and AML, leading to the identification of a number of candidate therapeutic targets. One of the great success stories of biologically-targeted therapy is the use of all-trans retinoic acid (ATRA) in the treatment of acute promyelocytic leukemia (APL). ATRA was initially integrated into therapeutic strategies based on the observations of clinical activity prior to the identification of a specific molecular mechanism of action. Based on clinical observations, investigators in China observed complete remission in patients with both newly diagnosed and relapsed acute promyelocytic leukemia (APML) (Huang etÂ€ al. 1988). ATRA is now thought to act by several mechanisms, one of which is to drive leukemic blasts to terminal differentiation, which in effect, targets the class II mutation patterns. ATRA has become part of standard therapy for APML, and since its inclusion in treatment regimens, the cure rates for APML have risen substantially. The use of ATRA in MDS is now evolving to single agent and combination studies, most typically added to a demethylating agent or a histone deacetylase inhibitor, which will be discussed below.

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Chromatin-Based Transcriptional Therapy Cancer cells contain complex genetic and epigenetic alterations leading to aberrant gene expression that result in the malignant phenotype. These changes characteristically involve global hypomethylation, promoter hypermethylation, and histone modification, leading to altered chromatin structure and function, as well as inappropriate gene silencing (Singal and Ginder 1999; Esteller etÂ€ al. 2001; Das and Singal 2004; Vidal etÂ€ al. 2007). As such, inhibition of DNA methyltransferase (DNMT) and histone deacetylation (HDAC) are the two most common therapeutic approaches to transcriptional therapy to date, and each will be discussed below more specifically. Other targets include the histone acetyltransferases, histone methyltransferases, and histone demethylases. Epigenetic and chromatin changes occur as a result of recruitment of multiprotein containing complexes to DNA (Cameron etÂ€ al. 1999; Rountree etÂ€ al. 2001; Rountree etÂ€al. 2000), and the interaction between DNA methylation and histone acetylation are thought to play a major role in both normal and oncogenic development (Jones and Wolffe 1999). Zhou and colleagues have shown that promoter DNA methylation changes are important in MDS and are associated with changes in gene expression patterns relevant to cancer development and progression, including among others, alterations in TNF superfamily, HOX B4, EVI1, and CEPBB genes (Zhou etÂ€al. 2008). In pediatric MDS, two genes, CALCA and CDKN2B, are reported to be frequently methylated (Vidal etÂ€al. 2007). This suggests that pediatric MDS displays aberrant methylation, and that methylation targeted therapies may be similarly appropriate for the treatment, as they are currently used in the adult population. The neuronatin (NNAT) gene on chromosome 20q11.2-q12 is associated with the loss of heterozygosity in pediatric MDS and AML. Differential methylation patterns in CpG islands of exon 1 of NNAT have been demonstrated (Kuerbitz etÂ€ al. 2002), and the treatment with 5-aza-2¢-deoxycytidine can reactivate NNAT expression and re-establish normal methylation patterns inÂ€ vitro. This may be a promising approach to patients with MDS who harbor abnormalities of NNAT. It is also of note that several AML-associated fusion proteins containing the leukemia zinc finger gene MOZ, including MOZ-CBP, MOZ-TIF2, and MOZ-p300, are thought to act by aberrant regulation of histone acetylation, thus suggesting a possible role for histone modification in the therapy of these leukemias (Imamura etÂ€al. 2003). TableÂ€3 summarizes the more common agents being explored in MDS.

DNA Methyltransferase (DNMT) Inhibitors DNA methylation in promoter regions of genes is a control mechanism for gene transcription. When cytosine methylation occurs, promoters are suppressed, as is gene transcription. DNA hypermethylation in promoter regions and consequent inactivation of tumor suppressor genes, including p15INK4B and p21WAF1/CIP1, is thought to play a role in the pathogenesis of MDS and AML (Vidal etÂ€al. 2007;

Immunomodulatory agents

Hydroxamic acid derivative Cyclic tetrapeptide Synthetic benzamide derivative Benzamide

Butyric acid pro-drug Polar hybrid hydroxamic acid derivatives

Short chain fatty acids

Histone deacetylase inhibitors Target

Thalidomide Lenalidomide (CC-5013, Revlimid®) Pomalidomide CC-4047)

Valproic acid Sodium phenylbutyrate AN-9 (Pivanex) Hexamethylene bisacetamide Vorinostat (SAHA, Zolinza®) Belinostat (PXD101) Panobinostat (LBH589) Trichostatin A Depsipeptide (FK228, Romidepsin™) Entinostat (MS275, SNDX 275) CI-994 MGCD0103

Agent

TableÂ€3â•… Methylation and histone-targeted agents of interest in myelodysplastic syndrome Target Agent Methylation targeted agents Hypomethylated DNA 5-azacitidine (Vidaza®) Methylated DNA, Hypomethylated DNA 5-aza-2¢-deoxycitidine (Decitabine®) Binds 3¢ untranslated region DNA MG-98 oligonucleotide methyltransferase mRNA; growth inhibition Riboside pro-drug; cytidine deaminase inhibitor Zebularine

uM uM nM uM nM uM nM

I, II, IV I, II, IV I, II, IV I, II, IV I I I

I, IIa I, IIa

Range of reported inÂ€vitro inhibition mM mM uM

HDAC inhibition specificity

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Uchida etÂ€al. 1997; Jones and Laird 1999; Jiang etÂ€al. 2009). DNA methylation inhibitors are incorporated as cytosine bases, irreversibly binding DNA methyltransferase I (DNMT1), and thus preventing DNA methylation, which ultimately results in altered gene transcription. Several DNA methylation inhibitors, including 5-azacytidine (Vidaza®), decitabine (5-aza-2-deoxycitadine, Dacogen®), and zebularine, are being studied in adults with MDS and acute leukemia. Combinations of the DNA methylase and histone deacetylase inhibitors (HDACi) are being extensively studied in clinical trials in adults, and results have been found to be superior to the prior approach of best supportive care methodologies, but not necessarily superior to single agent treatment (Garcia-Manero etÂ€al. 2006; Scott etÂ€al. 2007; Shaker etÂ€al. 2003; Silverman etÂ€al. 2006). Zebularine inhibits cytosine deaminase and DNA cytosine methyltransferase, but inÂ€vivo did not demonstrate hypomethylating activity when compared to azacitidine or decitabine (Flotho etÂ€al. 2009). Use of any of these agents has not advanced significantly in pediatric MDS to date.

Histone Deacetylases (HDACs) and HDAC Inhibitors (HDACi) Histone deacetylases catalyze the removal of acetyl groups from lysine residues on substrate proteins, thus regulating activity and/or function of a variety of cellular components. Core histones and non-histone proteins are substrates for HDACs. Deacetylation of the core histones is associated with regions of chromatin that are transcriptionally silenced or repressed. Acetylation of the non-histone substrate proteins regulates the activity of a variety of other effector proteins. HDACs show increased expression over normal progenitors are mutated or deleted in a variety of human malignancies. Proteins implicated in tumorigenesis, such as p53, Bcl6, Hsp90, Stat3, and E2F, as well as a variety of fusion proteins interact with HDACs to mediate inappropriate repression of target genes. HDAC inhibition likely leads to changes in transcription of genes important in cell cycle and apoptosis regulation, including p21WAF1/CIP1, p53, RB, bcl2, bcl6, bclxl, and mcl-1. Several HDAC inhibitors, including valproic acid, suberanilohydroxamic acid (SAHA, vorinostat, ZolinzaTM), entinostat (MS-275, now SNDX-275), and depsipeptide (FK228, RomidepsinTM), are under wide-spread investigation in MDS and the hematologic malignancies, both alone and in combination (Kuendgen etÂ€al. 2006; Kuendgen etÂ€al. 2005; Rosato etÂ€al. 2003); their evaluation has been more limited in pediatric applications. A phase I study of depsipeptide in pediatric patients with refractory solid tumors established the maximum tolerated dose to be 13Â€ mg/m2 when administered as a weekly 4-h infusion for 3 weeks on a 28-day cycle (Fouladi etÂ€al. 2006). To date, no studies in pediatric patients with hematological malignancies or MDS have been conducted. A phase I study of vorinostat in pediatric patients has been completed, but is not yet published. A combination study of vorinostat and etoposide in children with refractory cancers, including hematologic malignancies is under way.

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Maturation-Directed Therapies Treatment with ATRA leads to dissociation of the HDAC complexes and eventual transcriptional activation of the repressed genes, leading to resumption of cellular differentiation. This has been a particularly effective treatment for APML, and early phase clinical trials have incorporated ATRA into combination therapy for adults with MDS (Murgo 2001). Pediatric use of ATRA is commonplace in APML; however, the knowledge about dosing and tolerability gained in the leukemia experience has generally not been incorporated into MDS treatment.

Immunomodulatory Drugs (IMiDs) and Anti-angiogenic Agents Inhibition of angiogenesis leading to growth arrest of MDS cells has been evaluated clinically with several agents. Thalidomide, while shown to induce major erythroid lineage response in adults with MDS, has also been associated with significant neurological toxicities as well as the known teratogenic effects that have limited its use. IMiDs, immunomodulatory agents that are structurally and functionally similar to thalidomide, are thought to act by anti-inflammatory, antiproliferative, and proapoptotic mechanisms, and are under intense investigation. Lenalidomide (Revlimid®, CC-5013) is a less neurotoxic second-generation immunomodulatory agent that is thought to induce direct cytotoxic effect on the del(5q31) clone, and significant clinical response in patients harboring 5q31 deletions have been noted (Sekeres and List 2005; Sekeres and List 2006; Sekeres etÂ€al. 2008; Chang etÂ€al. 2006; List etÂ€al. 2006). Lenalidomide received FDA approval for use in transfusion-dependent adult patients with 5q-MDS after a pivotal study demonstrated significant improvement in red blood cell transfusion-independence (Raza etÂ€ al. 2008), and current National Comprehensive Cancer Network Clinical Practice Guidelines recommend the use of lenalidomide for lower-risk MDS patients with del(5q) and transfusion-dependent anemia. Lenalidomide is also approved for use in combination with dexamethasone in adults with multiple myeloma who have received at least one prior therapy, with a significant improvement in complete and partial response rates, and in time for progression of disease when compared to dexamethasone alone. Other immunomodulatory affects of this class of agents includes anti-inflammatory effects and decrease in TNF-alpha, co-stimulatory affects of anti-CD3 stimulated T-cells (including enhanced T-cell proliferation), augmentation of NK cell cytotoxic activity against tumor cells, and inhibition of T-regulatory cell proliferation and suppressor function. Pomalidomide (CC-4047) is a second-generation IMiD in early development. While there are phase II data published on the use of thalidomide in a variety of pediatric diseases, including Crohn’s disease, systemic lupus erythematosis, Behcet’s disease, and a variety of pediatric malignancies (Gilheeney etÂ€al. 2007; Kieran etÂ€al. 2005; Lopez-Aguilar etÂ€al. 2008), there are no published data on the pharmacokinetics or tolerability of lenalidomide or pomalidomide in pediatric patients to date.

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Other Targeted Therapies A subset of adults with MDS, AML, CML, and ALL over-express the WT1 gene, and current immunotherapy-based approaches, including WT1-specific cytotoxic lymphocyte generation, WT1 anti-sense, and a WT1 vaccine, are being explored (Rosenfeld etÂ€al. 2003). Alterations of WT1 in pediatric MDS have not been described. Small molecule tyrosine kinase inhibitors (TKIs) have revolutionized the therapy of CML, Philadelphia positive ALL, renal cell carcinoma, pancreatic, colon, and lung cancers among others. Boehrer and colleagues have demonstrated that the use of erlotinib, a small molecule inhibitor of the epidermal growth factor receptor (EGFR), can induce antineoplastic activity in MDS and AML cells via off-target inhibition of JAK2 leading to apoptosis, cell-cycle arrest, and differentiation (Boehrer etÂ€al. 2008). This illustrates the possibility of broadened use of TKIs in MDS and other hematologic malignancies. Another TKI, imatinib mesylate, has shown activity in patients with MDS who have t(5;12) and deletion of 4q clones (Galili etÂ€al. 2007), presumably due to its inhibitory activity against c-KIT and the known involvement of c-KIT in these patients.

Combination Therapies Significant numbers of clinical trials are focusing on combining one or more of the above classes of agents. The major efforts were initially to couple a methylationtargeted agent with a histone deacetylase inhibitor. There are now completed studies with a variety of combinations of demethylating agents (azacitidine, decitabine, and zebularine) with HDAC inhibitors, including valproic acid, vorinostat, entinostat, LBH589, PXD101, depsipeptide, CI-994, MG-98, and MGCD0103, in adults. Other combinations focus on a differentiating agent plus another agent, often a demethylating and/or HDAC inhibitor, such as decitabine and valproic acid, azacitidine and entinostat, azacitidine and ATRA, valproic acid and ATRA, ATRA and arsenic trioxide, or a triplet of ATRA, arsenic trioxide and valproic acid, or azacitidine, valproic acid and ATRA (Soriano etÂ€al. 2007). A combination study of vorinostat and cis-retinoic acid was performed in pediatric patients with selected solid tumors. The dose limiting toxicities noted when vorinostat was dosed at 180Â€mg/m2 daily were neutropenia, thrombocytopenia, anorexia, and hypophosphatemia. Grade 3 and 4 transaminitis, leucopenia, and lymphopenia were also noted. There were no objective response, but three patients, with pineoblastoma, neuroblastoma, and medulloblastoma, had stable disease for 5, 6, and 7 cycles, respectively. The patient with medulloblastoma had to be removed from the treatment due to persistent and profound thrombocytopenia. The MTD for the combination of vorinostat and 13 cRA was vorinostat 180Â€mg/m2 per day 4â•›×â•›per week, with 13 cRA given at 80Â€mg/m2/dose bid days 1 to 14 (Fouladi etÂ€al. 2008). One study of azacitidine in combination with valproic acid allowed children over the age of two to enroll, and there are anecdotal reports of the use of this combination in children with a variety of refractory cancers, although no formal studies have been published to date.

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The epigenetic regulation of normal DNA structure and function affects scaffolding proteins during DNA assembly. The control of complex protein folding is known to be dysregulated in a variety of malignancies. The proteasome inhibitors, which affect ubiquitin-mediated protein degradation, and the heat shock protein (HSP) inhibitors, which alter chaperone protein function, have been evaluated in clinical trials. A phase I study of the first-in-class proteasome inhibitor bortezomib has been completed in pediatrics, and it is now being incorporated into combination therapy in the pediatric acute leukemias. HSP90, HSP70, and HSP27 show increased expression in ALL, AML, and MDS cells compared to controls. In addition, the combination of etoposide and 17-AAG, an HSP90 inhibitor, shows synergistic cell killing against FLT3+ leukemias inÂ€vitro (Yao etÂ€al. 2007). 17-AAG has been evaluated in two pediatric phase I studies, and demonstrated reasonable safety and tolerability but no substantial antitumor activity (Weigel etÂ€al. 2007; Bagatell etÂ€al. 2007). Three pediatric patients studied by Bagatell etÂ€al. with refractory AML/MDS (2) and ALL (1) were treated with single agent 17-AAG in a separate study arm and demonstrated stability of disease for at least 8 weeks. These data suggest the possibility of combining an HSP90 inhibitor together with etoposide and a DNMT-targeted agent in pediatric patients with MDS to affect methylation and chaperone protein interactions in a complementary fashion. In pediatrics, there has been significant lag in developing combination studies for MDS outside of the phase III setting when MDS was formerly included in AML trials, in part due to the limited numbers of patients available for such enrollment, and with relatively little interest in such a small patient population on the part of pharmaceutical manufacturers. Contemporary phase III AML studies do not include MDS patients, and there are no current phase III trials evaluating deÂ€novo MDS in children. Another problem in the process of analyzing these agents in pediatrics has been the lack of biologic correlative studies performed within the context of a clinical trial to show target modulation. This is critical to better evaluate the array of new and older agents, both alone and in combination.

Stem Cell Transplantation for Myelodysplastic Syndromes To date, the only truly curative therapy for MDS has been allogeneic stem cell transplantation. A variety of conditioning regimens have been evaluated in adults, including myeloablative, non-myeloablative, or reduced-intensity conditioning. This is in part due to the fact that most adults going to SCT have more co-morbidities than pediatric patients with the same disease. These concomitant medical conditions and the associated cumulative organ dysfunction compromise the chance of successful transplantation outcomes. In addition, pediatric patients who develop MDS have typically been taken to transplant relatively soon after their initial diagnosis without being treated with chemotherapy or MDS directed therapies, such as demethylating agents. In adults, treatment with demethylating agents is more common, since those with MDS are often elderly and not good candidates for stem cell transplantation. Many adult transplant programs now incorporate the IPSS and WHO classification schemes into their decision-making regarding timing of transplant, and most centers

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restrict transplant for MDS to patients age 50 to 55 and under due to the excess morbidity and mortality noted in older patients. In contrast, most pediatric transplant centers assume that the patient with a diagnosis of MDS will undergo transplant as soon as feasible following diagnosis; the IPSS or other risk-based classification schemes do not usually play a major role in decision-making. The exception to this is the child who is highly transfusion-dependent or who has increasing evidence of clonality or leukemic progression on serial bone marrow evaluations, who is usually prepared for transplant more urgently. The principle limitation to transplant in pediatrics is most often limited to finding a suitable HLA-matched donor. With the increasing use of cord blood, and in some centers, HLA haploidentical individuals as a source of donor cells, the pool of available donors has expanded significantly. Overall and event free survival for pediatric MDS patients with SCT are reported to range from approximately 10 to 52%, with multiple variables affecting outcome (Stary etÂ€al. 2005; Woodard etÂ€al. 2006; Kindwall-Keller and Isola 2009). Similarly, the range of transplant-related morbidities, incidence of graft vs. host disease (GVHD), and transplant-related mortality range even more widely. Patients who have a higher bone marrow blast involvement and those with t-MDS/t-AML appear to have poorer outcomes, most often due to death from refractory or recurrent disease (Woodard etÂ€ al. 2006). The poorer outcome for patients with higher blast involvement suggests that perhaps these patients should undergo pretransplant therapy aimed at reducing the clonal population. Several studies in the adult transplant literature have evaluated either a demethylating agent or lenalidomide (Platzbecker etÂ€al. 2007) prior to allogeneic SCT, and shown this approach to be both safe and feasible. Efforts at post-transplant immunomodulation for relapse have also included epigenetic modifiers (Kroger 2008). Attempts to abrogate the severity of graft-vs.-host disease have been investigated using vorinostat (Reddy etÂ€ al. 2004). Czibere and colleagues describe the use of azacitidine with donor lymphocyte infusion for the relapse of AML or MDS following allogeneic transplant (Czibere etÂ€ al. 2006). One compelling approach for MDS would be to combine the epigenetic modifiers and/or immunomodulatory agents prior to and during stem cell transplantation, and then to use post-transplant maintenance therapy with epigenetic modulators as clinically and biologically indicated based on donor chimerism and molecular response. This could potentially provide definitive curative therapy with transplantation, even in the setting of reduced intensity conditioning regimens, while offering pre- and post-transplant maintenance medical therapy, to reduce disease burden prior to transplantation, and to sustain epigenetic and/or immunomodulatory effects following transplant.

Conclusions A wide variety of targeted agents are being studied for the treatment of MDS and the consequent leukemias in adult patients, and early results are promising, particularly in the realm of epigenetic modification of the cells of interest. While supportive

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care efforts have improved and newer agents are available to enhance support and decrease side effects for patients with MDS, these are unlikely to alter the biological features that drive ultimate disintegration into a fatal disease. Further, molecular characterization of the group of syndromes collectively known as MDS will be important in the development of tailored therapy. Taken together, there are a large number of biologically compelling approaches to the treatment of MDS. A significant challenge is to determine the optimal combinations of one or several agents in conjunction with more traditional chemotherapy that will improve cure rates and decrease short- and long-term morbidity associated with the treatment for these diseases. Unfortunately, many of these combination trials have been slow to be tested in children, leaving the pediatric clinician with few options in an otherwise active area of investigation. Multi-institutional and international collaborations for the MDS, due to their rarity in children, will be of paramount importance to better gauge utility and effectiveness of such approaches in this population (Kroger 2008).

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Yao Q, Weigel B, Kersey J. Synergism between etoposide and 17-AAG in leukemia cells: critical roles for Hsp90, FLT3, topoisomerase II, Chk1, and Rad51. Clin Cancer Res 2007:13(5): 1591–1600. Weigel BJ, Blaney SM, Reid JM, etÂ€al. A phase I study of 17-allylaminogeldanamycin in relapsed/ refractory pediatric patients with solid tumors: a Children’s Oncology Group study. Clin Cancer Res 2007:13(6):1789–1793. Bagatell R, Gore L, Egorin MJ, etÂ€al. Phase I pharmacokinetic and pharmacodynamic study of 17-N-allylamino-17-demethoxygeldanamycin in pediatric patients with recurrent or refractory solid tumors: a pediatric oncology experimental therapeutics investigators consortium study. Clin Cancer Res 2007:13(6):1783–1788. Stary J, Locatelli F, Niemeyer CM. Stem cell transplantation for aplastic anemia and myelodysplastic syndrome. Bone Marrow Transplant 2005:35 Suppl 1:S13–S16. Woodard P, Barfield R, Hale G, etÂ€ al. Outcome of hematopoietic stem cell transplantation for pediatric patients with therapy-related acute myeloid leukemia or myelodysplastic syndrome. Pediatr Blood Cancer 2006:47(7):931–935. Kindwall-Keller T, Isola LM. The evolution of hematopoietic SCT in myelodysplastic syndrome. Bone Marrow Transplant 2009:43:597–609. Platzbecker U, Mohr B, von Bonin M, etÂ€al. Lenalidomide as induction therapy before allogeneic stem cell transplantation in a patient with proliferative CMML-2 and del(5q) not involving the EGR1 locus. Leukemia 2007:21(11):2384–2385. Kroger N. Epigenetic modulation and other options to improve outcome of stem cell transplantation in MDS. San Francisco, CA: American Society of Hematology; 2008. Reddy P, Maeda Y, Hotary K, etÂ€al. Histone deacetylase inhibitor suberoylanilide hydroxamic acid reduces acute graft-versus-host disease and preserves graft-versus-leukemia effect. Proc Natl Acad Sci USA 2004:101(11):3921–3926. Czibere A, Graef T, Lind J, etÂ€al. 5-Azacitidine in combination with donor lymphocyte infusion for treatment of patients with MDS or AML relapsing after allogeneic stem cell transplantation. Blood 2006:108:5341. Morerio C, Rapella A, Tassano E, etÂ€al. Gain of 1q in pediatric myelodysplastic syndromes. Leuk Res 2006:30(11):1437–1441. Mogul MJ, Brady K, Brothman AR, etÂ€ al. Myelodysplastic syndrome presenting with clonal rearrangement isolated to chromosomal region 1q. Cancer Genet Cytogenet 1997:95(2): 210–212. Lahortiga I, Agirre X, Belloni E, etÂ€al. Molecular characterization of a t(1;3)(p36;q21) in a patient with MDS. MEL1 is widely expressed in normal tissues, including bone marrow, and it is not overexpressed in the t(1;3) cells. Oncogene 2004:23(1):311–316. Horsman DE, Massing BG, Chan KW, etÂ€al. Unbalanced translocation (1;7) in childhood myelodysplasia. Am J Hematol 1988:27(3):174–178. Stark B, Jeison M, Shohat M, etÂ€al. Involvement of 11p15 and 3q21q26 in therapy-related myeloid leukemia (t-ML) in children. Case reports and review of the literature. Cancer Genet Cytogenet 1994:75(1):11–22. Rubin CM, Larson RA, Anastasi J, etÂ€al. t(3;21)(q26;q22): a recurring chromosomal abnormality in therapy-related myelodysplastic syndrome and acute myeloid leukemia. Blood 1990:76(12): 2594–2598. Dalla Torre CA, de Martino Lee ML, Yoshimoto M, etÂ€al. Myelodysplastic syndrome in childhood: report of two cases with deletion of chromosome 4 and the Philadelphia chromosome. Leuk Res 2002:26(6):533–538 Pellier I, Le Moine PJ, Rialland X, etÂ€al. Myelodysplastic syndrome with t(5;12)(q31;p12-p13) and eosinophilia: a pediatric case with review of literature. J Pediatr Hematol Oncol 1996:18(3): 285–288. Wong WY, Wu SQ. Pentasomy 8 in pediatric myelodysplastic syndrome. Cancer Genet Cytogenet 2000:121(2):220–222. Taki T, Sako M, Tsuchida M, etÂ€al. The t(11;16)(q23;p13) translocation in myelodysplastic syndrome fuses the MLL gene to the CBP gene. Blood 1997:89(11):3945–3950.

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New Therapeutic Frontiers for Childhood Non-Hodgkin Lymphoma Megan S. Lim and Mitchell S. Cairo

Introduction Childhood non-Hodgkin lymphoma (NHL) accounts for approximately 6 to 7% of all childhood cancers diagnosed below the age of 15 in the United States and consists of four major histological subtypes of both intermediate and high-grade NHL including Burkitt lymphoma (BL), lymphoblastic lymphoma (LL), diffuse large B-cell lymphoma (DLBCL), and anaplastic large cell lymphoma (ALCL) (Cairo etÂ€al. 2005). While there are a large number of other histological subtypes of NHL that may occur in children, they only account for <5% of all cases of NHL diagnosed in the United States and Western Europe. Posttransplant lymphoproliferative disease (PTLD) may also occur in children following either solid organ transplantation or mismatched or T-cell depleted allogeneic stem cell transplantation and will be the subject of a separate chapter in this monograph. We will focus our discussion on the four major histological subtypes that account for >95% of all NHL in children (Cairo etÂ€al. 2005; Pinkerton 2005). BL accounts for approximately 40% of NHL diagnosed in children, and with the recent use of short and intensive multi-agent chemotherapy, the prognosis has improved dramatically over the past two decades (children with localized disease experience a ³95% 5-year event-free survival (EFS) and patients with more advanced disease a 60 to 90% EFS) (TableÂ€1) (Cairo etÂ€al. 2005; Pinkerton 2005). LL is the second most common histology of NHL diagnosed in children and accounts for approximately 30% of all cases and in patients with localized disease, depending on the intensity of therapy administered, 60 to 90% 5-year EFS and patients with more advanced disease, 80 to 90% 5-year EFS (TableÂ€ 1) (Cairo etÂ€ al. 2005; Pinkerton

M.S. Cairo (*) Division of Pediatric Blood and Marrow Transplantation, New York-Presbyterian Morgan Stanley Children’s Hospital, Columbia University, 3959 Broadway, CHN 10-03, New York, NY 10032, USA e-mail: [email protected]

P.J. Houghton and R.J. Arceci (eds.), Molecularly Targeted Therapy for Childhood Cancer, DOI 10.1007/978-0-387-69062-9_10, © Springer Science+Business Media, LLC 2010

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178 TableÂ€1â•… Childhood non-Hodgkin lymphoma Histology Frequency (%) Burkitt lymphoma (BL) 40 Lymphoblastic lymphoma (LL) 30 Diffuse large B-cell lymphoma 20 (DLBCL) Anaplastic large cell lymphoma 10 EFS event free survival

M.S. Lim and M.S. Cairo

Localized % EFS 95 60–90 95

Advanced % EFS 60–90 80–90 80–90

80–90

60–80

2005). DLBCL occurs in approximately 20% of all children diagnosed with NHL under the age of 15. Patients with localized disease have a >95% 5-year EFS and patients with more advanced disease, 60 to 80% 5-year EFS (TableÂ€1). ALCL is the least common form of childhood NHL and accounts for approximately 10% of all cases of NHL diagnosed in children under 15 years of age and has a 5-year EFS for localized disease of 80 to 90% and in patients with more advanced disease a 5-year EFS of 60 to 80% (TableÂ€1) (Cairo etÂ€al. 2005; Pinkerton 2005). Despite these high cure rates with intensive multi-agent chemotherapy, there is still an emergent need to identify new targets and pathways that can be strategically exploited for more rational therapeutic approaches. Pinkerton (2005) in a more recent review, noted there are still significant challenges in the diagnosis and treatment of childhood NHL. In patients with an excellent prognosis following multi-agent and intensive chemotherapy, there is still a high risk of morbidity, some mortality, prolonged hospitalization and late effects. This group of patients may benefit more from a more targeted therapeutic approach that could potentially reduce the above complications. Secondly, there is a group of patients who still only have 60 to 80% chance of being cured with intensive multi-agent chemotherapy and there is considerable room to improve upon the prognosis in this subgroup of patients. Lastly, in patients who relapse after front line therapy for childhood NHL, the prognosis is generally considered dismal with overall survival rates ranging only between 10 and 30%, and therefore, new therapeutic modalities need to be developed to retrieve this poor risk group of relapsing or refractory children with NHL (Cairo etÂ€ al. 2005; Pinkerton 2005). We recently summarized state of the science of what is known regarding the biology of childhood NHL. In the following discussion, we will focus on the potential molecular and pathway targets that could be potentially exploited for new rational drug design and therapeutics for childhood NHL.

Diffuse Large B-Cell Lymphoma DLBCL accounts for approximately 20% of all childhood and adolescent NHL. In the last international French-American-British (FAB) study in children and adolescents with newly diagnosed B-NHL, we reported a 80 to 90% 4-year EFS in

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children with DLBCL, depending on stage, LDH, primary site location, and response to initial induction therapy (Cairo etÂ€al. 2007; Patte etÂ€al. 2007). Significant progress has occurred in the last 5 years in the understanding of molecular pathogenesis of adult DLBCL and only recently have we identified similar findings in childhood DLBCL. Rosenwald etÂ€al. (2002) has identified three genetic subtypes of adult DLBCL including germinal center (GC-derived), activated B-cell-like (ABC) and type 3, all of which have a differential prognosis following standard CHOP therapy (Fig.Â€1a). Furthermore, this same group of investigators also demonstrated five distinct molecular signature patterns that identified four subquartile prognostic groups who had differential outcomes ranging from 10 to 70% following standard CHOP therapy (Fig.Â€1b) (Rosenwald etÂ€al. 2002) We recently examined the genetic origin of children with DLBCL treated on the FAB/lymphoma malignancy B (LMB) 96 international B-NHL study and demonstrated that >80% of children and adolescents with DLBCL had the GC phenotype, which may in part account for the

Fig.Â€1â•… (a) Gene expression profiles of subgroups of adult DLBCL and expression of signature genes. Level of expression of variables in the outcome predictor and the scores in the three subgroups of lymphoma. (b) Gene expression profile signature score predicts for overall survival in adult DLBCL. Kaplan-Meier estimates of overall survival among all patients. Rosenwald etÂ€al. 2002. Copyright © 2002 Massachusetts Medical Society. All rights reserved

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improved prognosis in children versus adults with DLBCL (Miles etÂ€al. 2007). A fourth subtype of DLBCL includes the primary mediastinal B-cell lymphomas (PMBL). The prognosis of PMBL in children and adolescents is significantly inferior compared to other forms of childhood and adolescent B-NHL with only an approximately 65% 4-year EFS following therapy on the most recent international FAB/LMB 96 trial (Patte etÂ€al. 2007). More recently, gene expression profiling of adult PMBL was compared to adult GCB and ABC DLBCL and was demonstrated to have a significantly different molecular signature pattern (Fig.Â€2a) (Rosenwald etÂ€al. 2003). Furthermore, the molecular signature of adult PMBL in fact resembled more that of Hodgkin disease (HD) than the other forms of adult DLBCL (Fig.Â€2b) (Rosenwald etÂ€al. 2003; Savage etÂ€al. 2003). Shipp etÂ€al. has recently proposed that PMBL, otherwise known as mediastinal large B-cell lymphoma (MLBCL), is part of a developmental continuum between classical HD (cHD) and classical DLBCL (Savage etÂ€al. 2003; Abramson and Shipp 2005). One of the most commonly analyzed molecular targets in DLBCL has been the BCL-6 gene, particularly in the subtype of germinal center-derived DLBCL

Fig.Â€2â•… (a) Identification of a PMBL gene expression. Hierarchical clustering identified a set of 23 PMBL signature genes that were more highly expressed in most lymphoma with a clinical diagnosis of PMBL than in lymphomas assigned to the GCB or ABC DLBCL subgroups. Each row presents gene expression measurements from a single Lymphochip microarray feature representing the genes indicated. Each column represents a single lymphoma biopsy sample. Relative gene expression is depicted according to the color scale shown. (b) Relationship of PMBL to Hodgkin lymphoma. Relative gene expression is shown in primary PMBLs (average of all biopsy samples), the PMBL cell line K1106, three Hodgkin lymphoma (HL) cell lines, and six GCB DLBCL cell lines, according to the color scale shown in (a). Reproduced from Rosenwald etÂ€al. 2003. Copyright 2003 The Rockfeller University Press

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Fig.Â€2â•… (continued)

(GC DLBCL). BCL-6 gene is localized on chromosome 3q27 and is genetically rearranged in over 35% of adults with the GC DLBCL subtype (Ye etÂ€al. 1993; Lo Coco etÂ€al. 1994). The BCL-6 protooncogene is a nuclear phosphoprotein belonging to the POZ/Zinc finger (ZF) family of transcription factors that encodes for a transcriptional repressor required for development of germinal centers and has been implicated in the pathogenesis of GC-derived DLBCL (Chang etÂ€al. 1996). Deregulation of BCL-6 expression recapitulates the pathogenesis of human DLBCL in mice (Cattoretti etÂ€al. 2005). A variety of stimuli, including B-cell receptor (BCR) activation, mitogenic stimulation, and CD40 receptor engagement, appear to downregulate BCL-6 expression (Allman etÂ€ al. 1996). Transcriptional regulation of the BCL-6 gene may be disrupted by somatic hypermutation in the five noncoding regions of

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the BCL-6 gene in GC-derived DLBCL (Migliazza etÂ€al. 1995; Pasqualucci etÂ€al. 1998) or may occur due to tumor-associated chromosomal translocations, which juxtapose heterologous promoters to the coding exons of BCL-6, causing its deregulated expression by a mechanism previously described as “promoter substitution” (Ye etÂ€al. 1995). BCL-6 is thought to function in GC formation presumably by its ability to repress the transcription of specific target genes. BCL-6 is believed to repress STAT 6 mediated, IL4-induced expression of Ig germ line transcripts, therefore, presumably modulating the isotype switch toward IgE inÂ€ vitro and inÂ€vivo. Additionally, a number of other putative BCL-6 network genes involved in apoptosis, cell cycle proliferation, cell differentiation and activation, and inflammation have also been proposed as potential targets (Harris etÂ€al. 1999; Baron etÂ€al. 2002). More recently, BCL-6 has been identified to control the expression of CD80, a costimulatory molecule, in normal GC B-cells (Niu etÂ€ al. 2003). We recently analyzed BCL-6 interacting proteins by tandem mass spectrometry by coimmunoprecipitation with an anti-BCL-6 antibody in a GC-derived DLBCL line (Miles etÂ€al. 2005a). Importantly, BCL-6 was identified to bind to a number of proteins including several histone deacetylators, including histone deacetylase (HDAC) 1,2,4,5,7 and 9, and a number of other important proteins, including BCL-11A, IRF4, JunB, c-Jun, among 61 other proteins, from a variety of functional categories including transcriptional regulators, binding activity, signal transduction activities, etc. (Fig.Â€3) (Miles etÂ€al. 2005a).

Fig.Â€3â•… Cellular location and molecular function of BCL-6-interacting proteins. The relative proportions of identified proteins are shown for functional classification. Miles etÂ€al. 2005a. Copyright © 2005 American Society for Biochemistry and Molecular Biology

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BCL-6 appears to be regulated by a similar set of enzymatic reactions that control chromatin remodeling. Histones similar to regulating chromatin remodeling also appear to influence the regulation of BCL-6. Histone acetyltransferases (HATs) regulate open chromatin structures allowing transcriptional activation while deacetylation by HDAC promotes chromatin condensation and transcriptional inactivation. Histone deacetylase inhibitors (HDACIs) inhibit the removal of the acetyl groups from the lysine tails of the histone and help maintain DNA and an open chromosome structure thereby promoting transcriptional activation (O’Connor 2005). Similarly, BCL-6 is subject to acetylation by a p300 acetylase (HAT), which completely abrogates its transcriptional repressor function, and thereby promotes a number of BCL-6 dependent genes to be transcribed and activated (Bereshchenko etÂ€al. 2002). Alternatively, deacetylation of BCL-6 by HDACs promotes BCL-6 to serve as a transcriptional repressor and inhibit the activities on a number of BCL-6 dependent genes (O’Connor 2005; Bereshchenko etÂ€al. 2002). Recent studies have demonstrated that HDACIs promote growth inhibition, apoptosis, and anti-tumor activity in BCL-6 genetic rearranged DLBCL (O’Connor 2005, Bereshchenko etÂ€al. 2002). Therefore, HDACIs can potentially modulate gene expression in GC-derived DLBCLs by one of two mechanisms, including influencing the aceytlation state of BCL-6, and second, by modulating the balance of open and closed chromatin available for transcription (O’Connor 2005). There have been a number of Phase 1 and Phase 2 clinical trials in adults with advanced cancer including those with DLBCLs that have investigated the safety and efficacy of HDACIs (O’Connor 2005; Piekarz etÂ€al. 2001; Kelly etÂ€al. 2003a; Prince etÂ€al. 2007 (abstract)). There are currently four major classes of HDACIs, including short-chain fatty acids, which include valproic acid, hydroxamic acids including suberoylanilide hydroxamic acid (SAHA) (vorinostat), cyclotetrapeptides including depsipeptide and benzamides (MS-275). Future studies will be required to test the safety, efficacy, and mechanism of anti-tumor activity of these HDACIs in GC-derived DLBCL. A number of these agents including depsipeptide and SAHA are already in Phase I investigation in children with a variety of solid tumors (Fouladi etÂ€al. 2006, 2007). An alternative approach to inhibiting BCL-6 is the use of specific BCL-6 peptide interference peptides (Polo etÂ€al. 2004). Polo etÂ€al. developed a peptide that binds to BCL-6 and blocks corepressor recruitment and disrupts BCL-6 mediated repression and reactivates BCL-6 target genes. Peptides were generated against the BTB binding domain (BBD motif) that is critical in BCL-6 interaction with other key target genes involved in transcriptional repression. BBD peptides were demonstrated to induce apoptosis, cell cycle arrest, suppress normal germinal center formation, and inhibit growth of B-cell lymphomas inÂ€vivo (Polo etÂ€al. 2004). We recently identified that only a minority of children and adolescents with DLBCL treated on the FAB/LMB96 international B-NHL trial were derived from the ABC subtype of DLBCL (Miles etÂ€al. 2005b (abstract)). In adults with ABCDLBCL, there is a significant difference in their gene expression signature compared to the more common GC-DLBCL subtype (Fig.Â€ 2a) (Rosenwald etÂ€ al. 2003). Recently, Ngo in the Staudt laboratory utilized a doxycycline inducible retroviral vector for the expression of small hairpin RNAs (shRNAs) to construct a library

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targeting 2,500 human genes, and subsequently utilized this library to infect cell lines representing both ABC-DLBCL and GC-DLBCL subtypes (Ngo etÂ€al. 2006). In this shRNA-transduced study, CARD11 shRNAs specifically targeted genes of the NF-kB pathway following engagement of MALT 1 and BCL10 (Ngo etÂ€al. 2006). These studies identified that CARD11 was the key upstream signaling component likely responsible for the ongoing constitutive NF-kB activity previously demonstrated in ABC-DLBCL (Ngo etÂ€al. 2006). CARD11 and other associated genes are considered to be attractive targets for ABC-DLBCL since inhibition of CARD11 signaling would likely only affect the lymphoid system (TableÂ€2) (Ngo etÂ€al. 2006; Thome 2004). Although there are no inhibitors of CARD11 available for clinical use today, these studies suggest that future development of CARD11 inhibitors could potentially be utilized in targeted therapy for ABC-derived DLBCL. Besides CARD11, there are a number of other targets in the NF-kB pathway that can be potentially inhibited to downregulate NF-kB gene expression and transcriptional activation of a variety of genes promoting cell proliferation and survival (TableÂ€2) (Karin 2006; Van Waes 2007). There are a number of signaling pathways that induce NF-kB activation and translocation to the nucleus to induce gene activation (Fig.Â€4) (Van Waes 2007). In the classical pathway of NF-kB activation, NF-kB homodimers or heterodimers are retained in the cytoplasm by binding to specific inhibitors named the inhibitors of NF-kBs (IkBs). Briefly, the IkB complex is formed by a number of IkB kinases (IKKs) and a number of NF-kB dimers, including RelA, cRel, RelB, p50/NF-kB1, and p52/NF-kB2. These heterodimeric and TableÂ€2â•… Molecular targeting of DLBCL and BL Subtype Gene structure Function GC-DLBCL

BCL-6 26-S proteasome

Transcriptional repressor BCL-6 interference peptide Ubiquitin-mediated protein degradation

ABC-DLBCL

BLIMP-1 CARD11 NF-kB

Tumor suppressor Transcriptional activator Transcriptional activator

PMBL-DLBCL BL

NF-kB C-MYC

Transcriptional activator Proto-oncogene, Transcriptional activator

Inhibitors/modifier HDACs[Vorinostat, Depsipeptide] BBD motif inhibitor Proteosome inhibitor â•… [Bortezomib] Unknown Unknown Rituximab Bortezomib Small IKK inhibitors Small IKK inhibitors Benozochazepine (Bz-423) GSI Anti-sense oligonucleotides SiRNAs, or ShRNAs

DLBCL diffuse large B-cell lymphoma, BL Burkitt lymphoma, GC-DLBCL germinal cell-Diffuse large B-cell lymphoma, ABC-DLBCL activated B-cell like-diffuse large B-cell lymphoma, PMBL-DLBCL primary mediastinal B-cell lymphoma-diffuse large B-cell lymphoma, BL Burkitt lymphoma, HDACs histone deacetylase inhibitors, BBD BTB binding domain, GSI Gamma secretase inhibitor

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Fig.Â€4â•… NF-kB activation in pathogenesis and therapy of cancer. (a) NF-kB activation in cancer development is linked with chronic exposure to bacteria, certain viral products, chemical promoters, and carcinogens and reactive oxygen species, which cause repeated DNA damage. Induction also occurs in response to cytotoxic chemotherapy and ionizing radiation. (b) classic NF-kB pathway activation occurs in response to these bacterial, viral, chemical, or physical stimuli; by aberrant cytokine, integrin, and growth factor ligand/receptor activation (e.g., TNFR, IL-1R, integrin a6h4,EG FR,H ER-2/neu, and other serum factors); expression of activating fusion proteins following translocations (e.g., BCR-ABL and MALT1); or aberrant activation by intermediate kinases (e.g., P I3K, CK2, and AKT). Intermediate kinases convey signals to the IkB complex formed by IKKa, IKKb, and IKKg, and IKKb and CK2 phosphorylate IkB, marking it for ubiquitination by E3 ligase bTrCP (SCF) and proteasome degradation. P105/RELA or cREL is processed to NF-kB1 (p50)/RELA or cREL heterodimers, which translocate to the nucleus and bind promoters of genes regulating proliferation, apoptosis, migration inflammation, angiogenesis, and innate immunity. (c) Alternative pathway. The alternative pathway may be activated by other TNF family members via the NF-kB inducing kinase and involves IKKa/IKKa homodimers, which activate NF-kB2/p100 for processing into p52/RelB heterodimers. The RelB/p52 heterodimer then translocates into the nucleus to bind the promoter of genes whose products are important for the malignant phenotype in some cancers and B-cell development and adaptive immunity. (d) In certain leukemias, overexpression of BCL3 can activate NF-nB2. E, repression of gene activation can occur in the presence of intact p14ARF and p53, which favor replacement of CBP/p300 with histone deacetylases. Red highlighted inhibitors of NF-kB activation under clinical and preclinical investigation include proteasome, bTrCP and IKK antagonists, and inhibitors of receptor and intermediate kinases involved in activation. Abbreviations: NIK NF-kB inducing kinase, IL-1R interleukin 1 receptor, EGFR epidermal growth factor receptor, TNFR TNF receptor, TRAF TNF receptor-associated factor, TAK transforming growth factor-b-activated kinase, FAK focal adhesion kinase, CK2 casein kinase 2, PI3K phosphatidylinositol 3-kinase, PDK 3-phosphoinositidedependent protein kinase. Originally published in Van Waes 2007. Copyright © 2007 American Association for Cancer Research

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homodimeric combinations of NF-kB are initially expressed in an active form in the cytoplasm. However, upon IKK phosphorylation, the NF-kB bound IkB proteins are targeted for polyubiquitination and rapid degradation by the ubiquitin ligase complex allowing NF-kB dimers to translocate to the nucleus where they promote the transcriptional activation of several hundred target genes (Fig.Â€ 4) (Karin 2006; Van Waes 2007). A number of drugs are in development, particularly the IKK small molecule inhibitors, which in preclinical studies appear to promote tumor cell apoptosis. Some of the more recent IKK selective antagonists, PS1145 or ML120b derivatives, have already demonstrated significant antiproliferative, cytotoxic, and anti-tumor effects (Van Waes 2007; Lam etÂ€al. 2005). There are a number of new drugs in development that are also capable of targeting NF-kB activation and may be useful in the future for treatment of ABC-derived DLBCL and potentially PMBL, as discussed below. An important component of NF-kB activation is the polyubiquitination of IkBs that occurs within the proteasome. A novel class of proteasome inhibitors for cancer therapy has been in development over the past decade and the prototype, bortezomib, was recently approved by the FDA for the treatment of multiple myeloma (TableÂ€ 2) (Voorhees and Orlowski 2006). Bortezomib was demonstrated to have broad anti-tumor activity in a number of tumor cell lines and in several murine xenograft models (Adams etÂ€al. 1999). Proteasome inhibitors block the degradation of IkB, thereby sequestering NF-kB in the cytoplasm and downregulating its transcriptional activity (Voorhees and Orlowski 2006; Traenckner etÂ€al. 1994). Phase 2 studies of bortezomib in adult B-NHL have already been completed and bortezomib is currently under investigation in combination with CHOP therapy in adult patients with DLBCL (Goy etÂ€ al. 2005; O’Connor etÂ€ al. 2005; Mounier etÂ€ al. 2007). It remains to be determined whether bortezomib will be selectively active in subtypes of DLBCL that have an overexpressed NF-kB signal transduction pathway including ABC-derived DLBCL and PMBL (TableÂ€2). Another agent that may be useful in targeting subtypes of DLBCL that have an overexpression of the NF-kB signal transduction pathways is the anti-CD20 antibody, rituximab. The mechanism of action of rituximab is believed to be multiÂ�factorial and possibly includes complement-dependent cytotoxicity (CDC), antibody-dependent cellular cytotoxicity (ADCC), and direct induction of tumor cell apoptosis. The specific mechanism by which rituximab may affect the NF-kB signaling pathway has recently been elucidated (Jazirehi etÂ€al. 2005). Rituximab appears to upregulate the Raf-1 kinase inhibitor protein (RKIP). RKIP physically associates with NF-kB inducing kinase (NIK), tumor growth factor-B activating kinase 1 (TAK1), and IKK, preventing them from signaling further downstream (Jazirehi etÂ€al. 2005). Upregulation of RKIP expression interferes with NF-kB signaling, leading to the diminished expression of the Bcl-xL gene product, which normally has an anti-apoptotic role (Jazirehi etÂ€al. 2005). Therefore, rituximab may have several mechanisms of action with respect to subtypes of DLBCL, including inducing CDC and ADCC as well as inhibiting NF-kB signaling (Jazirehi etÂ€al. 2005). Recently, B lymphocyte-induced maturation protein 1 (BLIMP-1), which has previously been demonstrated to be a transcriptional repressor expressed in germinal

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center B-cells and in plasma cells, is required for terminal B-cell differentiation into plasma cells (Turner etÂ€ al. 1994; Angelin-Duclos etÂ€ al. 2000). Pasqualucci etÂ€ al. (2006) recently demonstrated that the BLIMP 1 gene is inactivated by structural alterations in approximately 25% of ABC-derived DLBCLs. These studies suggest that BLIMP 1 may function as a tumor suppressor gene whose inactivation may contribute to ABC-derived DLBCL lymphomagenesis by inhibiting post-GC differentiation of B-cells into plasma cells. Although at the present time there are no known mediators to induce BLIMP 1 expression or activation, possibly in the future there may be the potential for developing a specific drug that can upregulate BLIMP 1 in patients with ABC-derived DLBCL and turn on the tumor suppressor property of BLIMP 1 (TableÂ€2) (Pasqualucci etÂ€al. 2006). The last major subtype of DLBCL is the PMBL, which accounts for <5% of all subtypes of DLBCL in children and adolescents (Cairo etÂ€ al. 2005; Pinkerton 2005). Recent genetic profiling studies in PMBL suggest that PMBL resembles HD more and resembles the other two subtypes of DLBCL less, including ABC and GC derivatives (Fig.Â€2a) (Rosenwald etÂ€al. 2003). Differential expression of PMBL, GC and ABC-DLBCL demonstrates a significant upregulation of a number of NF-kB target genes in PMBL (Fig.Â€5a) (Feuerhake etÂ€al. 2005). Subsequently, Lam etÂ€ al. (2005), utilizing several small IKK inhibitors such as PS1145 and MLX105, demonstrated selective toxicity in the subgroup of DLBCL that had overexpression of genes within the NF-kB signal transduction pathway (Fig.Â€5b). These studies and the studies mentioned with ABC-derived DLBCL suggest that

Fig.Â€ 5â•… (a) Differential expression of NF-kB target genes in an independent series of primary MLBCLs and DLBCLs. The independent data set includes all primary MLBCLs (38 tumors) and DLBCLs (26 tumors: 13 GC-type and 13 ABC-like) that were made available at the NIH Lymphoma/ Leukemia Molecular Profiling Project website. MLBCLs versus DLBCLs. This independent series of primary MLBCLs had significantly higher expression of 79% (26/33) of the NFkB target genes that were identified in our series and represented on both platforms; and GC-type versus ABC-like DLBCLs. This research was originally published in Blood. Feuerhake etÂ€al. 2005. (b) Gene expression profiling of ABC DLBCL and PMBL cells treated with IKK inhibitors. Genes that were downregulated in K1106 cells treated with 25Â€Amol/L MLX105 for the indicated times. Color intensity, ratio of gene expression in MLX105 treated versus untreated cells. Originally published in Lam etÂ€al. 2005. Copyright © 2007 American Association for Cancer Research

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specific inhibitors of the NF-kB signal transduction pathway may potentially have a selective and targeted toxic effect in subtypes of DLBCL, including ABC and PMBL (TableÂ€2).

Burkitt Lymphoma The prognosis for childhood and adolescents with BL has improved dramatically over the last 25 years (Cairo etÂ€al. 2003). Results from the most recent FAB/LMB 96 international childhood and adolescent B-NHL study demonstrated a 95 to 99% 5-year EFS in patients with localized disease and a 60 to 90% 5-year EFS in patients with advanced disease (Cairo etÂ€al. 2007; Patte etÂ€al. 2007). However, there are subgroups of children and adolescents with BL that respond poorly to standard therapy, including those newly diagnosed patients who have a poor response to reduction COP therapy, those patients with initial central nervous system and bone marrow involvement, and those patients who relapse off standard therapy (Cairo etÂ€al. 2007). Additionally, the morbidity of short and intense multi-agent chemotherapy is extremely high in this population, with 65 to 70% of patients developing Grade 3 and 4 infections and/or mucositis with prolonged hospitalizations (Cairo etÂ€al. 2007). Therefore, new targeted therapeutic modalities for childhood and adolescent BL is needed not only for newly diagnosed patients with either a poor prognosis or for those patients that are experiencing high morbidities with current therapy and is definitely needed for patients whose disease reoccurs, who have a <15% chance of overall survival (Cairo etÂ€al. 2007; Patte etÂ€al. 2007). Although almost 100% of childhood and adolescents with BL have an associated 8q24 (C-MYC) gene rearrangement, there are a number of other cytogenetic aberrations that we recently identified in this patient population in the most recent FAB/ LMB 96 international B-NHL study (Poirel etÂ€al. 2003 (abstract)). Abnormalities in 1q+ was the most common chromosomal aberration. However, in a multivariable analysis, controlling for stage, LDH, and group classification deletion of 13q and 7q+, each were associated with significantly poor outcome compared to the remainder of all patients treated with the same therapy (Poirel etÂ€ al. 2003 (abstract)). Therefore, specific cytogenetic aberrations are associated with a poor prognosis in childhood BL and may help us identify specific molecular targets that can be utilized for therapeutic purposes. Recently, a large group of adults and a small group of children with BL were analyzed by oligonucleotide microarray analysis (Dave etÂ€ al. 2006). Dave etÂ€ al. (2006) demonstrated significant genomic differences between BL and all subtypes of DLBCL with a significant increase in the BL subgroup in C-MYC targeted genes and GC-derived targeted genes but with a concomitant significant decrease in MHC-1 and NF-kB target genes compared to DLBCL (Fig.Â€6). C-MYC, a well known protooncogene and transcription factor, is a major regulator of a large network of genes that promote cell proliferation and survival. In a recent highly sophisticated analysis using an algorithm for the reconstruction of

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Fig.Â€6â•… Differential gene signatures of C-MYC (a), MHC class I (c), and NF-kB (d), target genes between BL and subtype of DLBCL. The average relative expression of genes that distinguish Burkitt lymphoma from each subgroup of diffuse large-B-cell lymphoma (activated B-cell–like, germinal-center B-cell–like, and primary mediastinal) are categorized into gene expression signatures: C-MYC and its target genes (a); MHC class I genes (c); and genes targeted by the NF-kB signaling pathway27 (d). Relative gene expression is depicted according to the color scale shown. Dave etÂ€al. 2006. Copyright © 2006 Massachusetts Medical Society. All rights reserved

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accurate cellular networks (ARACNe), Basso etÂ€ al. elucidated a hierarchical organized subnetwork of genes that interact with C-MYC (Basso etÂ€al. 2005). Three hundred and thirty-six expression profiles from different subpopulations of B-cell phenotypes were utilized to deconvolute the C-MYC cellular network. In this analysis, Basso etÂ€ al. (2005) identified that the C-MYC subnetwork included 56 genes that were directly connected to C-MYC and another 2,007 genes that were connected through an intermediate that they labeled secondary neighbors (Fig.Â€7a, b). As illustrated in Fig.Â€7a, there are over 2,000 genes that are connected through the C-MYC subnetwork and, in Fig.Â€7b, is a representation of the first 500 genes including the 56 first neighbors and 44 of the most statistically significant second neighbors (Fig.Â€ 7a, b) (Basso etÂ€ al. 2005). These results suggest that it may be possible to develop a molecular targeted approach to specific genes downstream from C-MYC for tumors such as BL, which are highly regulated by overexpression of C-MYC. One of the most notable C-MYC subhubs and a recently known C-MYC target identified in the analysis was BYSL (Basso etÂ€al. 2005). A preliminary analysis suggested that the BYSL network may regulate cell proliferation, nucleic acid, metabolism, and ribosomal biogenesis and could serve as a molecular target in future studies (Basso etÂ€al. 2005). Basso etÂ€al. (2005) suggest that the structure of the network indicates that the subhubs are crucial mediators of regulatory function and should be considered in the future as priority targets for molecular targeted cancer therapy. To date, there are no specific approved drugs that target C-MYC or any of the critically important genes in the subhub network of C-MYC. However, there are several developments both preclinical and in early clinical trials that may provide a targeted approach for C-MYC in BL. A novel benzodiazepine, Bz-423 was demonstrated to induce apoptosis and inhibit proliferation in both normal and transformed lymphoid cells (Blatt etÂ€al. 2002; Boitano etÂ€al. 2003). Initial investigations of this agent targeted lymphocytes in a mouse model of lupus and arthritis and demonstrated significant therapeutic benefits in autoimmune disease (Bednarski etÂ€ al. 2003). More recently, Bz-423 has been demonstrated to rapidly and specifically deplete C-MYC protein in a post-translational mechanism of inducing anti-proliferation and apoptosis (Sundberg etÂ€al. 2006). Sundberg etÂ€al. demonstrated that Bz-423

Fig.Â€7â•… (continued)â•… genes connected through an intermediate (second neighbors). For representation purposes, only the first 500 genes are shown, including all 56 first neighbors and the 444 most statistically significant second neighbors. Red or pink nodes represent first neighbor target genes for which ChIP data is available or not available, respectively; yellow and light yellow nodes represent second neighbor target genes for which ChIP data is available or not available, respectively; MYC is shown in green; white nodes represent genes for which no MYC-related information is available. The complete list of genes, including gene symbol, Affymetrix ID and LocusLink ID, is given in the Supplementary TableÂ€4 online (see original manuscript). (b) The first neighbors of the MYC subnetwork. The size of each circle is proportional to the number of gene interactions. For hubs with more than 100 interactions, the exact number of first neighbors is shown beside the gene symbol. Reprinted by permission from Macmillan Publishers Ltd: Basso etÂ€al. 2005, copyright 2005

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Fig.Â€7â•… (a, b) The MYC subnetwork. (a) A MYC-specific subnetwork was obtained by including all the genes that have pâ•›<â•›10−7 based on their pairwise mutual information with MYC. The faster bin-counting estimator was used with an error tolerance e = 0.15. The MYC subnetwork includes 56 genes directly connected to MYC (first neighbors; represented by larger circles) and 2,007

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inhibited f1f0-ATPase activity, which inhibited respiratory chain function in generating superoxide and thereby led to proteasomal degradation of C-MYC protein. The effects of Bz-423 not only decreased C-MYC protein, but also modulated expression of several cell cycle regulatory proteins dependent on C-MYC expression (TableÂ€2) (Sundberg etÂ€al. 2006). It is well known that NOTCH1 signaling is required for the regulation and maintenance of hematopoietic stem cells and is critically important to the development of T-cells (Allman etÂ€al. 2002). NOTCH1 signaling appears to regulate a number of processes including proliferation, cell growth, differentiation, and apoptosis. Activating NOTCH mutations have recently been identified in over 50% of human T-acute lymphoblastic leukemia (ALL) (Weng etÂ€al. 2004). Small molecule inhibitors of the gamma-secretase complex (GSI) inhibit NOTCH1 signaling and promote apoptosis and cell cycle arrest in T-ALL leukemia cells (O’Neil etÂ€al. 2006). Palomero etÂ€al. (2006) demonstrated that C-MYC is a critical mediator of NOTCH1 signaling and that NOTCH1 activation appears upstream of C-MYC and in part regulates cell proliferation and growth by directly regulating C-MYC activation. In vitro studies with NOTCH1 signaling inhibitors such as GSIs significantly downregulate C-MYC expression (Palomero etÂ€al. 2006). However, the slow kinetics of C-MYC downregulated by GSI treatment suggest that NOTCH1 probably only regulates a fraction of C-MYC expression, and therefore, GSI-type inhibitors may only partially inhibit C-MYC induced cell proliferation and growth (TableÂ€2) (Palomero etÂ€al. 2006). A third potential approach to target C-MYC in BL is the use of antisense oligodeoxynucleotides specifically designed against specific regions within the C-MYC gene. In previous inÂ€ vitro studies, phosphodiester and phosphorothioate anti-sense oligodeoxynucleotides induced significant inhibition in BL cell growth (Williams etÂ€al. 1997). We further demonstrated that the addition of cationic lipids significantly reduces the time and the dose required of C-MYC antisense oligodeoxynucleotides to inhibit BL inÂ€vitro growth (Williams etÂ€al. 1996). There has been, however, little development of C-MYC antisense oligodeoxynucleotides for a variety of reasons, including different C-MYC point rearrangements in different subtypes of BL, and the difficulty of delivering antisense oligodeoxynucleotides clinically, and more importantly, the uptake of antisense deoxyolidonucleotides in BL cells inÂ€vivo. However, further development is underway for exploring similar approaches using small hairpin or small interference RNAs that also target specific regions of the C-MYC gene (TableÂ€2).

Anaplastic Large Cell Lymphoma ALCL is a distinct subtype of aggressive CD30-positive peripheral T-cell NHL that makes up about 10 to 15% of pediatric NHL (Drexler etÂ€al. 2000; Perkins 2000). The World Health Organization (WHO) classification of lymphomas recognizes two subtypes of ALCL, a primary cutaneous ALCL that forms part of a clinical spectrum of CD30-positive cutaneous T-cell lymphoproliferative disorders and

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systemic ALCL (Greenland etÂ€ al. 2001; Kadin and Carpenter 2003). Systemic ALCLs are further subdivided into two clinically significant subtypes based on expression of anaplastic lymphoma kinase (ALK) (Drexler etÂ€al. 2000; Kadin and Carpenter 2003; Falini 2001). In the pediatric population, primary cutaneous and ALK negative ALCL are very rare (Tomaszewski etÂ€al. 1999). The majority of pediatric ALCL (90 to 95%) express ALK due to specific genetic events that lead to overexpression and constitutive activation of ALK (Kutok and Aster 2002). In about 80% of tumors, ALK overexpression arises due to a t(2;5)(p23;q35) translocation that juxtaposes the ALK gene on chromosome 2p23 to the nucleophosmin (NPM) gene on 5q35. This fusion gene encodes a chimeric, constitutively activated tyrosine kinase, NPMALK, consisting of the N-terminal portion of the NPM fused to the catalytic domain of ALK (Fig.Â€8). The fusion with NPM results in constitutive activation of the ALK kinase in a deregulated and ectopic manner (Drexler etÂ€al. 2000; Pulford etÂ€al. 2004).

Fig.Â€8â•… Chromosomal translocation t(2;5)(p23;q35) leads to NPM-ALK. The chromosomal translocation involving 2p35 (ALK) and 5q35 (NPM) results in the fusion gene product, NPM-ALK, which encodes a chimeric tyrosine kinase. The c-terminal portion of ALK tyrosine kinase fuses to the N-terminal portion of NPM, which leads to constitutive expression and activation of the NPMALK oncogenic protein. The fusion protein can be rapidly detected in routine tissue sections using the ALK-1 antibody, which demonstrates the fusion protein in the nucleus and the cytoplasm of tumor cells

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Over 13 alternative fusion partners for ALK have been identified and involve clathrin heavy polypeptide-like gene (CLTC) (Bridge etÂ€al. 2001), nonmuscle tropomyosin 3 gene (TPM3) (Lamant etÂ€al. 1999), nonmuscle tropomyosin 4 (TPM4) (Meech etÂ€ al. 2001), moesin (MSN) (Tort etÂ€ al. 2001), Trk fusion gene (TFG) (Hernandez etÂ€ al. 1999), cysteinyl-tRNA synthetase (CARS) (Cools etÂ€ al. 2002), nonmuscle myosin heavy chain (MYH9) (Lamant etÂ€al. 2003), RAN binding protein (RANBP2) (Ma etÂ€al. 2003), and 5-aminoimidazole-4-carboxamide ribonucleotide formyltransferase/IMP cyclohydrolase gene (ATIC) (Ma etÂ€al. 2000). Most of the partner proteins contain an oligomerization domain that is required for constitutive activation of the ALK kinase (Drexler etÂ€al. 2000; Lamant etÂ€al. 1999; Meech etÂ€ al. 2001). The downstream signaling pathways induced by the variant partner gene products are not known, although gene expression profiling studies comparing TPM3-ALK positive and NPM-ALK positive ALCL indicate overlapping pathways that may be shared (Bohling etÂ€al. 2008). The overexpression of ALK secondary to the creation of a fusion protein is detectable by immunohistochemistry on tissue biopsies, providing a very rapid and effective means to evaluate the presence of ALK translocation in over 90% of cases. This provides a rapid and routine method of identifying tumors that may be amenable to molecular targeted therapies. ALK expression has been strongly correlated with clinical outcome. Patients with ALK-positive ALCLs have a favorable prognosis and favorable clinical features including low age at diagnosis, low stage, low risk IPI staging, and 80 to 90% 5-year survival, although there is an increased incidence of late (2 years or later after therapy) relapse. In contrast, ALK-negative ALCL usually occurs in older adults and is associated with high stage disease and an approximately 40% 5-year survival (Kadin and Carpenter 2003; Falini 2001). Constitutive activation of the oncogenic ALK tyrosine kinase leads to activation of a multiple, diverse downstream signaling cascade involved in cell proliferation, survival, transformation, and anti-apoptosis (Fig.Â€9). It is believed that these signaling cascades sustain survival and viability of NPM-ALK-positive tumor cells (Drexler etÂ€al. 2000; Pulford etÂ€al. 2001; Pulford etÂ€al. 2004). This enables the application of two general strategies for development of molecularly targeted therapeutic agents in ALCLs. The first strategy is to target the ALK expression or activity. The other is to target any one of several downstream signaling pathways that are activated by ALK. Indeed, most studies to date have focused on targeting downstream signaling pathways, which will be discussed in the subsequent sections. A variety of approaches to ablate the expression or function of the ALK fusion protein have been attempted, including hammerhead ribozyme-mediated cleavage of the NPM-ALK fusion gene product (Hubinger etÂ€al. 2003) and siRNAmediated ablation (Ritter etÂ€al. 2003). Although both hammerhead ribozyme and siRNA effectively decreased the expression and activity of ALK and its mediators, such as AKT and ERK in cell lines (HeLa and 293) cotransfected with ALK, the results were not as significant in cell lines that expressed endogenous NPM-ALK with repeated transfections over 8 days leading to significant reduction of NPMALK protein but without induction of apoptosis. These results are attributed to the long half life of the NPM-ALK protein (48Â€hours). More recently, Piva etÂ€al. (2006)

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Fig.Â€9â•… Activation of multiple signaling pathways by NPM-ALK. The NPM-ALK fusion tyrosine kinase recruits a number of adaptor proteins via interactions with phosphorylated tyrosine residues. A complex network of signaling pathways including RAS/MAPK, JAK/STAT and PI3-K/ AKT are activated as a result of the activation of NPM-ALK. These signaling pathways play a critical role in enhancing the survival and proliferation of ALCLs

demonstrated a more effective approach to ablation of ALK by using lentiviral transduction of shRNA, targeting a sequence coding for the catalytic domain of ALK. Lentiviral transduction of human ALCL cells with shRNA against ALK led to G1 cell cycle arrest and apoptosis inÂ€ vitro and tumor growth inhibition and regression inÂ€vivo. These results indicate that sustained ablation of ALK expression is required for efficacious therapy of ALCLs and clinical application of this approach may have limited value at this time as a single modality therapeutic option. Indeed, the downregulation of NPM-ALK by siRNA has been shown to augment the chemosensitivity to chemotherapeutic agents such as doxorubicin (Hsu etÂ€al. 2007). Another strategy involves the use of small molecule inhibitors that may be delivered orally with increased bioavailability and better pharmacokinetics. Clearly, the benefit of this type of agent has been demonstrated by the immense success of imatinib for the treatment of chronic myeloid leukemias with BCR-ABL translocations. Indeed, there is clinical and therapeutic rationale to identify potent, selective, and efficacious inhibitors of all of the 75 to 80 known tyrosine kinases in the human genome. Furthermore, because the ALK tyrosine kinase is not normally expressed in many cell types, inhibition of the cancer-specific tyrosine kinase by small molecule

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inhibitors would be an attractive option for tumor-specific therapy. The development of small molecule kinase inhibitors for ALK has been somewhat slow in gaining enthusiasm by pharmaceutical companies. This is attributed to the low incidence of the disease and thus, low economic potential. However, several groups have recently reported the development of small molecule inhibitors of ALK (Wan etÂ€al. 2006; Galkin etÂ€ al. 2007) that have varying degrees of potency, selectivity and efficacy. One group of quinazoline-type compounds, designated WHI-P131 (4-(4¢hydroxyphenyl)amino-6,7-dimethoxyquinazoline) and WHI-P154 (4-[(3¢-bromo4¢-hydroxyphenyl)amino]-6,7-dimethoxyquinazoline) inhibited proliferation and induced apoptotic cell death of the ALK-positive T-cell lymphoma cells (Marzec etÂ€ al. 2005). These compounds abrogated STAT3 tyrosine phosphorylation in a Jak3-independent manner and directly inhibited the kinase activity of ALK with IC50 ranging from 5 to 10Â€mM. Another group of compounds developed by Cephalon represent cell-permeable fused pyrrolocarbazole (FP)-derived small molecule inhibitors of ALK. CEP-14083 and CEP-14513 exhibited potent ALK inhibitory activity in both inÂ€ vitro enzymatic assays (IC50â•›<â•›5Â€ nM) and cell-based assays of ALK tyrosine phosphorylation (IC50â•›<â•›30Â€nM) (Wan etÂ€al. 2006). Treatment of CEP14513 or CEP-14083 led to cell-cycle G1 arrest, followed by accumulation of subG1 population in ALK-positive ALCL cells, including Sudhl-1 and Karpas-299 cells. The G1 arrest that occurred as a result of ALK inhibition correlated with the inhibition of PI3K/AKT and STAT3 activation. These compounds, however, have unfavorable physical properties, and thus, are not suitable for inÂ€vivo use. Another promising group of compounds, identified through a cellular screen used to search for compounds that are cytotoxic to NPM-ALK transfected cells, is NVP-TAE684 (Galkin etÂ€al. 2007). This compound blocked the growth of ALCL-derived cell lines with IC50 of 2 to 10Â€nM. Furthermore, it was effective in rapid and sustained inhibition of ALK phosphorylation and its downstream effectors and resulted in cell cycle arrest and cellular apoptosis. More importantly, the compound also inhibited the development of tumors in xenograft models of ALCL. The favorable pharmacokinetic properties of NVP-TAE684 in mice, including high bioavailability, half-life, and tissue distribution, support future efforts in evaluating these small molecule kinase inhibitors for treatment of ALCLs. Activated ALK induces multiple downstream signaling pathways that represent potential targets for therapy. The most well-studied of these are the PI3K/AKT pathway and the JAK3/STAT3 pathway. ALK exerts anti-apoptotic activity via activation of PI3K/AKT pathway, leading to AKT-mediated phosphorylation of Bad proteins and inhibition of Bad pro-apoptotic activity (Bai etÂ€al. 2000). ALK also results in activation of STAT 3 by either direct phosphorylation by ALK or via JAK 3 activation that leads to upregulation of survivin and Bcl-XL, which results in promotion of cell survival (Zamo etÂ€al. 2002; Amin etÂ€al. 2003; Khoury etÂ€al. 2003). More recent evidence indicates that other pathways including FRAP/mTOR (Vega etÂ€ al. 2006; Marzec etÂ€ al. 2007a), MEK/ERK (Marzec etÂ€ al. 2007b), and HSP90 (Bonvini etÂ€al. 2002) are important mediators of ALK signaling and contribute to tumor cell survival in ALCLs. Because of the observed activation of both proliferation and anti-apoptotic functions by ALK, it has been hypothesized that therapeutic

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approaches that block ALK effects on signaling pathways may be effective. This has been attempted in experimental systems by a variety of approaches, including selective inhibition of STAT3 by adenoviral transfection of a mutated STAT3 molecule (Amin etÂ€ al. 2004), small molecular inhibition of JAK3/STAT3 using cucurbitacin I (JSI-124) (Shi etÂ€ al. 2006), and more recently, ablation of STAT3 expression by antisense oligonucleotides (Chiarle etÂ€al. 2005), which impaired the growth of human and mouse NPM-ALK tumors inÂ€vivo. The inhibition of the PI3K by wortmannin and the inhibition of AKT by small molecule inhibitors (Marzec etÂ€al. 2007a) resulted in decreased proliferation and increased apoptosis of ALCLs. In addition, selective inhibition of the mTOR pathway, which is downstream of AKT by rapamycin and mTOR-specific siRNAs resulted in cell cycle arrest and cellular apoptosis (Vega etÂ€al. 2006). A group of proteasome inhibitors, particularly the benzoquinone ansamycin geldanamycin (GA), have also been identified as agents that interfere with signal transduction of HSP90 client proteins that require HSP90 for the correct confirmation and stability. NPM-ALK is a client protein of HSP90. A recent study demonstrated that an analog of GA, 17-AAG, disturbed NPM-ALK/HSP90 complex formation, thereby destabilizing and degrading the NPM-ALK fusion protein (Bonvini etÂ€al. 2004). Our recent study evaluating the proteomic consequences of inhibition of HSP90 chaperone function in ALCL (Schumacher etÂ€al. 2007) indeed demonstrated the efficacy of GA in decreasing the expression of NPM-ALK and inducing cell cycle arrest in a dose and time-dependent manner. GA is currently being evaluated in phase I clinical trials at the National Cancer Institute for advanced epithelial cancer, malignant lymphoma, and sarcoma. Another group of potential therapeutic targets are represented by the adaptor proteins that are recruited to the autoactivated NPM-ALK chimeric fusion protein. p130Cas (Ambrogio etÂ€ al. 2005) and pp60c-src (Cussac etÂ€ al. 2004) represent two adaptor proteins that bind to phosphorylated and active NPM-ALK and mediate cellular transformation. Inhibition of Src-kinase by small molecules (PPI) or siRNA-mediated ablation of pp60c-src resulted in decreased cell viability of ALCLs (Cussac etÂ€al. 2004). Cytokine and growth factor receptors, which are upregulated in ALCLs, represent another group of therapeutic targets. ALCLs express CD30, a cytokine receptor that is a member of the tumor necrosis factor receptor (TNFR) family (Gruss and Dower 1995; Schneider and Hubinger 2002). Expression of CD30 has been associated with increased proliferation and decreased apoptosis via activation of the nuclear factor kB (NFkB) pathway in Hodgkin lymphoma, but to a much lesser degree in ALCL (Horie etÂ€ al. 1999; Mir etÂ€ al. 2000). A soluble form of CD30 (sCD30) has been detected in the serum of patients with ALCL (Gause etÂ€al. 1992; Nadali etÂ€ al. 1995), and is thought to arise from proteolytic cleavage of the cell surface CD30 by a zinc metalloproteinase (Hansen etÂ€al. 1995). Elevated levels of sCD30 at the time of diagnosis in patients with ALCL are associated with high tumor burden, a lower relapse-free survival, and overall survival, suggesting that this may be a specific indicator for a higher risk of treatment failure (Zinzani etÂ€al. 1998). Furthermore, the expression of CD30 is regulated by ALK.

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Because of its effects on cell proliferation and apoptosis, immunomodulatory therapy using antibodies directed at the ligand binding site for CD30 have been suggested as a means to transduce the apoptotic effects of CD30 binding. Two murine monoclonal antibodies directed against the ligand binding region of CD30 have shown anti-tumor effects in animal models (Tian etÂ€al. 1995). A newly developed human monoclonal antibody has shown anti-tumor activity and is currently entering clinical trials (Borchmann etÂ€al. 2003). SGN-30, a chimeric monoclonal antibody that binds to CD30 and inhibits growth of ALCLs, is currently being evaluated in a Phase I/II pilot study in children with CD30-positive recurrent ALCL (Children’s Oncology Group Trial ANHL06P1). CD52, Campath-1 protein, is another target that has been proposed as a possible target for antibody-mediated therapy. Initial studies on small numbers of pediatric ALK-positive ALCL suggest that the majority (70%) of tumors do express CD52 (Perkins etÂ€ al. 2004 (abstract)) and may respond to use of anti-CD52 immunotherapy. Our recent cDNA microarray analysis of ALCL-derived cell lines and tissue biopsies of pediatric ALCLs indicated the overexpression of IL-2 receptor (CD25) relative to reactive T-cells (Fillmore etÂ€al. 2002; Lim and Elenitoba-Johnson 2006). Additional studies demonstrate that CD25 expression is highly correlated with ALK expression in ALCLs (Lim and Elenitoba-Johnson 2006) and is often accompanied by high levels of serum IL-2 receptors (Janik etÂ€al. 2004). This suggests that use of immune-based therapies such as denileukin diftitox or humanized monoclonal anti-CD25 (daclizumab or basiliximab) would provide an alternative therapeutic approach. There are limited reports of this approach in ALK-positive ALCLs. InÂ€ vitro studies from our laboratory indicate that the aberrant overexpression of IL-2 receptor in ALCLs provides a rational target for therapy using denileukin diftitox. Treatment of ALCL-derived cell lines with denileukin diftitox resulted in a dose- and time-dependent decrease in cell viability, which was associated with increased cellular apoptosis (Lim et al. 2006). The authors are currently evaluating the clinical therapeutic utility of denileukin diftitox in a Phase II pilot study in children, adolescents, and young adults with relapsed or refractory ALCL.

Lymphoblastic Lymphoma LLs represent genetically and clinically heterogeneous neoplasms of immature or precursor lymphoid cells that comprise approximately 30% of the NHLs that occur in children and young adults. The majority of LLs arise from immature T-cells (Crist etÂ€ al. 1988; Uckun etÂ€ al. 1997), while fewer than 10% of LLs are of B-precursor cell origin. The predominant sites of disease in T-LL are the anterior mediastinum and supradiaphragmatic lymph nodes, whereas B-precursor LL is more commonly localized to peripheral lymph nodes and extranodal sites such as skin, soft tissues, and bone, with a predilection for the head and neck regions (Soslow etÂ€al. 1999; Lin etÂ€al. 2000; Neth etÂ€al. 2000; Maitra etÂ€al. 2001). A higher

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incidence of bone marrow and central nervous system involvement is seen in T-LL compared to B-precursor LL. Precursor T-LLs are characterized by a cortical thymocyte immunophenotype. Whereas most T-cell leukemias arise from earlier stages of intrathymic T-cell differentiation (stage I prothymocytes), the majority of T-cell LLs arise from intermediate (stage II) thymocytes (Crist etÂ€al. 1988). Precursor B-LLs most often display an early pre-B or pre B-phenotype (CD19, CD10, and TdT with variable CD20, CD22, HLA-DR and cytoplasmic immunoglobulin expression) (Perkins 2000; Maitra etÂ€al. 2001). ALL and LL are thought to represent a spectrum of a single disease entity, which is termed T (or B) lymphoblastic leukemia/lymphoma in the REAL (Harris etÂ€al. 1994) and WHO (Jaffe 2001) classification schemes. By convention, bone marrow involvement by the tumor must be <25% in LL. When the extent of marrow involvement is >25%, the disease is classified as ALL. Clinically, both B-precursor and T-cell LLs are effectively treated using ALLbased therapies, which include multidrug systemic chemotherapy and central nervous system prophylaxis (Reiter etÂ€al. 2000; Hoelzer etÂ€al. 2002; Goldberg etÂ€al. 2003). Long-term survival rates of 85 to 90% for localized disease and 65 to 85% for advanced stage disease have been achieved, paralleling those seen in ALL (Goldberg etÂ€al. 2003; Link etÂ€al. 1997; Patte etÂ€al. 2001). Although not observed in all studies, evidence suggests that early consolidation with high-dose methotrexate may also be beneficial in both adult and pediatric patients with T-LL (Goldberg etÂ€al. 2003; Asselin 2001; Thomas etÂ€al. 2004). LL and ALL are characterized by many of the same underlying cytogenetic abnormalities, which occur with a frequency of 50 to 90% (Thomas and Kantarjian 2001). As a part of developing antigen recognition capabilities, T and B-lymphocytes rearrange a series of V (variable), D (diversity), J (joining), and C (constant) genetic regions to produce unique antigen receptors. The antigen binding domains of T-cell receptors (TCR) and immunoglobulins (Ig) are unique and serve as clonal markers. Chromosomal translocations are the most frequent cytogenetic abnormalities that occur in T-LL (TableÂ€3). These chromosomal translocations frequently juxtapose promoter and enhancer elements from TCR genes next to transcription factors genes such as HOX11, TAL1, and LYL1, which are normally transcriptionally silent in normal T-lymphocytes (Sandlund etÂ€ al. 1996; Goldsby and Carroll 1998; Heerema etÂ€ al. 1998; Ferrando etÂ€ al. 2002).The most common translocations in T-LL involve TCRad (14q11), TCRb (7q32-36), and TCRg (7p15), with the more immature cellular phenotypes favoring the TCRg or TCRd loci (TableÂ€3). While the prognostic significance of these cytogenetic features is unknown in LL, many of these same cytogenetic findings have not been shown to have prognostic significance in pediatric T-ALL (Heerema etÂ€al. 1998). Recent developments in molecular cytogenetics including fluorescent in situ hybridization and mutation analysis revealed cytogenetically cryptic aberrations in most cases of T-ALL. Gene expression profiling studies using DNA microarrays have revealed that T-ALLs recapitulate specific stages of thymocyte maturation and have also identified potential targets for molecular therapies. Furthermore, there is support for the accumulation of multiple genetic abnormalities in the pathogenesis

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TableÂ€3â•… Cytogenetic and molecular changes in lymphoblastic lymphomas Involved Function of fusion gene or gene(s) Protein(s) expressed oncogene Translocations involving TCR genes t(7;10)(q34;q24) and TLX1 t(10;14)(q24;q11) (HOX11) t(1;14)(p32;q11) and t(1;7)(p32;q34) t(7;9)(q34;q32) t(7;19)(q34;p13) t(11;14)(p15;q11) t(11;14)(p13;q11) and t(7;11)(q35;p13) t(1;7)(p34;q34) Formation of fusion genes t(10;11)(p13;q14) (often cryptic)

Transcription factor

TAL1

Class II homeodomaincontaining HLH type II

Transcription factor

TAL2 LYL1 LMO1 LMO2

HLH type II HLH type II LIM-only domain LIM-only domain

Transcription factor Transcription factor Protein–protein interaction Protein–protein interaction

LCK

SRC family of tyrosine kinase

Signal transduction

CALM/

ENTH motif containing Zinc fingers/ leucine zippercontaining Homeodomain protein HLH type II

AF10

t(1;9)(q23;p13)

PBX1

21q addition

E2A Unknown

Transcription factor

Transcription factor Transcription factor

TCR T-cell receptors, ENTH epsin N-terminal homology, HLH helix-loop-helix

of T-ALL/LL. Gene expression studies performed to identify global expression signatures associated with T-ALL (Ferrando etÂ€al. 2002) have identified major oncogenic pathways associated with HOX11, TAL1, and LYL1 activation and help define molecular subtypes within the morphologically homogeneous disease (Fig.Â€ 10) (Ferrando and Look 2003). These seminal observations indicated the overexpression of key oncogenes in the majority of T-ALL and occurred frequently in the absence of translocations suggesting alternate mechanism of oncogene activation. Distinct molecular subtypes with varying treatment outcomes were identified based on the microarray analyses. Those with HOX11 expression were associated with a 92â•›±â•›8% 5-year survival, whereas those with TAL1 and LYL1 exhibited a much lower survival rate at 43â•›±â•›19% and 33â•›±â•›19%, respectively. Furthermore, subtypes with specific oncogene activation were associated with specific states of thymocyte differentiation. Those with expression of HOX11 tended to arise from earlier cortical thymocytes, whereas those associated with TAL1 expression possessed mature, late cortical thymocyte differentiation (Fig.Â€11) (Ferrando and Look 2003). To elucidate the genetic basis for T-LL, Raetz and coworkers (2006) compared the global gene expression profiles of T-ALL and T-LL using an Affymetrix U133A

New Therapeutic Frontiers for Childhood Non-Hodgkin Lymphoma TableÂ€4â•… Molecular targets for ALCL and LL Gene product or Subtype pathway Cellular function ALCL ALK Tyrosine kinase

201

Inhibitors/modifier

NVP-TAE684 CEP-11988 WHI-131, WHI-154 Wortmannin, LY PI3K/AKT Protein kinase ShRNA STAT3 Transcription factor AG490, Curcumin JAK3 Tyrosine kinase 17-AAG and Geldanamycin HSP90 Chaperone PPI pp60SRC Protein kinase Rapamycin, CCI-779 Protein kinase mTOR U01206 Protein kinase MAPK/ERK Denileukin diftitox, Growth cytokine receptor IL-2R Daclizumab SGN-30 Cytokine receptor CD30 Compound E Protooncogene LL NOTCH Benozochazepine (Bz-423) Protooncogene C-MYC Rapamycin, CCI-779 Protein kinase mTOR ALCL anaplastic large cell lymphoma, LL lymphoblastic lymphoma, ALK anaplastic lymphoma kinase, PI3K phosphatidylinositol-3-kinase, STAT3 signal transducer and activator of transcription 3, JAK3 janus kinase 3, HSP90 heat shock protein 90, mTOR mammalian target of rapamycin, MAPK/ERK mitogen-activated protein kinase/extracellular signal-regulated kinase, IL-2R interleukin-2 receptor, ShRNA small hairpin RNA, 17-AAG 17-N-allylamino-17-demethoxygeldanamycin, PPI protein–protein interaction, CCI-779 cell cycle inhibitor-779, SGN-30 Seattle Genetics’ anti-CD30 monoclonal antibody

GeneChip composed of over 22,283 probe sets. Unsupervised hierarchical clustering of ten T-ALL bone marrow samples and nine T-LL tissue samples led to the complete segregation of T-ALL and T-LL into distinct groups. Furthermore, they identified over 200 genes, which were able to differentiate between the diseases using significance analysis of microarrays. The results indicate the clear differences in the gene expression profiles between T-ALL and T-LL, suggesting the underlying differences in the biology of the two entities. Future studies in the identification of downstream target genes of the commonly overexpressed oncogenes will lead to identification of potential targets for therapy. The main categories of functional molecular abnormalities identified in LL and those that represent potential targets for therapy are discussed below. Defects in cell cycle control due to genetic loss of INK4 locus (p16/p14) and aberration of Rb1 and p53 are common in T-LL (Okuda etÂ€al. 1995; Cayuela etÂ€al. 1996). In addition, gene rearrangements can result in deregulated expression of transcription factors that lead to aberrant cell cycle control. For example, CALM-AF10 T-ALLs overexpress BMI1 that leads to suppression of p16. Another frequent underlying genetic event in T-LL is dysregulation of TAL1 (1p32), which is estimated to occur with a frequency of 30% (Baer 1993; Bash etÂ€al. 1995). Tumor-specific alterations in TAL1 occur by distinctive mechanisms. In a small percentage of cases, TAL1 is juxtaposed next to

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Fig.Â€10â•… Hierarchical cluster analysis of T-ALL gene expression data. The tree (top) shows the relatedness of gene expression among samples and is color-coded according to the expression of three major T-ALL transcription factor oncogenes, TAL1, HOX11, and LYL1. Each column represents a T-ALL mRNA sample and each row one of the genes in the microarray, selected based on a permutation test for genes best distinguishing among HOX11+, TAL1+, LYL1+, and unclassified samples (Other). Gene expression values are color-coded, as indicated by the scale beneath the graph. HOX11+, TAL1+, and LYL1+ cases are grouped in major branches of the dendrogram, while the unclassified cases are split into two groups, one related to HOX11+ cases (HOX11-like, branch H2) and the other related to TAL1+ cases (TAL1-like, branch T2). Three cases in the H2 cluster express high levels of HOX11L2, a HOX11-related gene, demonstrating the ability of the hierarchical analysis to cluster together cases with related mechanisms of transformation. Three cases expressing the MLL-ENL fusion oncogene were clustered together based on the gene expression signature as a major independent group (branch M). Numerous cytogenetic and molecular abnormalities (discrete symbols at the bottom of the tree) are found in cases that were clustered by the analysis, supporting the hypothesis that the gene expression profiles define major subgroups of T-ALL that arise from different multistep molecular pathogenetic pathways. From Ferrando and Look 2003. Copyright Elsevier, used with permission

TCR promoter/enhancer sequences as the result of t(1;14) or t(1;7) chromosomal translocations. More commonly, deletions of a regulatory region of TAL1 are observed that position TAL1 next to the promoter of the active SIL gene (Brown etÂ€al. 1990; Janssen etÂ€al. 1993). In both cases, deregulated expression of the full-length TAL1 gene occurs. TAL1 is a basic helix-loop-helix DNA binding protein that heterodimerizes with E2A proteins (E47 and E12), regulating transcription of target

New Therapeutic Frontiers for Childhood Non-Hodgkin Lymphoma

MLL-ENL LYL1+

γδ CD34, BCL2 CD7, IL7R

HOX11+/ HOX11L2+

TAL1+

203 CD8+ CD4-

TCRD TCRG

αβ

CD4- CD8+

Double Negative Thymocytes

CD10, CD1A CD1B, CD1C CD45

CD6, LCK CD3D, CD3E TCRB, TCRA

Late Cortical

Early Cortical CD4+ CD8+

CD8CD4+

Mature Single Positive T-cells

Double Positive Thymocytes Fig.Â€ 11â•… Correspondence of gene expression signatures of LYL1+, MLL-ENL+, HOX11+ and TAL1+ T-ALL samples with recognized stages of thymocyte differentiation. LYL1+ cases have a gene expression signature corresponding to that of the most immature normal T-cell precursors (CD4/CD8 double-negative cells), which express CD34 but not CD4, CD8, or CD3. Normal thymocytes can become committed to either the gamma-delta or the alpha-beta lineages of T-cell maturation. MLL-ENL+ T-ALL appears to correspond to thymocytes with aberrant differentiation along the gamma-delta lineage. As thymocytes within the alpha-beta lineage mature, they lose CD34 expression, while gaining CD4 and then CD8, eventually becoming double-positive thymocytes. HOX11+ cases have gene expression signatures corresponding to early double-positive cells that express CD1 and CD10 (early cortical thymocytes). TAL1+ corresponds to thymocytes that express both T-cell receptor RNAs and CD3 (late cortical thymocytes). Normal thymocytes that have undergone both positive and negative selection can then proceed through a final step of differentiation, in which they downregulate the expression of either CD4 or CD8 to become mature single-positive T cells, ready for export from the thymus. From Ferrando and Look 2003. Copyright Elsevier, used with permission

genes involved in cell growth and differentiation. TAL1 is normally expressed in megakaryocyte, mastocyte, and eyrthroid lineages but not in normal T-cells, and has been shown to have critical functional roles in early hematopoiesis (Delabesse etÂ€al. 1997; Hall etÂ€ al. 2003). Recent studies have shown that TAL1, similar to PMLRARa and AML-1/ETO, contributes to leukemogenesis by repressing gene expression and inducing a differentiation arrest in developing thymocytes (O’Neil etÂ€ al. 2004). TAL1 overexpression negatively regulates the p16 gene through interference with the E boxes sequences of the p16 promoter (Hansson etÂ€al. 2003). E2A, which is deregulated as a result of t(1;19)(q23;p13) in B-LL, positively regulates several cyclin-dependent kinase inhibitors’ promoters and negatively affects cell growth. Furthermore, HOX11 can act on the cell cycle by its interactions of protein serine/ threonine phosphatases such as PP2A and PP1 (Kawabe etÂ€al. 1997). Although the use of inhibitors of transcription factors has not been well developed, cell cycle regulators can provide targets for therapy (Fahraeus etÂ€al. 1996). Mutations in several key oncogenes have been identified in T-ALL. Gain of function NOTCH1 mutations have been recently identified as one of the most

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Â� common genetic abnormalities in T-ALL, occurring in more than 50% of cases (Weng etÂ€ al. 2004). NOTCH1 encodes a transmembrane receptor that regulates normal T-cell development. Activated NOTCH1 has been shown to potently induce T-ALL in murine models, and in a recent analysis of 96 diagnostic bone marrow samples from children with T-ALL, at least one activating mutation in the extracellular heterodimerization domain and/or the C-terminal PEST domain of NOTCH1 was present in 54 cases (56.2%). NOTCH1 mutations were observed in all molecularly defined subtypes of T-ALL, characterized by HOX11, TAL1, and LYL1 activation. Furthermore, recent studies using expression-profiling screens have led to the identification of C-MYC as a direct target of NOTCH in NOTCH-dependent T-ALL cell lines. Inhibitors of C-MYC interfered with the growth promoting effects of activated NOTCH1. C-MYC appears to be an important developmentally regulated mediator of I signaling in certain stages of thymocyte development. The discovery of aberrant NOTCH signaling highlights the potential use of gamma secretase inhibitors that interfere with NOTCH signaling as a potential therapy for T-LL. Furthermore, immunomodulatory agents such as benzodiazepine Bx-423, which target C-MYC protein for rapid and specific degradation (Sundberg etÂ€ al. 2006), may have a role in therapy of T-LL. A number of tyrosine kinase genes are activated as a result of chromosomal translocations or by gene mutation. Although rare, activating mutations (internal tandem duplications or point mutations) of FLT3, a receptor tyrosine kinase important in the development of hematopoietic stem cells, occur in T-ALL, with very immature phenotype expressing LYL1, LMO2, and the KIT receptor (Paietta etÂ€al. 2004). Similarly activating mutations of LCK and ABL1 interfere with preTCR and TCR signaling and provide a survival and proliferative advantage to T-ALL. ABL1 is a ubiquitously expressed cytoplasmic tyrosine kinase which plays a role in TCR signaling and is involved in the NUP214-ABl1 fusion found in approximately 6% of T-ALL. This fusion gene is expressed in amplified episomes, which are not cytogenetically visible. The resultant constitutive activation of ABL1 leads to activation of survival and proliferation pathways, which are sensitive to imatinib (Griesinger etÂ€ al. 2002). Although they are rare and occur in only 10 to 20% of T-ALL, they represent a very viable mode of targeted therapy using selective small molecule kinase inhibitors that are effective and improve bioavailability and pharmacokinetic properties. Future studies will determine whether T-LLs harbor activating mutations of FLT3, ABL1, LCK, and NOTCH1. Another downstream target of NOTCH activation identified by an innovative approach, utilizing protein microarrays composed of 108 epitopes representing 82 phosphorylated signaling proteins (Chan etÂ€ al. 2007), is the mTOR pathway. Simultaneous blockade of the mTOR pathway by rapamycin and the NOTCH pathway using GSI resulted in synergistic suppression of T-ALL growth. Similarly, the activation of phosphatidylinositol 3-kinase (PI3-K) (Barata etÂ€al. 2004), Akt (Sade etÂ€ al. 2004), extracellular signal regulated kinase ½ (Talora etÂ€ al. 2003) STAT5 (Kelly etÂ€ al. 2003b), and nuclear factor kB (Bellavia etÂ€ al. 2000) in T-ALL, all represent targets for combinatorial therapeutic targeting as a novel therapeutic approach for T-LL.

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Cytogenetic and molecular abnormalities in B-LL are less well-characterized. Classical chromosomal translocations which occur in B-precursor ALL, such as hyperdiploidy, t(12;21), t(1;19) and t(9;22), appear to occur less frequently in B-precursor LL (Maitra etÂ€ al. 2001; Head and Behm 1995). Reported cytogenetic abnormalities in B-precursor LL include additional material from the 21q locus (Maitra etÂ€al. 2001). As expected, precursor B-LLs will usually show monoclonal Ig gene rearrangements and lack evidence of somatic hypermutation (Hojo etÂ€al. 2001).

Summary and Future Directions The prognosis for childhood and adolescent NHL is excellent to outstanding with multi-agent chemotherapy without concomitant surgery and radiotherapy. The major future challenges are mainly to discover the molecular and cellular pathogenesis of malignant transformation (lymphomagenesis) to develop strategies of prevention, early identification, and more selective targeted therapies. A significant increase in new knowledge about the genetic basis for malignant transformation and prognosis has been identified in the last 10 years in adult DLBCL. It remains to be determined whether childhood and adolescent DLBCL has similar or disparate genetic and cellular mechanisms as adult DLBCL. While we have commonly treated BL and DLBCL under similar childhood chemotherapy regimens, biologically the two histological subtypes are dramatically different and further investigations will be required to identify the exact genetic differences that may lead to different therapeutic approaches. Primary mediastinal large B-cell lymphoma is likely a distinct genetic entity from other forms of DLBCL and may genetically resemble HD more than DLBCL. These childhood and adolescent primary mediastinal B-large cell lymphomas require more intense genetic and cellular investigations to help determine the correct therapeutic approach. Significant progress has been made in the identification of molecular targets for therapy in ALCLs. The challenges for the future of molecular targeted therapeutics in ALCLs lie in deciphering the complexity and diversity of the signaling pathways that are activated as a result of the NPM-ALK overexpression. Determining which of the multiple overlapping and redundant pathways are critical in promoting cell survival, proliferation, adhesion, and invasive phenotype is not clear and remains technically challenging with the model systems available. It would seem logical that inhibition of multiple pathways with either nonselective small molecular inhibitors or multiple inhibitors that target multiple proteins would represent reasonable approaches. Enhanced chemosensitivity that is observed with many signal transduction inhibitors in combination with routine chemotherapeutic agents would provide another alternative approach. On a socio-economic level, obtaining financial support to carry out clinical trials on a disease group that is considered an “orphan” or “rare” disease is another significant challenge and one that would benefit from international multi-institutional collaborative efforts and support from the Children’s Oncology Group.

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There is still a paucity of information on the genetic basis of LL-associated lymphomagenesis. Do the same genetic and cellular events that regulate T-ALL occur in T-LL? Are the transcription factors identified in the leukemogenesis process of T-ALL similar or disparate in T-LL? Few targets have been identified in T-LL compared to B-NHL and ALCL. New target identification in T-LL will facilitate the development of selective targeted therapy and hopefully reduce the morbidity currently associated with 2 years of prolonged therapy in T-LL. The integration of new agents into treatment approaches is needed to improve outcome for patients with a poor prognosis. Moreover, it is anticipated that future development of agents specifically targeted to underlying transforming pathways may replace more toxic conventional chemotherapeutic drugs. Acknowledgmentsâ•… The authors would like to thank Erin Morris for her assistance in the development of this manuscript and Sean Park for his preparation of the ALCL figures. Supported in part by grants from the Pediatric Cancer Research Foundation, Andrew Gargiso Foundation, Sonia Scaramella Fund and National Institutes of Health.

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Molecular Targeting of Post-transplant Lymphoproliferative Disorders Michael Wang and Thomas G. Gross

Introduction Post-transplant lymphoproliferative disorders (PTLD) represent a heterogeneous group of life-threatening lymphoproliferative disorders that can be observed in a transplant recipient. PTLD can occur in patients after solid organ transplantation (SOT) because of immunosuppression to prevent graft rejection (Penn etÂ€al. 1969), and continues to be a major cause of morbidity and mortality seen in about 10% of pediatric SOT recipients. There is a higher incidence in children following SOT than in adults (Ho etÂ€al. 1988; Swerdlow etÂ€al. 2000), with highest incidence of 20% following heart-lung transplant. PTLD occurs in hematopoietic stem cell transplantation (HSCT) recipients secondary to the immunosuppression of pre-HSCT preparative regimens, and the post-HSCT immunosuppression to prevent graft vs host disease (GVHD). PTLD in HSCT occurs at a lower rate than following SOT (approximately 1%), with the vast majority occurring within 6Â€months following HSCT (Bhatia etÂ€al. 1996; Curtis etÂ€al. 1999). Accordingly, few cases of PTLD have been reported after autologous HSCT (Lones etÂ€al. 2000; Nash etÂ€al. 2003). PTLD is associated with Epstein–Barr virus (EBV) and inadequate EBV immunity in the majority of cases. PTLD following HSCT is essentially all EBV-associated. EBVnegative PTLD occurs following SOT in as many as 30% of cases. (Leblond etÂ€al. 2001). The pathogenesis, treatment strategies and outcome differ from EBVpositive PTLD, as EBV-negative disease tends to require more aggressive therapy and portends a worse prognosis. This chapter will focus on EBV positive PTLD and molecularly targeted therapies in its prevention and treatment. The World Health Organization classification of PTLD contains three categories (Harris etÂ€al. 1999). Early lesions, which are usually felt to not represent “true PTLD,” are characterized by reactive plasmacytic hyperplasia with normal tissue architecture retained. When normal tissue architecture is disrupted by the lymphoproliferative T.G. Grossâ•›(*) Division of Hematology/Oncology/BMT, The Ohio State University College of Medicine, 700 Children’s Drive, Columbus, OH 43205, USA e-mail: [email protected]

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process, then the diagnosis of PTLD is appropriate. Polymorphic PTLD comprises different cells types, B cells, T cells, and histiocytes, and can be either monoclonal or polyclonal. Monomorphic PTLD is diagnosed when the lesions contain a more homogeneous population of transformed lymphocytes and resemble frank lymphoma such as B cell lymphoma (Burkitt, Burkitt-like lymphoma, diffuse large B cell lymphoma, or plasma cell myeloma), T cell lymphoma (peripheral T cell lymphoma, gd T cell lymphoma, hepatosplenic T cell lymphoma, T/NK lymphoma), and other types, such as Hodgkin disease-like and plasmacytoma-like lymphomas. Of interest, lymphoblastic and anaplastic large cell histologies are not associated with PTLD or immunodeficiency. Following SOT, PTLD are usually recipient in origin; however, following HSCT, PTLD is usually donor origin. Risk factors for developing PLTD after SOT include: type and intensity of immunosuppressive therapy, transplant from an EBV-seropositive donor into a seronegative recipient, and small bowel or heart-lung transplant (Opelz and Dohler 2004). HSCT PTLD risk factors are: T cell depletion, ATG as prophylaxis or therapy for GVHD, HLAmismatched transplant, and immunodeficiency as the primary diagnosis (Gross etÂ€al. 1999; Baker etÂ€al. 2003). The signs and symptoms of PTLD reported by patients are diverse and a high index of suspicion is necessary to make the diagnosis (Collins etÂ€al. 2001). Symptoms may include those suggestive of acute mononucleosis, but PTLD can involve almost any organ system, including the central nervous system. Fulminant disease can mimic sepsis, GVHD, or a hemophagocytic syndrome. Most PTLD presents as lymphoproliferation with extranodal involvement being the rule, not the exception. Recently, several investigators have demonstrated that EBV-PTLD is associated with increase in EBV DNA load in the peripheral blood of patients following SOT and HSCT (Stevens etÂ€al. 2001; Holmes etÂ€al. 2002; Wagner etÂ€al. 2004; Greenfield etÂ€al. 2006). Polymerase chain reaction (PCR) used to measure EBV DNA has been suggested as a method to make a preemptive diagnosis of PTLD. However, using quantitative real-time PCR assays relies on the assumption that growth of EBVinfected B cells correlates with increases in peripheral blood EBV DNA. This assumption has been demonstrated to be true with CMV monitoring in HSCT patients (Boeckh etÂ€al. 2003). The main difference is that CMV monitoring reflects active viral replication, whereas EBV-PTLD is associated with latent virus. Thus, detection of elevated levels of EBV DNA may point toward a diagnosis of PTLD, but signs and symptoms along with correlative imaging and tissue biopsy, if at all possible, must be analyzed before therapy is started. New approaches to EBV monitoring include assessing EBV-specific cytotoxic T cells (CTLs). One method measures interferon-g using either Elispot or flow cytometry assaying intracellular cytokine. The use of Elispot in combination with EBV DNA viral load has been reported to have a very high positive predictive value in predicting PTLD development (Smets etÂ€al. 2002). MHC-class I peptide tetramers can quantify CTLs-specific EBV antigens, both lytic and latent viral antigens, and have been used to measure the recovery of EBV-specific CTL after HSCT. The predictive value of developing PTLD appears much higher with low numbers of EBV-specific CTL rather than with high levels of EBV DNA (Meij etÂ€al. 2003).

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Current strategies to treat EBV-PTLD have largely involved the experience of the clinician or retrospective case series of patients. There is no clear consensus therapy as the disease is heterogeneous and patient co-morbidities may limit therapeutic options. Most first-line treatment strategies involve reducing or changing the type of immunosuppressive medications. This has been most effective in patients who have polymorphic PTLD or early stage disease; however, it may be effective even in monoclonal or disseminated disease. Surgery and radiation have also shown good efficacy for localized disease, though this is feasible only in a minority of patients. For patients who do not respond to reduction of immunosuppression, especially those who have monomorphic PTLD or lymphoma, chemotherapy has been used. Standard lymphoma regimens are associated with increased toxicities in the transplant population. In children with PTLD following SOT, it appears that a low-dose regimen, that is, cyclophosphamide and prednisone, may be as effective as standard lymphoma regimens without as much toxicity (Gross etÂ€al. 2005). A number of targeted therapies are currently being evaluated and will be the focus of discussion of this chapter.

Epstein–Barr Virus Infection of Humans To discuss potential preventative and treatment strategies of PTLD, a review of human EBV infection is necessary (Dolcetti and Masucci 2003; Young and Rickinson 2004). EBV is a human gamma-herpes virus that establishes latency in about 90% of the adult population. EBV infects naïve B cells by entry through the gp350-CD21 interaction causing a polyclonal expansion of EBV-transformed lymphoblasts in oropharyngeal lymphoid tissue. This leads to a lytic infection usually in tonsillar B cells and serves as the site of production of infectious EBV particles for spread of the virus to subsequent hosts. In most B cells that disseminate throughout the body, viral latency is determined by one of three latent gene transcription programs. The first is the “growth” program, type III latency, where all of the latent genes are expressed: Epstein–Barr nuclear antigens (EBNAs -1, -2, -3A, -3B, -3C, -LP), latent membrane protein (LMP1, LMP2A and LMP2B), polyadenylated viral RNAs (EBER1 and EBER2), and BamH1 RNAs (BARTs). Latency type III promotes proliferation and survival of infected naïve B cells without differentiation. This is accomplished in part by the expression of EBNA-2, which mimics Notch signaling (Hofelmayr etÂ€al. 2001). The control of this EBV-driven B cell expansion is by T cells, with both CD4(+) and CD8(+) cells playing important roles. This interplay and ultimate establishment of latency represents a careful co-evolution of the human immune system and EBV (Thorley-Lawson 2001). After exiting the cell cycle, EBNA-2, the main latent gene transactivator of the latency type III program and inhibitor of B cell differentiation, is turned off. These B cells now enter type II latency where B lymphoblasts migrate to lymphoid follicles and differentiate into a germinal center phenotype. Differentiation of B cells is now driven by LMP1 and LPM2A independent of antigen signals from follicular dendritic cells or T cell help. LMP2A suppresses B cell receptor signaling but

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enhances signaling through Syk and Src – family kinases for B cell survival (Dykstra etÂ€al. 2001). The survival signals normally provided by T cell help come from LMP1, which is a functional mimic of CD40. Both LMP2A and LMP1 are transmembrane proteins, ligand-independent and constitutively active, organizing themselves in lipid rafts (Kilger etÂ€al. 1998). B cells that survive type II latency join the pool of long-lived memory B cells where EBV persists for the lifetime of the host. Type I latency is characterized by the absence of EBV gene expression except for the periodic expression of EBNA-1 that allows the viral episome to be passaged extrachromosomally during cell division. A latency 0 stage has been suggested where EBER 1 and 2 and possibly BARTs are expressed, but the remainder of the EBV genome is silenced. With the lack of EBV gene expression in latency stages 0 and 1, host immune memory established after the primary infection with EBV would be unable to detect EBV infected cells, and EBV can persist by evading immune detection, and hence, establishing a lifelong infection (TableÂ€1).

Host Immune Response Against EBV Much of the understanding and monitoring of primary EBV infection is derived from early serological work that investigated IgM and IgG titers to EBNAs expressed in latently infected cells. Most of these data today are derived from patients with infectious mononucleosis (IM) (Pedneault etÂ€al. 1998). When patients present with IM, they usually have high titer IgM to viral capsid antigen (VCA) and rising IgG titers to VCA and early antigen (EA). The IgG anti-EA response falls more rapidly and to lower levels than the IgG anti-VCA. In addition, IgG responses to EBNA2 occur in the acute phase, while IgG anti-EBNA-1 responses occur in late in convalescence. Antibody responses to LMP1 or LMP2 are not seen in IM. Healthy EBV carriers are IgG anti-VCA, anti-gp350 neutralizing antibody and, IgG anti-EBNA-1 positive, but can also carry stable antibody reactivity against other EBV proteins such as EA (Rickinson and Kieff 2007). Serologic studies can be misleading in transplant patients due to the immunosuppression and/or passive antibody transfer from blood products or supplemental gammaglobulin. Cellular immune responses are vital for controlling primary EBV infection and subsequent EBV reactivation. The importance of the innate and adaptive cellular immune system is best exemplified through understanding genetic defects in patients susceptible to EBV disease, such as X-linked lymphoproliferative disorder (XLP), or signaling lymphocyte activation molecule (SLAM)-associated protein deficiency (Veillette etÂ€ al. 2007), and X-linked inhibitor-of-apoptosis (XIAP) (Rigaud etÂ€al. 2006). The defect in innate cellular immunity caused by mutations in these genes has been shown to alter normal development of NKT cells, likely due to abnormal peripheral homeostasis. Following EBV infection, patients with these diseases also demonstrate abnormalities in adaptive immune responses. SAP mutations have been shown to influence the ability of CD4(+) T cells to secrete certain cytokines

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TableÂ€1â•… Potential targeted treatment strategies for EBV associated PTLD Target Desired result Potential agents Potential obstacles EBV latent genes BHRF1 (Bcl-2 homologue) Enhance None currently Delivery & tumor apoptosis specificity LMP1 (upregulates NF-Kb Enhance None currently Delivery & tumor gene expression) apoptosis specificity LMP2A (increases LMP1 Enhance None currently Delivery & tumor half-life via RAS/PIK/akt) apoptosis specificity EBNA1 (can inhibit p53) Enhance None currently Delivery & tumor apoptosis specificity EBV lytic genes BCRF1 (IL-10 homologue) Enhance cellular None currently Delivery & tumor specificity immune response BARF1 Enhance cytokine None currently Delivery & tumor response specificity None currently Delivery & tumor Enhance viral gp350, ZEBRA VCA, specificity & potential replication & thymidine kinase viral of increase infected cell lysis DNA polymerase B-cell pool and future PTLD risk Cellular proteins Death receptors (TRAIL, Enhance Monoclonal Tumor specificity etc) apoptosis antibody Tumor specificity & Monoclonal Tumor cell surface antigens Enhance tumor activity in immune kill antibody compromised host (apoptosis, ADCC, etc) Growth signaling pathways Growth inhibition Sirolimus, Efficacy and toxicities everolimus remain (mTOR, etc) to be determined Enhanced immune response to EBV Humoral response vaccine Inhibit EBV Anti-gp350 Does not prevent infection infection & activity in immune compromised host None currently Activity in immune Cellular response vaccine Enhance control compromised host of EBV B-cell proliferation Limited to few Activity in immune Adoptive EBV-specific Enhance control centers compromised host cellular therapy of EBV B-cell proliferation

and mediate B cell help (B cell antibody production). In addition, SAP mutations decrease the proliferation, cytokine production, and cytotoxicity of CD8(+) T cells. The main defect in XIAP deficiency patients is increased activation-induced cell death of B cells, CD4(+) and CD(8+) T cells. Cellular responses to EBV infection in immunocompetent individuals have been well characterized (Callan etÂ€ al. 1996; Rickinson etÂ€ al. 2000; Hislop etÂ€ al. 2007).

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CD8(+) T cells undergo a multifold expansion during IM. In some individuals, this can be seen as large expansions of Vb-restricted CD8(+) T cells to epitopes from both latent and lytic cycle proteins. Lytic epitopes have been shown by tetramer staining to represent up to 50% of the CD8(+) T cell pool, whereas latent epitopes accounted for less than 5% (Callan etÂ€al. 1998). The lytic response is directed against immediate early antigens (BZLF1 and BRLF1) and early antigens (BMLF1, BMRF1, BALF2, and BALF5). Most responses directed against latent epitopes include EBNA3A, -3B, -3C, LMP2 and EBNA1. Rarely is a response seen against EBNA-2, EBNA-LP, or LMP1. The pool of antigen expanded T cells undergo contraction, and after 1 to 2Â€years, CD8(+) T cells specific for lytic antigens represent 2 to 5% of the T cell pool and have an effector memory T cell phenotype (Callan etÂ€al. 1998; Hislop etÂ€al. 2002). In contrast, CD8(+) T cells specific directed against latent antigens only represent 0.5 to 2% of peripheral T cells but have a central memory phenotype (Catalina etÂ€ al. 2001). CD4(+) T cell immunity has been less characterized than CD8(+) T cells (Maini etÂ€al. 2000; Williams etÂ€al. 2005). Clones that recognize lytic-antigens BZLF1, BHRF1, gp350 and gp110 have been characterized. CD4(+) T cell memory to latentantigens has been more extensively studied, and the hierarchy of immunodominance is different from that of CD8(+) T cells (Woodberry etÂ€ al. 2005). Most memory responses are focused on EBNA-1, EBNA-2, and EBNA-3C proteins. These clones secrete IFN-g and display a TH1 phenotype. Whether they have T cell effector function inÂ€vivo is unknown. NK cells also play a role in EBV immunity by linking adaptive and cellular immunity. It is likely that increased NK cell numbers function to initially control primary EBV infected B cells and protect humans during early infection. Additionally, they secrete cytokines that enhance T cell immunity, which ultimately resolves the primary EBV infection (Williams etÂ€al. 2005). EBV evades the immune system by cytokine modulation and the production of a viral homologue of IL-10 (vIL-10, the product of EBV gene BCRF1) during the lytic infection. vIL-10 binds to the IL-10 receptor with reduced affinity and is associated with decreased secretion of IL-12 and interferon-g (Moore etÂ€ al. 2001). This may result in suppression of Th1 T cell responses as infected cells transition from lytic to latent infection (Bejarano and Masucci 1998). Viral IL-10 also downregulates the expression of transporters associated with antigen presentation (TAP) proteins and interferes with CTL recognition of EBV-infected B cells (Zeidler etÂ€al. 1997). Finally, during lytic infection, a soluble receptor for colony stimulating factor 1, BARF1, is expressed. BARF1 may inhibit cellular immune function by blocking antiviral cytokine (interferon-a) release from monocytes (Cohen and Lekstrom 1999).

EBV Targets Apoptotic Resistance Apoptosis is potential target of EBV. Early studies revealed that expression of EBV latency genes could prevent death from serum withdrawal (Gregory etÂ€ al. 1991). BHRF1 is a Bcl-2 homologue that inhibits apoptosis induced by Fas ligand and TRAIL (Kawanishi etÂ€ al. 2002). LMP1 upregulates multiple anti-apoptotic genes (bfl-1, mcl-1, A20, clAP2) that are downstream targets of NF-kb (Young etÂ€ al.

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1999), and Bcl-2 (Henderson etÂ€ al. 1991). LMP1 via phosphoinositide-3 kinase (PI3K) (Lambert and Martinez 2007) induces IL-10 production that alters T cell immunity. LMP2A signaling can constitutively activate the RAS/PI3K/Akt pathway and prolongs the half-life of LMP1. It is assumed that the major function EBNA1 is maintenance of the EBV genome in latency 1 by binding ubiquitin-specific protease 7 (USP7) to prevent p53 stabilization by deubiquitination (Saridakis etÂ€ al. 2005). EBNA2 can block apoptosis by binding Nur77 and inhibiting cytochrome C release from mitochondria after it translocates out of the nucleus into the cytoplasm (Lee etÂ€al. 2002). Finally, death receptors may play a role as EBV lymphoblasts derived from PTLD patients exhibit resistance to TRAIL-induced apoptosis (Snow etÂ€ al. 2006). To date, there are no clinical data on the use of anti-apoptosis agents to treat EBV disease of EBV-PTLD, though there are several interesting potential targets.

Molecular Targets for EBV-PTLD Vaccines Targeting EBV antigens with vaccines is an attractive therapeutic option for all malignancies associated with EBV, including nasopharyngeal carcinoma and endemic Burkitt lymphoma. Recently, an EBV recombinant gp350 vaccine completed a phase II, randomized, double-blind, placebo controlled trial for IM (Sokal etÂ€al. 2007). The rationale for choosing gp350 as an immunologic target was that EBV predominantly infects B cells through binding of gp350 to CD21 (Nemerow etÂ€al. 1987). Therefore, a neutralizing anti-gp350 antibody response should theorectically prevent EBV infection of B cells. This study showed that the recombinant gp350 vaccine decreased the number of symptomatic infections from 10% in the control group to 2% in the vaccine treated group. However, it did not reduce the incidence of asymptomatic EBV infections. The utility of vaccines that induce a humoral response to EBV for PTLD development remains to be determined. It is unclear if this vaccination approach would decrease the risk of PTLD for HSCT or SOT recipient who are EBV naïve prior to transplant. It is possible that gp350 vaccine might decrease EBV viremia and hence reduce the risk of EBV related posttransplant disease even in EBV positive patients. Vaccines to produce effective cellular immune responses should have greater utility in prevention of EBV related disease; however, progress in the development of a vaccine that produces an effective cellular response to latent virus infection has been slow in coming.

Anti-Viral Medications The role of anti-viral medications has not been substantiated even as prophylaxis of patients at risk for developing EBV-PTLD as thymidine kinase inhibitors do not suppress the proliferation of EBV-infected B cells during latency. These medications

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cannot eradicate latently infected lymphocytes as EBV-thymidine kinase and BGLF4 gene product, which are necessary to phosphorylate gancyclovir and convert it to its active cytotoxic form, are expressed only during the lytic infection. One potential novel therapy to enhance the efficacy of gancyclovir would be to intentionally induce EBV lytic infection. This approach takes advantage of the expression of virus encoded kinases (thymidine kinase) during lytic infection. Arginine butyrate can induce viral thymidine kinase and has been shown to treat patients with refractory PTLD when used with gancyclovir (Mentzer etÂ€ al. 2001). A recent phase I/II trial supports this hypothesis (Perrine etÂ€al. 2007). Additionally, inÂ€vitro, gemcitabine and doxorubicin have been shown to induce EBV lytic infection, and when used with gancyclovir inhibit EBV-lymphoproliferative disease in SCID mice (Feng etÂ€ al. 2004). Similar synergy between rituximab, dexamethasone, and gancyclovir inÂ€vitro has been shown to induce EBV lytic infection (Daibata etÂ€al. 2005). The histone deactylase inhibitor valproic acid when given with chemotherapy can induce EBV lytic infection and increased chemotherapy tumor, killing inÂ€vitro and inÂ€vivo; however, it has not been used with an anti-viral such as gancyclovir (Feng and Kenney 2006).

Immunosuppression The more T cell immunosuppressive a regimen, including such agents as calcineurin inhibitors, OKT3 and anti-thymocyte globulin, the higher the risk of PTLD. Withdrawing immunosuppression has been an effective treatment and the “gold standard” for PTLD; however, this potentially jeopardizes the allograft. Recently, the role of proliferation signal inhibitors has been studied in order to better understand their role in malignancies post-transplantation (Pascual 2007). The mTOR pathway is nearly ubiquitously activated in PTLD (El-Salem etÂ€ al. 2007). Everolimus and sirolimus are macrolide antibiotics that are immunosuppressive. They are proliferation signal inhibitors that have been shown to inhibit the growth of PTLD-like cell lines inÂ€vitro and inÂ€vivo (Majewski etÂ€al. 2000), and may have the potential to prevent PTLD in high risk populations (Majewski etÂ€al. 2003). A potential mechanism for the efficacy of proliferation signal inhibitors is that they inhibit the IL-10 transduction pathway (Nepomuceno etÂ€al. 2003). In patients with PTLD who experienced graft rejection, sirolimus has been able to rescue the graft and decrease EBV viremia (Sindhi etÂ€al. 2001). Proliferation signal inhibitors offer potential as not only immunosuppression but, additionally, prophylaxis for patients at increased risk to develop PTLD.

Monoclonal Antibodies Monoclonal antibodies that recognize antigens (CD20, CD21, CD24) on B cells have been used in patients who fail a trial of reduction of their immunosuppression. Now that rituximab, a monoclonal antibody that recognizes CD20, has become

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commercially available and licensed to treat lymphoproliferative disease, the approach to treat PTLD has changed dramatically. Rituximab is an IgG1-kappa chimeric monoclonal antibody that is made from the variable regions from heavy and light chains of a murine anti-CD20 antibody and human IgG1 and kappa constant regions (Reff etÂ€al. 1994). CD20 may play a role in the development and differentiation of B-cells into plasma cells. It is expressed on over 80% of EBV-PTLD (Swerdlow 1992). In vitro, rituximab induces both complement and antibody dependent cytotoxicity, apoptosis, and enhances the cytotoxic effects of chemotherapy (Demidem etÂ€al. 1997). It is an attractive therapy to choose because it is easy to administer, has limited co-morbid side effects and does not limit EBV-CTL function; however, the cost of rituximab is significant and long-term suppression of B-cells has been observed with it use (Gross 2007). Rituximab has been successfully used to treat PTLD in children and adults, and response rates have ranged from 44 to 65%, even as a single agent (Lee etÂ€ al. 2007). The largest prospective study using rituximab as a single agent included 43 SOT patients with PTLD (Choquet etÂ€al. 2006). Three-fourths of the patients had advanced stage disease at the time of treatment. The overall response rate at 80Â€days was 44% and the overall survival at 1 year was 67%. The median survival was 15Â€ months. A smaller phase II trial found similar results; however, EBVpositivity and a shorter time from transplantation to diagnosis correlated with response to rituximab. In this study, none of the EBV-negative PTLD patients responded to rituximab (Oertel etÂ€al. 2005). Data regarding the use of rituximab with chemotherapy are limited. A phase II study using the low-dose regimen of cyclophoshamide and prednisone plus rituximab in children, adolescents, and young adults with PTLD after SOT is currently being conducted by the Children’s Oncology Group.

T Cell Adoptive Immunotherapy EBV-CTL can be used to contain the virus or potentially kill virus-infected tumor cells; therefore, EBV-specific viral antigens recognized by host T cells in EBVPTLD are an attractive target for cellular immunotherapy (Bollard etÂ€ al. 2003; Fujita etÂ€al. 2008). Autologous and allogeneic EBV-CTLs have been evaluated by numerous groups (Savoldo etÂ€ al. 2006; Haque etÂ€ al. 2007). The advantages of autologous EBV-CTL are that they do not increase graft rejection and they can increase EBV-specific cellular immunity. A major limitation of T cell adoptive immunotherapy is our understanding of how to maintain long-term memory pools of infused T cells in the SOT recipients. Additionally, it takes 4 to 6Â€weeks to generate EBV-CTL, which may be problematic for patients with EBV-PTLD, and it is possible, though difficult, to generate EBV-CTL from EBV-seronegative recipients pretransplant, the highest risk population. This technology requires significant commitment of resources and expertise and is highly regulated to administer, making it unavailable but to a few centers.

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Conclusion PTLD is a heterogeneous group of diseases that has multiple potential molecular targets. Determining the right combination of therapies for any given patient with EBV-PTLD remains a major clinical problem. Simply reducing immunosuppression may no longer be sufficient and the use of proliferation signal inhibitors may improve both graft function, spare calcineurin inhibitor use, and treat PTLD. Many patients who develop EBV-PTLD cannot tolerate multi-agent chemotherapy or low-dose chemotherapy approaches may not establish long-term remissions for all patients. The use of antiviral medications will not likely be as effective as agents that can initiate EBV lytic infection. Early studies suggest this to be a promising therapeutic approach. EBV-specific vaccines may one day show efficacy in EBVnegative patients, but vaccines that stimulate an effective cellular response have been difficult to produce. New and more specific monoclonal antibodies look promising since they do not interfere with graft function, but long-term immunological consequences need to be evaluated. T cell therapy has shown the most promise, but remains non-feasible for the majority of patients. Other strategies of enhancing EBV-specific T cell immunity that would provide viral clearance and/or protective immunity against EBV-PTLD would be of great benefit. Though there are multiple anti-apoptotic targets, both cellular and viral genes, to date no agents have been identified as being feasible for clinical trials. To improve outcome of EBV-PTLD, as in most cancer, combination modality therapy with noncross reacting toxicity and complementary mechanism of tumor cell clearance will be required.

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Part II

Solid Tumors

Molecularly Targeted Therapies for Astrocytomas Ian F. Pollack

Introduction Astrocytomas constitute the largest group of central nervous system (CNS) neoplasms during childhood, and incorporate tumors with diverse histological features and biological characteristics (Pollack 1994). Whereas patients with superficial low-grade gliomas are often cured with surgery alone, patients with deep-seated or malignant gliomas continue to have a suboptimal prognosis, despite recent improvements in surgery and adjuvant therapy. The prognosis remains particularly grim for patients with malignant gliomas, such as glioblastoma multiforme (GBM) and anaplastic astrocytoma, which generally lead to death within several years after diagnosis (Finlay etÂ€ al. 1995). The poor response of these tumors to conventional therapies reflects a resistance of malignant glioma cells to undergo apoptosis in response to DNA damage, which may result from mutations of tumor suppressor and cell cycle control genes and aberrant activation of growth and survival signaling pathways. Although molecular pathways leading to tumorigenesis have been clearly established for adult malignant gliomas (Louis 1997), the involvement of such pathways in pediatric glial neoplasia remains inferential. For example, because in adult gliomas, platelet-derived growth factor receptor (PDGFR) and epidermal growth factor receptor (EGFR) have been observed to play important roles in tumor proliferation, and have therefore constituted logical targets for molecularly targeted therapies, such approaches have concurrently been explored in pediatric gliomas. This chapter reviews the molecular features of childhood gliomas associated with disease progression and prognosis, and discusses recent molecularly targeted therapeutic strategies.

I.F. Pollack (*) Department of Neurosurgery, Children’s Hospital of Pittsburgh, 3705 Fifth Avenue, Pittsburgh, PA, USA e-mail: [email protected]

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Molecular Features of Tumorigenesis Low-Grade Gliomas Gliomas are broadly classified as low-grade (grades 1 and 2) and high-grade (grades 3 and 4) (Kleihues etÂ€al. 1993). Juvenile pilocytic astrocytomas are the most common low-grade gliomas, and frequently occur in the cerebellar and cerebral cortices, where they are generally amenable to complete surgical resection. However, these lesions can arise from midline structures, such as the hypothalamus, where they are not amenable to gross total resection without unacceptable morbidity. Studies within the last few years have demonstrated that pilocytic astrocytomas commonly exhibit molecular changes leading to constitutive activation of the BRAF gene, which may constitute a promising therapeutic target (Jones et al. 2008). Although other consistent molecular abnormalities have not been observed in these tumors, a subset of pilocytic astrocytomas exhibits deletions of the chromosome 17q (Von Deimling etÂ€al. 1993a), the site of the neurofibromin gene that is commonly altered in patients with NF1. Patients with NF1 have a significantly increased frequency of pilocytic astrocytomas (Guttmann etÂ€al. 2000), and NF1-deleted transgenic mice have been used as an animal model for low-grade gliomas (Dasgupta etÂ€al. 2005). However, an association between NF1 loss and tumor development in sporadic pilocytic astrocytomas has not been confirmed. Recently, Wong etÂ€al. (2005) using microarray-based screening identified in a small institutional series of low-grade gliomas, identified a series of genetic alterations in tumors that had progressed after initial therapy. Non-pilocytic low-grade astrocytomas include a diverse group of lesions, the most common being grade II fibrillary astrocytomas, which resemble the infiltrative low-grade gliomas commonly seen in adults. Whereas adult low-grade gliomas often exhibit deletions of chromosome 17p, the site of the TP53 gene, as well as mutations of the TP53 gene (James etÂ€al. 1989), this pattern has not been frequently observed in childhood lesions (Lang etÂ€al. 1994; Griffin etÂ€al. 1988; Cheng etÂ€al. 2000). These findings suggest that lesions in these two age groups may arise from a different series of molecular events. This distinction also fits with the dramatically different prognoses observed in childhood versus adult low-grade gliomas. Whereas the latter frequently progress to grade III and IV lesions over time, this pattern of disease progression is less common in childhood gliomas.

High-Grade Gliomas Pediatric high-grade astrocytomas include anaplastic astrocytoma and oligoastrocytoma (grade III) and GBM (grade IV). These lesions have historically had a poor prognosis with conventional therapies, including surgery, irradiation, and cytotoxic chemotherapy (Finlay etÂ€al. 1995). In contrast to the extensive efforts that have been directed at characterizing the patterns of genomic abnormalities in adult

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high-grade gliomas (Louis 1997; Ichimura etÂ€al. 2000), there is comparatively little information regarding the molecular features of pediatric malignant gliomas. One clinical pathway that has been observed in adults involves so-called “deÂ€novo” or primary development of a glioblastoma, which is frequently associated with amplification and/or rearrangement of the EGFR gene, but without mutation of the TP53 gene. These tumors also characteristically show loss of chromosome 10q involving the region of the PTEN gene (von Deimling etÂ€al. 1993b; Louis 1997; Collins 1999). Studies from our group and others indicate that EGFR amplification and PTEN deletions are observed in only about 10% of pediatric malignant gliomas, suggesting that this pathway is less commonly implicated than in adults (Cheng etÂ€al. 1999; Raffel etÂ€al. 1999; Sung etÂ€al. 2000; Pollack etÂ€al. 2006a). In a recent analysis of the cohort from the Children Cancer Group 945 (CCG-945) study, mutations of PTEN were observed in only 1 of 62 tumors, and amplification of EGFR was almost equally uncommon (Pollack etÂ€al. 2006a). However, expression of high levels of the EGFR protein product was commonly observed (Bredel etÂ€al. 1999b), suggesting that this receptor may play a role in tumor growth, despite the lack of amplification. Similarly, loss of heterozygosity involving chromosome 10 in the vicinity of the PTEN locus was observed in a subset of these tumors (Pollack etÂ€al. 2006a), suggesting that PTEN may be inactivated by a mechanism other than mutation. More recently, studies in adult primary malignant gliomas indicate that these tumors may include several molecularly defined subsets of lesions, although the applicability of these subsets to the childhood age group remains uncertain (Verhaak et al. 2010). A second clinical pathway of tumorigenesis involves the development of a malignant astrocytoma from a pre-existing low-grade glioma, which evolves into an anaplastic astrocytoma, and ultimately becomes a GBM (von Deimling etÂ€ al. 1993b; Louis 1997; Collins 1999). Such tumors characteristically exhibit mutations of the TP53 gene, overexpression of PDGFRa, and a low frequency of EGFR amplification. It is of interest that recent analyses of childhood malignant gliomas have identified TP53 mutations in 40% of tumors and an even higher incidence in grade 4 lesions (Sung etÂ€al. 2000; Pollack etÂ€al. 2001b), comparable to the frequency in secondary adult malignant gliomas. However, such lesions appear to arise deÂ€novo in children, rather than progressing from a pre-existing low-grade lesion, which calls into question whether pediatric malignant gliomas are truly analogous to the adult secondary gliomas from a molecular and biological standpoint. In that regard, our observation in both an institutional cohort (Pollack etÂ€al. 1997) and the CCG-945 study group (Pollack etÂ€al. 2002) of a worse prognosis in children whose malignant gliomas had TP53 mutations than those with non-mutated tumors contrasts with the lack of such an association in adult lesions. In addition, secondary adult malignant gliomas have been noted to frequently have mutations in the IDH1 gene, changes that are relatively uncommon in pediatric malignant gliomas (Parsons et al., 2008; Hartmann et al., 2009), further suggesting that these groups of tumors are biologically distinct. A third group of adult gliomas arises by a distinct pathway, with characteristic deletions of chromosomes 1p and 19q (Ino etÂ€ al. 2000). Such tumors often have oligodendroglial features and carry a better prognosis than other groups of malignant

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gliomas, although the genetic loci that confer the more favorable prognosis remain undefined. Although 1p and 19q alterations are observed in about 20% of childhood malignant gliomas, a favorable association between 1p/19q loss and outcome was not observed in the CCG-945 cohort (Pollack etÂ€al. 2003). One consequence of the genetic alterations seen in malignant gliomas is that these tumors exhibit abnormalities in signal transduction that drive invasiveness, proliferation, and cell survival. A significant percentage of tumors exhibit autocrine stimulation via receptors for EGF, PDGF, (Nistér etÂ€al. 1991), Â�insulin-like growth factor (Glick etÂ€al. 1989), and fibroblast growth factor (FGF) (Gross etÂ€al. 1990) by producing both the growth factor and its receptor. These tumors also promote their growth by paracrine interactions, secreting proteins, such as vascular endothelial growth factor (Plate etÂ€al. 1992), FGF, and PDGF, which stimulate adjacent tumor cells and support proliferation of endothelial cells, contributing to an angiogenic phenotype. Cell survival is further promoted by dysregulation of PI3K/Akt signaling, stimulated both by upstream receptor signaling and loss of PTEN expression, disinhibiting Akt activation. As with the above molecular data, it is important to recognize that these observations have largely been made in adult high-grade gliomas, and work is ongoing to determine whether similar changes occur in pediatric gliomas. In parallel with their autocrine and paracrine stimulation of cell proliferation, malignant gliomas in adults characteristically exhibit dysregulation of cell cycle control gene products as a result of homozygous deletion, mutation, or epigenetic silencing of p16 or p15, which encode inhibitors of cyclin-dependent kinases; amplification of cyclin-dependent kinase 4; or deletion or mutation of the retinoblastoma susceptibility gene RB1 (Collins 1999). Such molecular alterations have also been observed in pediatric malignant gliomas (Cheng etÂ€al. 1999). Another molecular feature associated with malignant gliomas in both children and adults is the acquisition of a drug-resistant phenotype, which in part accounts for the poor response of these tumors to many conventional chemotherapeutic agents, particularly alkylating agents. Several studies have noted that both adult and pediatric malignant gliomas commonly express high levels of alkylguanine DNA alkyltransferase (AGT), the proximal mechanism of resistance to alkylators, such as the nitrosoureas and temozolomide (Friedman etÂ€al. 1998b; Pollack etÂ€al. 2006b). Recent studies have noted that the response of these tumors to such agents correlates with levels of AGT expression and activity (Jaeckle etÂ€al 1998; Esteller etÂ€al. 2000; Hegi etÂ€al. 2005; Pollack etÂ€al. 2006b). The association between tumor AGT expression status and outcome was demonstrated in children with malignant gliomas in the CCG-945 cohort (Pollack etÂ€ al. 2006b), in which those with high levels of AGT expression had a substantially worse progression-free survival after nitrosourea-based therapy than those with low levels. These results were independently validated in the ACNS0126 study of the Children’s Oncology Group (COG) among children treated with temozolomide during and after irradiation. In addition to AGT, a number of other factors have been associated with resistance to temozolomide, including deficiency of mismatch repair (MMR) protein expression (Friedman etÂ€al. 1998b) and alterations of base-excision repair function.

Molecularly Targeted Therapies for Astrocytomas TableÂ€1â•… Molecularly targeted therapies Inhibitors of growth factor receptors PDGF inhibition â•… Imatinib â•… Sunitinib â•… AZD2171 EGFR inhibition â•… Gefitinib â•… Erlotinib â•… Lapatanib â•… Cetuximab Inhibitors of downstream signaling Farnesyl transferase inhibition â•… Tipifarnib â•… Lonafarnib PKC inhibition â•… Enzastaurin MAPK cascade/Raf inhibition â•… Sorafenib PI3K/Akt pathway (mTOR) inhibition â•… Sirolimus â•… Temsirolimus â•… Everolimus Angiogenesis inhibition VEGFR/multitargeted kinase inhibition â•… SU5416 â•… Valatinib â•… ZD6474 â•… Sunitinib â•… CEP-7055 â•… Sorafenib â•… AZD2171 VEGF inhibition â•… Bevacizumab Integrin inhibition â•… Cilengitide Other putative antiangiogenics â•… Thalidomide â•… Lenalidomide â•… Cox-2 inhibitors â•… Rofecoxib â•… Endostatin â•… Angiostatin

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Counteracting drug resistance phenotype AGT inhibition â•… O6-Benzylguanine PARP Inhibition â•… INO-1001 ABT888 HDAC Inhibition â•… SAHA â•… Depsipeptide â•… Valproic acid HSP Inhibition â•… Geldanamycin â•… 17-AAG Proteosome inhibition â•… Bortezomib Cell cycle modulation â•… UCN-01 â•… CYC202 â•… Flavopiridol Immunologic or ligand-based therapies Vaccine-based targets Antibody therapy â•… Cetuximab â•… Bevacizumab Radiolabeled intracavitary antibody therapy â•… Anti-EGFR â•… Anti-Tenascin Immunotoxins â•… Tf-CRM107 â•… IL13-PE38QQR â•… TP-38 â•… IL4-PE

Molecularly Based Therapeutic Strategies The availability of novel agents for therapy of gliomas has dramatically increased during the last decade. The portfolio of available compounds can be broadly categorized into the following major groups: (1) inhibitors of growth factor receptors expressed on glioma cells; (2) inhibitors of signal transduction pathway intermediates;

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(3) inhibitors of angiogenesis; (4) agents that counteract drug resistance; and (5) immunological or ligand-based agents targeted against molecules overexpressed in gliomas (TableÂ€ 1). Illustrative examples of such agents are discussed subsequently, and results (in most cases preliminary) are presented, where available.

Inhibitors of Growth Factor Receptors Because growth factor receptors, particularly EGFR and PDGFR, were recognized to be commonly overexpressed or amplified in malignant gliomas, these have constituted a major target for drug development. A variety of strategies have been used in preclinical studies for growth factor receptor blockade, including neutralizing antibodies, antisense techniques, and dominant negative mutants. For example, Nitta and Sato (1994) observed that c-sis antisense oligonucleotides inhibited growth inÂ€vitro of A172 glioma cells, which express large quantities of PDGF-BB and the b receptor. Similarly, growth of U87 and U343 glioma cells transfected with a dominant negative construct for PDGF-A, was suppressed inÂ€vitro, and these cells exhibited diminished tumorigenicity in a nude mouse model (Shamah etÂ€al. 1993). Our previous studies have also demonstrated significant antiproliferative activity inÂ€ vitro using a PDGF neutralizing antibody (Pollack etÂ€ al. 1991). Other groups have noted significant activity of antibodies designed to neutralize EGFR or to target the most common, constitutively active EGFR mutant (EGFR vIII) (Mishima etÂ€al. 2001). Similarly, in non-CNS tumors, monoclonal antibodies directed against EGFR (e.g., C225, Cetuximab, ImClone Systems) have demonstrated efficacy in preclinical models as well as clinical studies (Baselga etÂ€al. 2000). Although the above results confirm that blocking critical growth factor pathways can interfere with cell proliferation in selected tumor cell lines, the applicability of these results for glioma therapeutics is limited by the challenges imposed by the blood–brain barrier, which impairs drug access to the tumor after systemic administration. An alternative approach involves the use of “small molecule” inhibitors of receptor signaling. During the last several years, molecular modeling approaches have allowed the development of “designer inhibitors,” which selectively target one or more tyrosine kinase receptors by competitive inhibition of ATP-binding sites involved in receptor activation. A brief summary of several promising agents is provided below.

Platelet-Derived Growth Factor Receptor as a Target PDGF was originally identified as a potent mitogen for fibroblasts, glial cells, and smooth muscle. The ligand is a disulfide-linked dimer composed of A, B, C, and D polypeptide chains, the B chain exhibiting homology to the v-sis oncogene isolated from simian sarcoma virus-transformed cells (Lokker etÂ€ al. 2002). The various PDGF isoforms (AA, AB, BB, CC, DD) bind with differential affinity to two cellsurface PDGF receptors (Nistér etÂ€al. 1991; Lokker etÂ€al. 2002). Concurrent expression

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of one or more of these ligands and their receptors has been observed in a high percentage of malignant gliomas and the acquisition of PDGF receptor overexpression represents a potential step in the transition from grade II to grade III gliomas in adults (Hermanson etÂ€al. 1996), which is supported by studies in transgenic mouse glioma models (Dai etÂ€al. 2001) showing that overexpression of PDGF alone leads to low-grade gliomas, whereas combined PDGF overexpression with other genetic changes leads to a more malignant phenotype. From a mechanistic perspective, PDGF and its receptors have been implicated in autocrine and paracrine growth stimulation in malignant gliomas (Nistér etÂ€al. 1991; Lokker etÂ€al. 2002). Ligand binding to the receptor leads to receptor dimerization, autophosphorylation of tyrosine residues, and phosphorylation of a series of signaling intermediates, such as RAS, phospholipase C-gamma (PLCg), and phosphatidylinositol 3-kinase (PI3K). Previous studies have indicated that PDGF is a potent mitogen for malignant glioma cells (Pollack etÂ€ al. 1991), and that inhibition of PDGFR activation inhibits glioma growth in preclinical models (Nitta and Sato 1994). In addition, because of the role of PDGF in supporting glioma-induced angiogenesis (Wang etÂ€ al. 1999), inhibition of PDGFR may provide a means for simultaneously blocking tumor growth and angiogenic activation. A number of PDGFR inhibitors are in various phases of clinical development. One agent that has been extensively tested is STI571 (CGP57148B, also known as Gleevec or Imatinib, Novartis Pharmaceutical Corp). This compound initially became a focus of clinical interest because of its potent inhibition of BCR-ABL, associated with Philadelphia chromosome-positive leukemias. Because of the dramatic hematologic and cytologic responses observed in preliminary studies (Druker etÂ€al. 2001), phase III trials were conducted, which demonstrated superiority of this agent in terms of complete cytogenetic responses and freedom from disease progression in comparison to conventional therapy (O’Brien etÂ€al. 2003). A second target for STI571 is c-kit, constitutively activated in a substantial percentage of gastrointestinal stromal tumors. Initial studies demonstrated striking efficacy in patients with advanced disease (Joensuu etÂ€al. 2001). Because imatinib was also extremely potent in blocking PDGFR signaling (Buchdunger etÂ€al. 2000), attention was focused on the application of this agent in tumors with PDGFR-driven proliferation. Preliminary studies in glioma cell lines demonstrated inhibition of proliferation inÂ€vitro and delay of tumor growth inÂ€vivo (Kilic etÂ€al. 2000), although the results were less striking than those noted earlier for c-kit and BCR-ABL-dependent tumors. Based on these results, dose escalation studies in children and adults with malignant gliomas were initiated by several cooperative groups, including the North American Brain Tumor Consortium (NABTC) and Pediatric Brain Tumor Consortium (PBTC). An unexpected finding from several of these studies was that a subset of patients experienced intratumoral hemorrhage during treatment (Wen etÂ€al. 2006). The etiology of this effect remains to be determined, but one potential mechanism relates to known effects on perivascular cells (Pietras etÂ€al 2001). This observation, coupled with the fact that a paucity of responses or long-term survivors were noted in the pediatric phase I trial (Pollack etÂ€al. 2007) or the NABTC study, limited enthusiasm for a pediatric phase II study.

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In contrast to these observations, a study from Europe detected intratumoral hemorrhage in only 1 of 51 patients, and four patients had partial radiological responses (Raymond etÂ€al. 2004). In a second study, prolonged disease control was achieved in 3 of 6 high-grade glioma patients younger than 45Â€years of age versus only one of nine older than 45Â€years (Katz etÂ€al. 2004). Combinations of STI571 and conventional chemotherapeutic agents have also been examined. Although the optimal agent to employ in this regard remains uncertain, promising results have been reported with hydroxyurea (1,000Â€mg/day) combined with 400Â€mg/day of STI571, with objective disease regression in 5 of 26 patients (Dresemann 2005). In a separate phase II study of this combination conducted by Reardon etÂ€ al. (2005a), 9% of GBM patients achieved radiographic responses, while 35% had stable disease. The progression-free survival rate at 6Â€months was 26.3%, which compared favorably with results using single-agent cytotoxic chemotherapy. Combinatorial application for pediatric gliomas has, to date, not been systematically evaluated.

Epidermal Growth Factor Receptor as a Target EGFR, also known as erbB1, the protein product of the cellular homolog of the v-erbB oncogene, is one of a family of receptors that also includes erbB2 (HER2/neu), erbB3 (HER3), and erbB4 (HER4). The ligands for these receptors include EGF and TGFa, among others (Schlessinger 2000). EGFR is normally activated by ligand binding to its extracellular receptor domain (Ullrich and Schlessinger 1990). However, in some tumors with EGFR amplification, the gene is also rearranged, leading to constitutively active mutants (Libermann etÂ€al. 1985; Wong etÂ€al. 1992). The most common mutant, the EGFRvIII variant, is caused by deletions of exons 2 to 7. Because activated EGFR induces tyrosine phosphorylation of substrates that contribute to cell proliferation, excessive activation of this protein, either by ligand binding or mutation-induced constitutive signaling, may provide cells with a growth advantage under certain conditions. Although pediatric malignant gliomas do not share the high incidence of EGFR amplification noted in primary adult malignant gliomas, they commonly exhibit high levels of EGFR expression (Bredel etÂ€al. 1999b), which suggests that excessive EGFR signaling may contribute to cell proliferation in these tumors as well. The important role of EGFR in brain tumor development is supported by the fact that targeted inhibition of this receptor, using dominant negative constructs, interfered with glioma proliferation (O’Rourke etÂ€al. 1997). Similarly, antibody-based therapies targeted at wild-type or mutant EGFR family members have been shown to have efficacy in preclinical glioma models (Mishima etÂ€al. 2001), which provided a rationale for small molecule-based strategies. One such agent to be tested in pediatric malignant gliomas was ZD 1839 (Iressa, Gefitinib, AstraZeneca), which is effective in blocking EGFR autophosphorylation and EGFR-dependent cell signaling inÂ€vitro in cell lines that rely heavily on EGFR activation for proliferative stimulation, and tumor growth inÂ€vivo in EGFR-dependent xenograft model systems (Wakeling etÂ€al. 2002). Initial clinical data indicated that

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gefitinib was well tolerated at effective doses (Albanell etÂ€al. 2002), with the most common toxicity being an acneiform skin rash, and diarrhea being a common doselimiting toxicity. Results from several clinical studies for non-central nervous system solid tumors demonstrated activity of gefitinib as a single agent (Baselga etÂ€al. 2002; Fukuoka etÂ€al. 2003), although phase III studies in patients with advanced small cell lung cancer failed to demonstrate a convincing benefit of adding this agent to regimens, including either gemcitabine/cisplatin or paclitaxel/carboplatin (Herbst etÂ€al. 2004). Although these results were initially a source of disappointment, recent reports have demonstrated that response to gefitinib is strongly influenced by tumor EGFR status, with a high percentage of objective responses among patients with gain-of-function mutations of the EGFR gene (Lynch etÂ€al. 2004). These observations emphasize the importance of studies of tumor genotype and phenotype in considerations of study design and response analysis. With these issues in mind, Phase I/II studies of gefitinib were initiated within the NABTC (for adults with malignant glioma) and the PBTC (for children with recurrent malignant glioma and newly diagnosed brainstem malignant glioma). These studies included analyses of receptor expression and mutational status, in an effort to correlate molecular features with treatment responses. In the phase II component of the NABTC study, 7 of 55 patients were noted to have partial tumor regression, although the median times to progression were not superior to historical controls (Lieberman etÂ€al. 2004). In the pediatric study (PBTC007), gefitinib was administered with irradiation to patients with newly diagnosed malignant gliomas with separate strata for children with brainstem gliomas and non-brainstem high-grade gliomas. After completing the phase I component of the study, which established the maximal tolerated dose, a phase II component was initiated. The results are currently pending. Clinical trials for gliomas have also been completed using other EGFR inhibitors. As with gefitinib, OSI-774 (Erlotinib, Tarceva, Genentech) reversibly inhibits EGFR by competition with the ATP binding site (Pollack etÂ€al. 1999). In phase I studies, toxicities were comparable to ZD1839, with rash and diarrhea as common events (Hidalgo etÂ€ al. 2001). Phase II studies in patients with non-CNS tumors demonstrated activity in terms of tumor response (Perez-Soler 2004), as did a trial in adults with malignant glioma, conducted by the NABTC (Prados etÂ€al. 2006). However, as with gefitinib, median time to progression for the latter cohort was no better than historical control data. A phase I trial of erlotinib with RT in patients with GBM determined the toxicity and maximum-tolerated dose (Krishnan etÂ€al. 2005). The median time to progression was 161Â€days, and the median survival time was 386Â€days. The combination of erlotinib with temozolomide and concurrent RT was also well tolerated, although it is unclear if this improved outcome (Peereboom etÂ€al. 2006). A phase I study of this agent alone and in conjunction with temozolomide for children with recurrent disease of a variety of histologies was conducted in the Children’s Oncology Group (ADVL0214), although activity was insufficient to support a phase II study of this combination in gliomas. Because responses were observed to both gefitinib and erlotinib in studies for adults with malignant gliomas, efforts were made to determine whether a molecular

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signaling profile could distinguish responders from non-responders. Although one analysis failed to note a clear association between EGFR status and response (Lassman etÂ€al. 2005), a subsequent study noted that responses were typically seen in tumors that had amplification of EGFR and expression of the EGFRvIII variant in conjunction with preservation of PTEN (Mellinghoff etÂ€al. 2005), thereby suppressing constitutive Akt activation. An attempt to correlate response with EGFR amplification and expression is also planned in the pediatric glioma study. One other selective, reversible EGFR inhibitor that is in early phase trials is GW572016 (Lapatinib, GlaxoSmithKline, Research Triangle Park, NC) (Burris 2004), which has demonstrated activity against erbB2-expressing tumors (Rusnak etÂ€ al. 2001), and has been tested in a study of the PBTC (PBTC-016), including patients with recurrent malignant gliomas, ependymomas, and medulloblastomas. After completing the phase I portion of the trial to define the maximal tolerated dose (MTD), a phase II study was initiated, which incorporated a molecular biology component for patients in whom a resection was planned. In such patients, lapatinib was administered for several days prior to surgery and then a portion of the resected tumor tissue was analyzed for suppression of the receptor target and other downstream molecular features. The results of this study will soon be available.

Inhibitors of Downstream Signaling Inhibition of intermediate and downstream components of growth factor signaling pathways, such as RAS, PI3K, PKC, and mTOR, is a promising strategy for interfering with the proliferation of malignant gliomas and other brain tumors (Pollack etÂ€al. 1998). Because the number of inhibitors and targets that are being examined preclinically and in early clinical studies is vast, we focus below on the approaches that have been most intensively considered to date.

Inhibition of RAS Processing The response of cells to growth factors, such as EGF and PDGF, is mediated by cell-surface receptors that contain an extracellular domain that interacts with a ligand, a transmembrane domain that anchors the receptor to the cell membrane, and an intracytoplasmic domain that interacts with downstream signaling components. Activation induces conformational changes within the receptor and/or receptor cross-linking, and phosphorylation of tyrosine residues within their cytoplasmic domains (Ullrich and Schlessinger 1990). Phosphorylation exposes Src homology 2 (SH2) domains, binding sites for intracellular adapter molecules, such as Grb2 (McCormick 1993). The adapter proteins in turn associate with guanine nucleotide exchange factors (Pawson 1995), which facilitate the activation of RAS family guanine nucleotide triphosphatases by exchanging GDP for GTP. Excessive activation of

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these pathways has been implicated in the development of a variety of human tumors. In certain tumor types, mutation of one of the RAS genes to a constitutively active protein product has been associated with tumorigenesis (Peddanna etÂ€ al. 1996). Although such mutations are rare in gliomas, RAS activity is markedly elevated as a result of deregulation of upstream signaling elements, such as growth factor receptors (Prigent etÂ€al. 1996; Guha etÂ€al. 1997). These observations suggest that targeted inhibition of RAS-dependent signaling may constitute a therapeutically useful strategy for malignant gliomas. Because RAS is synthesized as a pro-peptide and undergoes post-translational modifications to associate with the inner surface of the plasma membrane, one strategy for blocking RAS activity has involved interference with those modifications. Inhibition of farnesylation has been one of the principal strategies used to date (Bredel etÂ€al. 1998; Rowinsky etÂ€al. 1999). Although farnesyl transferase inhibitors (FTIs) inhibit RAS farnesylation, their antiproliferative effects are not exclusively due to the effects on RAS as they target other G-proteins, including Rho-B (Lebowitz and Prendergast 1998; Jiang etÂ€ al. 2000). Feldkamp etÂ€ al (1999) and Bredel etÂ€al. (1998) demonstrated that astrocytomas are amenable to growth inhibition by FTI’s, through a combination of antiproliferative and pro-apoptotic effects. Based on encouraging preclinical data, clinical trials have been initiated with several FTIs. Tipifarnib (R115777, Zarnestra; Johnson and Johnson) is a non-peptidomimetic FTI with inÂ€ vivo activity against many human cancer cell lines and xenograft models (End etÂ€ al. 2001). Although some activity was observed in patients with hematological malignancies (Alsina etÂ€al. 2004), the results of Phase II and III trials in adults with solid tumors have been less promising (Rao etÂ€ al. 2004; Van Cutsem etÂ€al. 2004). In a recent phase II trial in adult malignant gliomas, tipifarnib showed a low level of activity, with 5 of 89 patients with GBM achieving partial responses (PRs) (Cloughesy etÂ€al. 2006). Similar results were observed in a pediatric trial of the COG (ACNS0226) for children with progressive tumors. Among 31 evaluable patients with progressive high-grade glioma, 1 patient had a confirmed PR and 3 patients continued on therapy for at least 4 cycles. Among the 35 patients with brainstem glioma, 1 patient had a PR, and 4 patients continued on therapy with stable disease for at least 4 cycles (11%). The 6-month progressionfree survival was 14â•›±â•›6% for high-grade glioma and 3â•›±â•›3% for brainstem glioma. A trial of this agent with radiotherapy was recently completed for children with malignant brainstem gliomas (PBTC-014). Results are pending. A second FTI that was examined in the PBTC (PBTC003) was Lonafarnib (SCH66336, SARASARTM, Schering-Plough) (Kieran etÂ€al. 2007), which has previously been evaluated in a variety of tumor types alone and in conjunction with conventional chemotherapeutic agents (Sharma etÂ€al. 2002; Kim etÂ€al. 2005). Fifty-three children with diverse tumor tumors were treated in the PBTC study: high-grade glioma (19), brain stem glioma (7), medulloblastoma (3), PNET (4), ependymoma (9), refractory low-grade glioma (5), meningioma (3), sarcoma (1), and gliomatosis cerebri (1). Forty-eight patients were evaluable for response: one with anaplastic astrocytoma demonstrated a PR lasting 13 courses and 12 demonstrated stable disease for 2 to 18 courses. Seven patients remained on therapy for at least 1Â€year.

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Inhibition of Protein Kinase C Another important signaling pathway in gliomas involves the activation of protein kinase C (PKC), which comprises a family of serine/threonine kinases that constitute an element of the signaling cascade of several growth factors that stimulate glioma cell proliferation, such as EGF and PDGF (Fields etÂ€al. 1990; Nishizuka 1992). Activation of PKC leads to diverse biological effects, depending upon which isoforms are involved. These include phosphorylation of other effectors, such as RAF and MAPK, and influencing the activation status of RAS ((Kolch etÂ€ al. 1993; Marais etÂ€ al. 1998), which together contribute to the transduction of a proliferative signal to the nucleus. Levels of PKC activity have been noted to correlate with proliferative status in neoplastic astrocytes (Couldwell etÂ€al. 1991). In addition, astrocytoma cells express levels of PKCa and e that are up to tenfold higher than in normal astrocytes. Glioma mitogens, such as PDGF and EGF, produce elevations in PKC activity that parallel increases in DNA synthesis (Couldwell etÂ€al. 1992). In addition, pharmacological and antisense agents that target PKC isoforms overexpressed in astrocytomas diminish glioma proliferation inÂ€ vitro (Pollack etÂ€al. 1996; Yazaki etÂ€al. 1996) and induce apoptosis (Bredel etÂ€al. 1999a). LY317615 (enzastaurin, Eli Lilly and Company), is a bisindolylmaleimide-derived PKC inhibitor that has demonstrated antiangiogenic activity (Keyes etÂ€al 2004) as well as antitumor activity in U87 glioma xenografts and a variety of other solid tumors (Graff etÂ€al. 2005). Enzastaurin and its active metabolites prevent substrate phosphorylation by competing with the enzyme’s ATP binding site. The inÂ€ vitro IC50s for PKCb inhibition is approximately 5Â€nM, whereas inhibition is 3- to 8-fold less potent for PKCa and 10- to 20-fold less potent for PKCg and PKCe. Additionally, this compound inhibits signaling through the PI3 kinase/AKT signaling pathway. Accordingly, inhibition of the PKC and AKT signaling pathways by enzastaurin suppresses phosphorylation of glycogen synthase kinase 3b, induces apoptosis and inhibits proliferation in cultured cell lines from a variety of human cancers, including gliomas, and also has antiangiogenic activity. In single-agent phase I studies, enzastaurin was well tolerated (Carducci etÂ€ al. 2006). Fine and colleagues recently reported preliminary results of a phase II trial of enzastaurin in adult patients with recurrent high-grade gliomas (Fine etÂ€al. 2005). Of the 87 patients evaluable for response, 22% were reported to have objective radiographic responses, and 5% had stable disease. The drug was well tolerated, and a maximum tolerated dose was not determined. Intra-tumoral hemorrhages were noted in seven patients; however, six of the seven had progressive disease at the time of the bleed. Based on these encouraging results, a phase III study was initiated in adult patients with GBM and lymphoma. A phase I study of this agent in children with recurrent brain tumors is also in progress (PBTC023).

MAPK Cascade Inhibitors Following the activation of the membrane-associated components of the various signaling pathways, downstream signals reach the nucleus by a variety of mechanisms.

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One of the most critical pathways involves the mitogen-activated protein kinase (MAPK) cascade. This cascade involves multiple separate protein kinases. The most proximal kinase in the cascade is the RAF (MAPKKK) family, which includes at least three members. RAF, a serine/threonine kinase, is recruited to the cell membrane, stabilized by interaction with other proteins, and phosphorylated to an active form (Freed etÂ€ al. 1994). Activated RAF phosphorylates and activates MAP/ERK kinase (MEK, also known as MAPKK), which subsequently activates MAPK (also known as ERK (extracellular signal-regulated kinase)) (Howe etÂ€al. 1992). MAPKs are serine/threonine kinases that activate a number of additional downstream mediators that regulate transcription, protein translation, and cytoskeletal rearrangement. In an effort to block this signaling pathway, a number of agents that function as RAF kinase inhibitors have been developed. These include sorafenib (BAY 43-9006; Bayer Pharmaceuticals) (Wilhelm etÂ€al. 2004; Jane etÂ€al. 2006), which has been a focus of combinatorial studies for malignant gliomas within the NABTC. Results are pending. This agent has also been examined in a phase I study for recurrent pediatric malignancies in the Children’s Oncology Group, and a phase II study for brain tumors is under consideration. Because this agent also inhibits VEGF receptor activation, it may have applicability as an antiangiogenic agent (discussed below), independent of any direct cytotoxic activity against glioma cells.

Inhibitors of PI3K/Akt Pathways Another major pathway implicated in growth factor receptor-mediated signaling involves activation of AKT via PI3K. PI3K is a phospholipid kinase that contains a regulatory subunit, p85, and a catalytic subunit, p110. Upon cell-surface activation, PI3K phosphorylates phosphatidylinositol 4,5-biphosphate (PI4,5P2, PIP2) to form PI3,4P2, and PIP3. PIP3 leads to translocation of PDK1 and AKT. Akt is a serine/ threonine kinase that is phosphorylated at threonine 308 by PDK1 and at serine 473 by a second kinase. Activated AKT phosphorylates several proteins involved in cell survival signaling, such as BAD, forkhead transcription factor, glycogen synthase kinase, and the mammalian target of rapamycin (mTOR) (Cross etÂ€ al. 1995; Cardone etÂ€al. 1998). Under normal conditions, Akt activation is inhibited by PTEN, a phosphatase that converts PIP3 to PIP2 (Li and Sun 1998; Cantley and Neel 1999). However, PTEN is mutated in at least 40% of grade IV gliomas (Parsons et al. 2009), particularly in primary GBMs. The importance of this pathway in glioma development is highlighted by the fact that transfer of a wild-type PTEN gene to the PTEN-deleted U87 glioma cell line suppressed tumor growth, leading to cell cycle arrest (Li and Sun 1998). Conversely, greater PI3K/AKT activity has been associated with resistance to irradiation (Nakamura etÂ€al. 2005). Based on these observations, inhibitors of the PI3K/AKT pathway appear to be logical therapeutic agents for GBM. To date, inhibitors of PI3K, such LY294002 (Nakamura etÂ€al. 2005), have demonstrated promising activity in preclinical models,

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but the profile of toxicity has precluded their clinical use. Accordingly, interest has focused on blocking downstream targets of the Akt pathway. In particular, several inhibitors of the mTOR are being investigated in clinical trials. These include sirolimus (rapamycin, Rapamune®; Wyeth), temsirolimus (CCI-779; Wyeth), and everolimus (RAD-001, everolimus, Certican®; Novartis), all of which inhibit glioblastoma proliferation in culture and intracerebral xenografts. Temsirolimus demonstrated modest activity in recurrent gliomas in recent phase I/II studies (Chang etÂ€al. 2005). Everolimus has also undergone phase I testing for children with recurrent malignant brain tumors and a phase II study is being developed. In a recent phase I study, administration of temsirolimus to patients with progressive GBM was well tolerated, with some objective responses (Galanis etÂ€al. 2005). High levels of phosphorylated p70s6 kinase, as determined by immunohistochemistry in baseline tumor samples, seemed to correlate with response to treatment.

Inhibition of Angiogenesis VEGFR Inhibition In addition to direct effects of growth factor receptor inhibition on tumor proliferation, there is compelling evidence that these agents may have indirect effects on angiogenesis, which may be relevant for brain tumor therapeutics. Recent studies have confirmed that the secretion by tumor cells of vascular endothelial growth factor (VEGF), the most potent endothelial cell mitogen, depends heavily on EGFRmediated signaling (Maity etÂ€ al. 2000) and that a significant component of the therapeutic efficacy of EGFR-targeting agents reflects this secondary effect on tumor angiogenesis (Ciardiello etÂ€al. 2001; Huang etÂ€al. 2002). Similarly, PDGF has been demonstrated to stimulate tumor angiogenesis (Yancopoulos etÂ€ al. 2000) in addition to supporting the growth and survival of vascular pericytes and promoting glioma VEGF secretion (Wang etÂ€al. 1999). In view of these paracrine interactions, an important element in evaluating the therapeutic utility of EGFR- and PDGFRtargeted inhibitors must focus on understanding the effects of these agents on not only the tumor cells themselves, but also on the surrounding vasculature (Ciardiello etÂ€al. 2001; Pietras etÂ€al. 2001). Because VEGF represents a major stimulatory factor for the initiation of angiogenesis, inhibition of ligand/receptor interactions has been a focus of recent attention for malignant gliomas. SU5416 (Sugen, Pfizer) was the first antiangiogenic agent studied in the PBTC, although development plans were ultimately halted after a negative phase III study for patients with colorectal carcinoma. Subsequently, a number of other agents have been examined in adult patients with GBM, including PTK787/ZK 222584 (valatinib, Novartis Pharmaceuticals and Schering AG), ZD6474 (Zactima™; AstraZeneca Pharmaceuticals), and CEP-7055 (Sanofi-Aventis), based on encouraging preclinical data in glioma xenografts in nude mice (Goldbrunner etÂ€al. 2004; Reardon and Wen 2006). Clinical trials of other multitargeted kinase inhibitors that

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block VEGF receptors among other kinase targets, such as sorafenib, AZD2171 (AstraZeneca), sunitinib (SU11248, Pfizer), have also been conducted in adults with malignant gliomas. A phase I study of sorafenib has been conducted in the Children’s Oncology Group, although there was not a specific focus on gliomas, and a phase II study is being considered. A phase I study of AZD2171 is currently ongoing in the Pediatric Brain Tumor Consortium (PBTC021).

VEGF Blockade A particularly promising agent is bevacizumab (Avastin®; Genentech, Inc.), a recombinant, humanized monoclonal antibody targeting VEGF (Ferrera etÂ€ al 2005). This agent has been shown to cause decreased vascular permeability and increased apoptosis in intracranial xenografts of human glioblastoma and synergism has been observed with several chemotherapeutic agents (Ignoffo 2004). In a phase III placebo-controlled trial of 815 patients with metastatic colorectal cancer who were randomized to receive either bevacizumab plus irinotecan/5-FU/leucovorin or irinotecan/5-FU/leucovorin plus placebo, bevacizumab produced a significantly better rate and duration of response compared to placebo (Hurwitz etÂ€ al. 2004). Bevacizumab also prolonged the time to progression compared with placebo for patients with metastatic renal cell cancer (Yang etÂ€al. 2003). Stark-Vance (2005) has also treated adult patients with recurrent malignant glioma with bevacizumab plus irinotecan and reported 1 patient with a complete response (CR), 8 patients with PR, 11 patients with stable disease, among 21 patients treated. On the basis of the preclinical and clinical efficacy demonstrated in adult studies of recurrent solid tumors, a phase II study was initiated for patients with recurrent malignant glioma. In a preliminary communication, 20 of the first 32 patients (63%) reportedly demonstrated an objective response (19 PR and 1 CR) to treatment (Vredenburgh etÂ€al. 2006) with a 6-month progression-free survival rate of 39%, which provided a basis for a host of additional studies in adults. These results also provided a basis for a phase II study of bevacizumab plus irinotecan initiated by the PBTC (PBTC022) for patients with recurrent malignant gliomas and those with brainstem malignant gliomas.

Other Angiogenic Agents In addition to inhibition of promoters of VEGF secretion, such as EGFR and PDGFR, and direct inhibition of VEGF receptors and VEGF itself, evaluation of a number of other agents that have affects on angiogenesis has been conducted in adult and pediatric malignant gliomas. Monotherapy with thalidomide (Thalomid®; Celgene Corporation) has been investigated for the treatment of GBM and malignant brainstem glioma because of its antiangiogenic effects. Although this agent has had

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only modest activity in this setting, with 4 of 36 objective radiologic responses in one study (Fine etÂ€ al. 2000), more recent studies have examined lenalidomide (CC-5013, (Revlimid®; Celgene Corporation)), a more potent analog of thalidomide. A study of this agent for patients with recurrent brain tumors is being conducted by the PBTC (PBTC-018). Long-term stable disease has been observed in several patients with gliomas, and a phase II study is being considered. In addition, it has been observed that the combination of thalidomide and chemotherapy appears to be more active in patients with recurrent gliomas than either approach alone (Fine etÂ€al. 2003), and combinatorial studies with this agent as well as CC-5013 are being considered. Other agents that are being examined as potential angiogenesis inhibitors include the avb3 integrin inhibitor cilengitide (EMD 121974; EMD Pharmaceuticals) and the previously mentioned PKC inhibitor enzastaurin (LY317615), which among its other effects has been noted to decrease VEGF levels in a mouse tumor model (Taga etÂ€al. 2002; Keyes etÂ€al. 2004). A phase I trial of cilengitide was conducted in the PBTC (PBTC-012), and the MTD was carried forward in a Phase II trial being conducted within the Children’s Oncology Group (ACNS0621). As noted above, a phase I trial of enzastaurin is in progress within the PBTC (PBTC023). Other angiogenic inhibitors of interest include cyclooxygenase 2 (COX-2) inhibitors, based on the association between COX-2 expression and angiogenesis (Reardon and Wen 2006). Both celecoxib (Celebrex, Pfizer Pharmaceuticals) and rofecoxib (Vioxx, Merck) have been combined with conventional chemotherapeutic agents in studies for both children and adults with recurrent brain tumors, including gliomas (Reardon etÂ€al. 2005b; Tuettenberg etÂ€al. 2005). Other agents include endostatin and angiostatin, which are natural product inhibitors of angiogenesis, and Atrasentan, a selective inhibitor of the endothelin A receptor, which has been proposed to be involved in regulating glioma-induced angiogenesis (Barnett etÂ€ al. 2004; Phuphanich etÂ€ al. 2005). In a phase I study of this agent, outcome results were disappointing with a median progression-free survival time of only 1.5Â€months.

Interference with Growth Factor Receptor and Survival Signaling May Potentiate Other Therapies Although signal transduction inhibition may have activity in decreasing proliferation and inducing apoptosis in a subset of tumors, the efficacy of these approaches may be enhanced by combining them with other therapeutic strategies. Preclinical data suggest potentiation of the activity of cytotoxic drugs by growth factor receptor inhibitors. EGFR-targeted monoclonal antibodies have been noted to enhance the effects of cisplatin (Baselga etÂ€al. 2000), topotecan, gemcitabine (Bruns etÂ€al. 2000), and taxol. In addition, marked radiosensitization has been achieved by EGFRspecific monoclonal antibodies (Huang etÂ€al. 1999; Milas etÂ€al. 2000) and dominant negative transfection (O’Rourke etÂ€al. 1998). Recent studies also suggest that small molecule inhibitors of EGFR kinase activity, such as gefitinib (Ciardiello etÂ€al. 2000;

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Huang etÂ€ al. 2002), and PDGFR kinase activity, such as imatinib (Topaly etÂ€ al. 2001; Pietras etÂ€al. 2002), may achieve similar potentiation of conventional therapies. The application of such combinatorial approaches in brain tumor therapeutics is already under way. Both adult and pediatric brain tumor trials have examined the combination of erlotinib and temozolomide, and pediatric studies in brainstem glioma have evaluated the combination of gefitinib and concurrent irradiation (PBTC007), and imatinib immediately following irradiation in newly diagnosed patients (PBTC006), with outcome comparisons versus historical control groups treated with irradiation alone (Pollack etÂ€al. 2007). In addition to the applicability of combining growth factor receptor-targeted inhibitory strategies with conventional chemotherapy, recent studies indicate that combinations of growth factor receptor inhibitors with other molecularly targeted approaches may potentiate efficacy by circumventing resistance mechanisms (Bianco etÂ€ al. 2003). For example, Bianco etÂ€ al. (2003) observed that treatment resistance to EGFR inhibitors in tumors with PTEN deletions could be counteracted by independent inhibition of Akt signaling, suggesting a role for agents, such as rapamycin, that block downstream Akt signaling, in conjunction with EGFRtargeted therapies. In that context, preliminary data from a phase I trial of gefitinib in combination with rapamycin for the treatment of patients with recurrent malignant glioma have been reported (Reardon etÂ€al. 2006b). The optimal agent(s) to combine with individual growth factor receptor inhibitors is a topic of intense research interest. Similarly, combinations of agents may be needed to effectively block angiogenic signaling. The combination of endostatin and SU5416 has been reported to achieve superior tumor growth inhibition in preclinical models, compared to treatment with either agent alone (Abdollahi etÂ€al. 2003). Combinations of antiangiogenic agents with conventional chemotherapeutic agents have also shown promise. For example, patients with recurrent GBM who received thalidomide and carmustine had a response rate of 24%, which compared favorably with carmustine alone (Fine etÂ€al. 2003). Moreover, the combination of thalidomide and temozolomide in patients with GBM was more effective than thalidomide alone with respect to survival and response (Baumann etÂ€al. 2004). Recent studies for patients with recurrent brain tumors, including children, have incorporated multiple antiangiogenic agents in a “cocktail,” in an effort to simultaneously block several targets potentially contributing to tumor-induced neovascularization.

Counteracting the Drug Resistance Phenotype Alkylguanine DNA Alkyltransferase Inhibition Drug resistance, either intrinsic or acquired, is frequently observed in tumors of the CNS, and many different mechanisms have been described. One particularly important mechanism involves the reversal of DNA damage induced by alkylating agents,

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such as BCNU and temozolomide. These agents cause alkylation at the O6-position of guanine, which can be repaired by alkylguanine-DNA alkyltransferase (AGT), also known as methylguanine DNA methyltransferase (MGMT), a DNA-binding protein with an alkyl group acceptor site. This protein has some characteristics of an enzyme; however, unlike a true enzyme, the receptor site on AGT becomes irreversibly alkylated and the AGT is permanently inactivated and rapidly degraded (Dolan etÂ€al. 1993; Hotta etÂ€al. 1995). Once the enzyme is depleted, further repair by this mechanism requires deÂ€novo synthesis of the protein. Therefore, the ability to repair O6-alkylation of DNA is related to the number of AGT molecules and the rate of protein resynthesis (Belanich etÂ€al. 1996). Although endogenous AGT levels vary by organ and cell type, many tumors express AGT levels much higher than their corresponding normal tissue, suggesting that this protein provides an acquired mechanism for drug resistance. Whereas brain tissue expresses low or undetectable levels of AGT, significant levels of expression have been observed in most brain tumor specimens from both children and adults, in many cases several orders of magnitude higher than normal brain (Silber etÂ€al. 1993). Several groups have noted that AGT levels correlated inversely with response to alkylator-based therapy in preclinical models (Schold etÂ€al. 1989; Hotta etÂ€al. 1995). Moreover, several investigators have confirmed an adverse association between elevated AGT levels and clinical response to nitrosoureas and temozolomide (Belanich etÂ€al. 1996; Esteller etÂ€al. 2000; Jaeckle etÂ€al. 1998; Hegi etÂ€al. 2005; Pollack etÂ€al. 2006b). Although the preferred substrate for AGT is O6-methylguanine on double-stranded DNA, the protein will target other O6-alkylated substrates, which if present in excess, can effectively deplete AGT activity. In particular, O6-benzylguanine (O6-BG) has been shown to reverse AGT-mediated resistance to alkylating agents in a variety of tumor cell lines inÂ€vitro by saturating AGT (Dolan etÂ€al. 1991; Gerson etÂ€al. 1993), and inÂ€vivo studies have confirmed that O6-BG potentiates the therapeutic effect of BCNU and other alkylating agents. In a clinical trial in adult patients with gliomas who received O6-BG as a bolus dose of 100Â€ mg/m2, AGT levels within the tumor 18Â€hours after O6-BG administration were below the level of detection in 11/11 patients (Friedman etÂ€al. 1998a). No serious side effects were observed either with O6-BG alone or in combination with low doses of BCNU (Friedman etÂ€al. 1998a). In a recent pediatric study, P9871, a fixed dose of 06-BG was administered with escalating doses of BCNU (dose levels: 25 to 78Â€ mg/m2). Although there were a number of objective responses, it was unclear whether this exceeded the rate that might have been observed with BCNU alone. In addition, a recently completed phase I study of the NABTT, involving 41 patients undergoing Gliadel wafer implantation, demonstrated that administration of a continuous infusion of 30Â€mg/m2/day O6-BG following the initial bolus maintained an extended interval of AGT suppression. Following confirmation of AGT suppression to unmeasurable levels in 11 of 13 patients who received a 2-day infusion prior to tumor resection and Gliadel placement, the postsurgical infusion duration was increased to 2Â€weeks (Weingart etÂ€al. 2007). Although this was tolerated without excessive toxicity, it remains to be determined whether combining this approach with wafer implantation improves tumor response rate or progression-free survival

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compared to Gliadel delivery alone. Such an approach was also initiated in the PBTC (PBTC-009), but in view of the high frequency with which tumor resections necessitated large openings in the ventricular wall, accrual of eligible patients was challenging, and the study ultimately closed. More recently, attention has been directed at combining O6-BG with temozolomide. 6 O -BG has been noted to increase tumor cell sensitivity to temozolomide by 2.5- to 10-fold inÂ€vitro and to potentiate sensitivity to this agent in malignant glioma xenograft models (Wedge and Newlands 1996). This enhanced efficacy is to some extent counterbalanced by increased toxicity against normal tissues, such as hematopoietic progenitor cells (Fairbairn etÂ€al. 1995), necessitating dose reductions of temozolomide. A Phase I trial of O6-BG and temozolomide on a 5-day schedule in pediatric patients has recently been completed. Patients received O6-BG IV over 60Â€ min, followed 30Â€ min later by oral temozolomide, dailyâ•›×â•›5Â€ days every 4Â€ weeks. This study had a two-part dose-escalation scheme. Initially, the dose of O6-BG was escalated from 60 to 90 to 120Â€mg/m2/dayâ•›×â•›5, while the temozolomide dose was held at 28Â€mg/m2/day. In subsequent cohorts, the dose of temozolomide was escalated while the dose of O6-BG was held constant at 120Â€mg/m2/day. Forty-one patients were entered and 32 were evaluable. The MTD was defined as an O6-BG dose of 120Â€mg/m2/day and a temozolomide dose of 75Â€mg/m2/day. Dose-limiting toxicity was myelosuppression. Several patients had objective responses or prolonged stable disease. Based on these data, the PBTC opened two phase II studies with temozolomide and O6-BG. One study (PBTC-015) used the above MTD daily for 5 days of each 28-day cycle, for patients with recurrent malignant gliomas and malignant brainstem gliomas. A second study (PBTC-005), used an O6-BG bolus with escalating doses of temozolomide, followed by a continuous O6-BG infusion for 48Â€hours.

Base-Excision Repair Inhibition Although AGT is a proximal mechanism of resistance to alkylating agents, other mediators are also involved. Friedman etÂ€al. (1998b) noted that defects in mismatch repair can contribute to temozolomide resistance. For example, O6-methylguanine pairs with thymine rather than guanine, leading to repetitive cycles of futile mismatch repair, which results in growth arrest and/or apoptosis (D’Atri etÂ€al. 1998). A deficiency of DNA mismatch repair bypasses this step and thereby renders cells resistant to the apoptotic effects of temozolomide inÂ€ vitro (Liu etÂ€ al. 1996) and inÂ€ vivo (Friedman etÂ€ al. 1997). Additional mechanisms of resistance are also involved, including alterations in the DNA base excision repair (Liu etÂ€al. 1999) and apoptotic signaling pathways (Bocangel etÂ€al. 2002). The importance of recognizing these alternative resistance mechanisms is that DNA mismatch repair deficiency can confer resistance to temozolomide, even after O6-BG mediated AGT inhibition (Friedman etÂ€ al. 1997; Bocangel etÂ€ al. 2002). Likewise, a series of studies have shown that two temozolomide-initiated adducts,

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N7-methylguanine and N3-methyladenine, are not susceptible to AGT demethylation and thus produce cytotoxicity independently of AGT or DNA mismatch repair activity (Tentori etÂ€al. 2003). These lesions are repaired by the base excision repair pathway, involving the actions of N-methylpurine-DNA glycosylase, AP endonuclease, poly (ADP-ribose) polymerase (PARP), DNA polymerase b, X-ray repair cross complementing protein 1 (XRCC1), and ligase III. Tumor cells resistant to temozolomide due to DNA mismatch repair deficiency have been rendered susceptible by inhibition of base excision repair. One strategy to accomplish this has involved inhibition of PARP, one of the most abundant proteins in the nucleus, which binds to damaged DNA through a zinc finger domain, catalyzing the cleavage of NAD+ into nicotinamide and ADP-ribose and using the latter to synthesize branched nucleic acid polymers, which are covalently attached to nuclear acceptor proteins (Liu etÂ€al. 1999; Tentori etÂ€al 2002, 2003). Tentori etÂ€al. (2002, 2003) have demonstrated that PARP inhibition increases susceptibility to apoptosis induced by temozolomide inÂ€vitro and increases the antitumor activity of temozolomide treatment in mice with orthotopic xenografts of human GBM. INO-1001 (Inotek Pharmaceuticals Corporation) has recently been identified as a potent PARP inhibitor, and preclinical studies of this agent for malignant gliomas were conducted at the Duke University Medical Center. The combination of this agent with temozolomide was evaluated in athymic mice carrying AGT-negative, MMR-deficient or MMR proficient D-245 malignant glioma xenografts. Groups of ten mice carrying these xenografts were treated with varying doses of INO-1001 and temozolomide. Higher doses of temozolomide and INO-1001 resulted in significant tumor regression in MMR-deficient xenografts compared to temozolomide alone, suggesting that INO-1001 was able to reverse MMR deficiency-related resistance. A phase I clinical trial of this combination was recently initiated in adults with malignant gliomas and consideration has been given to a separate pediatric study. Studies with an alternate PARP inhibitor, ABT888, are also under development.

Other Contributors to Drug Resistance Recent studies have indicated that alterations in the patterns of gene expression and protein processing may influence the growth promoting and apoptosis-resisting phenotype of malignant glioma cells, which has suggested the use of several strategies. There is evidence that histone processing is altered in malignant gliomas. The acetylation and deacetylation of histone proteins of the nucleosomes in chromatin play an important role in the regulation of gene expression (Gray and Ekstrom 2001). Acetylation of histone lysine residues is associated with a relaxation of the DNA wrapped around the core histones, enhancing access by the transcriptional machinery; conversely, deacetylation condenses the nucleosome structure, restricting access to the DNA (Marks etÂ€al. 2000). Accordingly, treatment of mammalian cells with inhibitors of histone deacetylase (HDAC) activity results in increased expression of a variety of genes. In recent years, HDAC inhibitors have been observed to have antitumor effects,

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inhibiting cell growth, inducing terminal differentiation and preventing the formation of tumors in murine models (Yoshida etÂ€al. 1995; Marks etÂ€al. 2000). One of the first HDAC inhibitors to be evaluated clinically was phenylbutyrate. In a phase I/II study in adults with recurrent solid tumors, Gilbert etÂ€ al. (2001) observed that 25% of patients had stable disease for >6Â€months. In addition, Baker etÂ€al. (2002) reported a complete response in a recurrent multicentric malignant glioma treated with phenylbutyrate. Other HDAC inhibitors have been observed to independently inhibit tumor growth inÂ€vitro and potentiate the activity of conventional cytotoxic agents (Marks etÂ€al. 2001; Sawa etÂ€al. 2004). As with phenylbutyrate, treated cells typically undergo cell cycle arrest with induction of p21 and, in some cases, apoptosis. Clinical trials of several histone deacetylase inhibitors, including valproic acid, depsipeptide, and suberoylanilide hydroxamic acid (SAHA), are currently under way or planned (Plumb etÂ€al. 2003). A second strategy for modulating the availability of proteins that may influence apoptosis resistance involves inhibition of members of the heat shock protein (HSP) family, which are involved in the conformational maturation of a variety of key growth stimulating and apoptosis-inhibiting proteins. Inhibitors of HSP90, such as geldanamycin and 17-AAG, have been observed to potentiate the efficacy of conventional cytotoxic chemotherapeutic agents against gliomas (Graner and Bigner 2005) as well as other signaling modulators (Premkumar etÂ€al. 2006), although their clinical applicability for gliomas has been limited by their extremely poor penetration of the blood–brain barrier. Other potential targets for reversing therapy resistance in malignant gliomas include inhibition of NF-kB, which is involved in counteracting downstream mediators of apoptotic signaling. In the absence of a clinically applicable direct pharmacological NF-kB inhibitor, this has been accomplished indirectly by inhibition of the ubiquitin/proteasome system, which regulates the post-transcriptional degradation mechanism of NF-kB, as well as proteins involved in cell cycle regulation, DNA transcription and repair, apoptosis, angiogenesis, and cell growth. Accordingly, there has been interest in drugs that target this system. One such agent, bortezomib (Velcade®; Millennium Pharmaceuticals, Inc.), has been noted to induce apoptosis in human GBM cell lines and primary GBM explants and is currently in preliminary testing in adult patients with recurrent or progressive gliomas (Yin etÂ€al. 2005), with the eventual goal of combining this agent with a conventional cytotoxic chemotherapeutic agent. More developmental strategies for potentiating apoptosis include stimulation of signaling from TNF-related apoptosis-inducing ligands, such as TRAIL/Apo2L. Although this strategy has shown significant promise in glioma preclinical models in several laboratories (Pollack etÂ€ al. 2001a; Walczak etÂ€ al 1999), clinical applicability of this approach remains to be confirmed. Finally, recent studies have explored the use of inhibitors of cell cycle regulatory proteins, in order to either (1) promote passage of injured cells through the cell cycle, thereby bypassing damage repair steps or (2) block cell cycle progression at phases of the cell cycle that confer vulnerability to various classes of chemotherapeutic agents. In the former category are agents, such as 7-hydroxystaurosporine (UCN-01), which interfere with Chk1 kinase function. By blocking cdc25 phosphorylation,

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UCN-01 secondarily disinhibits cell cycle progression through the G2/M checkpoint, thereby abrogating the potential for cell cycle arrest and DNA repair after treatment with agents such as temozolomide (Hirose etÂ€al. 2001). Conversely, other inhibitors, such as cyc202 (Cyclacel), flavopiridol, and numerous recently characterized small molecular inhibitors, block various members of the cyclin-dependent kinase family, thereby promoting cell cycle arrest, which potentially causes cells to remain in portions of the cell cycle that are selectively vulnerable to adjuvant modalities, such as irradiation and conventional chemotherapy.

Immunological or Ligand-Based Therapies Vaccine-based (active) immunotherapy against protein targets expressed in gliomas is a potentially promising immunological approach that has recently transitioned from preclinical to pilot clinical studies. As a cellularly mediated strategy, it is beyond the scope of the current chapter, but is nonetheless relevant from the standpoint of molecularly targeted approaches. An array of pharmacologically oriented immunotherapy approaches has also been developed in recent years. Although systemic application of antibody-based inhibitor strategies to neutralize or block receptor-mediated signaling in malignant gliomas is challenged by the presence of a blood–brain barrier, one strategy for circumventing this limitation has involved the use of antiangiogenic antibodies targeted to the tumor vasculature, such as bevacizumab, which obviates concerns about blood vessel penetration. A second strategy has involved intracavitary delivery of antibodies directed against proteins overexpressed in the tumor cells, such as tenascin or EGFR, linked to radioisotopes or toxins (Lorimer etÂ€al. 1996; Reardon etÂ€al. 2006a). Recent studies have suggested a potential survival benefit in appropriately selected patients using such intratumoral delivery approaches (Quang and Brady 2004). A third approach for achieving delivery of a high-molecular weight agent into the tumor involves convectional-enhanced delivery, which employs positive-pressure infusion of microliter volumes over a period of several days through stereotactically placed catheters (Bobo etÂ€ al. 1994) to achieve bulk flow directly into the brain tumor or peritumoral brain. In preclinical studies, this approach allowed for the distribution of an active agent over wide areas of the brain in rodent and primate models (Laske etÂ€al. 1997a). Subsequent clinical studies confirmed the feasibility of drug distribution over a wide area around a tumor, along the white matter tracts that provide principal pathways for tumor expansion. A common strategy for applying these techniques has been to use ligands or antibodies targeted against receptors overexpressed on brain tumor cells relative to normal brain, and coupled to a mutated toxin, such as pseudomonas exotoxin or diphtheria toxin. To date, a variety of receptors have been targeted, including EGFR, the interleukin 4 (IL4), and 13 (IL13) receptors, and the transferrin receptor (TfR). By eliminating or inactivating the receptor binding moiety of the native toxin and conjugating the active component of the toxin to a ligand or antibody for the tumor-associated receptor, the immunotoxin is selectively transferred into receptor-expressing tumor cells, but excluded from

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normal cells, thereby facilitating tumor-specific cell killing (Pastan and FitzGerald 1991; Greenfield etÂ€ al. 1987). Adverse effects have consistently related to edema or necrosis within peritumoral brain, rather than systemic toxicity, which calls attention to the importance of defining volumetric and concentration-related delivery parameters. A discussion of several of the convection-enhanced delivery approaches that have been examined in malignant gliomas is provided below.

Tf-CRM107 The first immunotoxin to undergo clinical testing for patients with brain tumors was Tf-CRM107, a conjugate of a mutated diphtheria toxin linked to human transferrin (Laske etÂ€al. 1997b). Diphtheria toxin has A and B subunits, with the A subunit catalyzing the transfer of ADP ribose to elongation factor-2, thereby blocking the transfer of peptidyl-tRNA on ribosomes, which inhibits protein synthesis and kills the cell. The B subunit is responsible for toxin binding to the cell surface. In CRM107, the B chain is mutated, which decreases binding activity by four orders of magnitude, but leaves the toxin functions intact. Because transferrin receptors are highly expressed on rapidly dividing cells, such as malignant glioma cells (Recht etÂ€al. 1990), in comparison to low levels of expression on normal glial cells, selective tumor targeting is feasible with local drug delivery. Preclinical studies demonstrated the efficacy of this toxin in inhibiting protein synthesis (Weaver and Laske 2003) with IC50 values in the low picomolar range. Activity of the conjugate was demonstrated against subcutaneous and intracranial glioma xenografts (Laske etÂ€al. 1994). These results provided a foundation for a phase I clinical study of this agent in patients with recurrent malignant brain tumors to determine the maximal tolerated drug concentration and infusate volume (Laske etÂ€ al. 1997b). All patients underwent stereotactic biopsy to confirm the presence of tumor, followed by implantation of 1 to 3 silastic infusion catheters into the tumor. Nine of 15 evaluable patients demonstrated more than a 50% decrease in tumor volume and two had complete responses. Although transient worsening in neurological status occurred during 3 of 44 infusions, this was controllable with steroids and hyperosmolar therapy. A subsequent phase II study used the maximal tolerated dose established in the above study followed by a second infusion cycle 4 to 10Â€weeks later. Forty-four patients were enrolled, and of the 34 evaluable for efficacy, five had complete responses and seven had partial responses. These encouraging results provided a rationale for a larger phase III trial in adults with recurrent malignant gliomas as well as studies for newly diagnosed patients. A pediatric trial has been considered but not undertaken.

IL13-PE38QQR IL13-PE38QQR (Neopharm) is a tumor-targeted cytotoxin that combines human IL13 and an enzymatically active portion of Pseudomonas exotoxin (PE). This exotoxin contains three domains: domain Ia binds to the PE receptors, domain II

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catalyzes the translocation of the toxin into the cytosol, and domain III contains the toxin moiety (Pastan and FitzGerald 1991). The immunotoxin is formed by expression from a chimeric gene that fused the human gene for IL13 with a mutated PE gene harboring amino acid substitutions in the receptor binding domain (Debinski etÂ€ al. 1995). The rationale for developing this combination was the observation that malignant glioma cells express the IL13 receptor (IL13R) at high density, while normal brain tissue expresses little if any IL13R (Debinski etÂ€al. 1999; Joshi etÂ€al. 2000). In vitro studies have demonstrated that most malignant glioma cell lines are sensitive to IL13-PE38QQR at concentrations in the picomolar range, with inhibition of protein synthesis and loss of clonogenicity. In vivo glioma model studies in nude mice demonstrated significant activity with intratumoral delivery in an IL13R-dependent fashion. Following toxicology studies in rodent and primate models (Husain and Puri 2003), a series of phase I and II clinical trials were initiated in patients with recurrent malignant gliomas by the NABTT, the NABTC, and a multinational consortium. The NABTT study (Weingart etÂ€al. 2003) involved the administration of the drug over 96Â€hours via two intratumoral catheters in two infusions for patients with unresectable malignant gliomas, with escalation of the infusion concentration. The NABTC study assessed the histological effects and safety of pre- and postoperative infusions of IL13-PE38QQR in patients with recurrent resectable malignant glioma. In the first stage of this study (Prados etÂ€ al. 2002), after biopsy and catheter placement into the tumor on day 1, the drug was delivered via one intratumoral catheter for 48Â€hours on days 2 to 4. Resection was conducted on day 8, with a goal of en bloc removal of the tumor and catheter to determine the histologically effective dose to the primary tumor site. Following resection, two or three catheters were placed in the peritumoral brain, and the immunotoxin was administered for 96Â€hours, at a range of doses to assess toxicity in peritumoral brain. Not unexpectedly, the intratumoral preresection dose was effective in inducing tumor necrosis immediately around the infusion catheter, and given the lack of penetration into the surrounding brain and the fact that the treated area was resected, it had relatively low toxicity. Subsequently, in the second stage of the study, the preresection dose was eliminated and the postresection dose was escalated. Objective responses and long-term survival have been noted in patients with appropriately placed peritumoral catheters (Kunwar etÂ€al. 2007). A third multinational study used a single infusion, with escalation of the infusion duration at a constant intratumoral infusion rate and concentration, followed by resection at week 3. After establishment of the maximally tolerated infusion duration, IL13-PE38QQR concentration was escalated to define the maximal tolerated concentration. Taken together, these studies provided a basis for a phase III randomized study comparing IL13-PE38QQR with GLIADELTM; unfortunately, preliminary results were reportedly disappointing. A pediatric trial (PBTC011C) had been developed, although its opening was placed on hold based upon preliminary analysis of the aforementioned adult trial.

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TP-38 Human malignant gliomas commonly overexpress EGFR relative to normal astrocytes. As with the IL13-PE38QQR, TP-38 (IVAX Research, Inc) was designed to couple a receptor ligand, in this case TGFa, an endogenous ligand for EGFR, with a mutated form of the pseudomonas exotoxin. The interactions between TGFa and EGFR provide the basis for internalization of the toxin. Following animal toxicology and efficacy results, pilot studies of this agent in adults were conducted at Duke University and the University of California at San Francisco (Sampson etÂ€al. 2003). Catheters were placed stereotactically with radiographic confirmation of catheter position prior to infusion. TP-38 was infused over 50Â€h at a fixed flow rate and volume, with escalation of the concentration and dose in successive cohorts. Patients were stratified based on the presence or absence of residual disease at the time of infusion. Toxicity was solely neurologic. In an initial report of this series, 2 of 15 patients with residual disease had objective responses and four remained progression-free for 1Â€year. Based on these promising results, further studies were initiated and a pediatric trial was briefly opened (PBTC-013), although closed secondary to funding constraints prior to achieving the study objectives. In addition, studies of another cytokine receptor targeted immunotoxin approach, using a circularly permutated IL4 conjugated to a mutated pseudomonas exotoxin have also been initiated in adult patients, based on encouraging preclinical data (Puri etÂ€al. 1996) and evidence of activity in an initial pilot study (Weber etÂ€ al. 2003). Because of these promising findings, a multicenter dose escalation study was planned followed by a phase II efficacy study.

Future Directions and Challenges Because of the poor responsiveness of malignant gliomas to conventional therapies, there is a pressing need to implement new approaches to improve the outcome of patients with these tumors. Studies in many laboratories have demonstrated that the proliferation and survival of malignant glioma cells is strongly influenced by a number of molecular pathways that stimulate tumor growth, promote angiogenesis, and inhibit apoptosis induction. These pathways have therefore emerged as promising targets for therapy, which have been effectively addressed by small molecule inhibitors as well receptor-directed cytotoxins. The major ongoing challenges are the identification of tumor genotypic and phenotypic features that predict response to individual agents and the discovery of rational combinations of agents to potentiate therapeutic efficacy. Given the molecular diversity and heterogeneity of malignant gliomas, it is unlikely that any single agent will have efficacy in more than a subset of tumors. Moreover, responses to individual drugs, if any, are likely to be transient at best. Accordingly, the appropriate use of these agents will likely require combinations of molecularly targeted compounds and conventional therapies or of several molecularly targeted agents

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administered as a “cocktail,” incorporating a tumor-tailored therapy paradigm based on the molecular features of the lesion to guide agent selection. Acknowledgmentâ•… This work was supported in part by NIH grant NSP0140923.

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Targeted Therapy in Medulloblastoma in Molecularly Targeted Therapy for Childhood Cancer Yoon-Jae Cho and Scott L. Pomeroy

Introduction Medulloblastomas are primitive embryonal tumors of the central nervous system arising exclusively in the cerebellum. They have a tendency to disseminate via CSF spaces throughout the brain and spine and are the most common malignant brain tumors in children (CBTRUS 2005). There has been considerable progress in our understanding of the mechanisms driving these tumors through the study of normal cerebellar development, familial cancer syndromes, genome-based analyses of human primary tumor samples (Table 1) and genetic mouse models. These studies have provided a framework for the development of several targeted therapies. This chapter focuses on our current molecular understanding of medulloblastoma and the targeted therapies in development and clinical trials.

Histopathological and Clinical Considerations Medulloblastomas are a heterogeneous group of small blue cell tumors. The 2007 World Health Organization (WHO) classification differentiates subtypes of medulloblastoma by specific, sometimes subtle, and histological features. The major recognized subtypes are classic, desmoplastic/nodular, and medulloblastoma with extensive nodularity, large cell, and anaplastic variants (Louis etÂ€ al. 2007). Rarer subtypes include melanotic and medullomyoblastoma. Classic medulloblastomas are composed of sheets of small round blue cells with high nuclear to cytoplasmic ratios and, typically, uniform nuclei set in a fibrillary background. There may be signs of neuronal differentiation in the form

S. L. Pomeroy (*) Department of Neurology, Children’s Hospital Boston, 300 Longwood Avenue, Enders 270, Boston, MA 02115, USA e-mail: [email protected]

P.J. Houghton and R.J. Arceci (eds.), Molecularly Targeted Therapy for Childhood Cancer, DOI 10.1007/978-0-387-69062-9_13, © Springer Science+Business Media, LLC 2010

267

Nijmegen breakage syndrome

NBS1

8q21

Medulloblasotma, lymphoma, leukemia, rhabdomyosarcoma

TableÂ€1â•… Cancer predisposition syndromes associated with medulloblastoma Syndrome Gene Locus Tumor spectrum Gorlin syndrome PTCH1 9q22.3 Medulloblastoma, basal cell carcinoma Turcot syndrome APC 5q21 Medulloblastoma, colorectal adenoma Li Fraumeni syndrome TP53 17p13.1 Medulloblastoma, leukemia, sarcomas, carcinoma Fanconi anemia, type D BRCA2 13q12.3 Medulloblasotma, leukemia, breast cancer, Wilms’ tumor

References Hahn etÂ€al. (1996), Johnson etÂ€al. (1996) Hamilton etÂ€al. (1995) Pearson etÂ€al. (1982), Barel etÂ€al. (1998), Guran etÂ€al. (1999) de Chadarevian etÂ€al. (1985), Ruud and Wesenberg (2001), Offit etÂ€al. (2003), Hirsch etÂ€al. (2004), Tischkowitz etÂ€al. (2004) Bakhshi etÂ€al. (2003), Distel etÂ€al. (2003)

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of Homer-Wright rosettes or occasional groups of ganglion cells. To a small degree, differentiation along glial lineages as well as nuclear pleomorphism can be seen. However, when nuclear pleomorphism is abundant, with cell molding, numerous mitotic figures and apoptotic bodies, the tumor is considered an anaplastic variant. Large cell medulloblastomas contain groups of cells with round nuclei, open chromatin, and a single nucleolus and often contains regions similar to the anaplastic phenotype (Louis etÂ€al. 2007). Due to the considerable overlap of large cell and anaplastic medulloblastomas, these two subtypes are often thought of as a continuum of the same tumor. Medulloblastomas may also show varying degrees of a nodular architecture which largely defines the desmoplastic/nodular and medulloblastoma with extensive nodularity subgroups. These tumors are characterized by scattered nodules of neurocytic cells among desmoplastic regions containing larger and more pleomorphic nuclei. In the desmoplastic subtype, the “pale islands” of tumor cells are surrounded by a dense fibrous, reticulin rich stroma (McManamy etÂ€ al. 2007). Medulloblastomas with extensive nodularity in comparison have less of the dense reticulin but instead a markedly expanded lobular architecture due to the fact that the reticulin-free zones become unusually large and rich in neuropil-like tissue. This confers a “grape-like” appearance on neuroimaging and gross pathology.

Risk Stratification Several studies have associated histological subtype with outcome, in particular, desmoplastic tumors with good prognosis and large cell/anaplastic variants with poor prognosis (Pomeroy etÂ€al. 2002; Gajjar etÂ€al. 2006). However, intratumoral heterogeneity is not uncommon and a single tumor may have regions suggestive of one subtype and other regions suggestive of a different subtype. This often makes definitive subtyping via traditional histopathology techniques difficult. Various molecular and/or cytogenetic features have been assigned to help define histological subtypes and although they are increasingly incorporated into diagnostic schemas, they are neither uniformly used nor standardized. Thus, current risk stratification schemas are based solely on clinical criteria. This includes the age at diagnosis, the extent of surgical resection, and the presence or absence of tumor dissemination at diagnosis. Specific multimodal treatment regimens based on this clinical stratification include surgical resection, age appropriate craniospinal irradiation, and conventional chemotherapy. This schema has afforded 5-year survival rates of up to 85% for standard risk patients and 60 to 80% for high risk groups (Crawford etÂ€ al. 2007). Survivorship, however, almost universally comes at some cost with long-term neurological and neurocognitive deficits resulting from aggressive therapies on the developing nervous system. Thus, emphasis is

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placed on the identification of molecular targets to which less toxic targeted therapies can be developed and applied.

Cellular Origins of Medulloblastoma As medulloblastomas arise exclusively in the cerebellum, studies of cerebellar development have provided significant insight into its origin. Fate-mapping experiments in the mouse and chick reveal most, if not all, cell types in the mature Â�cerebellum are derived from two germinal zones: the ventricular zone and the rhombic lip (Hatten etÂ€ al. 1997; Lin and Cepko 1999; Machold and Fishell 2005; Wang etÂ€al. 2005a). The ventricular zone generates Purkinje neurons and various deep cerebellar nuclei, including the dentate nucleus. In addition, this region contributes Bergmann glia and other astroglial derivatives (Sottile etÂ€al. 2006). Precursors from the rhombic lip, however, appear to be more restricted in their differentiation potential and are fated to become granule cell neurons, the most abundant neuronal population in the mature central nervous system (Alder etÂ€al. 1996; Goldowitz and Hamre 1998). During development, granule neuron precursor cells stream from the rostral portion of the rhombic lip around the outermost boundary of the cerebellum forming the external granular layer (EGL). Here, they undergo tremendous proliferation and upon exiting the cell cycle migrate to the internal granular layer, where they become mature granule cell neurons. Mouse studies have shown Sonic Hedgehog (SHH) expressed from Purkinje neurons that regulate the proliferation of granule cell progenitors (Wallace 1999). This proliferation is completed within the first 3Â€weeks in the postnatal mouse, and the EGL is largely gone by 1Â€month of age in mouse and 1 to 2Â€years of age in humans (Wechsler-Reya and Scott 1999). Their abundance and prolonged proliferative phase during cerebellar development make granule neuron precursors an attractive candidate as the cell of origin in medulloblastoma. This is especially the case for desmoplastic medulloblastomas whose gene-expression patterns indicate dysregulated SHH signaling, a key regulator of granule cell proliferation and specification (Pomeroy etÂ€al. 2002). The cell of origin for the majority of classic and large cell/anaplastic medulloblastomas is less clear. Conceivably, these subgroups might arise from granule cell precursors through mutations involving other molecular pathways. Alternatively, they might arise from different cells of origin (Katsetos etÂ€ al. 1995). Indeed, the Wingless (WNT) signaling pathway is important in the support and maintenance of ventricular zone precursors and is pathologically activated in a subset of classic medulloblastomas (Ellison etÂ€al. 2005; Clifford etÂ€al. 2006; Salsano etÂ€al. 2007). Recently, there has been great interest in characterizing subpopulations of tumor cells which have stem cell-like properties. These “cancer stem cells” have an inher-

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ent capacity for self-renewal and multipotentiality and are proposed to serve as the root of cancers. They, therefore, represent a very attractive therapeutic target. The evidence for the existence of such cancer stem cells in medulloblastoma comes from the identification of a defined population of medulloblastoma cells expressing the neural stem cell marker, CD133 (Singh etÂ€al. 2003, 2004; Peterson etÂ€al. 2005). These CD133+ cells have an increased ability to propagate or “self-renew” in primary and secondary neurosphere cultures and xenograft assays when compared to CD133- medulloblastoma cells. In addition, the stem cell factor BMI1 is highly expressed in some medulloblastomas (Singh etÂ€al. 2003, 2004). Mice lacking Bmi1 have decreased pools of progenitor cells in the subventricular and proliferative regions of the cerebellum with a distinct lack of robust neurosphere growth ex vivo (Leung etÂ€al. 2004; Bruggeman etÂ€al. 2005; Wiederschain etÂ€al. 2007). The real-world implications for targeting cancer stem cells and the mechanisms that maintain their “stemness” still need to be tested in proof of principle studies. To our knowledge, there are no currently available therapies that directly target cancer stem cells in medulloblastoma. However, several pathways implicated in medulloblastoma pathogenesis, such as SHH signaling, WNT signaling, and NOTCH signaling, have also been implicated in the maintenance of normal stem cell compartments (Ruiz i Altaba etÂ€al. 2002; Radtke and Clevers 2005; Reya and Clevers 2005; Androutsellis-Theotokis etÂ€ al. 2006; Ward etÂ€ al. 2006; Dreesen and Brivanlou 2007).

Molecular Features of Medulloblastoma and Targeted Strategies Several molecular pathways when dysregulated have been identified as pathogenic in medulloblastoma, either causally or in a supportive role. Current targeted strategies employ agents directed toward a single or a number of gene products. Alternatively, compounds that affect broader cellular functions have been developed (Fig.Â€1). The most prolific work involves targeting of the SHH pathway in medulloblastoma or pathways that cooperate with SHH activation in promoting tumorigenesis. This is largely due to the availability of robust preclinical mouse models to study, design, and test targeted therapies, and the discovery of cyclopamine, a plant derived SHH pathway inhibitor. Thus, current targeted therapies are somewhat biased toward the treatment of SHH-activated or desmoplastic/nodular subtypes of medulloblastoma. Some of these therapies have been proposed to work across all medulloblastoma subtypes; however, this has not been definitively proven. Small molecule screens have also generated numerous drugs directed at specific tyrosine kinase inhibition. Several of these compounds are in clinical trials and even clinical use for various types of cancer. Given many of these receptor tyrosine kinases are activated in medulloblastoma, these small molecule kinase inhibitors are currently being developed for trials in medulloblastoma as well.

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Y.-J. Cho and S.L. Pomeroy Cyclopamine HhAntag

R1507 Ab

CXCL12

NVP-AEW541

TrkC EGFR/ERBB IGF1R PDGFR CXCR4

AMD3100 AMD3465

Lapatinib Erlotinib

PI3K

WNT

SHH

FZD

PTCH1

GLI Fused SUFU

GSK3B

+ VPA / Vorinostat

BMP2

differentiation apoptosis

Rapamycin Everolimus

AKT

NOTCH

γ-secretase

DSH

PtdIns

13-cis-retionic acid

SMO

APC β-catenin

DAPT

AXIN

mTOR

β-catenin

c-MYC TCF/LEF

N-MYC GLI

PROLIFERATION, SURVIVAL

Fig.Â€1â•… Molecular pathways and targeted therapies in medulloblastoma. Current pathways implicated in medulloblastoma tumorigenesis and reagents shown to inhibit proliferation and/or tumor cell survival inÂ€vitro and in mouse models are outlined

Sonic Hedgehog The SHH pathway was first implicated in medulloblastoma tumorigenesis through the association of medulloblastoma with Gorlin syndrome, an autosomal dominant disorder caused by germline mutation of the SHH receptor PTCH1 (Hahn etÂ€ al. 1996; Johnson etÂ€al. 1996) Table 1. Approximately 2 to 5% of patients with Gorlin syndrome develop medulloblastoma, specifically the desmoplastic variant (Evans etÂ€al. 1991a, b). Overall, activation of SHH signaling is seen in 20 to 30% of medulloblastomas with the majority of these tumors harboring inactivating mutations in the PTCH1 tumor suppressor gene (Pietsch etÂ€al. 1997; Raffel etÂ€al. 1997). PTCH1 is a multipass membrane protein which binds SHH. In the absence of SHH, PTCH1 sequesters and prevents the activation of Smoothened (SMO) at the cell membrane. When bound by SHH, PTCH1 releases SMO to transactivate the Glioma-associated Oncogene Homolog (GLI) family of transcription factors via Suppressor of Fused (SUFU), resulting in upregulation of transcriptional targets such as MYCN and Cyclin D1 (Pomeroy etÂ€al. 2002; Kenney etÂ€al. 2003; Oliver etÂ€al. 2003) TableÂ€2. Somatic activating mutations in SMO have been found in medulloblastomas, although with very low frequency, whereas upregulation of GLI family members is uniformly seen in desmoplastic medulloblastomas (Lam etÂ€al. 1999). Conversely, truncating mutations in SUFU, which normally acts to sequester GLI in a cytoplasmic

Targeted Therapy in Medulloblastoma in Molecularly Targeted Therapy TableÂ€2â•… Mutations demonstrated in sporadic medulloblastoma % Mutation # of cases in study SHH signaling pathway PTCH1 10 7 of 68 20 5 of 24 10 5 of 46 SMO 4 1 of 21 6 1 of 15 SUFU 9 4 of 46 3 2 of 77 0 132 GLI3 0 12 WNT signaling pathway b-catenin 5 3 of 80 9 4 of 46 4 3 of 67 10 5 of 51 10 7 of 77 25 8 of 32 25a 27 of 109 APC 2 2 of 46 4 3 of 77 AXIN1 4 1of 23 0 0 of 46 1 1 of 86 5 2 of 39 AXIN2 1 1 of 116 Other TRP53 0 0 of 12 11 1 of 9 23 5 of 22 11 2 of 19 c-MYC genomic 16 4 of 24 amplification 6 4 of 69 5 4 of 77 1 1 of 71 6 4 of 71 N-MYC genomic amplification 21 4 of 19 13 2 of 15 6 4 of 77 a Nuclear localization of b-catenin

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References Pietsch etÂ€al. (1997) Raffel etÂ€al. (1997) Thompson etÂ€al. (2006) Lam etÂ€al. (1999) Reifenberger etÂ€al. (1998) Taylor etÂ€al. (2002) Thompson etÂ€al. (2006) Koch etÂ€al. (2004) Erez etÂ€al. (2002) Koch etÂ€al. (2001) Huang etÂ€al. (2000) Zurawel etÂ€al. (1998) Eberhart etÂ€al. (2000) Thompson etÂ€al. (2006) Clifford etÂ€al. (2006) Ellison etÂ€al. (2005) Huang etÂ€al. (2000) Thompson etÂ€al. (2006) Yokota etÂ€al. (2002) Thompson etÂ€al. (2006) Dahmen etÂ€al. (2001) Baeza etÂ€al. (2003) Koch etÂ€al. (2007) Saylors etÂ€al. (1991) Adesina etÂ€al. (1994) Badiali etÂ€al. (1993) Ohgaki etÂ€al. (1991) Rossi etÂ€al. (2006) Ebinger etÂ€al. (2006) Aldosari etÂ€al. (2002) De Bortoli etÂ€al. (2006) De Bortoli etÂ€al. (2006) Bayani etÂ€al. (2000) Michiels etÂ€al. (2002) Aldosari etÂ€al. (2002)

ubiquitination/degradation complex, have been reported in as many as 9% of sporadic medulloblastomas (Taylor etÂ€al. 2002). Preclinical mouse models of SHH-activated medulloblastomas which faithfully recapitulate the human condition in many regards have been developed TableÂ€ 3. Mice heterozygous for mutation of the murine homolog of the SHH receptor PTCH1 develop medulloblastomas at a low frequency (10 to 20% depending on

274 TableÂ€3â•… Mouse models of medulloblastoma Mouse model Type Ptch1+/− Germline knockout Germline knockout Ptch1+/−;trp53−/− DNAlig4−/−;trp53−/− Germline knockout Parp1−/−;trp53−/− Germline knockout Brca2−/−;trp53−/− Conditional Xrcc4−/−;p53−/− Conditional Rb−/−;trp53−/− Conditional Ink4c−/−;trp53−/− Conditional Ku80−/−;Rag1−/−;trp53−/− Germline knockout Sufu+/−;trp53−/− Germline knockout ND2-SmoA1 transgenic Transgene (NeuroD2 promoter) SHH with AKT and/or IGF2 Retroviral infection at E13.5

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References Goodrich etÂ€al. (1997) Wetmore etÂ€al. (2001) Lee and McKinnon (2002) Tong etÂ€al. (2003) Frappart etÂ€al. (2007) Yan etÂ€al. (2006) Marino etÂ€al. (2000) Uziel etÂ€al. (2006) Holcomb etÂ€al. (2006) Lee etÂ€al. (2007) Hallahan etÂ€al. (2004) Rao etÂ€al. (2004)

background strain). When crossed into a Trp53−/− background, latency of tumor formation is dramatically reduced and penetrance increases to 100% (Goodrich etÂ€al. 1997). Another mouse model overexpressing an activated form of Smo under the control of a NeuroD2 promoter also develops medulloblastoma in 48% of mice regardless of Trp53 status (Hallahan etÂ€ al. 2004). In utero injection of a SHH expressing retrovirus in E13.5d embryos results in medulloblastoma in as many as 76% of infected mice (Weiner etÂ€al. 2002). There are two well-published inhibitors of the SHH pathway that work at the level of SMO: cyclopamine and HhAntag (Cooper etÂ€al. 1998; Romer etÂ€al. 2004). Inhibitors that target the downstream SHH signaling mediator GLI have also been developed but have yet to be studied in medulloblastomas specifically (Lauth etÂ€al. 2007). Cyclopamine (11-deoxojervine) is a naturally occurring chemical that belongs to the group of steroidal jervatum alkaloids. It is derived from the corn lily Veratum californicum and was discovered in 1957 as a causative teratogen in endemic cases of cyclopic/holoprosencephalic lambs born to pregnant sheep grazing on the corn lily plant in Idaho pastures (Keeler 1970, 1978). HhAntag, on the other hand, was discovered by a high-throughput cell-based screening assay designed to identify small-molecule inhibitors of the SHH pathway (Williams etÂ€al. 2003). HhAntag, a benzimidazole derivative, has a higher affinity for Smo than cyclopamine and blocks SHH signaling at greater than tenfold lower concentrations (Romer etÂ€al. 2004). Both cyclopamine and HhAntag were very efficient at inhibiting medulloblastoma formation in Ptch1+/−;Trp53−/− mice. In addition, the treatment of mice which had spontaneous tumors or subcutaneously allografted tumors, inhibited tumor growth and promoted regression via induction of differentiation or apoptosis. Although lower doses neither completely nor permanently eliminated medulloblastomas in the Ptch1+/−;Trp53−/− mice, higher doses and longer treatments with HhAntag often resulted in complete tumor eradication and overall event free survival was markedly prolonged (Romer and Curran 2004). Importantly, both cyclopamine and HhAntag appeared to be very well tolerated and no noticeable adverse reactions were reported in mice, even at the highest doses

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of HhAntag (Berman etÂ€al. 2002; Romer and Curran 2004). However, further studies demonstrated that transient inhibition of the Hedgehog pathway by HhAntag caused permanent defects in bone growth (Kimura etÂ€al. 2008). At the time of this writing, several SMO inhibitors have been recently developed and are in early phase clinical trials for adult and pediatric malignancies, including medulloblastoma.

WNT/b-Catenin Activation of the Wnt/beta-catenin pathway in medulloblastoma was first established by the association of medulloblastoma with Turcot syndrome, also referred to as Brain Tumor Polyposis syndrome. Turcot syndrome results from mutations in the tumor suppressor gene, adenomatous polyposis coli (APC) (Hamilton etÂ€ al. 1995). APC is part of a multimeric complex which functions to sequester b-catenin in the cytosol, preventing its nuclear translocation where it normally interacts with the T-Cell Factor (TCF)/LEF family of transcription factors to transcriptionally regulate WNT target genes such as c-MYC, MYCN, and Cyclin D1(Gordon and Nusse 2006). Mutations found in APC prevent this sequestration of b-catenin allowing for its nuclear translocation and transcriptional activity. Activation of the WNT/b-catenin pathway as inferred from the nuclear localization of b-catenin occurs in 15 to 20% of medulloblastoma (Eberhart etÂ€al. 2000; Salaroli etÂ€al. 2007). However, mutations in the APC gene in sporadic medulloblastoma are rare, occurring in only 1 to 2% of tumors (Huang etÂ€ al. 2000). Mutations in the b-catenin gene contribute to 5 to 10% of medulloblastomas (Clifford etÂ€al. 2006). Mutations in other genes involved in the positive or negative regulation of the WNT pathway have been reported with rare frequency (Baeza etÂ€ al. 2003; Koch etÂ€al. 2007). Mutations found in the b-catenin gene confer stability to this oncogene by preventing its ubiquitination and thus degradation/downregulation. This results in stabilization of b-catenin, constitutive nuclear localization and thus transcriptional activation (Eberhart etÂ€ al. 2000; Koch etÂ€ al. 2001; Clifford etÂ€ al. 2006). Interestingly, aCGH and SNP microarray studies have identified a subset of medulloblastomas in which the only cytogenetic feature is the numerical loss of one chromosome 6 copy. All of these tumors are of the classic hitological subtype and on immunohistological staining exhibit nuclear localization of b-catenin (Clifford etÂ€al. 2006; Thompson etÂ€al. 2006). Two reports have suggested better the overall survival of patients with this subtype of tumor, but larger scale analyses are necessary to substantiate these findings (Clifford etÂ€al. 2006; Thompson etÂ€al. 2006). Nonetheless, these findings clearly identify a subset of medulloblastomas with WNT/b-catenin pathway activation which would be amenable to targeted therapy of this pathway. Although there are currently no mouse models of medulloblastoma derived from aberrant WNT pathway activation, targeted therapies designed against the WNT pathway are being developed for use in other types of cancers. Preclinical studies have shown some promise of ZTM000990 and PKF118-310, and lead compounds

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targeting the canonical WNT signaling cascade. In addition, anti-WNT1 and anti-WNT2 monoclonal antibodies show inÂ€ vitro effects in various tumor types (Barker and Clevers 2006).

NOTCH NOTCH family members have important roles in a variety of developmental processes via regulation of cell–cell interactions. The NOTCH proteins function as receptors for membrane bound ligands (Lutolf etÂ€ al. 2002). On interaction with these ligands, the NOTCH protein is cleaved in two places. The first, just outside the membrane, releases the extracellular portion of Notch, which remains bound to the ligand and is endocytosed by the ligand-expressing cell. The second cleavage occurs just inside the inner leaflet of the cell membrane and is mediated by g-secretase (Shih Ie and Wang 2007). This releases the intracellular portion of the NOTCH protein, which then moves to the nucleus upregulating various genes, including HES1 and HES5. The NOTCH family member, NOTCH2, and downstream signaling partners are upregulated in some primary medulloblastomas suggesting their role in medulloblastoma pathogenesis. The upregulation of these genes is particularly evident in medulloblastomas derived from SHH pathway activation (Hallahan etÂ€al. 2004; Dakubo etÂ€al. 2006). Inhibitors of g-secretase have been developed to specifically target the NOTCH endoproteolytic cleavage step which is necessary for downstream NOTCH signaling (Dovey etÂ€ al. 2001; Sastre etÂ€ al. 2001; Shih Ie and Wang 2007). Treatment of medulloblastoma cell lines with N-S-phenyl-glycine-t-butyl ester (DAPT) in particular resulted in 40 to 60% decrease in total viable cell number after 48Â€hours of treatment (Hallahan etÂ€ al. 2004). Primary mouse tumors derived from NeuroD1Smo transgenic mice and human primary tumor cell line xenografts had equal responses to DAPT or cyclopamine. Moreover, combined SHH and NOTCH pathway inhibition had an additive effect in these models suggesting the importance of NOTCH signaling in the setting of SHH activation. However, antitumoral effects afforded by DAPT diminished after 2Â€weeks, presumably due to an increase in drug metabolism (Hallahan etÂ€ al. 2004). Regardless, these studies establish NOTCH signaling as an important regulator of medulloblastoma tumorigenesis and identify it as a target for therapies. Currently, there are no NOTCH pathway inhibitors in clinical trials for medulloblastoma, however several inhibitors are currently in development or in early phase trials (MK0752) for other tumor types.

IGFR The insulin-like growth factor-1 receptor (IGF1R) is a membrane-associated tyrosine kinase which together with its corresponding ligands, IGF1 and IGF2, is expressed during embryonic and early postnatal cerebellar development (Rotwein

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etÂ€al. 1988; Baserga etÂ€al. 1994; Lee etÂ€al. 1995). This expression is restricted to granule neuron precursor cells and decreases significantly during postnatal development (LeRoith etÂ€al. 1993). IGF1R, like other receptor tyrosine kinases, signals through several cascades, including phosphatidylinositol-3¢ kinase (PI3K) and its second messengers. High-throughput gene expression profiling of primary medulloblastomas has shown increased expression of IGF2, mainly in desmoplastic/nodular tumors (Glick etÂ€al. 1993; LeRoith etÂ€al. 1993; Pomeroy etÂ€al. 2002). Moreover, homozygous deletion of the IGF2 gene completely eliminates tumor formation in Ptch1+/− mice, suggesting IGF2 and its downstream signaling partners are essential for SHH mediated medulloblastoma tumorigenesis (Hahn etÂ€al. 2000). In addition, retroviral transduction with combinations of SHH, IGF2, and activated AKT into Nestin-expressing neural progenitors induced tumor formation in mice, with the combination of SHH/AKT (48%) having the strongest oncogenic transformation effect compared to SHH/IGF2 (39%) and SHH alone (15%) (Rao etÂ€al. 2004). Importantly, neither IGF2 nor AKT caused tumors when expressed independently emphasizing SHH signaling as the dominant pathway in medulloblastoma pathogenesis. Induced tumors showed upregulation of insulin receptor substrate-1 (Irs1) expression and phosphorylation of IGF1R and AKT, consistent with activated IGF signaling seen in primary human medulloblastomas (Del Valle etÂ€al. 2002). Studies implementing anti-IGF1 and anti-IGF1R neutralizing antibodies, dominant negative mutants, and RNA interference have demonstrated efficacy of IGF1R targeting in inhibiting proliferation and tumorigenesis inÂ€vitro (Dunn etÂ€al. 1998; Reiss etÂ€al. 1998; Liao and Wang 2005; Wang etÂ€al. 2005b; Araki etÂ€al. 2006). Currently, several highly specific anti-IGF1R antibodies are in the early phases of clinical trials. The use of a blocking monoclonal anti-IGF1R antibody has previously been shown to decrease the growth of the Daoy medulloblastoma cell line (Chin etÂ€ al. 1996). Although this Phase I trial has not yet opened, patients with recurrent or refractory advanced solid tumors, including medulloblastoma, are expected to be enrolled in a trial to evaluate one such antibody, R1507. This study is entitled “Multiple Ascending Dose (MAD) Phase I Study of the IGF1R Antagonist R1507 Administered as an Intravenous Infusion in Children and Adolescents With Advanced Solid Tumors”. In addition, several small molecule inhibitors of IGF1R, which specifically inhibit IGF-mediated IGF1R autophosphorylation and abrogate its downstream signaling cascade, have been developed (Garcia-Echeverria etÂ€al. 2004; Scotlandi etÂ€al. 2005). Preclinical studies using NVP-AEW541, a pyrrolo[2,3-d]pyrimidine derivative, show growth inhibition of mouse and human medulloblastoma cell lines inÂ€ vitro (Urbanska etÂ€ al. 2007). Interestingly, this antitumoral effect of NVPAEW541 was enhanced in anchorage-independent culture conditions and was potentiated by inhibition of GSK3b constitutive phosphorylation (which is coincidentally observed when mouse medulloblastoma cells are deprived of attachment). The authors, therefore, proposed that the use of this inhibitor in combination with agents activating GSK3b (dephosphorylation) might be particularly effective

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against disseminated medulloblastoma (Urbanska etÂ€ al. 2007). Although clinical trials have not formally been introduced for medulloblastoma specifically, many of these IGF1R tyrosine kinase inhibitors are in various phases of clinical trials for other cancers and diseases associated with increased IGFR signaling.

CXCR4 The chemokine receptor CXCR4 is essential for the normal development of the cerebellar cortex and targeted gene deletion leads to mislocalization and limited proliferation of granule neuron precursor cells (Stumm etÂ€al. 2003; Vilz etÂ€al. 2005). In addition, increased expression of CXCR4 has been documented in desmoplastic medulloblastomas, medulloblastomas with extensive nodularity, and medulloblastomas generated in Ptch1+/− mice (Rubin etÂ€ al. 2003; Schuller etÂ€ al. 2005; Yang etÂ€al. 2007). Targeting of CXCR4 with the specific antagonists AMD3100 and AMD3465 inhibits the growth of medulloblastoma cell line xenografts, reportedly increasing apoptosis and decreasing the proliferation of tumor cells (Rubin etÂ€al. 2003; Yang etÂ€al. 2007). It was also shown that the CXCR4 ligand, CXCL12, mediated tumor growth through sustained inhibition of cyclic AMP (cAMP) production, and AMD3465 blocks this cAMP suppression. Given these observations, the phosphodiesterase inhibitor (PDI), Rolipram, was used to elevate pharmacologically the levels of cAMP in medulloblastoma cell line-xenografted mice. The effect was comparable to AMD3465 but only resulted in 58% of tumor regression of the medulloblastoma cell line-xenograft. However, the combined effect of another PDI and CXCR4 antagonist (caffeine and AMD3465, respectively), resulted in an 85% reduction in tumor size in this same xenograft model. These data indicate the clinical use of PDIs and CXCR4 inhibitors that might be an effective strategy in the treatment of medulloblastoma (Yang etÂ€al. 2007). Currently, there are no clinical trials offering PDI or CXCR4 inhibitor in the treatment of medulloblastoma.

PI3K/AKT/mTOR The mammalian target of rapamycin (mTOR) pathway is a central integrator of signals from nutrients, energy status, and growth factors. This pathway regulates growth and proliferation by modulating numerous cellular functions, including autophagy, ribosome biogenesis, and metabolism (Sabatini 2006; Guertin and Sabatini 2007). mTOR is a serine/threonine kinase that is activated by various receptor tyrosine kinases via phosphorylation and activation of PI3K and AKT. Briefly, the activation of PI3K generates phosphatidylinositol (3,4,5) trisphosphate which recruits AKT to the plasma membrane. AKT phosphorylation allows for the subsequent activation of mTOR through several intermediates. Tyrosine kinase receptors such as IGFR, PDGFR, TRKC, NGFR, and EGFR, are activated in

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medulloblastoma primary tumors and cell lines and phosphorylated forms of AKT and PI3K have been demonstrated (Wlodarski etÂ€al. 2006). Together, this indicates the potential utility of mTOR inhibitors in medulloblastoma treatment. Several mTOR inhibitors, such as rapamycin, RAD001 (Everolimus), and CCI779 (Temsirolimus), are being evaluated as novel agents for cancer therapy including medulloblastoma (Geoerger etÂ€ al. 2001; Bjornsti and Houghton 2004; Beuvink etÂ€ al. 2005; O’Reilly etÂ€ al. 2006). Phase I and II cancer trials have shown that mTOR inhibitors are well tolerated by patients, with minimal adverse effects being reported. In particular, chronic immunosuppression has not been reported with the dose schedules used in current clinical trials (Yee etÂ€al. 2006). However, rapamycin alone produces only partial responses in medulloblastoma (Geoerger etÂ€al. 2001). Indeed, most Phase II cancer trials to date have shown limited effectiveness of rapamycin and its analogs as single agent therapy (Chang etÂ€al. 2005; Galanis etÂ€al. 2005). Thus, it has been suggested that rapamycin analogs should be used in conjunction with conventional chemotherapeutic agents or radiation therapy to increase efficacy. One alternative approach in mouse models combined rapamycin with intratumoral injection of oncolytic viruses. This combination was effective in promoting tumor regression in cell-line derived medulloblastoma xenografts (Lun etÂ€al. 2007). Currently, a clinical trial entitled “Phase I/II Trial of RAD001 (Everolimus) in Pediatric Patients With Recurrent Refractory Solid Tumors or Brain Tumors With Phase II Limited to Recurrent or Refractory Rhabdomyosarcomas and Non Rhabdomyosarcomatous Soft Tissue Sarcomas” is open for enrollment.

EGFR Family The EGFR family members, ERBB1–ERBB4, have been shown to be differentially expressed in medulloblastoma cell lines and primary tumors. Overexpression of ERBB2, in particular, was evident in a large number of medulloblastomas and also reported as a negative prognostic factor (Gilbertson etÂ€ al. 1992, 1995, 1997). Several inhibitors of EGFR family members have been developed as it is an important regulator in different types of cancer (Dancey 2004; Ciardiello 2005). These include Gefitinib and Lapitinib, which specifically inhibit ERBB1 and ERBB2; Herceptin, a monoclonal antibody to ERBB2; and Erlotinib, a specific inhibitor of ERBB1. Erlotinib and Lapitinib are currently in Phase I and II trials, respectively, for patients with medulloblastoma. The first trial entitled “Phase I Studies of TARCEVA™ (ERLOTINIB HYDROCHLORIDE, OSI-774) as Single Agent in Children With Refractory and Relapsed Malignant Brain Tumors and in Combination With Irradiation in Newly Diagnosed Brain Stem Glioma” is active but no longer recruiting patients. A second Phase II clinical trial entitled “A Molecular Biology And Phase II Study Of Lapatinib (GW572016) In Pediatric Patients With Recurrent Or Refractory Medulloblastoma, Malignant Glioma Or Ependymoma” is currently open for enrollment.

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PDGFR Activation of PDGFRb signaling was published as a marker for metastatic medulloblastomas in studies utilizing high-throughput gene expression arrays (MacDonald etÂ€ al. 2001; Gilbertson and Clifford 2003). PDGFR is an attractive candidate for molecular targeting as Imatinib (Gleevec), the prototypical small molecule inhibitor, inhibits both the a and b isoforms of this receptor tyrosine kinase. In preclinical studies, blocking monoclonal antibodies to PDGFR and a small molecule inhibitor to MAP2K1, MAP2K2, and MAPK1/3, mediators of the PDGFR signaling cascade, were shown to inhibit migration of medulloblastoma cells inÂ€vitro (MacDonald etÂ€al. 2001; Gilbertson and Clifford 2003). Currently, there are no clinical trials of PDGFR inhibitors for use in patients with medulloblastoma.

TRKC TrkC is the preferred receptor for NT3, a neurotrophin that regulates granule cell development. TrkC is expressed in granule cell neurons where it promotes axonal maturation. It is also expressed in subsets of medulloblastomas and importantly was the first receptor tyrosine kinase to be associated with clinical significance in embryonal tumours (Segal etÂ€al. 1994; Eberhart etÂ€al. 2004). In particular, elevated expression in medulloblastomas is associated with favorable clinical outcome, presumably through the induction of apoptosis via NT3-TrkC signaling mechanisms (Kim etÂ€al. 1999). Currently, there are no TrkC agonists or NT3 analogs in development or clinical trials.

HDAC Inhibitors and Retinoic Acid There is some evidence in cell culture and xenograft models suggesting the utility of histone deacetylase (HDAC) inhibitors in treating primary medulloblastomas (Li etÂ€ al. 2005; Shu etÂ€ al. 2006; Spiller etÂ€ al. 2006, 2008). Valproic acid (VPA) and suberoylanilide hydroxamic acid (SAHA, marketed as Vorinostat) represent two HDAC inhibitors that are currently in early phase clinical trials. The suggested antitumoral mechanism in medulloblastoma appears to be sensitization of cells to retinoic acid receptor pathways. These have independently been shown to induce terminal differentiation and apoptosis in medulloblastoma cell lines and xenograft models through upregulation of BMP2 and caspase activity (Spiller etÂ€ al. 2006, 2008). Thus, clinical trials have been designed combining the treatment of HDAC inhibitors with 13-cis-retinoic acid. Currently, there are two clinical trials utilizing this approach. The first, entitled “A Phase I Study of Valproic Acid in Children With Recurrent/Progressive Solid Tumors Including CNS Tumors” is no longer open for enrollment, but is still active.

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The second, entitled “A Phase I Study of SAHA (NSC#: 701852 IND#: 71976) in Pediatric Patients With Recurrent or Refractory Solid Tumors (Including Lymphomas) and Leukemia Followed By a Phase I Study of SAHA in Combination With 13-Cis-Retinoic Acid for Patients With Selected Recurrent/Refractory Solid Tumors” is currently open for enrollment.

Future Directions and Challenges There is clear evidence that currently available targeted therapies for medulloblastoma could offer alternatives or adjuvants to chemo and radiotherapies. Moreover, high-throughput screening methods for drug discovery and development present increasing opportunities to rapidly take biological discoveries from the bench to preclinical testing and ultimately to clinical trials and the bedside. However, several challenges will need to be addressed prior to such treatments arriving at the bedside. First, the establishment of a definitive molecular taxonomy for medulloblastoma is required. A move toward a more stringent molecular definition for each subtype of medulloblastoma will be the first step in transitioning targeted therapies into real world personalized applications. This type of schema is needed to identify patients for which current and future targeted therapies will work. Although some molecular markers and cytogenetic features have been in use for several years, they are neither uniformly used nor standardized. Whether newer markers involve the use of gene “signatures” generated from array-based studies or more predictive single or sets of biomarkers remain to be seen. Validation through CLIA certification will be necessary. Refinement of the current histopathological schema for medulloblastoma is an alternative, but will be challenged with the subjective and inconsistent nature of this diagnostic methodology. Secondly, our understanding of the biology of medulloblastomas is incomplete. Much of the current literature and development of targeted therapies is heavily weighted toward the desmoplastic/nodular class of medulloblastomas due to the informativity of the Ptch1+/− mouse model. In vivo models of medulloblastoma driven by mechanisms unrelated to SHH is somewhat lacking. There are several DNA repair deficient mice that generate medulloblastomas (Lee and McKinnon 2002; Tong etÂ€al. 2003; Holcomb etÂ€al. 2006; Yan etÂ€al. 2006; Frappart etÂ€al. 2007). However, these genes have yet to be identified as pathogenetically mutated in primary human medulloblastomas other than very rare familial case reports such as Nijmegen Breakage Syndrome and Fanconi Anemia Type D (Ruud and Wesenberg 2001; Bakhshi etÂ€al. 2003; Distel etÂ€al. 2003; Offit etÂ€al. 2003; Hirsch etÂ€al. 2004; Tischkowitz etÂ€al. 2004). Several medulloblastoma cell lines have been instrumental in early stages of drug discovery and preclinical testing. However, it is clear from genomic comparisons of these cell lines to primary medulloblastomas that they have diverged over repeated passages making them less representative of their primary state (Cho and Pomeroy, unpublished data). The use of xenografts and neurosphere

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cultures derived from freshly resected primary tumor is increasingly utilized and may provide an important alternative for use in preclinical testing of new therapies. Studies in such systems will be greatly benefited by our ability to rapidly assign a molecular phenotype to a tumor soon after its resection. Third, incorporating new therapies into current treatment protocols letÂ€ alone implementing targeted therapies as first line treatment will remain challenging. As conventional multimodal therapies achieve survival rates close to 85% in some series, introducing new targeted drugs as an alternative may be difficult, perhaps unethical. Conversely, the addition of a targeted therapy to conventional treatments may confound efficacy results (Crawford etÂ€al. 2007). Thus, in order to realize the true potential of targeted treatments, carefully designed trials with the emphasis of eradicating unacceptable toxicities of current treatment regimens will need to be developed. Finally, the collaborative, multidisciplinary care of the patient diagnosed with medulloblastoma will be particularly tested as targeted therapies evolve. The care team will need to coordinate proper acquisition and transfer of the clinical sample from the operative suite to the laboratory, proper processing of the sample in the laboratory for DNA and RNA based studies, and proper interpretation of these results by the patient’s neurologist, oncologist, and pathologist. Definitive molecular phenotyping will need to be performed in a timely manner if this process is to dictate clinical management. Despite these challenges, development and implementation of targeted therapy for medulloblastoma shall continue with the hope of eradicating this lethal childhood disease.

References Adesina, A.M., Nalbantoglu, J., and Cavenee, W.K. 1994. p53 gene mutation and mdm2 gene amplification are uncommon in medulloblastoma. Cancer Res 54: 5649–5651. Alder, J., Cho, N.K., and Hatten, M.E. 1996. Embryonic precursor cells from the rhombic lip are specified to a cerebellar granule neuron identity. Neuron 17: 389–399. Aldosari, N., Wiltshire, R.N., Dutra, A., Schrock, E., McLendon, R.E., Friedman, H.S., Bigner, D.D., and Bigner, S.H. 2002. Comprehensive molecular cytogenetic investigation of chromosomal abnormalities in human medulloblastoma cell lines and xenograft. Neuro Oncol 4: 75–85. Androutsellis-Theotokis, A., Leker, R.R., Soldner, F., Hoeppner, D.J., Ravin, R., Poser, S.W., Rueger, M.A., Bae, S.K., Kittappa, R., and McKay, R.D. 2006. Notch signalling regulates stem cell numbers inÂ€vitro and inÂ€vivo. Nature 442: 823–826. Araki, K., Sangai, T., Miyamoto, S., Maeda, H., Zhang, S.C., Nakamura, M., Ishii, G., Hasebe, T., Kusaka, H., Akiyama, T., etÂ€al. 2006. Inhibition of bone-derived insulin-like growth factors by a ligand-specific antibody suppresses the growth of human multiple myeloma in the human adult bone explanted in NOD/SCID mouse. Int J Cancer 118: 2602–2608. Badiali, M., Iolascon, A., Loda, M., Scheithauer, B.W., Basso, G., Trentini, G.P., and Giangaspero, F. 1993. p53 gene mutations in medulloblastoma. Immunohistochemistry, gel shift analysis, and sequencing. Diagn Mol Pathol 2: 23–28. Baeza, N., Masuoka, J., Kleihues, P., and Ohgaki, H. 2003. AXIN1 mutations but not deletions in cerebellar medulloblastomas. Oncogene 22: 632–636.

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Future Treatments of Ependymoma Richard J. Gilbertson

Introduction Although ependymoma is the third most common form of central nervous system (CNS) tumor, it is a poorly understood disease. The variable patterns of histology and clinical presentation, lack of consensus regarding appropriate means to stage and grade the disease, and the separation of pediatric and adult oncology services, have each hindered efforts to advance understanding of ependymoma biology and treatment. As a result, no new therapeutic approaches have been identified to treat ependymoma during the last 20Â€years and up to 40% of patients remain incurable (Brandes etÂ€al. 2005; Merchant and Fouladi 2005). The remarkable insensitivity of ependymoma to most nonsurgical conventional therapies has contributed further to this impasse in clinical management. While ependymoma remains a challenging disease to treat, recent laboratory and clinical research has resulted in important discoveries that are likely to provide new direction for patient management. The efficient integration of these discoveries into treatment protocols will require an understanding of contemporary therapies as well as novel mechanisms to target molecular alterations. This chapter will review current and emerging understanding of ependymoma biology and treatment as well strategies to integrate this knowledge within clinical management protocols.

R.J. Gilbertsonâ•›(*) Neurobiology and Brain Tumor Program, St. Jude Children’s Research Hospital, Memphis, TN, USA e-mail: [email protected] P.J. Houghton and R.J. Arceci (eds.), Molecularly Targeted Therapy for Childhood Cancer, DOI 10.1007/978-0-387-69062-9_14, © Springer Science+Business Media, LLC 2010

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What Do We Know About Ependymoma? Clinical Issues Epidemiology and Histopathology Approximately 900 new cases of ependymoma are diagnosed each year in the US and Europe; most often in children aged less than 4Â€years (CBTRUS 2006). These CNS ependymal tumors originate from the wall of the ventricular system and the spinal canal, and display moderate cellularity, ultrastructural properties of ependymal cells, and express markers of glial differentiation (Kleihues etÂ€al. 2002). Although ependymomas are thought to arise from cells adjacent to the ventricle, 10% are positioned distal from the ventricular system within the brain parenchyma. The incidence of ependymoma does not appear to vary by geographic region or socioeconomic class, but the disease may be more common in Caucasians (Gurney etÂ€al. 2001; Alston etÂ€al. 2007). Children and adults are predisposed to develop ependymoma in different parts of the CNS (Moynihan 2003): posterior fossa tumors present most often in patients aged less than 10Â€years; supratentorial ependymomas tend to occur in older children and adults; and spinal forms of the disease are confined largely to adults (CBTRUS 2006). There are no pathonemonic histologic features of ependymoma, but these tumors are characterized by perivascular collections of tumor cells termed pseudorosettes. Ependymoma cells typically display a glial immunophenotype and ultrastructural properties of ependymal cells. A number of ependymoma histologic subtypes are recognized. Myxopapillary and sub-ependymomas occur almost exclusively in the cauda equina and are relatively benign tumors (WHO grade I). Classic ependymomas can display papillary or clear cell features (WHO grade II), and are contrasted with anaplastic ependymoma that displays increased cellularity, cytologic atypia, and microvascular proliferation (WHO grade III). Ependymoblastomas are now considered a form of primitive neuroectodermal tumor. The degree to which ependymoma histology grade correlates with patient prognosis remains controversial and current histologic criteria are of limited use for predicting patient outcome. Therefore, collaborative efforts between European and American neuropathologists are underway to establish a more robust tumor grading system for ependymomas. There is also no official staging classification of ependymoma, but the diagnosis of metastatic disease is thought to be important for assigning appropriate treatment. However, measures to detect disseminated disease that include neuraxis MRI and CSF cytology are unreliable and are associated with significant rates of false positive and false negative results (Butler etÂ€al. 2002; Kumar etÂ€al. 2007).

Current Therapy Surgery and radiation have served as the mainstay of ependymoma therapy for more than 40Â€years (Kricheff etÂ€al. 1964). Total tumor resection remains the most

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important treatment for patients with localized ependymoma (Rousseau etÂ€al. 1994; Duffner etÂ€al. 1998; Robertson etÂ€al. 1998). Indeed, data suggest that patients who achieve a complete surgical resection may require no further therapy (Hukin etÂ€al. 1998). This approach is to be tested in one arm of the forthcoming Phase III ependymoma trial to be conducted within the Children’s Oncology Group (COG). If a gross-total resection is not achieved at first surgery, then careful consideration should be given to the use of further surgery. Second surgery may be accomplished safely, and in most cases, has been shown to be effective at achieving a gross-total resection ((Korshunov etÂ€al. 2004) and Osterdock, Sanford, and Merchant, unpublished data). Most ependymomas are localized at diagnosis, and are therefore treated with 54 to 59.4Â€Gy of focal conformal adjuvant radiation targeted to the postoperativelydefined tumor bed (Merchant etÂ€al. 2004). Although most patients can be spared whole neuraxis irradiation (Merchant etÂ€al. 1997; Paulino etÂ€al. 2002), seven percent of ependymomas are disseminated throughout the neuraxis at diagnosis, and craniospinal irradiation is the required treatment for these patients. Despite this aggressive therapy, disseminated ependymoma is extremely difficult to cure and long-term survival rates for this population are likely to be less than 25%. Children with recurrent ependymoma after prior irradiation have few treatment options and the majority will die within 2Â€ years. Treatment failure may include local progression, neuraxis dissemination, or a combination of the two. There are limited data suggesting that re-irradiation may be an effective treatment option for children with recurrent ependymoma when combined with resection of locally recurrent or metastatic disease. Although ependymoma is sensitive to conventional chemotherapy, there is no convincing evidence that this treatment improves overall patient survival. Conventional chemotherapies including cisplatin (Khan etÂ€al. 1982; Sexauer etÂ€al. 1985; Walker and Allen 1988), carboplatin (Gaynon etÂ€ al. 1990; Friedman etÂ€ al. 1992), ifosfamide (Chastagner etÂ€ al. 1993) and etoposide (Davidson etÂ€ al. 1993; Needle etÂ€al. 1997) have each demonstrated modest activity against the disease; but combination chemotherapy regimens have proven ineffective at improving the cure rates achieved with surgery and radiation therapy alone (Evans etÂ€ al. 1996; Robertson etÂ€ al. 1998; Timmermann etÂ€ al. 2000). While not generally effective, chemotherapy may have a role in delaying CNS irradiation of very young patients. The Pediatric Oncology Group (POG) 8,633 study, reported a 48% response rate to a combination of vincristine, cyclophosphamide, cisplatin, and etoposide among 25 children with residual tumor after initial surgery (Duffner etÂ€ al. 1993). Similar results have been reported with other conventional chemotherapy regimens developed within the European consortia (Grill etÂ€al. 2001; Grundy etÂ€al. 2007). These data are important since exposure of the developing brain to radiation results in long-term endocrine and cognitive deficits (including learning, memory attention, and behavior disorders). Thus, ependymoma is a clinically heterogeneous disease for which there are few conventional treatment options. If the tumor is totally resected, then the disease can be controlled; however, disseminated or relapsed disease is usually fatal. There is general consensus that better understanding of disease biology will lead to more

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effective treatments for these patients. However, in contrast to astrocytic and primitive neuroectodermal tumors that have served as the focus of most brain tumor biology studies, very little is known about the biology of ependymoma.

Tumor Biology Genetics Progress in understanding the biology of ependymoma has been limited since most research has been conducted by isolated groups studying the sequence and expression of a handful of genes within a small number of locally and retrospectively collected tumors. Consequently, knowledge of the genetic alterations in ependymoma has been limited largely to lists of large chromosomal gains and losses, for example, +1q, −6q, +7p, −9p, −16q, and −22q (Reardon etÂ€al. 1999; Hirose etÂ€al. 2001; Ward etÂ€al. 2001; Carter etÂ€al. 2002; Dyer etÂ€al. 2002; Grill etÂ€al. 2002; Jeuken etÂ€al. 2002; Koschny etÂ€al. 2002). Studies of heritable forms of ependymoma have identified a very small number of tumor suppressor genes (TSG). Patients with Neurofibromatosis type 2 are predisposed to spinal ependymoma (Rouleau etÂ€ al. 1993) and somatic mutations in NEUROFIBROMIN 2 (NF2, 22q12.2) occur in approximately 25% of sporadic SP tumors (Rubio etÂ€al. 1994; Ebert etÂ€al. 1999). Ependymomas have also been reported in two patients with Turcot’s syndrome (germline mutation in APC) (Torres etÂ€al. 1997; Mullins etÂ€al. 1998), and in one patient with Li Fraumeni Syndrome (germline mutation in TP53) (Metzger etÂ€al. 1991). However, mutations in CTNNB1, APC or TP53 occur in less than 1% of sporadic ependymomas (Ohgaki etÂ€ al. 1991; Gaspar etÂ€ al. 2006; Onilude etÂ€ al. 2006). In contrast to TSG, there are no known oncogenes of ependymoma, although overexpression of members of the Epidermal Growth Factor Receptor (EGFR) family has been reported in aggressive forms of the disease (Gilbertson etÂ€ al. 2002; Mendrzyk etÂ€al. 2006). Similarly, high tumor cell expression of the catalytic unit of telomerase (hTERT) has been associated with poor clinical outcome among patients with ependymoma (Tabori etÂ€ al. 2006). Identifying oncogenes and other factors such as hTERT that contribute to transformation is particularly important for advancing treatment since these genes are likely to include the most useful drug targets (Druker etÂ€al. 2001; Vogel etÂ€al. 2002; Lynch etÂ€al. 2004; Paez etÂ€al. 2004).

Genomics The lack of knowledge regarding the key causative molecular alterations of ependymoma has hindered efforts to develop new treatments of the disease. However, tools that detect genome-wide patterns of gene expression and chromosomal

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alteration are now being applied to the study of large cohorts of ependymoma. Of particular note, these studies have shown that ependymomas from the different regions of the CNS are molecularly distinct diseases (Korshunov etÂ€ al. 2003; Ammerlaan etÂ€al. 2005; Taylor etÂ€al. 2005; Mendrzyk etÂ€al. 2006; Modena etÂ€al. 2006). For example, while the great majority of cerebral ependymomas activate the NOTCH signal pathway and delete the INK4A/ARF TSG locus, spinal ependymomas express high levels of members of the HOX gene family (Taylor etÂ€al. 2005; Modena etÂ€ al. 2006). These data are likely to be of great clinical significance; implying that ependymomas should be regarded as distinct site-specific entities that are likely to respond differentially to molecular targeted therapies. Ongoing genomic studies are identifying amplicons in ependymomas that contain oncogenes, for example, VAV1 (19p13.3) and hTERT (5p15.33) (Ammerlaan etÂ€al. 2005; Taylor etÂ€al. 2005; Mendrzyk etÂ€al. 2006). These data provide proof-ofprincipal that genomic technologies are valuable mutation-discovery tools with which to study ependymoma. Most of the amplicons identified so far extend across megabases of chromosomal DNA and contain many genes (Ammerlaan etÂ€al. 2005; Taylor etÂ€ al. 2005; Mendrzyk etÂ€ al. 2006). Therefore, pinpointing specific oncogenes and TSG of ependymoma will require the analysis of much larger numbers of samples with higher resolution DNA mapping tools. Efforts are now underway to apply technologies of higher resolution (500Â€K single nucleotide polymorphism mapping arrays) to pinpoint specific oncogenes and TSG among large cohorts of ependymoma.

Cancer Stem Cells and The Origins of Ependymoma Considerable excitement has surrounded the recent discovery of cancer stem cells (CSC) (Clarke and Fuller 2006). CSC make up just a small fraction of the total population of the malignant cells in many solid tumors and leukemias. However, evidence indicates that these self-renewing and multipotent stem cell-like cells generate all of the phenotypically diverse cells that populate tumors (Lapidot etÂ€al. 1994; Bonnet and Dick 1997; Al-Hajj etÂ€al. 2003; Singh etÂ€al. 2004; Taylor etÂ€al. 2005). The discovery of CSC has therefore provided researchers with a practical point of focus for studying the natal cellular and molecular events of tumorigenesis. The identification of CSC is likely to have important implications for the treatment of cancer. If tumors are derived entirely from CSC, then it would follow that to be curative cancer, treatments should disable or destroy these cells. Indeed, drugs that are designed to kill CSC could prove highly effective treatments of cancer. As outlined above, ependymoma subsets have been shown to exhibit distinct patterns of gene expression and regions of chromosome gain and loss that correlate with the anatomic location of the tumor (supratentorial region, posterior fossa or spine). Taylor etÂ€al. (2005) demonstrated that the gene expression signatures that most discriminate supratentorial, posterior fossa, and spinal ependymoma included

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many genes that are known regulators of neural precursor cells in the corresponding region of the CNS. Indeed, using in situ hybridization and immunofluorescence to map the site of expression of ependymoma signature genes in the developing mouse, this study went on to identify embryonic radial glia (RG) – that are neural progenitor cells – as candidate cells of origin of ependymoma. Importantly, the study showed also that self-renewing and multipotent CSC isolated from fresh samples of ependymoma are bipolar RG-like cells; express the CD133+/Nestin+/ RC2+/BLBP+ immunophenotype of RG; and are both required and sufficient to generate tumors inÂ€vivo. Interestingly, pilocytic astrocytomas have been shown also to share the gene expression profiles of normal primary astrocytes and neural stem cells from the corresponding region of the brain (Sharma etÂ€ al. 2007). Together, these data suggest a new hypothesis for the origin of ependymoma and other glial tumors; progenitor cells in different parts of the CNS are predisposed to acquire distinct genetic abnormalities that transform these cells into CSC of supratentorial, posterior fossa, and spinal tumors (Gilbertson and Gutmann 2007).

Ependymoma Stem Cell Niches Stem cells of organisms across the plant and animal kingdoms reside within stem cell niches, highlighting the fundamental importance of these specialized microenvironments for normal stem cell biology (Scheres 2007). The central structural element of the neural stem cell niche is provided by capillaries (Riquelme etÂ€al. 2007). This organization places the stem cells in close proximity to endothelial and other vascular cells, facilitating communication among these cell types. Calabrese and colleagues recently provided compelling data that stem cells from a variety of brain tumors, including ependymoma, are maintained within vascular niches that mimic the neural stem cell niche (Calabrese etÂ€ al. 2007). Using co-immunofluorescence and multi-photon laser scanning microscopy, they showed first that CD133+, Nestin+ cells within sections of human ependymomas are located in close proximity to tumor capillaries. They then demonstrated that CD133+, Nestin+ cells, but not other cells isolated from ependymomas, migrate to interact intimately with the vascular tubes that are formed by endothelial cells in three-dimensional cultures. The self-renewal and proliferation of these ependymoma stem cells in culture was maintained by factors secreted by endothelial cells. Importantly, by co-transplanting brain tumor stem cells and endothelial cells into immunocompromised mice, the investigators showed that endothelial-derived factors also accelerate the initiation and growth of tumors in the brain. Finally, the investigators demonstrated that the anti-VEGF antibody bevacizumab can halt the growth of brain tumors by disrupting the perivascular niche and depleting the cancer stem cell fraction. Although additional studies will be required to understand better the ependymoma stem cell niche, these early data have exposed a previously unknown vulnerability in brain tumors for therapeutic attack (Gilbertson and Rich 2007).

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Prior Testing of Molecular Targeted Therapies in Ependymoma Given the paucity of information regarding the mutations that cause ependymoma and the relative rarity of the disease, it is not surprising that few molecular targeted therapies have been tested in this tumor. Those drugs that have been tested have been selected empirically, often in the context of Phase I studies that have enrolled patients with a variety of recurrent or progressive CNS tumor. For example, the US Pediatric Brain Tumor Consortium (PBTC) recently tested in Phase I the farnesyltransferase inhibitor lonafarnib (SCH66336). Radiographic responses or stable disease were reported in 10 of 53 patients with recurrent or refractory brain tumors including one child with ependymoma (Kieran etÂ€al. 2007). Although this approach is useful for gathering drug toxicity, dose and pharmacokinetic data, more efficient mechanisms will be required to develop new therapies of ependymoma. Ongoing clinical trials are targeting the EGFR family in ependymoma (e.g., PBTC016 study of Lapatinib) that has been reported to be overexpressed in poor prognosis ependymoma (Gilbertson etÂ€al. 2002; Mendrzyk etÂ€al. 2006); however, a causative role for EGFR family signaling in ependymoma remains to be demonstrated. Anecdotal reports of the therapeutic activity of other growth factor receptor inhibitors (e.g., Imatinib) in ependymoma have also been reported but remain to be confirmed (Fakhrai etÂ€al. 2004).

How Can We Improve the Treatment of Ependymoma? There is a dire need for new treatment approaches of ependymoma. Identifying these treatments will require a comprehensive and collaborative research process that builds on what we know about the disease, and continually integrates new understanding of disease biology and clinical management. This process should account for a number of key issues.

Ependymoma Is not a Single Disease The observation that ependymoma comprises subgroups of clinically and molecularly distinct diseases is a critical finding of recent studies. These data suggest strongly that cerebral, posterior fossa, and spinal tumors should be treated separately. Efforts should be made to continually integrate understanding of the genetic alterations in these disease subgroups with attempts to develop molecular targeted therapies. For example, gene expression profile studies have identified high expression of the NOTCH ligands JAGGED 1 and 2 in supratentorial tumors (Taylor etÂ€al. 2005). Binding of JAGGED ligands to NOTCH leads to g-secretase mediated cleavage of the NOTCH receptor and activation of signaling (Radtke and Raj 2003). Thus, inhibitors of g-secretase that are being considered for clinical trials in leukemia could have utility in the treatment of supratentorial ependymoma (Huntly and Gilliland 2005). Importantly, JAGGED1 and

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2 are not expressed highly in posterior fossa or spinal ependymomas, suggesting that inhibitors of g-secretase might be less effective against these tumors. Integrated analyses of molecular data with central clinical and histologic data should also transform the classification of ependymoma, and provide a new clinico-molecular disease-risk stratification system for this disease. This new disease-risk stratification system may allow greater tailoring of conventional therapies among patients as well as identify new targets for molecular based therapies. Defining ependymoma subgroups may improve understanding of the disease and the efficiency with which we use conventional treatments; but it will also convert an already uncommon disease into a group of extremely rare tumors: this will present a major challenge to the conduct of randomized clinical trials. It will only be possible to conduct these trials through larger international collaborations. Thus, we will need to forge close links between North American and European consortia. One such collaboration is already under the leadership of groups at St Jude Children’s Research Hospital and MD Anderson Cancer Center (www.CERN-Foundation.org). This privately funded consortium has established an adult and pediatric clinical trials network as well as comprehensive projects in histopathology, genomics, drug discovery, and ependymoma stem cells. The support of the national cancer institutions in the US (e.g., NCI and NINDS) and Europe (e.g., NICR and CRUK) will also play important roles in the collaborative process. The international pediatric neuro-oncology community would be wise to give early consideration to establishing the international links necessary to complete these studies.

Some Ependymomas May Be Cured with Minimal Conventional Therapy It is important to recognize that an as yet undefined group of patients with ependymoma can be cured with minimal conventional therapy. In the case of completely (macro- and microscopically) resected intracranial disease, this may involve surgery alone. Better histologic and molecular classification systems of ependymoma may allow prospective identification of these patients. In the absence of alternative therapies, postoperative focal radiation is likely to continue to be used to treat patients undergoing sub-total resections. These patients are still at risk of distantmetastatic spread and new methods are required to define residual disease at the primary site or evidence of metastatic dissemination.

Curing All Patients with Ependymoma Will Require New Treatments While improvements may be made in the use of conventional treatments, these will not cure all patients with ependymoma. There are at least three areas that could provide fertile ground for developing new treatment approaches. First, seminal studies of a variety of hematologic and solid tumors have identified

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gene mutations as targets of effective new anti-cancer therapies (Druker etÂ€al. 2001; Vogel etÂ€al. 2002; Lynch etÂ€al. 2004; Paez etÂ€al. 2004; Romer etÂ€al. 2004). Thus, ongoing genomic efforts to characterize oncogenic mutations in ependymoma could lead to the development of new treatments. At present there are relatively few widely available cell line or mouse models of ependymoma that can be used for pre-clinical drug development. Thus, efforts to understand the causative mutations of ependymoma should generate also preclinical models that can be used to develop treatments that target these alterations. Second, the recent discovery that ependymomas are derived from radial glia-like CSC that reside in perivascular niches (Taylor etÂ€al. 2005; Calabrese etÂ€al. 2007) has identified a series of completely new drug targets to treat this disease (Gilbertson and Rich 2007). Drugs that target CSC or their niches in ependymoma could prove highly effective treatments; however, the similarities between normal and malignant neural stem cells predict that such treatments may also possess significant toxicities. The development of anti-CSC therapies for ependymoma will require the identification of factors that maintain ependymoma CSC, but not normal neural stem cells. Third, it may prove possible to develop strategies that increase the overall sensitivity of ependymoma to conventional treatments. These strategies are likely to target the distinct populations of cells that exist within tumors, including the CSC. Glioma stem cells have been shown to be inherently radio – and chemoresistant (Bao etÂ€al. 2006) and might be protected further from conventional therapies by factors within the vascular niche. Treatments that disrupt aberrant vascular stem cell niches might therefore enhance sensitivity to conventional treatments. Indeed, clinical trials that include a combination of the antiangiogenic drug Bevacizumab and the cytotoxic irinotecan have revealed remarkable synergy between these two drugs in the treatment of glioblastoma (Vredenburgh etÂ€al. 2007). It is possible that this combination therapy disrupts the vascular niche, exposing both glioblastoma stem cells and the remaining tumor cells to the cytotoxic effects of the conventional chemotherapy.

Future Directions and Challenges In 2008, ependymoma remains a poorly understood disease with few treatment options. The outlook is especially dismal for patients with disseminated disease. Advances in genomics, stem cell biology, and developmental therapeutics hold great promise to break the deadlock in the hunt for new treatments of ependymoma. International collaboration between medical, scientific, and government personnel will be critical if this effort is to succeed.

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Development of Targeted Therapies for Rhabdoid Tumors Based on the Functions of INI1/hSNF5 Tumor Suppressor Ganjam V. Kalpana and Melissa E. Smith

Introduction Rhabdoid tumors (RTs) are rare, but highly aggressive and mostly incurable Â�pediatric malignancies (Strother 2005; Biegel 2006). RTs occur in children younger than 5 years of age and the peak incidence is between birth and 3 years of age (Biegel 2006). RTs were originally described in kidneys and termed Malignant Rhabdoid Tumors (MRT), and were subsequently found in the central nervous system (Atypical Teratoid and Rhabdoid Tumors, AT/RT), and soft tissues (extrarenal Rhabdoid Tumors) (Biegel 2006). Irrespective of their location, all RTs are characterized by the presence of sheets or nests of rhabdoid cells. Recent molecular genetic studies have established that RTs are distinguished from other tumors by the presence of recurrent biallelic deletions and/or mutations in the INI1/hSNF5 gene, located at chromosome 22q11.2, providing a definitive diagnostic criteria (Versteege etÂ€al. 1998; Biegel 1999; Biegel etÂ€al. 1999; Sevenet etÂ€al. 1999). Current therapeutic regimens for RT involve empirically selected combinations of chemotherapeutic agents that are highly toxic and rarely curative, as such, the survival rate for children with RTs remains poor (Packer etÂ€al. 2002; Reddy 2005; Strother 2005; Biegel 2006; Yamamoto etÂ€al. 2006). Thus, there is a dire need to develop novel therapeutic strategies for RTs, preferably based on the understanding of the molecular factors responsible for the genesis, growth, and survival of these tumors. In the following sections, we will describe features of RTs, current therapeutic practices, the molecular basis of rhabdoid tumorigenesis, and development of potential targeted therapies based on these understandings.

G.V. Kalpana (*) Department of Molecular Genetics and Albert Einstein College Cancer Center, Albert Einstein College of Medicine of Yeshiva University, 1300, Morris Park Ave., Ullman 821, Bronx, NY 10461, USA e-mail: [email protected]

P.J. Houghton and R.J. Arceci (eds.), Molecularly Targeted Therapy for Childhood Cancer, DOI 10.1007/978-0-387-69062-9_15, © Springer Science+Business Media, LLC 2010

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General Features of Rhabdoid Tumors Pathology RTs arise most commonly in the kidney and these MRTs comprise from 1.5 to 4% of malignant renal tumors (Mitchell etÂ€ al. 2000; Tomlinson etÂ€ al. 2005). Additionally, AT/RTs account for approximately 1 to 2% of pediatric brain tumors and 10% of CNS tumors arising in infants (Biegel 2006). Although AT/RTs arise within the entire CNS, approximately 50% of these tumors are present within the posterior fossa (Packer etÂ€ al. 2002). In this location, there is preference for the cerebellopontine angle and the tumor often invades adjacent structures such as meninges and ependyma (Packer etÂ€ al. 2002). Imaging of these tumors reveals mixed cellularity, hemorrhage, necrosis, and cysts (Packer etÂ€al. 2002). Computed tomographic (CT) images of AT/RT show a hyperdense mass with enhanced intensity (Packer etÂ€ al. 2002). The T1-weighted magnetic resonance images (MRI) visualize the tumor as being isointense often with hyperintense foci due to intratumoral hemorrhage and the T2 images are heterogeneous with hypo-, iso-, and hyper-intense foci (Packer etÂ€al. 2002).

Diagnosis RTs were first described as a separate entity in 1978 (Beckwith and Palmer 1978). Until that time, they were considered an aggressive “rhabdomyosarcomatoid” variant of Wilms’ tumor and were often misclassified. MRTs have been commonly misdiagnosed as rhabdomyosarcomas, Wilms’ tumors, choroid plexus carcinoma (CPC), germ cell tumors, or ependymomas while AT/RTs are mistaken for glioblastoma, medulloblastoma (MB), or primitive neuroectodermal tumors (PNET) (Biegel 2006). One reason for this misdiagnosis is that AT/RTs display nonspecific radiologic features. AT/RTs appear as sections of increased density on nonenhanced CT scan, and with MRI, there is decreased signal intensity on various images due to hypercellularity, features that do not allow for the differential diagnosis of AT/RTs versus PNET, MB, CPC, teratoma, astrocytoma, or ependymoma (Fenton and Foreman 2003). As studies of RTs progressed, advanced diagnostic methods became available based on cellular, genetic, and molecular features of these tumors. Histologically, all RTs contain sheets or nests of rhabdoid cells alongside primitive neuroepithelial, epithelial, and/or mesenchymal elements (Fenton and Foreman 2003). Rhabdoid cells are large polygonal cells with eccentric nuclei, a prominent nucleolus, and juxtanuclear eosinophilic cytoplasmic inclusions (Packer etÂ€ al. 2002; Hoot etÂ€ al. 2004). Though varied, the immunophenotypic profile of RT cells can support diagnosis. The most consistently expressed markers within RTs are vimentin, epithelial membrane antigen, smooth muscle actin, and cytokeratin (Bruch etÂ€al. 2001; Hoot

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etÂ€ al. 2004; Hauser etÂ€ al. 2001). Among these, positive staining for epithelial membrane antigen often distinguishes RTs from other CNS tumors (Biegel 2006). Defining the molecular genetics of these rare tumors (discussed further in detail below) has provided additional tools for diagnosis. In brief, identification of the INI1/hSNF5 gene, located at chromosome 22q11.2, as a critical tumor suppressor inactivated in a majority (>95%) of RTs has facilitated definitive diagnosis for identification of RTs (Versteege etÂ€al. 1998; Biegel 2006). Immunohistochemical analysis for loss of INI1/hSNF5 expression has been shown to effectively distinguish MRTs and extra-renal RTs from other pediatric soft tissue tumors (Hoot etÂ€al. 2004). Additional studies demonstrated that fluorescence in situ hybridization (FISH) for chromosome region 22q11.2 could be used to visualize loss of INI1/hSNF5 and to effectively distinguish AT/RTs from other CNS tumors (Bruch etÂ€al. 2001).

Current Therapies for Rhabdoid Tumors RTs are aggressively treated with multiple modalities including surgery, chemotherapy, and radiation therapy. Recent strategies to intensify treatments for RTs have led to isolated cases of long-term survival (Madigan etÂ€al. 2007). Despite such aggressive treatment strategies, mean survival rates remain extremely low, and it has been reported that the mean survival with surgical intervention alone is only 3 months and increases slightly to 8 months with adjuvant chemotherapy and radiotherapy (Fenton and Foreman 2003). Surgical intervention is used as a primary treatment strategy in order to remove bulk tumor mass. Chemotherapy with or without radiation is used as adjuvant therapy and some patients with advanced disease stage receive hematopoietic stem cell rescue (HSCT) (Madigan etÂ€al. 2007). Those patients most likely to experience long-term survival present with localized disease, allowing for complete surgical resection (Madigan etÂ€al. 2007). Since MRTs were initially misclassified as an aggressive, “rhabdomyosarcomatoid” variants of Wilms tumor, they were originally treated using Wilms tumor protocols (Madigan etÂ€al. 2007). These protocols, involving vincristine, actinomycin-D, doxorubicin, and cyclophosphamide have proven to be ineffective in the treatment of MRT (Gururangan etÂ€al. 1993). Some disease responses have resulted, however, with combinations of cisplatin, doxorubicin, and imidazole carboxamide (DTIC) (Roper etÂ€al. 1981; Kent etÂ€al. 1987). Other instances using chemotherapeutic regimens containing vincristine, dactinomycin, doxorubicin, and cyclophosphamide, however, did little to improve mortality rates (Tomlinson etÂ€al. 2005). Such poor responses to conventional chemotherapies led to more aggressive chemotherapeutic treatments, which are often supplemented with intrathecal chemotherapy in order to reduce cerebral spinal fluid (CSF) seeding of AT/RT cells (Hilden etÂ€al. 2004). Radiation therapy has been used in addition to surgery and chemotherapy in attempts to increase treatment efficacy. Defining the role of radiotherapy in increasing patient survival is difficult as its use is limited in younger children due to subsequent neurological defects. Therefore the population of patients likely to

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receive significant doses of radiation therapy is older patients, who have a more favorable initial prognosis. Analysis of a patient group with disregard to age has shown that patients receiving 25 Gy or more of radiation appear to have a better disease outcome (Tomlinson etÂ€ al. 2005). A more extensive study provided that patients receiving radiation doses higher than 30 Gy exhibited an increased mean survival rate of 16 months compared to 6 months survival for those treated with lower doses or no radiotherapy (Chen etÂ€al. 2005). With and without radiation therapy, some success has been found for patients 3Â€years and older with treatments involving surgical resection and chemotherapy using ifosfamide, carboplatin, and etoposide (ICE) (Tekautz etÂ€al. 2005). Successful treatment of three young patients with MRT was achieved using a similar strategy involving surgical resection, radiation, and chemotherapy using ICE alternating with rounds of vincristine, doxorubicin, and cyclophosphamide (VDCy) (Wagner etÂ€ al. 2002; Yamamoto etÂ€ al. 2006). VDCy was originally used to treat Ewing Â�sarcoma patients (Yamamoto etÂ€al. 2006). Along with total surgical resection and 30.6 Gy radiation therapy, a similar chemotherapeutic regimen of VDCy alternating with ifosfamide and etoposide (IE) was able to successfully treat an infant with extrarenal RT of the chest wall (Hosoi etÂ€al. 2007). Another intensive therapeutic program that was used with success on three AT/RT patients employed rounds of cisplatin, doxorubicin, vincristine, actinomycin, cyclophosphamide, cisplatin, and vincristine and adriamycin, cyclophosphamide, followed by triple intrathecal therapy and radiotherapy (Chen etÂ€al. 2005). Ifosfamide is common to many of these therapeutic regimens. Gururangan etÂ€al. treated patients with ifosfamide alone or in combination with carboplatin and etoposide, bleomycin, cyclophosphamide, doxorubicin, methotrexate, or prednisolone. From these studies, they concluded that transient partial responses can be observed in some patients treated with ifosfamide alone or in combination with carboplatin and etoposide (Gururangan etÂ€al. 1993).

Ongoing Clinical Trials for RTs A number of clinical trials have been completed recently or are underway in attempts to define improved treatment strategies for RTs. An ongoing phase I study has tested the efficacy of intensive chemotherapy and peripheral blood stem cell rescue in infants with malignant brain or spinal cord tumors (including, but not limited to RTs). This study will test the efficacy of treatment with cisplatin, vincristine, cyclophosphamide, and etoposide followed by treatment with filgrastim and peripheral blood stem cell harvest. The study’s second phase involves treatment with carboplatin and thiotepa followed by peripheral blood stem cell transplantation (trial #NCT00003141). A recently completed phase I trial with a more simplistic treatment strategy tested irinotecan in pediatric patients with various malignancies including AT/RTs. This study defined the maximum tolerated dose for irinotecan and showed that four cycles of 30Â€ mg/m2 irinotecan was able to induce stable Â�disease in one AT/RT patient (Blaney etÂ€al. 2001).

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A number of phase II trials are currently recruiting new patients. One such study is focused specifically on treating AT/RT with intrathecal combination therapy (methotrexate, cytarabine and hydrocortisone), systemic chemotherapy (various alternating combinations of vincristine, carboplatin, cyclophosphamide, etoposide, dactinomycin, dexrazoxane, temozolomide, and filgrastim), and radiotherapy (trial #NCT00084838). Another phase II trial is focused on determining the use of myeloablative chemotherapy with peripheral blood stem cell rescue and its ability to increase survival rates of patients with newly diagnosed high-risk CNS tumors, including, but not limited too AT/RT (trial #NCT00179803). One phase II trial testing different drugs is currently underway to test the ability of antineoplastons A10 and AS2-1 in children with AT/RT (trial #NCT00003469). One phase II clinical trial that has recently been completed was focused on the use of oxaliplatin in pediatric patients with recurrent or refractory AT/RT, MB, or supratentorial PNET (trial #NCT00047177). Oxaliplatin was administered as a 2-h infusion every 21 days (one round). One out of five patients with AT/RT experienced stable disease after 17 rounds. This study provided that oxaliplatin is relatively well tolerated in children, but had limited activity in patients with recurrent CNS tumors that were treated previously with platinum compounds (Fouladi etÂ€al. 2006). Most trials currently focus on the treatment of AT/RT; however, one phase II trial is focused solely on the treatment of high-risk kidney tumors, including MRT. This study, proposed by Children’s Oncology Group, will combine surgical resection, chemotherapy (rounds of VDCy alternating with rounds of cyclophosphamide, etoposide, and carboplatin along with filgrastim, and in some cases, additional rounds of Â�vincristine and irinotecan), and radiotherapy (trial #NCT00335556). Though these clinical trials may define more effective treatment strategies, most are based upon multi-drug combinations involving chemotherapeutics that do not necessarily target factors directly involved in the survival and/or growth of RTs. Despite some success, recurrent or progressive RT often appears, even during active courses of chemotherapy and attempts to treat progressive RT have been reported as unsuccessful with additional therapy (Tekautz etÂ€al. 2005). Even with the use of potent chemotherapeutic, radiotherapeutic, and surgical interventions, the prognosis for RT patients remains poor, especially for children diagnosed with AT/RT before the age of 3 (Biegel etÂ€al. 2002; Packer etÂ€al. 2002). Current estimates suggest a 15% 2-year survival rate for children afflicted with AT/RT (Biegel 2006) and a 19 to 25% 5-year survival rate for those diagnosed with MRT (Yamamoto etÂ€al. 2006). Factors that further predict poor survival are younger age of tumor onset and higher stage of disease at the time of diagnosis (Tomlinson etÂ€al. 2005). One possible reason for the failure of current therapies may be because they involve treatment regimens based on other tumor types that are similar to, but are distinct from RTs. Since RTs have a unique identity, it may be necessary to develop selective therapeutic strategies that are specific to this tumor type. It is likely that understanding the molecular mechanism of rhabdoid tumorigenesis could pave the way to develop molecularly targeted therapies. Identification of INI1/hSNF5 as a critical tumor suppressor deleted in RTs has facilitated such development.

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The Â�following sections will summarize what is known about INI1-mediated tumor Â�suppression in RT cells, specifically in light of developing targeted therapy based on this understanding.

Mechanisms of INI1-Mediated Tumor Suppression INI1/hSNF5, also known as BAF47 and SMARCB1, was first isolated as an interacting protein for HIV-1 integrase via the yeast two-hybrid system, and subsequently, it was demonstrated to be a component of the human chromatin remodeling SWI/ SNF complex (Kalpana etÂ€al. 1994; Wang etÂ€al. 1996). SWI/SNF is an evolutionarily conserved, multi-subunit, high molecular weight (>2Â€ MDa) complex that remodels chromatin in an ATP-dependent manner. There are two broad classes of chromatin remodeling machines (CRM), including those that remodel chromatin by covalent modifications such as acetylation, deacetylation, and methylation, and those that modify chromatin by disrupting and/or repositioning the nucleosomes in an ATP-dependent manner. Currently, four different classes of ATP-dependent chromatin remodeling complexes have been identified (SWI/SNF, ISWI, Mi-1, and Ino80), each characterized by a unique subunit composition and defined by the presence of distinct ATPases (Fig.Â€1) (Martens and Winston 2003). SWI/SNF, a prototypical ATP-dependent remodeling complex, consists of at least nine subunits that are conserved among eukaryotes (Fig.Â€1) (Narlikar etÂ€al. 2001). ATP-dependent CRMs Key ATPase (human)

hSWI/SNF-A (BAF) BAF53 BAF155

OSA1 BAF170 b-Actin

ISWI

Mi-2

Ino80

SWI2/SNF2L BRG1 BRM

ISW1

Mi-2/CHD

Ino80

hSWI/SNF-B (PBAF)

HBrm

BAF180

BAF250 BAF60a

SWI/SNF

BRG1 Or BRM

INI1

BAF60a BAF155 BAF57 BAF170 b-Actin

HBrg1(I)

BAF250

BAF250 BAF53

BRG1

BAF60

BAF53 BAF155

BAF57 BAF170

INI1

BRM

INI1 P220

HBrg1(II)

HDAC1

Sin3A

RbAP48

BAF60 BAF155 BAF57

BAF170

BAF250 BAF155 BRG1

INI1 P220

BAF57 BAF170

BRG1

INI1

HDAC1

HDAC2

RbAP48

Sin3A

P66

RbAP48 HDAC2

BAF53

BAF53

P66

P66

Fig.Â€1â•… Association of INI1/hSNF5 with multiple SWI/SNF complexes in mammalian cells. CRM chromatin remodeling machine. Four ATP-dependent remodeling complexes have been identified in mammalian cells, which are distinguished by the key ATPase present within each complex. Different types of SWI/SNF complexes have been identified that differ in their subunit composition. However, INI1/hSNF5 is present in each one of these SWI/SNF complexes

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Among these are four core subunits required for chromatin remodeling, including SWI2/SNF2, the key ATPase subunit, and INI1/hSNF5. The Drosophila orthologue of SWI2/SNF2 is termed Brahma, from which the two human paralogues derive their names, hBRM and BRG1 (Brahma Related Gene). There are two functionally distinct classes of SWI/SNF complexes in mammalian cells, hSWI/SNF-A or BAF and hSWI/SNF-B or PBAF. Three additional complexes that contain a mixture of components derived from histone deacetylase complex (HDAC) and SWI/SNF are known as hBRM, hBRG1(I), and hBRG1(II) complexes (Fig.Â€ 1) (Martens and Winston 2003). The stoichiometry, subnuclear distribution, or exact functions of SWI/SNF complexes are not well defined in mammalian cells. It is important to note that while these complexes differ greatly in subunit composition, INI1/hSNF5 is present in each of the different SWI/SNF complexes, implicating its multiple and varied functions in mammalian cells.

Regulation of Transcription by INI1 and the SWI/SNF Complex Chromatin remodeling complexes are global regulators of cellular transcription. However, the activity of each particular complex is restricted to a subset of genes. The SWI/SNF complex has been shown to effect a subset of approximately 2 to 10% of cellular genes (Martens and Winston 2003). One mechanism by which SWI/SNF regulates only a small subset of genes is by selective targeting to specific gene promoters. SWI/SNF components have no sequence-specific DNA binding ability, and therefore, are recruited to specific promoters via protein–protein interactions with transcription factors that directly bind promoter elements (Fig.Â€ 2). In this regard, we have found that INI1/hSNF5 interacts with cMYC, a sequence-specific transcription factor, and that the SWI/SNF complex is required for the transactivation function of cMYC (Cheng etÂ€al. 1999). Determining the mechanism by which target specificity is achieved by INI1/hSNF5 and defining the subset of cellular genes affected will likely provide more insight into the mechanism of tumor suppression by INI1/hSNF5. SWI/SNF complexes are well characterized as transcriptional activators. Nevertheless, INI1/hSNF5 and these complexes are not only involved in activation, but also in repression of target genes (Martens and Winston 2003). The activation functions of SWI/SNF result directly from its ability to remodel chromatin at promoter regions. However, the mechanism by which SWI/SNF mediates repression is not completely understood. Recent evidence, including studies from our laboratory, indicates that repression is due to direct recruitment of INI1/hSNF5 and the SWI/ SNF complex to specific promoter regions (Fig.Â€2). This recruitment is mediated by SWI/SNF interaction with sequence-specific DNA-binding repressors such as E2F, co-repressors such as REST, components of the HDAC1 complex, or mammalian heterochromatin protein (HP1a) (Martens and Winston 2003). It is not clear however, how SWI/SNF complexes selectively activate some promoters while repressing others. One possible explanation involves the existence of distinct SWI/SNF

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a SWI/SNF BRG1

INI1 TF

Promoter

Activation

b INI1

HDAC1

TSA

TF

Promoter

Repression

Fig.Â€ 2â•… SWI/SNF complexes can both activate and repress transcription. (a) Absence of a sequence-specific binding subunit requires that the SWI/SNF complex be recruited to specific promoters by interaction of its components (such as INI1/hSNF5) with sequence-specific DNA binding transcription factors (TF). Remodeling or rearrangement of nucleosomes is likely to activate transcription. (b) One mechanism of SWI/SNF-mediated repression is by its association with members of the HDAC1 complex. Trichostatin A (TSA) is an inhibitor of HDAC1 that reverses transcriptional inhibition mediated by HDAC1 complex

complexes, some of which are associated with components of the HDAC1 repressor complex as described previously (Fig.Â€1). Understanding the function and specificity of each of these complexes in distinct cell types is necessary to fully comprehend the mechanisms by which INI1/hSNF5 mediates tumor suppression in RT precursor cells.

Pathways Affected by Loss of INI1 in Rhabdoid Tumors Based on the association of INI1/hSNF5 with the SWI/SNF complex and the requirement of this complex for varied cellular functions, it can be surmised that INI1/hSNF5 mediates tumor suppression in part by selective transcriptional Â�regulation of genes important for cell cycle, differentiation, senescence, or apoptosis. A hallmark of cancer cells is the lack of regulatory circuits to maintain normal cell division and homeostasis. Tumors cells are characterized by: (1) self sufficiency in growth signals, (2) insensitivity to growth inhibitory signals, (3) evasion of apoptosis, (4)

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limitless replicative potential, (5) sustained angiogenesis, and (6) potential for tissue invasion and metastasis [Hanahan and Weinberg (2000)]. These properties are often achieved by mutations in oncogenes and/or tumor suppressors; genes that directly or indirectly regulate these processes. Consistent with this hypothesis, it has been established that reintroduction of INI1/hSNF5 into RT cells leads to deregulation of various cellular pathways important for normal cell Â�division and homeostasis. These studies have been critical to understand the mechanism of tumor suppression by INI1/hSNF5.

Induction of G0/G1 Arrest by INI1/hSNF5 In vitro studies have established that reintroduction of INI1/hSNF5 into RT cells is sufficient to induce G0/G1 cell cycle arrest and flat cell formation, indicative of senescence (Zhang etÂ€al. 2002; Reincke etÂ€al. 2003; Oruetxebarria etÂ€al. 2004; Chai etÂ€ al. 2005). Induction of G0/G1 arrest and senescence by INI1 is correlated to transcriptional repression of cyclin D1 (Zhang etÂ€al. 2002) and activation of p16INK4a and p21CIP (Betz etÂ€al. 2002). Studies within our laboratory demonstrated that INI1/ hSNF5 directly represses cyclin D1 by recruiting the HDAC1 complex to its promoter (Fig.Â€2b) (Zhang etÂ€al. 2002). Repression of cyclin D1 by INI1/hSNF5 specifically occurred in rhabdoid cells but not in other cell types such as HeLa (Zhang etÂ€al. 2002). Among the three D-type cyclins (cyclin D1–D3), only cyclin D1 was repressed by INI1/hSNF5 in rhabdoid cells, while other D-type cyclins (cyclins D2 and D3) were unaffected (Tsikitis etÂ€al. 2005). Additionally, we demonstrated that co-expression of cyclin D1 from a heterologous promoter along with INI1/hSNF5 is sufficient to overcome INI1-mediated cell cycle arrest in RT cells (Zhang etÂ€al. 2002). These studies indicate that transcriptional regulation of a selective subset of genes in a cell-type specific manner is likely important for INI1-mediated tumor suppression in RT precursor cells and that cyclin D1 could be a critical downstream target of INI1-mediated repression.

Role of INI1 in Mitotic Spindle Check-Point INI1 is also involved in mitotic spindle checkpoint activation through the p16Cyclin D1/CDK4-pRb-E2F pathway (Vries etÂ€al. 2005). It was demonstrated that when reintroduced into rhabdoid cells, INI1/hSNF5 decreased the number of aberrant aneuploid cells and facilitated diploidization. Interestingly, it was found that reintroduction of cancer-associated INI1-mutants, P48S and R127G, failed to generate a diploid cell population and expression of S284L and S289A mutants strongly promoted polyploidization (Vries etÂ€ al. 2005). Gene expression profile analysis indicated that, in addition to activating p16INK4a, INI1 represses a high degree of mitotic genes including PLK1 (see below) (Morozov etÂ€al. 2007).

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INI1/hSNF5 Induces Interferon Signaling and Markers of Senescence Several cDNA microarray studies to investigate pathways downstream of INI1/ hSNF5 have been carried out. These studies revealed that expression of only a small percentage (approximately 2%) of genes is affected by INI1/hSNF5. Early studies compared gene expression patterns of RTs to other pediatric tumors (Pomeroy etÂ€al. 2002). These studies indicated that RTs have a distinct molecular signature despite their presence in varied anatomical locations (Pomeroy etÂ€al. 2002). Other studies have compared the changes in global expression patterns when INI1/hSNF5 was reintroduced into INI1-null rhabdoid cells (Medjkane etÂ€al. 2004; Vries etÂ€al. 2005; Morozov etÂ€ al. 2007). Others have explored INI1/hSNF5-mediated expression profile changes in normal mouse embryo fibroblasts (Isakoff etÂ€ al. 2005). Genes upregulated by INI1/hSNF5 appear to be anti-proliferative or involved in senescence or differentiation while genes repressed by INI1/hSNF5 are involved in cell cycle progression (Morozov etÂ€ al. 2007). Genes upregulated by INI1/hSNF5 and involved in senescence include MMP1 and PAI-1 (Reincke etÂ€al. 2003; Chai etÂ€al. 2005; Morozov etÂ€al. 2007). An overwhelming majority of mitosis-specific genes were downregulated by INI1/hSNF5 including PLK1, TOP2A, STK6, KIF2C, CENP-F, and Securin (Morozov etÂ€ al. 2007). Our studies indicated that INI1/ hSNF5 induced many interferon-stimulated genes (ISGs) at early time points and that interferon (IFN) signaling through its receptor was required for this induction of ISGs (Morozov etÂ€al. 2007). The microarray results are consistent with the functions of INI1/hSNF5 as a tumor suppressor and provided us with many pathways and genes to investigate as potential therapeutic targets for RT treatment, which will be discussed below.

Rhabdoid Tumor Cell of Origin and the Role of INI1 in Differentiation Reintroduction of INI1/hSNF5 into RT cells has an anti-proliferative effect. Contrarily, many studies have indicated that INI1/hSNF5 is required for the Â�survival of nonRT cells and normal cells based on the following reports. (1) Homozygous deletion of INI1/hSNF5 is embryonic lethal and Ini1−/− mouse fibroblasts undergo cell death in culture (Klochendler-Yeivin etÂ€al. 2000; Guidi etÂ€al. 2001), (2) Conditional deletions of Ini1/Snf5 in neonatal mice result in massive apoptosis and hemorrhage in the liver and other organs (Roberts etÂ€ al. 2002), (3) Knockdown of INI1/hSNF5 in HeLa cells leads to a senescence-like phenotype and cell death (Kato etÂ€al. 2007). These results indicate that INI1/hSNF5 has differential effects on the survival of RT cells versus nonRT or normal cells. One possible reason for this is that regulation of cellular pathways by INI1/hSNF5 is cell-type specific.

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Knowing the cell of origin of RTs may provide insight into the differential function and mechanism of INI1/hSNF5-mediated tumor suppression; however, the RT cell of origin remains undefined. Since RTs arise in many anatomical locations, several possible cellular progenitors have been proposed, including neural, neuroectodermal, myogenic, mesenchymal, and epithelial cell types (Ota etÂ€ al. 1993). The hypothesis of a mesenchymal cell of origin was supported by comparative global gene expression profile analysis, which revealed a common mesenchymal origin gene signature among RTs (Pomeroy etÂ€al. 2002). It is unclear however, if this gene signature is an acquired characteristic due to loss of INI1/hSNF5 or if it reflects the original cell lineage. Another hypothesis, based on studies using genetic crosses between TgT121 and Ini1+/− knockout mice suggested that RTs might arise from undefined neural progenitor cells (Chai etÂ€ al. 2007). Interestingly, loss of INI1/ hSNF5 was recently associated with schwannomatosis, a tumor arising from schwann cells, and therefore, ectodermal in origin. These results suggest that loss of INI1 leads to schwannomatosis from schwan cells (Hulsebos etÂ€al. 2007).

INI1’s Involvement in Cellular Differentiation Pathways The highly undifferentiated phenotype of RTs suggests that loss of INI1/hSNF5 impairs differentiation programs within RT precursor cells; an idea supported by various studies. Reintroduction of INI1/hSNF5 into DEV and MON RT cell lines induces differentiation into the neural lineage (Albanese etÂ€al. 2006). Furthermore, INI1/hSNF5 was found to be essential for SWI/SNF-dependent induction of neural differentiation programs as siRNA knock-down significantly impairs the ability of rat PC12 cells to differentiate into the neural lineage upon exposure to neural growth factor (Albanese etÂ€al. 2006). The involvement of INI1/hSNF5 in adipocyte differentiation pathways has also been demonstrated. Reintroduction of INI1/ hSNF5 into MON cells using an inducible expression system led to differentiation toward the adipocyte lineage (Caramel etÂ€al. 2007). siRNA knock-down of INI1/ hSNF5 in murine 3T3-L1 preadipocytes and human mesenchymal stem cells eliminates the ability of these cells to differentiate into the adipocyte lineage (Caramel etÂ€al. 2007). Mechanistic studies showed that INI1/hSNF5 cooperates with C/EBPb and PPARg2 to activate adipocyte-specific gene expression (Caramel etÂ€al. 2007). In addition to its involvement in directing differentiation into neural and adipocyte lineages, INI1/hSNF5 is also essential for hepatocyte differentiation. Inactivation of INI1/hSNF5 in the developing liver blocked the expression of many genes normally upregulated during liver development and severely impaired glycogen storage and epithelial morphogenesis, characteristics of hepatocyte differentiation (Gresh etÂ€al. 2005). These findings indicate that loss of INI1/hSNF5 in RT precursor cells Â�disrupts cell fate, causing the severely undifferentiated phenotype common to RT cells. These results provide useful, but limited information on the role of INI1/ hSNF5 during differentiation and on the possible cell of origin, but the relationship between its role in differentiation and tumor suppression remains unanswered.

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Therapeutic Targeting of Downstream Effectors of INI1/hSNF5 for Treatment of Rhaboid Tumors Though there is still a large deficit in knowledge regarding the RT cell of origin or the specific mechanism by which loss of INI1/hSNF leads to RT formation, the abundance of information about the pathways affected by INI1/hSNF5 have provided insight that may lead to the development of novel targeted treatment strategies for these tumors. The remaining sections of this review summarize various preclinical studies that explore the development of targeted therapies for RTs with the potential for having greatly improved efficacy compared to current treatments. Development of successful targeted therapies relies on knowledge of specific cellular factors or pathways that are both deregulated and required for the genesis, survival, or progression of RTs. While many factors are deregulated in RTs, only those known to be necessary for the growth of these tumors may serve as potentially effective therapeutic targets. As will be described, Cyclin D1 is a factor with such potential. Extensive research has demonstrated that INI1/hSNF5 suppresses the RT phenotype by repressing cyclin D1 and activating p16INK4a, indicating that targeting the Cyclin/cdk-axis may be a useful strategy for developing a selective therapy for RTs. Additional inÂ€ vitro studies have implicated other pathways downstream of INI1/hSNF5 and supported their possible use as therapeutic targets.

Hypothesis for the Mechanism of Tumor Suppression by INI1 The current observations suggest that the mechanism of tumor suppression by INI1/hSNF5 is complex, involving its ability to regulate multiple cellular pathways and affect expression of and genes. One hypothesis is that the rhabdoid precursor cell is destined to undergo terminal cell cycle exit due to stimuli leading to differentiation, and/or senescence (Fig.Â€3). INI1/hSNF5 may facilitate the terminal cell cycle exit, differentiation, and/or senescence by: (1) specific repression of genes important for cell cycle progression, (2) activation of cell cycle inhibitors, and (3) activation of genes involved in differentiation or senescence (Fig.Â€3, top panel). When INI1/hSNF5 is deleted in the precursor cell, its downstream pathways will be deregulated, thus leading to tumorigenesis by conferring unlimited proliferative capacity and by protecting the cells from senescence and apoptosis (Fig.Â€ 3, bottom panel). It follows that a useful treatment strategy for RTs is to mimic the effect of INI1/hSNF5. This can be done by treatment with small molecular weight inhibitors (drugs) or siRNA constructs that selectively affect pathways downstream of INI1/hSNF5 that are critically deregulated in RTs (Fig.Â€3, bottom panel). In the sections that follow, we will describe how we have tested this, using preclinical models.

Development of Targeted Therapies for Rhabdoid Tumors Based on the Functions

a

INI1/hSNF5

Rhabdoid precursor Cell

pRB

Cyclin D1/cdk4

p16/Ink4a

Senescence

b

Apoptosis

G1

E2F

S

G2

PLK1 and other Mitotic genes M

X

X

pRB

Cyclin D1/cdk4

Mitotic catastrophe Apoptosis

E2F

PLK1 and other Mitotic genes

?

X

Senescence

c

X

Apoptosis

G1

S

G2

Cyclin D1/cdk4

p16/Ink4a

pRB

E2F

?

Senescence

Apoptosis

M

X

G1 Mitotic catastrophe Apoptosis

Targeting G1 and Mitotic genes

Drugs/siRNA

X

G1

Rhabdoid Cell

INI1/hSNF5

p16/Ink4a

317

G1

S

G2

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M

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Fig.Â€3â•… Mechanism of INI1/hSNF5-mediated tumor suppression. (a) In a rhabdoid precursor cell (unknown) INI1/hSNF5 regulates transcription of key cell cycle regulatory genes. It activates p16, and represses cyclin D1 and mitotic genes, leading to G0–G1 arrest and mitotic arrest. Activation of p16 is associated with senescence and control of ploidy, and repression of cyclin D1 often inhibits apoptosis. (b) In a rhabdoid cell, loss of INI1/hSNF5 leads to transcriptional deregulation of genes involved in cell cycle – p16 is not activated and cyclin D1 and PLK1 are derepressed. As a result, cells escape cell cycle control and senescence and undergo uncontrolled cell division. (c) Use of drugs or siRNA that can inhibit genes or pathways (Cyclin/cdk and mitotic genes) normally inhibited by INI1/hSNF5 could be useful as molecularly targeted therapies for RTs

Critical Role of Cyclin D1 in Rhabdoid Tumor Formation and Survival As described, INI1/hSNF5 directly and selectively represses transcription of cyclin D1 in a cell cycle-independent manner. Further studies showed that Cyclin D1 is a critical downstream target of INI1/hSNF5 and that its over-expression promotes rhabdoid tumorigenesis. In vitro studies indicated that INI1/hSNF5 transcriptionally represses cyclin D1 by recruitment of the HDAC1 complex, and that INI1/

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hSNF5-mediated cell cycle arrest is at least partially overcome by co-expression of cyclin D1 from a heterologous promoter (Zhang etÂ€al. 2002). Consistent with INI1/ hSNF5’s ability to repress transcription of cyclin D1, we found that Cyclin D1 is de-repressed/over-expressed in both mouse and human primary RTs (Zhang etÂ€al. 2002; Fujisawa etÂ€al. 2005; Tsikitis etÂ€al. 2005; Donner etÂ€al. 2007). To unequivocally establish the critical role of Cyclin D1 in rhabdoid tumorigenesis, we utilized a genetically engineered mouse model of RTs. As indicated earlier, heterozygous Ini1+/− mice develop RTs at a high frequency, which mimic the etiology and characteristics of human RTs. We generated three cohorts of Ini1+/− mice harboring different cyclin D1 genotypes. We found that Ini1+/− mice with cyclin D1+/+ or +/− genotypes were able to develop RTs at the usual frequency of about 25%, while none of the Ini1+/− mice with a cyclin D1−/− background developed RTs (Tsikitis etÂ€ al. 2005). These striking results established that genetic ablation of cyclin D1 is sufficient to abrogate the genesis of RTs and provided proof that Cyclin D1 is a necessary and critical component for the formation and/or survival of RTs.

Development of Novel Therapeutic Strategies Against RTs by Targeting Cyclin/cdk Axis The exquisite dependence of RTs on Cyclin D1 suggests that targeting Cyclin D1 or its pathway is a practical option for developing a molecularly targeted therapy for RTs. It is important, however, to first demonstrate that Cyclin D1 is not only necessary for the genesis of RTs, but also for the survival of established RTs. This was accomplished by siRNA-mediated knockdown of cyclin D1 in RT cells, which induced G0–G1 arrest and apoptosis (Alarcon-Vargas etÂ€al. 2006). Taken together, the inÂ€vitro and inÂ€vivo data defining the role of Cyclin D1 in RTs provide compelling evidence that Cyclin D1 is required for rhabdoid tumorigenesis and survival. Cyclin D1 is overexpressed in several human tumors including approximately 30% of human breast cancers, as well as pituitary tumors, MBs, head and neck carcinoma, and esophageal squamous cell carcinoma. The oncogenic potential of cyclin D1 is in part due to its ability to facilitate cell cycle progression through G1 phase. Cyclin D1 binds to and activates Cyclin-dependent kinases (cdks) 4 and 6, which in turn phosphorylate retinoblastoma protein (Rb). Phosphorylation of Rb relieves E2F, allowing for activation of E2F-target gene transcription. In addition, overexpressed Cyclin D1 sequesters p21cip1 (cdk inhibitor), effectively activating the Cyclin E-cdk2 holoenzyme and resulting in G1–S transition. Cyclin D1 exhibits additional, cdk-independent functions, which are also important for tumorigenesis (Ewen and Lamb 2004; Fu etÂ€ al. 2004). Independent of interaction with cdks, Cyclin D1 modulates the activity of several transcription factors including estrogen receptor (ER), D1-interacting-myb-like protein 1(DMP1), androgen receptor (AR), and signal transducer and activator of transcription (STAT3). Taken together, these studies indicate that both cdk-dependent and -independent functions of Cyclin D1

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INI1

p

RNAPII Cyclin D1

p16

p Cyclin D1

(pTEFb)

CDK

Fenretinide

CyclinT CDK9

Degradation p16

CyclinD

CyclinB

M

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CDK4 CDK6 p21

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Flavopiridol

G1 CyclinA CDC2

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Fig.Â€4â•… Targeting the Cyclin/cdk pathway in rhabdoid tumors. INI1/hSNF5 represses cyclin D1 and activates p16 to control the Cyclin/cdk pathway. Drugs such as Fenretinide and Flavopiridol target Cyclins and cdks as part of their mechanism of action. Fenretinide is known to inhibit transcription of cyclin D1 and facilitate degradation of Cyclin D1. Flavopiridol is a pan-cdk inhibitor, which, in addition to inhibiting cdk activity also inhibits cyclin D1 transcription through inhibition of cdk9. Preclinical studies have demonstrated that these two drugs significantly inhibit RT growth inÂ€vitro and inÂ€vivo with their activity correlated to repression of Cyclin D1

are important for tumorigenesis, and therefore, targeting Cyclin D1 and/or the Cyclin/cdk axis may effectively inhibit RT growth. The following sections will describe preclinical studies that investigated the effects of two different chemotherapeutic agents, 4HPR (N-(4-hydroxyphenyl) retinamide, or Fenretinide) and Flavopiridol, and their ability to inhibit the Cyclin/cdk axis in RTs (Fig.Â€4).

4HPR as a Therapeutic Agent for Rhabdoid Tumors 4HPR is a synthetic retinoid shown to have a low toxicity profile and potent chemopreventive effects in various preclinical models. It exhibits inÂ€vitro cytotoxicity and

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suppresses tumor cell growth at low micromolar concentrations (IC50’s ranging from 1 to 10Â€mM) (Garaventa etÂ€al. 2003). 4HPR is an FDA approved drug that has shown much promise in clinical trials and continues to be under phase II trials for many cancers. In pediatric neuroblastoma patients, 4HPR has induced prolonged disease stabilization in pilot clinical studies (Reynolds etÂ€ al. 2003; Lovat etÂ€ al. 2004, 2005; Reynolds 2004). It has been largely studied as a chemo-preventive agent in animal models of carcinogen-induced epithelial tumors and in patients at risk for breast cancer (Fontana and Rishi 2002; Formelli etÂ€ al. 2003; Hail etÂ€ al. 2006; Zanardi etÂ€ al. 2006). A 15-year follow-up study of phase III trials using 4HPR to prevent second breast cancer has recently been completed. This follow-up indicated that 4HPR significantly reduces the risk of recurring breast cancer in premenopausal women (Bonanni etÂ€al. 2007). 4HPR induces apoptosis in tumor cell lines by various mechanisms including: (1) activation of retinoid receptors RAR b and g, (2) induction of ceramide-dependent cell cytotoxicity, (3) generation of radical oxygen species, (4) increase of nitric oxide synthase (NOS) resulting in increased NO-dependent cell cytotoxicity, and (5) increase of mitochondrial permeability transition (Fontana and Rishi 2002; Reynolds etÂ€al. 2003; Lovat etÂ€al. 2004, 2005; Hail etÂ€al. 2006). 4HPR also induces cell cycle arrest likely by down-modulating the expression or activity of proliferation-related factors such as c-Myc, telomerase, p34/cdc2 and cyclin D1 (Igawa etÂ€al. 1994; Delia etÂ€al. 1995; Bednarek etÂ€al. 1999; Sun etÂ€al. 1999, 2001; Reynolds and Lemons 2001; Soria etÂ€al. 2001, 2003; Bowman etÂ€al. 2002; Hiyama and Hiyama 2002; Christov etÂ€al. 2003; Reynolds etÂ€al. 2003). For example, 4HPR affects transcription and protein stability of Cyclin D1 in a concentration dependent manner (DiPietrantonio etÂ€ al. 1998; Panigone etÂ€ al. 2000; Christine Pratt etÂ€ al. 2003; Dragnev etÂ€al. 2004). Furthermore, it has been demonstrated that overexpression of Cyclin D1 sensitizes breast cancer cells to 4HPR (Pirkmaier etÂ€al. 2003). Although 4HPR has been reported to down-modulate Cyclin D1, it was never used to specifically target the Cyclin D1 pathway in cancer cells. The effect of 4HPR on Cyclin D1 transcription and stability prompted us to carry out a series of inÂ€vitro and inÂ€ vivo experiments testing its efficacy on RTs (Alarcon-Vargas etÂ€ al. 2006). Additionally, the effect of combining 4HPR with 4OH-Tamoxifen (4OH-Tam) was tested because of the reported synergism of these two drugs inÂ€ vitro (Wang etÂ€ al. 2003). Furthermore, suppression of tumor growth by 4OH-Tam has been associated with down-modulation of Cyclin D1 (Jang etÂ€al. 2001). This study demonstrated that 4HPR and 4OH-Tam synergistically inhibit the survival of RT cells, induce G1 arrest, and activate caspase 3/7-mediated apoptosis (Alarcon-Vargas etÂ€al. 2006). These cell culture studies were extended inÂ€vivo using a xenograft model of RTs and it was found that the effect of 4HPR and 4OH-Tam is as well synergistic in inhibiting RT growth inÂ€vivo (Alarcon-Vargas etÂ€al. 2006). Importantly, it was found that inhibition of RT cell growth, both inÂ€ vitro and inÂ€ vivo was associated with reduction of Cyclin D1 levels, and inÂ€vivo, with differentiation of xenograft tumors into bone and cartilage (Alarcon-Vargas etÂ€ al. 2006). These preclinical results support our hypothesis that down-modulation of Cyclin D1 is an effective therapeutic strategy for RTs and demonstrate that 4HPR and 4OH-Tam are effective chemotherapeutic agents for RTs.

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Flavopiridol as a Therapeutic Agent for Rhabdoid Tumors Another method of inhibiting the Cyclin D1 pathway is to use drugs that inhibit cdk activity. Flavopiridol is a pan-cdk inhibitor, and one of the first broad-spectrum cdkinhibitors to enter clinical trails. Flavopiridol is a competitive inhibitor of multiple cdks including cdk 1, 2, 4, 6, 7 and 9 (Chao and Price 2001). Its ability to inhibit cdks 1, 2, 4, and 6 directly affects the function of cyclin/cdk complexes resulting in cell cycle arrest. Furthermore, it inhibits cdk9 resulting in inactivation of P-TEFb (Cyclin T1/cdk9 complex), therefore blocking RNA polymerase II-mediated transcription of many genes, including cyclin D1 (Mani etÂ€ al. 2000; Chao and Price 2001; De Azevedo etÂ€al. 2002). Flavopiridol also indirectly inhibits TNF-mediated induction of NF-kB, which regulates Cyclin D1 (Takada and Aggarwal 2004). Furthermore, flavopiridol inhibits the activation of Akt leading to inhibition of forkhead proteins, resulting in decreased translation of Cyclin D1 protein (Schmidt etÂ€al. 2002; Cappellini etÂ€ al. 2003; Liang and Slingerland 2003). Recent studies using pharmacologically based schedules of flavopiridol administration have shown promising activity in clinical trials of chronic lymphocytic leukemia (CLL) (Byrd etÂ€al. 2007). The strong effect of flavopiridol on various aspects of the cyclin/cdk-axis have led us to test its ability to inhibit the growth of RTs (Smith etÂ€al. 2008). In vitro studies demonstrated that nanomolar concentrations of flavopiridol inhibited RT cell growth. Treatment with flavopiridol induced G1 or G2 arrest and apoptosis in a manner dependent on drug concentration and time of exposure. Interestingly, inhibition of rhabdoid cell proliferation at concentrations required for 50% cell killing (IC50) was apparently independent of Cyclin D1 while higher levels of cell killing (IC95) were correlated to down-modulation of Cyclin D1, upregulation of p21, and induction of caspase 3/7 activities. Furthermore, inÂ€vivo studies demonstrated that flavopiridol at 7.5Â€ mg/kg significantly inhibited the growth of xenografted RTs with effects correlated to upregulation of p21 and downmodulation of Cyclin D1 (Smith etÂ€al. 2008) These results demonstrated that, in addition to 4HPR and 4OH-Tam, flavopiridol is potentially a novel chemotherapeutic agent for RTs and strongly supports our hypothesis that therapeutically targeting the Cyclin–cdk axis is effective in inhibiting RTs.

Other Pathways Amenable for Developing Targeted Therapies for RTs Gene expression profile analysis and other studies have provided clues to many other pathways, apart from the Cyclin/cdk axis, that are amenable for therapeutic targeting in RTs. These include the mitotic spindle checkpoint, IFN, and Akt pathways. Preclinical studies investigating the relevance of these additional pathways are Â�currently ongoing. We will provide a brief account of what is known about the strategies for targeting these additional pathways.

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Targeting the Mitotic Spindle Checkpoint The mitotic spindle checkpoint ensures proper segregation of chromatids to daughter nuclei during mitosis and guards against the deleterious effects of aneuploidy. Many cancer cells have a weakened mitotic checkpoint due to overexpression of genes necessary for mitotic progression. Recent investigations have demonstrated that cancer-causing INI1/hSNF5 mutants induce aberrant chromosomal segregation and aneuploidy in RT cells (Vries etÂ€al. 2005). Furthermore, we have demonstrated that a high frequency of genes necessary for mitotic progression, including PLK1, Aurora, TOP2A, etc., are targets of INI1-mediated repression in rhabdoid cells. Immunohistochemical analysis indicated that PLK1 is highly expressed in the cytoplasm of cells in human and mouse RTs, independent of cell cycle stage, suggesting that PLK1 is deregulated in RTs (Morozov etÂ€al. 2007). Repression of PLK1 by INI1/hSNF5 and its overexpression in human and mouse RTs suggests that PLK1 is a potential target for therapeutic intervention for these tumors. PLK1, a crucial mitosis-promoting kinase, is currently an attractive target for developing anti-cancer therapies (McInnes etÂ€al. 2005). Overexpression of this protein is a prognostic marker for various cancers and is associated with aggressive stage and poor survival in cancer patients (Eckerdt etÂ€ al. 2005). Furthermore, it appears that tumor cells are sensitive to down-modulation of PLK1 as they succumb to mitotic catastrophe, while normal diploid cells tolerate its inhibition, possibly due to the presence of redundant mechanisms (Guan etÂ€ al. 2005; Liu etÂ€ al. 2006). Currently, there are several drugs in the developmental stage to target the activity of PLK1. As a first test of targeting PLK1 in RTs, RNA interference analysis (RNAi) was used to inhibit its expression in two rhabdoid cell lines (MON and G401). Transfection of RT cells with siRNA to PLK1 resulted in defective growth and enlarged cells with multiple and fragmented nuclei or aberrantly segregated chromatids (Morozov etÂ€al. 2007). Further characterization indicated that inhibition of PLK1 leads to decreased cell survival, increased Caspase 3/7 activity, and apoptosis (Morozov etÂ€al. 2007). Together, these results suggest that targeting PLK1 is potentially useful in inhibiting RTs. However, these are inÂ€vitro studies and further inÂ€vivo studies are required to facilitate the development of therapies by targeting PLK1 or other mitotic genes in RTs.

Targeting the Interferon Signaling Pathway Reintroduction of INI1 activates the expression of a high degree of IFN signal induced genes ISGs (Morozov etÂ€ al. 2007). Furthermore, addition of exogenous type I or type II IFNs to RT cells also induced ISGs and resulted in G0-G1 arrest and flat cell formation, a phenotype similar to that resulting from reintroduction of INI1/hSNF5 (Morozov etÂ€al. 2007). These results imply that addition of IFNs may

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mimick the effect of INI1/hSNF5. IFN treatment is an approved therapy and has been used as adjuvant therapy for a number of neoplasms including skin cancer, renal cell cancer, leukemia, liver cancer, and myeloma. IFNs have been shown to induce anti-proliferative effects in various cancer cell lines (Decatris etÂ€ al. 2002; Kirkwood 2002; Wall etÂ€al. 2003). Furthermore, induction of ISGs has been associated with inhibition of immortalization and induction of a senescence-like phenotype in certain cancer cells (Xin etÂ€al. 2003; Moiseeva etÂ€al. 2006; Pammer etÂ€al. 2006). Therefore, IFN therapy should be considered, at least as an adjuvant therapy for RTs. Studies on the use of IFNs as RT therapy are preliminary however, and further inÂ€vivo studies are necessary to establish their therapeutic efficacy. Several AT/RT or MRT tumor xenograft models have been included in the panel of pediatric solid tumors in preclinical studies testing efficacy of novel anticancer drugs. Efficacy of several drugs including but not limited to a-VEGF antibody (Soffer etÂ€ al. 2002), HDAC inhibitors (benzamide derivatives and desipeptides) (Graham etÂ€ al. 2006; Jaboin etÂ€ al. 2002), EGFR-tyrosine kinase inhibitors (gefitinib) (Kuwahara etÂ€al. 2004), fungal derivatives (Irofulven) (Leggas etÂ€al. 2002), and mTOR inhibitor (Rapamycin) (Houghton etÂ€al. 2007) have been tested in rhabdoid xenograft tumor models. Some of these drugs have demonstrated partial or complete response and further mechanistic studies are in progress to establish the basis of therapeutic efficacy of these drugs.

Future Directions and Challenges Analysis of the molecular pathways affected by loss of INI1/hSNF5 has provided insights for developing targeted therapies for RTs. The aggressive nature of RTs and their insensitivity to many potent chemotherapy regimens pose a challenge. The aggressiveness of RTs could be a consequence of deregulation of multiple cell cycle and growth control pathways. It is clear that INI1/hSNF5 regulates multiple downstream genes and pathways, which in turn influences its ability to mediate tumor suppression. Many of these genes/pathways repressed by INI1/hSNF5 are de-repressed in RTs and several of them are necessary for the genesis and survival of these tumors (Zhang etÂ€al. 2002; Tsikitis etÂ€al. 2005; Alarcon-Vargas etÂ€al. 2006; Morozov etÂ€al. 2007). Therefore, one can envision that a multi-targeted therapy may be required to effectively inhibit the growth of RTs. It is possible that simultaneously or sequentially targeting multiple INI1/hSNF5-downstream pathways is more effective in inhibiting RTs to ensure long term survival in those affected. While some of the current therapies have demonstrated promise, the mechanism of drug action or the overall effectiveness of these drugs against RTs is not completely understood. In cases of long-term survival, lack of understanding of molecular basis for these treatment strategies makes it hard to predict future outcomes in additional patients treated in the same way. In cases where the outcome is poor, the reasons for treatment failures could be multifold including development of drug resistance, suboptimal drug delivery to the tumor, constraints of the blood brain

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barrier, or drug related toxicities. None of these parameters have been studied in detail, partly due to the rarity of these tumors and lack of dedicated funding opportunities to study these tumors. Nevertheless, identification of the genetic basis of RTs with the discovery of INI1/hSNF5 as the critical tumor suppressor has provided a wealth of information to develop and investigate molecularly targeted therapies against these tumors. While preclinical studies have indicated promising leads, further clinical studies are needed to formulate these concepts into a therapeutic strategy that is both effective and that confers long lasting survival in children with RTs. Acknowledgmentsâ•… We apologize to all investigators whose work was not included in this article due to space limitation. Work in our laboratory is supported by grants from American Cancer Society, Children’s Tumor foundation, Mark Trauner faculty scholar and Irma T. Hirschl awards.

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Development of Targeted Therapies for Neurofibromatosis Type 1 (NF1) Related Tumors Brigitte Widemann

Introduction Neurofibromatosis Type 1 (NF1), previously referred to as von Recklinghausen disease, is a relatively common (1:2,500 to 1:3,000) autosomal dominant, progressive tumor predisposition syndrome characterized by manifestations in many organ systems including neurocutaneous findings and the propensity to develop tumors of the peripheral and central nervous system (Friedman 2002; Korf 2002; Ferner 2007). The natural history of NF1 is poorly understood, and for most NF1 related tumor manifestations the only standard treatment option is surgery (Korf 2001; Ferner etÂ€ al. 2007). Increasing knowledge of molecular and biologic pathways implied in the development of NF1 related tumors has resulted in the development of treatment trials with targeted agents (Packer etÂ€al. 2002). This chapter reviews NF1 related tumor manifestations, pathways implied in tumor development and progression, ongoing, and planned clinical trials for NF1 related tumors. Differences in the development of agents for NF1 related tumors and refractory cancers and resulting challenges toward the development of safe and effective therapies for NF1 related tumors are highlighted.

Genetics and Diagnosis of NF1 Neurofibromatosis type 1 is caused by a mutation in the NF1 tumor suppressor gene on chromosome 17q11.2 – 350Â€kb, 60 exons – (Friedman 2002; Ferner 2007). The gene product neurofibromin (2,818 amino acids) contains a domain with significant homology to RAS GTPase-activating proteins (GAP) and thus regulates

B. Widemannâ•›(*) Pharmacology and Experimental Therapeutics Section, National Cancer Institute, Pediatric Oncology Branch, 10 Center Drive, 10-CRC, Room 1-5750, MSC 1101, Bethesda, MD 20892, USA e-mail: [email protected] P.J. Houghton and R.J. Arceci (eds.), Molecularly Targeted Therapy for Childhood Cancer, DOI 10.1007/978-0-387-69062-9_16, © Springer Science+Business Media, LLC 2010

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RAS activity. RAS is a small GTPase and plays a central role in survival, proliferation, and differentiation by transducing responses to growth stimuli initiated in the cell surface to intracellular signaling molecules (Satoh and Kaziro 1992). RAS functions as a molecular switch in which it toggles between an inactive (GDP-bound) and active (GTP-bound) state. Neurofibromin accelerates RAS-GTP hydrolysis and thus functions as a potent negative regulator of RAS (Fig.Â€1). Lack of functional neurofibromin in NF1 therefore leads to dysregulated RAS and tumorigenesis (Cichowski and Jacks 2001). Neurofibromatosis type 1 has 100% penetrance, but features variable expressivity. A wide range of mutations have been described, and while mutation analysis of the NF1 gene allows identification of 95% of mutations to date, only two phenotype

Fig.Â€1â•… Schema of the NF1 pathway. The NF1 gene product neurofibromin functions as GTPase activating protein and thus facilitates turning RAS from the active GTP bound form to the inactive GDP bound form. Neurofibromin is dysfunctional in NF1 and thus results in increased activity of RAS

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genotype correlations have been described (Upadhyaya etÂ€al. 1998; Messiaen etÂ€al. 2000; Upadhyaya etÂ€al. 2007). The diagnosis of NF1 is thus based on clinical criteria including café-au-lait macules, axillary and inguinal freckling, presence of NF1 related tumors, a family history of NF1, and can typically be made by 6 years of age (NIH etÂ€al. 1988). NF1 is a progressive disorder, and manifestations in essentially every organ (skin, bone, cardio-vascular system, cognitive system) develop throughout life, which explains the need for a multidisciplinary approach in the care for NF1.

NF1 Related Tumor Manifestations Patients with NF1 have an increased risk of developing tumors of the central and peripheral nervous system including plexiform neurofibromas (PN) (25%), dermal neurofibromas (>99%), optic pathway gliomas (OPG) (15%), brain tumors (2 to 3%), malignant peripheral nerve sheath tumors (MPNST) (8 to 13%), juvenile myelomonocytic leukemia -JMML-, pheochromocytomas (2%), rhabdomyosarcomas (1.5 to 6%), glomus tumors, and benign hamartomas of the iris called Lisch nodules (>95%) (North 1997; Korf 2000, 2002; Evans etÂ€al. 2002). Highlighted below are tumor manifestations for which targeted clinical trials are ongoing or becoming available.

Dermal Neurofibromas The hallmark feature of NF1, typically develop in adolescence with continued development of new tumors throughout adulthood and during pregnancy (Huson etÂ€al. 1988; Huson etÂ€al. 1989). Surgical excision is sometimes required, but neurofibromas may grow back after surgery. No medication is available to control the growth of these tumors. While dermal neurofibromas rarely, if ever, progress to malignancy they impose a substantial cosmetic burden, which is exacerbated by the unpredictability of their growth and ultimate number.

Plexiform Neurofibromas These are benign nerve sheath tumors that grow along the length of nerves and involve multiple branches of a nerve (Korf 1999). These tumors are usually diagnosed early in life, but may develop throughout life. Morbidity from PN includes substantial disfigurement, compression of vital structures, progressive neurologic deficit, and often unremitting pain (Fig.Â€2). The only standard treatment for PN is surgery. However, given the location, infiltrative nature, high vascularity, and size of PN, complete surgical removal is usually not feasible, and up to 44% of tumors progress after the first surgery, most commonly in patients younger than 10 years

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Fig.Â€2â•… Progressive disfiguring plexiform neurofibroma in a young girl with NF1 demonstrating the need to develop effective medical interventions for young children

of age with head and neck tumors (Needle etÂ€al. 1997). There is no known effective medical treatment for patients with PN.

Malignant Peripheral Nerve Sheath Tumors Also called neurogenic sarcomas, malignant schwannomas, and neurofibrosarcomas, are soft tissue sarcomas which arise from a peripheral nerve or show nerve sheath differentiation and are associated with a high risk of local recurrence and hematogenous metastasis (Ferner and Gutmann 2002). They account for 10% of all soft tissue sarcomas, and half of these malignancies arise in patients with NF1 with a lifetime risk in NF1 of 8 to 13% (Evans etÂ€al. 2002). Early diagnosis of MPNSTs is crucial, as only complete surgical resection has been shown to be curative. However, the diagnosis of MPNSTs in NF1 is difficult to establish because clinical indicators of malignancy (mass and pain) may also be the features of preexisting benign PN from which most MPNSTs arise. For NF1 associated MPNSTs, younger age at diagnosis and decreased survival have been described (Evans etÂ€al. 2002; Ferner and Gutmann 2002; Carli etÂ€ al. 2005), and the largest retrospective analysis describes a lower response rate to chemotherapy in individuals with NF1 versus sporadic MPNST (Carli etÂ€ al. 2005). However, the role of chemotherapy in MPNST has not been assessed prospectively, and the response to standard chemotherapy agents used to treat pediatric and adult sarcomas is unknown and the objective of an ongoing trial of standard a sarcoma chemotherapy agents for high-grade unresectable MPNSTs.

Optic Pathway Gliomas These tumors develop in 15% of children with NF1 (King etÂ€al. 2003) and typically manifest clinically by an age of 6 years (Listernick etÂ€al. 1994). Only approximately 50% of children with NF1 related OPG will develop related signs or symptoms.

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Current recommendations are to treat NF1 associated OPG only if there is clear evidence of radiographic or ophthalmological (visual acuity, fields, color vision) progression (Listernick etÂ€al. 2007). Chemotherapy with vincristine and cisplatin is considered standard treatment for children with progressive disease, and targeted trials for refractory tumors are in development. Radiotherapy should be avoided because of the risk of malignancy and vascular problems, including moyamoya syndrome.

Juvenile Myelomonocytic Leukemia This is a rare myeloproliferative disorder of early childhood. Activating RAS mutations have been reported in 18 to 25% of children with JMML, 10 to 14% of children with JMML have a clinical diagnosis of NF1, and NF1 mutations are found in approximately 30% of patients with JMML. JMML cells are hypersensitive to GM-CSF, and GM-CSF stimulation is associated with increased levels of RASGTP in hematopoietic cell lines (Emanuel 2004). Chemotherapy followed by hematopoietic stem cell transplantation is the only treatment resulting in extended survival, but relapse rates are high ranging from 35 to 55% (Emanuel 2004).

Molecular Features of Tumorigenesis RAS Pathway The NF1 gene encodes neurofibromin, a GTPase-activating protein (GAP) for members of the p21RAS family, which negatively regulates RAS output by accelerating the conversion of active RAS-GTP to inactive RAS-GDP (DeClue etÂ€ al. 1991; DeClue etÂ€ al. 1992; Cichowski and Jacks 2001). Analysis of tumors from patients with NF1 has shown biochemical evidence of hyperactive RAS as well as frequent loss of the normal NF1 allele, consistent with its role as a tumor suppressor gene (Kluwe etÂ€al. 1999a; Kluwe etÂ€al. 1999b; Weiss etÂ€al. 1999; Serra etÂ€al. 2000; Sherman etÂ€ al. 2000; Perry etÂ€ al. 2002). RAS has three major isoforms (K-RAS, N-RAS, and H-RAS). Studies in NF−/− astrocytes demonstrated that activation of only K-RAS and not H-RAS accounted for the proliferative advantage in these cells, and that this could be corrected by a dominant inhibitory K-RAS (Dasgupta etÂ€ al. 2005a). Similarly, K-RAS was found to modulate the RAF/ mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase kinase (ERK) pathway and phosphatidylinositol 3¢-kinase (PI3K) pathway in wildtype NF+/− mast cells and genetic deletion of K-RAS reduced this gain of function (Khalaf etÂ€al. 2007). Introduction of the functional NF1-GAP-related domain could partially reverse the transformation of human NF1 tumor-derived Schwann cells, but was not sufficient to decrease the inÂ€ vitro angiogenic potential of these cells

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(Hiatt etÂ€al. 2001; Thomas etÂ€al. 2006). While NF1 likely serves functions outside of its role as RAS-GAP; for example, through regulation of cyclic adenosine monophosphate (Hegedus etÂ€al. 2007), its function as activator of RAS has most clearly been linked to tumorigenesis.

Cells of Tumor Origin and Tumor Environment Nearly all individuals with NF1 develop dermal neurofibromas, and some PN, which are composed of axons, Schwann cells, fibroblasts, perineurial cells, endothelial cells, and mast cells. Schwann cells are believed to be the primary pathogenic cell in neurofibromas because they show biallelic mutation of NF1 (Cichowski and Jacks 2001). Recent data support that tumors do not arise from neural crest stem cells, but from proliferation of fully differentiated nonmyelinated Schwann cells in a microenvironment with degeneration of normal nonmyelinated axon/Schwann cell relationships and mast cell infiltration (Joseph etÂ€al. 2008; Wu etÂ€al. 2008; Zheng etÂ€al. 2008) with the timing of loss of NF1 being critical in order for neurofibromas to develop (Wu etÂ€al. 2008). However, while loss of NF1 in the Schwann cell lineage was sufficient to generate tumors, complete NF1 mediated tumorigenesis required both a loss of NF1 in cells destined to become neoplastic as well as heterozygosity in nonneoplastic cell (Zhu etÂ€ al. 2002). For example, in inÂ€ vitro studies NF1 deficient Schwann cells were shown to be angiogenic and invasive and to secrete Kit ligand (Kim etÂ€ al. 1997; Yang etÂ€ al. 2003). This was shown to stimulate mast cell migration, and NF1+/− mast cells were shown to be hypermotile in response to Kit ligand and to secrete TGF-b (Yang etÂ€al. 2006). The addition of imatinib to these cultures blocked the activity of NF+/− mast cells on fibroblast proliferation, collagen remodeling, and fibroblast migration (Yang etÂ€al. 2006). These findings and studies in a genetically engineered NF1 mouse model (Zhu etÂ€al. 2002) indicate an important role of tumor microenvironment in neurofibroma development.

Angiogenesis Angiogenesis also contributes to tumor formation in NF1. RAS mutations can upregulate VEGF expression (Rak etÂ€al. 1995; Kranenburg etÂ€al. 2004) and vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (FGF) are also highly expressed in neurofibromas from patients with NF1 at the mRNA and protein level (Kawachi etÂ€al. 2003). Furthermore, VEGF expression and tumor vascularization significantly increased in MPNSTs (Angelov etÂ€ al. 1999). Use of a specific small molecular inhibitor of VEGF-receptor 2 (VEGFR2) in a mouse explant model of neurogenic sarcomas showed a reduction in tumor growth due to

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decreased tumor angiogenesis with subsequent reduction in tumor cell proliferation and an increase in apoptosis (Angelov etÂ€al. 1999). NF1 deficient Schwann cells induced FGF, platelet derived growth factor (PDGF), and midkine (MK). Midkine was found to stimulate human MPNST and fibroblastoid cells of neurofibromas (Mashour etÂ€ al. 2001). Subsequent studies demonstrated that MK and stem cell factor, but not epidermal growth factor (EGF) were substantially increased in serum of NF1 patients compared to healthy controls (Mashour etÂ€al. 2004).

Growth Factors and Growth Factor Receptors PNs have increased expression of growth factors and growth factor receptors, including PDGF receptors and VEGF (Kim etÂ€al. 1997; DeClue etÂ€al. 2000; Ingram etÂ€al. 2001; Mashour etÂ€al. 2001; Kawachi etÂ€al. 2003). In addition, epidermal growth factor receptor (EGFR) has been identified as an upstream activator of RAS in NF1. EGFR is not normally expressed by normal Schwann cells, but EGFR expression was demonstrated in benign neurofibromas, in MPNST cell lines established from NF1 patients (DeClue etÂ€al. 2000), and in tumor cell lines derived from NF1:TP53 mice. Cell lines responded to EGF by activation of the downstream signaling pathways MAPK/ERK, and phosphatidylinositol 3¢-kinase (PI3k)/AKT. The growth of these cell lines could be blocked by an EGFR antagonist (Li etÂ€al. 2002). The potential role of EGFR in peripheral nerve tumor formation was subsequently demonstrated in transgenic mouse Schwann cells expressing EGFR, which elicited features of neurofibromas. In addition, genetic reduction of EGFR in NF1+/− p53+/− mice that develop sarcomas significantly improved survival (Ling etÂ€al. 2005). Agents blocking Raf-MAPK-ERK and PI3K-AKT pathways in NF1 have been shown to block proliferation (Lau etÂ€al. 2000; Li etÂ€al. 2002; Mattingly etÂ€al. 2006a, b; Khalaf etÂ€al. 2007), and therefore may be rational targets in NF1 in addition to agents blocking upstream activation of RAS.

Mammalian Target of Rapamcyin (mTOR) Recent studies have demonstrated that NF1 regulates mTOR pathway activation. NF1 loss in mouse embryonic fibroblasts and primary mouse astrocytes resulted in RAS- and PI3 kinase/AKT dependent mTOR pathway activation, which could be inhibited with rapamycin (sirolimus) (Dasgupta etÂ€ al. 2005b; Johannessen etÂ€ al. 2005). Increased proliferation associated with loss of neurofibromin expression in human MPNST cell lines was also dramatically reduced by the treatment with sirolimus (Johannessen etÂ€al. 2005). Using a genetic mouse model of NF1-deficient malignant peripheral nerve sheath tumor – (MPNST) development, sirolimus completely inhibited the growth of these tumors inÂ€vivo (Johannessen etÂ€al. 2008). In addition, sirolimus treatment of optic gliomas developing in a genetically-engineered

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NF1 mouse model resulted in attenuated mTOR signaling inÂ€ vitro and inÂ€ vivo (Sandsmark etÂ€ al. 2007; Hegedus etÂ€ al. 2008). Similarly, the mTOR inhibitor RAD001 (everolimus) decreased growth in MPNST cell lines, and prevented the growth of subcutaneously implanted MPNST in mice (Johansson etÂ€al. 2008). A negative feedback loop between mTOR and Akt was not found in the NF1 optic glioma and MPNST preclinical models after the treatment with sirolimus (Hegedus etÂ€al. 2008; Johannessen etÂ€al. 2008).

Molecular Features for MPNSTs Genetic and molecular alterations in addition to NF1 loss and changes described above contribute to the progression of PN to malignancy. Immunohistochemical analysis and molecular studies (Kourea etÂ€ al. 1999; DeClue etÂ€ al. 2000; Li etÂ€ al. 2002; Perry etÂ€al. 2002; Zhou etÂ€al. 2003) have implicated TP53, EGFR p16INK4A, and p27 as potential contributors to malignant transformation in peripheral nerve sheath tumors. Further, highlighting the unique pathogenesis of this tumor type, both NF1 deletions and homozygous p16INK4A deletions appear to be relatively restricted to MPNSTs in comparison to other spindle cell sarcomas with overlapping morphologic features (Perry etÂ€al. 2002). Gene expression profiling, immunohistochemistry, and/or Western blot analysis of human MPNSTs showed expression/ overexpression of EGFR of human MPNSTs (Watson etÂ€al. 2004), PDGFRa and b (Badache and De Vries 1998; Holtkamp etÂ€al. 2006), and C-Kit (Dang etÂ€al. 2005; Holtkamp etÂ€ al. 2006) matrix metalloprotetinase 13 (MMP13) (Holtkamp etÂ€ al. 2007). In order to identify events contributing to malignant transformation a number of studies were performed comparing human Schwann cells and MPNST (Miller etÂ€al. 2006), and PN and MPNST (Skotheim etÂ€al. 2003; Holtkamp etÂ€al. 2004; Levy etÂ€al. 2004; Miller etÂ€al. 2006), and identified that MPNSTs differentially expressed neural crest stem cell markers SOX9 and TWIST1 (Miller etÂ€ al. 2006), genes involved in cell proliferation (MKI67, TOP2A, CCNE2), apoptosis (BIRC5/Survivin, TP73), extracellular matrix remodeling (MMP13, MMP9), genes involved in the RAS signaling pathway (RASF2, HMMR/RHAMM) and the Hedgehog-Gli signaling pathway (DHH, PTCH2). In addition, high resolution array comparative genomic hybridization was used to compare, dermal neurofibromas, PN, and MPNST and identified amplification of some genes, including PDGFRa, MET, TP73, and HGF, deletions in NF1, MMP13, p16INK4A, and TP53 demonstrating the potential of array CGH in identifying markers for MPNST (Mantripragada etÂ€al. 2008). Analysis for somatic mutations in MPNST from NF1 patients showed loss of heterozygosity (LOH) across the TP53 region and TP53 mutation in 14 of 20 tumors (Upadhyaya etÂ€al. 2008). However, while NF1 associated MPNST appears to have worse outcome compared to sporadic tumors, gene expression profiling of NF1 associated (nâ•›=â•›25) and sporadic (nâ•›=â•›17) MPNSTs did not identify a molecular signature that could reliably distinguish between both groups (Watson etÂ€al. 2004).

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Rationale of Pathways A number of targeted agents under development for the treatment of adults and children with refractory cancers have a good scientific rationale to be considered for development in NF1 related tumors. These include inhibitors of RAS, angiogenesis, growth factors, mast cell proliferation, and mTOR. Farnesyltransferase inhibitors (FTI) were among the first targeted agents to be evaluated for NF1 related tumors. They were developed to prevent posttranslational RAS processing and transduction of proliferative signals, which is required for the activity of mutant and wildtype RAS, and inhibition of RAS activity was the rationale for the evaluation of FTIs in cancer and NF1 (Rowinsky etÂ€ al. 1999). However, farnesylation is required for the activity of a number of additional proteins, including Rho-B, Rac, membrane lamins, and centromeric proteins that interact with microtubules to promote mitosis, which may contribute to the anti-tumor effects of FTIs in clinical trials (Lancet and Karp 2003; Santos etÂ€al. 2004). In addition, N-, and K-RAS can undergo an alternate lipid modification by geranyl-geranylation and thus overcome the effect of FTI (Rowinsky etÂ€al. 1999). This is critical in light of recent data pointing toward K-ras as a key mediator of RAS activity in NF1 tumors (Dasgupta etÂ€al. 2005a; Khalaf etÂ€al. 2007). Thus, additional strategies, such as novel RAS inhibitors blocking all RAS isoforms (Barkan etÂ€al. 2006) or agents blocking downstream pathways of RAS, are being considered for the development in NF1.

Current Strategies: Development of Targeted Treatments for NF1 Tumors Completed, ongoing, and planned clinical trials with targeted agents for NF1 related tumors are summarized in TableÂ€1. Most of these focused on PN and used oral agents, which were administered on a chronic schedule. Initial trials used similar trial designs and endpoints as used in trials for refractory cancers. However, differences between refractory cancers and NF1 required different approaches toward drug development for NF1 related tumors (TableÂ€ 2). This includes the requirement for more prolonged preclinical toxicology studies, including reproduction toxicology for NF1 trials given the near normal life expectancy of individuals with NF1. There are also important differences between individuals with NF1 related PN and refractory cancers, which is highlighted by the development of the FTI tipifarnib for NF1. In the phase I trial of tipifarnib, which was performed simultaneously in children with refractory cancers or NF1 related PN, the median age at trial entry was 7 years for NF1 (range 5 to 16 years) compared to 15 years (range 5 to 18 years) for refractory cancers (Widemann etÂ€al. 2006). In addition, the treatment duration for children with NF1 was longer (median cycle number 10, range 1 to 32 cycles) compared to children with refractory cancers, who were removed from treatment with tipifarnib after a median cycle number of 1 (range 1 to 4) for

FTase

Fibroblast

Immune modulation Angiogenesis

Tipifarnib

Pirfenidone

Peginterferon alfa-2b

Oralâ•›×â•›21d q 28d Oral continuous

II

Oral continuous SC q week

SC q week

II

II

I

Oral continuous

I

II

Oralâ•›×â•›21 d q 28d

I

Inoperable, symptomatic, or progressive PN

Progressive PN 3–21 years Inoperable PN 1.6–21 years

Inoperable PN 3–21 years

Progressive PN 3–25 years Inoperable or sympt. PN Adults

Inoperable PN 3–21 years

Who-3D MRI

3D MRI Clinical

TTP Response

3D MRI

3D MRI

3D MRI

3D MRI

WHO

MTD

TTP

Toxicity PK

Response

TTP

Toxicity PK, PD

TableÂ€1â•… Completed, ongoing, and planned clinical trials with targeted agents for NF1 related tumors Mechanism of Response Drug action/target Phase Schedule Eligibility Endpoint evaluation Plexiform neurofibroma Thalidomide Angiogenesis I Oral Progressive PN Toxicity WHO continuous >5 years

Ongoing, enrollment complete (nâ•›=â•›36) MTD 1Â€mcg/kg SC weekly Several clinical and 3D MRI responses Ongoing

Optimal dose 500Â€mg/ m2/d Median cy # at MTD 15#

Gupta etÂ€al. (2003)

Max dose given 200Â€mg/day Minor response and symptomatic improvement in few pts. MTD 200Â€mg/m2/d po BID ×21 d q28d Med cy #10 Ongoing, enrollment complete (nâ•›=â•›62) Volume decrease in 4 patients

–

Jakacki et al. (2008)

BabovicVuksanovic etÂ€al. (2006) BabovicVuksanovic etÂ€al. (2007) –

Widemann etÂ€al. (2006) –

Reference

Result

340 B. Widemann

C-Raf, B-Raf, VEGFR2, C-Kit, PDGFRb

AZD2171

Sorafenib

FTase

C-Raf, B-Raf, VEGFR2, C-Kit, PDGFRb C-Kit, PDGFRb, VEGFR1

Ranibizumab

Angiogenesis VEGF

Dermal neurofibromas

JMML Tipifarnib

Imatinib

Sorafenib

EGFR

VEGFR2

Sirolimus

MPNST Erlotinib

C-Kit, PDGFRb, VEGFR1 mTOR

Imatinib

Pilot

II window

Intratumor injection

Oralâ•›×â•›21 d q 28d, 2 cy.

Oral continuous

II

II

Oral continuous Oral continuous

Oral continuous

I

II

Oral continuous

Oral continuous

Oral continuous

II

II

II

Response

>18 years dermal neurofibromas

Newly diagnosed JMML

Tumor volume VEGF signaling

Response

Response

Response

Refractory adults

Refractory children and adults

Response

Refractory adults

TTP Inoperable or Response progressive PN children and adults Response Inoperable or progressive PN adults Inoperable PN Toxicity, 3–21 years PK, PD, DEMRI

Children and adults

Soon to open

3D MRI

WBC, Liver size

Albritton etÂ€al. (2006) Maki et al. (2009)

–

–

–

–

Ongoing

–

Response in WBC and Castleberry etÂ€al. organomegaly in (2005) 58% of pts.

Ongoing

Inactive TTP 2 cycles Inactive

Ongoing

3 D MRI

RECIST

Ongoing

Ongoing

3D MRI

RECIST

Development of Targeted Therapies for Neurofibromatosis Type 1 (NF1) Related Tumors 341

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TableÂ€2â•… Differences in clinical drug development for refractory cancers and NF1 Endpoint Type of study Objective Cancer NF1 Preclinical Safe start dose Toxicology in rodent and Requirement for chronic toxicity and nonrodent species reproduction toxicity studies for human trial Chronic dosing requires redefining Toxicity during cycle #1 Phase I MTD DLT and MTD Pharmaco- Cumulative toxicity not Monitoring for cumulative toxicity assessable kinetics Phase II

Activity

Response Time to progression

Phase III

Efficacy

Survival, QOL

Response may be unrealistic with targeted agents Progression difficult to measure with solid tumor response criteria Unknown natural history requires control group Near normal survival in NF1QOL

MTD, maximum tolerated dose; DLT, dose-limiting toxicity; QOL, quality of life; PK, pharmacokinetics

disease progression. Cumulative toxicity could thus not be assessed in children with refractory cancers, but could be assessed in NF1, which was critical for the development of a subsequent phase II trial. Standard two-dimensional measurements (WHO) to assess response (Miller etÂ€al. 1981) were inadequate to monitor changes in NF1 related PN. For subsequent clinical trials, a method of automated volumetric MRI analysis of PN was developed to sensitively and reproducibly monitor smaller changes in PN size (Solomon etÂ€al. 2004). This method is now used in most ongoing PN trials and defines disease progression based on much smaller changes than standard solid tumor response criteria, which allows to limit the time period exposed to potentially inactive or toxic agents (Tableâ•›3). For the phase II trial with tipifarnib time to progression (TTP) rather than response was chosen as primary trial endpoint, as the expected pharmacologic effect of a cytostatic drug, such as tipifarnib, would be a slowing of PN growth, rather than a measurable decrease in PN size. The absence of prior data about the time to disease progression of PN required a concurrent control population. Therefore, the phase II trial was designed as a randomized, placebo-controlled, double-blinded, crossover phase II trial with the primary objective of determining whether tipifarnib increases time to disease progression in patients with progressive PN. Subsequent phase II trials for PN (pirfenidone, pegintron, sirolimus) will use the placebo arm of the tipifarnib phase II trial for PN as historical control to define the effect of the agent under study on time to disease progression. Eligibility criteria for these trials are identical to ensure that the historical control from the tipifarnib arm is valid. Other endpoints incorporated in clinical trials include pharmacodynamic effects, quality of life, and pain. Targeted treatment trials have also become available for MPNSTs, and while the agent studied did not result in responses, the feasibility of performing histology specific trials within an acceptable time period has been confirmed (Albritton etÂ€al. 2006; D’Adamo etÂ€al. 2007).

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TableÂ€3â•… Equivalent percent increase in diameter (RECIST), product (WHO), and volume for spherical lesions. Bold typeface highlights the definition of progression by RECIST, WHO, and the ongoing tipifarnib phase II trial for NF1 PN Percent change in tumor size RECIST diameter (1D) WHO product (2D) Tipfarnib phase II volume (3D) 6% 12% 20%

13% 25% 44%

20% 40% 73%

Future Directions and Challenges Preclinical Evaluation of Agents Until recently, the evaluation of agents in preclinical models of NF1 was difficult due to the lack of models recapitulating the disease and the number of cell types involved in NF1 related tumors. However, inÂ€vitro (Ingram etÂ€al. 2000; Yang etÂ€al. 2003; Yang etÂ€al. 2006), xenograft (Johannessen etÂ€al. 2005; Johansson etÂ€al. 2008), and transgenic mouse models (Cichowski etÂ€al. 1999; Reilly etÂ€al. 2000; McClatchey and Cichowski 2001; Hegedus etÂ€al. 2008) of NF1 have become available, and are beginning to be validated for their value in predicting response in NF1 (Hegedus etÂ€al. 2008). These studies are resource intensive. Recently, the Children’s Tumor Foundation (former National Neurofibromatosis Foundation) proceeded to fund a preclinical NF consortium with the goal to accelerate the identification of effective therapies for NF1. Candidate drugs will be evaluated in multiple tumor models of NF1 with the goal to move the most promising agents to clinical trials similar to the preclinical pediatric testing program for pediatric cancers (Houghton etÂ€al. 2002; Houghton etÂ€al. 2008). This work will lead to more rational selection of agents for clinical trials. In addition, the hope is that these models will help identify the role of specific cancer genes, perform target validation, define steps in tumorigenesis, evaluate impact of the tumor environment, and identify tumor specific biomarkers and gene modifiers (Gutmann and Giovannini 2002).

Infrastructure for Clinical Trials Initial clinical trials were performed through collaboration of limited institutions with funding provided by the Department of Defense (DoD). In order to develop an effective trials program, the infrastructure to conduct clinical trials in sequence is required. Recently, the DoD initiated funding of a NF Consortium, with the goal to allow for timely development of clinical trials in order to develop more effective therapies. This Consortium has nine participating site nationwide, and just initiated the first clinical trial of sirolimus for PN.

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Development of Agents for Young Children Longitudinal volumetric MRI analysis of PN demonstrated that PNs grow more rapidly in younger children compared to older children (Dombi etÂ€ al. 2007). This raises the potential need for age stratification in clinical trials but also emphasizing the need to develop agents for young children. This is associated with multiple challenges. The lower age limit for most currently ongoing clinical trials for PN is 3 years due to the concern of toxicity in developing organs, particularly the nervous system. However, many young children may already have substantial PN burden at that time they become 3 years old and eligible for clinical trials (Fig.Â€1). Availability of age appropriate drug formulations and of trial designs, which provide for careful monitoring of toxicities in children will be critical to the conduct of trials in this population. This is exemplified by the development of angiogenesis inhibitors, which may have unique toxicities in children compared to adults. Inhibition of angiogenesis in growing but not aged animals resulted in the growth retardation and expanded growth plates (Gerber etÂ€al. 1999). While this was largely reversible, there is concern for this toxicity, particularly in young children who have near normal life expectancy as in NF1. This toxicity cannot be easily monitored in phase I trials for refractory childhood cancers, as patients are older and remain on trial for short time periods (Bender etÂ€ al. 2008). In a soon-to-open phase in trial of sorafenib for children with NF1 patients will be carefully monitored over multiple cycles for the development of unique toxicities including bony toxicity and growth plate changes using a newly developed method of automated volumetric MRI analysis of the growth plate. Observations from this and other future trial may thus not only inform for the drug development in NF1 but also for the drug development in childhood cancers.

Evaluation of Effect of Agents on Other NF1 Manifestations NF1 is a multisystem disorder and its manifestations in many organ systems may be, at least partially, the result of abnormal RAS function. While the primary goal in clinical trials directed at NF1 tumors is to monitor the effect on the tumor, agents under study may reverse deficits resulting from excessive RAS function in other organ systems. Thus, careful consideration should be given for the evaluation of other organ systems on clinical trials for NF1 related tumors.

Target Validation Access to tumor biopsies for target validation will be limited in NF1 related tumors given that many trials enroll children, and that biopsies frequently cannot be easily performed. Development of noninvasive assays, which allow monitoring of effects on desired targets should be an important goal for future NF1 trials.

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Combination Therapy Finally, progress in the treatment of childhood cancers has been made by combining chemotherapy agents with different mechanisms of action. Given that multiple pathways are implied in tumor development in NF1, the evaluation of combined therapies may be required for successful treatment, which poses another challenge for future trials.

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Molecular Therapy for Neuroblastoma Yaël P. Mossé and John M. Maris

Introduction Despite the generally held notion that embryonal cancers are simpler from a genetic standpoint than adult neoplasms, these cancers show multiple chromosomal rearrangements, suggesting a complicated series of acquired alterations during malignant evolution. This is best exemplified in neuroblastoma, an enigmatic and highly heterogeneous pediatric neoplasm that accounts for 15% of pediatric cancer mortality and remains an important clinical problem in which tumor genomics correlate with disease phenotype (George etÂ€al. 2007). Neuroblastoma represents a spectrum of diseases, and distinct patient subsets exist based on tumor biological features (Maris 2005). Low-risk neuroblastomas are likely characterized by mitotic dysfunction leading to a hyperdiploid modal karyotype with whole chromosome gains, but few, if any, structural cytogenetic rearrangements. These tumors may regress spontaneously, differentiate, or respond completely to modest doses of chemotherapy. In contrast, aggressive neuroblastomas are characterized by complex segmental chromosomal aberrations. Two main categories of unfavorable neuroblastomas exist, with a well-defined subset (approximately 40%) defined by deletion of the short arm of chromosome 1 and MYCN amplification. On the other hand, the majority of aggressive neuroblastomas show multiple other chromosomal rearrangements, most frequently involving chromosome 11q (Attiyeh etÂ€al. 2005). All high-risk patients are treated with dose-intensive induction chemotherapy and surgery, followed by myeloablative therapy with stem cell rescue, local radiation therapy and biological response modification using retinoids. Despite this aggressive approach, survival on recent high-risk studies remains <35% (Berthold etÂ€al. 2005; Matthay etÂ€al. 1999) with only modest improvement over the past few

Y.P. Mossé (*) Division of Oncology, Department of Pediatrics, Children’s Hospital of Philadelphia, University of Pennsylvania, 3615 Civic Center Blvd., ARC 907C, Philadelphia, PA, USA e-mail: [email protected]

P.J. Houghton and R.J. Arceci (eds.), Molecularly Targeted Therapy for Childhood Cancer, DOI 10.1007/978-0-387-69062-9_17, © Springer Science+Business Media, LLC 2010

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1990-1994 (N=575) 1995-1999 (N=345) 2000-2004 (N=856)

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Fig.Â€ 1â•… Overall survival probability in 5-year intervals for all high-risk neuroblastoma patients enrolled in the United States-based pediatric cooperative group clinical trials from 1990 to 2004. While the most recent Kaplan-Meier survival curve is significantly different from the others (Pâ•›<â•›0.001), the clinical significance of this improved outcome is debatable, especially with longer follow-up. Data courtesy of Dr. Wendy London, Children’s Oncology Group

years (Fig.Â€1). It is clear that major improvements in patient outcome will unlikely be realized by simply adding cytotoxic therapy to the already dose-intensive regimens prescribed for children with high-risk neuroblastoma. This chapter explores the potential of how we can rationally exploit known tumor-specific alterations toward current and future treatment strategies.

Risk Classification Risk assessment depends upon a number of clinical and biologic features. The Children’s Oncology Group currently stratifies patients into low-, intermediate-, or high-risk categories based upon the well-defined prognostic factors of age at diagnosis, disease stage, tumor histopathologic classification, DNA index, and MYCN amplification status. Although this system is useful, there are almost certainly misclassifications resulting in patients who are either over- or undertreated. Amplification of the MYCN oncogene is a strong prognostic factor, but over 60% of patients with high-risk neuroblastoma do not have MYCN amplification present in the diagnostic specimen. There is also very likely a subset of children currently classified as high-risk who do not require the extreme dose-intensive therapeutic approach currently prescribed. Because approaches to risk stratification have varied greatly throughout the world, efforts have recently been made to develop a consensus approach that will permit comparison of outcomes for patients with neuroblastoma

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treated in centers around the globe. In an effort to develop an International Neuroblastoma Risk Group (INRG) classification system, a working group, representing the major pediatric cooperative groups around the world, met in 2005 to review data collected on 11,054 patients treated in Europe, Japan, North America, and Australia between 1974 and 2002. Considerable progress has been made in the past decade toward understanding human neuroblastoma at a cellular and molecular level. Many studies clearly indicate that somatically acquired chromosomal aberrations in diagnostic neuroblastomas are intricately related to the clinical phenotype, and suggest that tumor genomics will provide a much more sensitive and specific indication of clinical phenotype and will greatly refine, or even replace, our current risk classification system.

Current Approach to Therapy Locoregional Tumors The majority of localized neuroblastomas have favorable biological features and most are successfully treated with surgery alone (Alvarado etÂ€ al. 2000; Berthold etÂ€al. 1982; Evans etÂ€al. 1996; Kushner etÂ€al. 1996b; Matthay etÂ€al. 1989). Studies suggest that a subset of localized tumors will spontaneously regress, and these patients can be safely observed without any treatment (Nishihira etÂ€al. 2000; Oue etÂ€al. 2005; Suita etÂ€al. 1996; Yamamoto etÂ€al. 1998). Local recurrences can typically be managed surgically. Metastatic recurrences are rare and often treated successfully with chemotherapy. The treatment of patients with localized tumors with unfavorable biological features, particularly MYCN amplification, remains controversial. Although these children have significantly worse outcome than patients with localized disease that lacks MYCN amplification, a subset may achieve longterm remission following surgery alone if a gross total resection is achieved (Alvarado etÂ€al. 2000; Cohn etÂ€al. 1995; Perez etÂ€al. 2000). These rare cases require continued prospective evaluation to clarify optimal management. The management of more invasive locoregional tumors (INSS stage 3) also remains controversial (Matthay etÂ€al. 1998b). Traditionally, patients with favorable biology INSS stage 3 tumors have received moderately intensive chemotherapy with the goal of facilitating subsequent surgical resection. Given the favorable outcome for these children, most investigators avoid the use of radical surgery or radiotherapy. In fact, there is a gradual movement toward reducing adjuvant therapy in these cases despite the presence of gross residual tumor (Kushner etÂ€al. 1996a), although these approaches require prospective validation. The current COG intermediate-risk clinical trial utilizes additional molecular genetic features (1p36 and 11q23 allelic status) and a response-based algorithm to attempt further reduction in chemotherapy exposure to these often very young children. In contrast, for invasive locoregional tumors with unfavorable biological features, intensive multimodality therapy is often required to achieve cure.

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Metastatic Tumors Treatment of older children with widely disseminated neuroblastoma (stage 4) remains one of the greatest challenges for pediatric oncologists. It is clear that very young children with metastatic disease frequently have a much less malignant disease, and again this is largely predictable by tumor biology (Schmidt etÂ€al. 2000, 2005). In recent years, attempts have been made to improve outcomes in high-risk patients (generally children older than 1 year of age at diagnosis with metastatic disease) by delivering intensive induction therapy. Commonly used agents include cisplatin, etoposide, doxorubicin, cyclophosphamide, and vincristine (Cheung and Cheung 2001). The combination of topotecan and cyclophosphamide has been used in the relapse setting for more than a decade, and a recent pilot study has demonstrated the feasibility of integrating this combination into an aggressive induction regimen. During induction therapy, stem cells are harvested in preparation for the consolidation phase of therapy. The goal of consolidation is to eliminate any remaining tumor, usually with myeloablative cytotoxic agents and stem cell rescue. Delayed surgical resection of the primary tumor and external beam radiotherapy to the primary and major metastatic sites are provided. The concept of eliminating resistant tumor clones with supralethal chemotherapy has been studied in neuroblastoma since the early 1980s, and the results of a randomized Children’s Cancer Group (CCG) Phase III cooperative group study demonstrated improved event-free survival with autologous transplantation (Matthay etÂ€ al. 1999). An intent-to-treat analysis of a randomized clinical trial of high-risk neuroblastoma patients conducted by the German Society of Pediatric Oncology and Hematology also demonstrated an improvement in 3-year event-free survival with myeloablative therapy and autologous stem cell rescue compared to maintenance chemotherapy (Berthold etÂ€ al. 2005). Similar to the CCG study, however, overall survival rates were not statistically different. Prolongation of the disease free interval is clinically important, but clearly much remains to be done to improve overall survival for high-risk patients. The COG now has an active Phase III study with a topotecan-containing induction and a randomization between single versus tandem myeloablative peripheral blood transplantation. As relapse is a frequent occurrence after autologous transplantation, biological therapy to treat persistent minimal residual disease has been added to current treatment regimens. Several novel agents specifically targeted to the unique biology of neuroblastoma may be effective in eliminating minimal residual disease. The retinoids are a class of compounds known to induce terminal differentiation of neuroblastoma cells inÂ€ vitro (Sidell 1982). The use of 13-cis-retinoic acid in the post-transplant setting was tested in a randomized Phase III trial conducted by the CCG. The cohort of patients assigned to receive post-transplant therapy with 13-cis-retinoic acid had a significantly improved event free survival and toxicity was acceptable (Matthay etÂ€al. 1999). Thus, retinoid-based biotherapy in the posttransplant setting is now widely used. Other retinoids such as 9-cis-retinoic acid, all trans-retinoic acid and/or fenretinide may also have activity for minimal residual

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disease in high-risk neuroblastoma patients and warrant further study. There has been extensive interest in monoclonal antibody-based therapy to clear minimal residual disease. Both murine and chimeric anti-GD2 agents have shown activity in refractory neuroblastoma, and the ch14.18 monoclonal antibody is currently being studied in a randomized Phase III trial in the COG in conjunction with 13-cisretinoic acid and cytokines (Berthold etÂ€al. 2005; Kushner etÂ€al. 2001; Ozkaynak etÂ€al. 2000).

Strategies to Identify Molecular Targets While further intensification of therapy as proposed in the next COG high-risk study (tandem myeloablative consolidation) may result in incremental improvement, it can be reasonably argued that no substantive advances in cure rates will be achieved until we can integrate radically new treatment strategies based on the fundamental molecular alterations present in neuroblastoma. A major effort has been mounted toward utilization of genomic technology to discover neuroblastomaspecific therapeutic targets. Strategies such as the neuroblastoma TARGET (Therapeutically Applicable Research to Generate Effective Treatments) initiative have been funded to perform a comprehensive assessment of the neuroblastoma genome designed to identify the critical genetic alterations and to leverage this information to develop targeted therapies. We anticipate that the combination of genomic (including noncoding RNAs), transcriptomic, and epigenomic data from carefully designed experiments will be synergistic in terms of our understanding of the biological basis of neuroblastoma, and that integration of these datasets will lead to the identification of rational therapeutic targets. We are also taking a global approach that takes into account that many oncogenic events will be silent at the DNA and RNA level. It is likely that overexpressed and constitutively active proteins resulting from activating mutations may be ideal therapeutic targets, and that high-throughput transient siRNA knockdown screens of druggable proteins (i.e., protein kinases) in representative neuroblastoma cell lines will enable us to identify the proteins essential for cellular survival. Finally, there is the pediatric preclinical testing program (PPTP), which was designed to evaluate the potential efficacy of new drugs in a manner that will allow direct translation to the clinic.

New Approaches to High Risk Disease While there are highly effective salvage options for patients with low- and intermediate-risk disease with local relapses, refractory high-risk disease remains a significant clinical challenge with no known curative approach. Over the past several years, an expanding portfolio of novel therapeutic agents with efficacy in the relapsed setting have been developed (TableÂ€1) and several are highlighted in this section.

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TableÂ€1â•… Investigational agents in early phase clinical development for neuroblastoma Mechanism of action/target Examples Reference Tubulin binding agents ABT-751 Fox etÂ€al. (2006) Proteosome inhibition Bortezomib Brignole etÂ€al. (2006), Houghton etÂ€al. (2007) VEGF inhibition Bevacizumab Dickson etÂ€al. (2007), Maris etÂ€al. (2008) AZD2171 VEGF TRAP Glutathione synthetase inhibitor Buthionine Anderson and Reynolds (2002) sulfoximine Trk tyrosine kinase inhibitor Lestaurtinib Evans etÂ€al. (1999), Marshall etÂ€al. (2005) SRC tyrosine kinase inhibitor Dasatinib Shah etÂ€al. (2004) Demethylation Decitabine Teitz etÂ€al. (2000) Histone deacetylase inhibitor Depsipeptide Marks etÂ€al. (2001), Coffey etÂ€al. (2001) Vorinostat SAHA BCL2 antisense Genasense Banerjee (2001) Histone deacetylase inhibitor Vorinostat Keshelava etÂ€al. (2007), Yang etÂ€al. (2007) EGFR tyrosine kinase inhibition Iressa Ho etÂ€al. (2005) Heat shock protein modulation 17-AAG Bagatell etÂ€al. (2007) MYCN inhibition Bisphosphonate Zoledronic acid Sohara etÂ€al. (2003) Mitotic spindle inhibitors Zhou etÂ€al. (1998) Centrosome inhibition Slack etÂ€al. (2007) 131 Norepinephrine receptor/targeted I-MIBG Matthay etÂ€al. (2006), (2007) radiotherapeutic Retinoids Fenretinide Maurer etÂ€al. (2000) Ceramide modulator Safingol Maurer etÂ€al. (1999), Reynolds etÂ€al. (2004) Anti-GD2 3F8 Berthold etÂ€al. (2005), Kushner etÂ€al. (2001), Ozkaynak etÂ€al. 14.18 (2000) ch14.18 Anti-anti-GD2 (anti-id antibody) mAb1A7 Yu etÂ€al. (2004) Humanized anti-GD2 and IL2 fusion hu14.18-IL2 Osenga etÂ€al. (2006) protein Immune response enhancer/iC3b Beta-d-glucan Cheung and Modak (2002) leukocyte receptor Immune modulation IL12 Lode etÂ€al. (1999), Siapati etÂ€al. (2003) Anti-IGF-I receptor monoclonal Ab IMC-A12 Miller and Yee (2005)

Cytotoxic Agents The topoisomerase 1 inhibitors, topotecan and irinotecan, are often used early in the relapse setting because of their proven efficacy and low toxicity profile (Langler etÂ€al. 2002; Saylors etÂ€al. 2001; Vassal etÂ€al. 2003; Wagner etÂ€al. 2004). Although

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both have efficacy as single agents, a recent randomized trial showed that topotecan combined with low dose cyclophosphamide was superior to topotecan alone (CRâ•›+â•›PR 31% vs. 19%) (Frantz etÂ€al. 2004). Irinotecan combined with temozolomide has been shown to be tolerable in the Phase I setting (Wagner etÂ€al. 2004), and the activity is currently being tested in a COG Phase II study. The most common toxicity related to Irinotecan is diarrhea, which can potentially be prevented with the concomitant use of oral antibiotics (Alimonti etÂ€al. 2003). The combination of irinotecan and topotecan has been studied in the Phase I setting, but unacceptable toxicity has limited further development (Rodriguez-Galindo etÂ€al. 2006). A promising novel cytotoxic agent is ABT-751, an oral tubulin-binding agent that is not a classic multidrug resistance pump substrate. Early clinical trials suggest an activity against refractory neuroblastoma (Cho etÂ€al. 2004), and ABT-751 is being studied in a COG Phase II trial.

Tyrosine Kinase Inhibitors Malignant transformation of neuroblasts may result in part from a failure to respond to normal differentiation signals. The factors responsible for regulating normal differentiation in the sympathetic nervous system are not completely understood, but they at least in part involve the neurotrophin receptor pathways (Brodeur 2003). Neurotrophins critical to these pathways include nerve growth factor (NGF) (Azar etÂ€al. 1990; Baker etÂ€al. 1989), as well as brain-derived neurotrophin growth factor (BDNF), neurotropin-3 (NT-3), and neurotrophin-4 (NT-4) (Brodeur etÂ€al. 1996). To date, three high affinity receptor tyrosine kinases for this family of Â�neurotrophins have identified. The genes TrkA, TrkB, and TrkC encode the primary receptors for NGF, BDNF, and NT-3, respectively (Brodeur etÂ€al. 1996). The primary receptor for NT-4 is not known, but it appears to function through TrkB. The expression pattern of the Trk neurotrophin receptors is correlated with biological and clinical features of neuroblastoma. High TrkA expression has been shown to be associated with younger age (<1 year), lower stage (stage 1, 2, and 4S), and a favorable outcome (Kogner etÂ€al. 1993; Nakagawara etÂ€al. 1993; Suzuki etÂ€al. 1993; Tanaka etÂ€al. 1998). In addition, Nakagawara and colleagues have demonstrated that there is an inverse correlation between TrkA expression and MYCN amplification and that the combined assessment of MYCN copy number and TrkA expression provides additional prognostic information over either variable alone (Nakagawara etÂ€ al. 1992, 1993). Furthermore, primary neuroblastoma cells with high TrkA expression differentiate in the presence of NGF inÂ€ vitro, whereas the same cells die in the absence of NGF (Nakagawara etÂ€al. 1993). Thus, the NGF/ TrkA pathway may explain the propensity for some neuroblastomas to differentiate or to regress spontaneously. The studies of TrkB and TrkC expression in neuroblastomas are more limited. TrkB is expressed in about a third of neuroblastomas (Nakagawara etÂ€al. 1994). The truncated form of TrkB, lacking the tyrosine kinase domain, is expressed predominantly

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in more differentiated tumors (ganglioneuroblastomas and ganglioneuromas), whereas the full-length TrkB transcript is expressed in tumors with MYCN amplification often in association with its cognate ligand BDNF (Nakagawara etÂ€al. 1994). Thus, the TrkB/BDNF pathway may serve as an autocrine or paracrine pathway to promote survival in MYCN-amplified, and perhaps other, high-risk tumors. In addition, constitutively active BDNF/TrkB signaling seems to confer (or at least be associated with) resistance to conventional chemotherapeutic agents (Ho etÂ€ al. 2002; Jaboin etÂ€al. 2002a). TrkC expression is present in about 25% of neuroblastomas tested, and its pattern of expression resembles that of TrkA (Ryden etÂ€ al. 1996; Yamashiro etÂ€al. 1996), and may represent an alternate or additional pathway for neuronal differentiation in these tumors. Evans and colleagues showed that small molecule inhibition of the Trk tyrosine kinases causes significant growth inhibition of neuroblastoma xenografts (Evans etÂ€al. 2001). CEP-701, a small molecule inhibitor of Trk tyrosine kinase, has been shown to have a significant growth inhibitory effect on neuroblastoma inÂ€vivo (Evans etÂ€al. 1999, 2001), providing the rationale for the ongoing Phase I clinical trial of this compound. Other tyrosine kinase inhibitors, including inhibitors of the epidermal growth factor receptor, may have activity against neuroblastoma (Ho etÂ€al. 2005), and are entering clinical trials. Imatinib mesylate has also been studied in neuroblastoma because some tumors appear to express c-KIT and/or PDGFR (Beppu etÂ€al. 2004; Vitali etÂ€al. 2003), but activating mutations in these receptors have not been reported. Screens of the neuroblastoma “kinome” for other potential drug targets, especially those in late stage clinical development for adult malignancies, are ongoing.

The Neuroblastoma Stem Cell Conundrum Evidence suggests that malignancies may arise or be maintained in a stem cell compartment with the attributes of limitless replication and self-renewal. The Notch, Sonic hedgehog and Wnt/b-catenin developmental programs play a critical role in stem cell determination and renewal in diverse tissues and misappropriation of these pathways appears to be a recurring theme in embryonal tumorigenesis (Allenspach etÂ€ al. 2002; Taipale and Beachy 2001; Giles etÂ€ al. 2003). b-Catenin signaling is involved in the maintenance and expansion of neural crest stem cells (Reya and Clevers 2005; Chenn and Walsh 2002) and neural progenitors (Zechner etÂ€al. 2003). An engineered gain-of-function b-catenin allele targeted to neural tissues causes marked neural progenitor expansion by promoting cell cycle entry at the expense of differentiation (Chenn and Walsh 2002). It is intriguing to speculate that this pathway may contribute to maintenance of neuroblastoma stem cells as well, raising the question of whether emerging inhibitors of this pathway might have therapeutic utility. Indeed, our own unpublished data suggest that aberrant b-catenin signaling may play a role in promoting a high-risk phenotype through induction of MYC and additional advantageous target genes in neuroblastomas without MYCN amplification.

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Stem cells also appear programmed to subvert many death stressors, sparing them from local insults to allow for tissue regeneration and repair. Neural stem cells promote resistance to cytokine and death receptor mediated apoptosis at least partly through developmentally down-regulated caspase-8 and upregulated PEA15. A significant proportion of primary neuroblastomas lack caspase-8 expression (Teitz etÂ€al. 2000), yet ectopic restoration of caspase-8 in deficient cells does not fully restore apoptosis. This suggests that inhibition of apoptosis occurs at multiple levels, and it is possible that the absent caspase-8 expression reflects a developmental program or sustained stem cell feature of neuroblasts rather than a tumor-specific somatic mutation (Goldsmith and Hogarty 2005). This distinction has important implications for experimental therapeutics targeting these latent death-signaling pathways.

Targeted Delivery of Radionucleotides Because neuroblastoma is a radiation sensitive, but also systemic tumor, there has been interest in targeting the delivery of radioactive molecules that are selectively concentrated in neuroblastoma cells. Approaches have included the attachment of radionuclide to metaiodobenzylguanidine (MIBG) (Garaventa etÂ€ al. 1999; Matthay etÂ€ al. 1998a; Tepmongkol and Heyman 1999), somatostatin analogs (Borgstrom etÂ€al. 1999; O’Dorisio etÂ€al. 1994; Wiseman and Kvols 1995), and anti-GD2 antibodies (Cheung etÂ€al. 1998; Kushner etÂ€al. 2001; Yu etÂ€al. 1998). The compound 131I-MIBG is furthest in clinical development. Low dose 131I-MIBG has been shown to be highly effective for disease palliation (Kang etÂ€al. 2003), and a Phase I dose escalation trial established 12Â€ mCi/kg of 131I-MIBG as the maximum tolerated dose with the only significant toxicity being hematopoietic (Matthay etÂ€ al. 1998a). This trial further showed that doses up to 18Â€mCi/kg are tolerable with stem cell support for the 1/3 of patients with protracted Grade 4 hematopoietic toxicity. A completed Phase II study showed an objective response rate of approximately 40% in heavily pretreated patients receiving 18Â€mCi/kg with acceptable toxicity (DuBois etÂ€al. 2004; Howard etÂ€al. 2005). Current clinical investigation is focusing on further dose intensification of MIBG, combining 131 I-MIBG with myeloablative therapies for high-risk patients with a poor response to initial treatment (Yanik etÂ€al. 2002), as well as combining 131I-MIBG with radiosensitizing agents. The European experience combining topotecan and 131I-MIBG has excellent preclinical activity in mouse xenograft models of neuroblastoma, and had no unexpected toxicities in a pilot clinical study. The New Approaches to Neuroblastoma Therapy (NANT) Consortium is leading an ongoing Phase I trial to test the safety of irinotecan and vincristine when given with 131I-MIBG.

Immunotherapy Immunotherapeutic strategies for treating neuroblastoma were originally postulated based on the observations of spontaneous regression, as well as tumor infiltrating

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lymphocytes in some favorable neuroblastomas, both suggesting a host response to tumor (Cooper etÂ€ al. 2001). Targeted molecules directed against neuroblastomaspecific cellular antigens are a strategy that seems promising for application during the minimal residual disease phase of therapy. Murine, chimeric, and humanized antibodies specific to the cell surface ganglioside GD2, highly expressed in the majority of neuroblastomas, either alone or with cytokines, have shown the activity in preclinical models (Barker etÂ€al. 1991; Cheung etÂ€al. 1986; Kushner and Cheung 1989; Lode etÂ€al. 1999), as well as in Phase I and Phase II clinical trials. Measurable responses have been observed in patients with refractory neuroblastoma (Cheung etÂ€ al. 1998; Murray etÂ€ al. 1994; Yu etÂ€ al. 1998), and the monoclonal antibody directed against the GD2 antigen is now furthest in clinical development with a current Phase III study looking at the chimeric monoclonal antibody ch14.18 in the treatment of minimal residual disease. To maximize the immunogenicity of monoclonal antibodies, particularly in heavily pretreated patients who have chemotherapysuppressed function of immune effector cells, immunocytokines or tumor-specific monoclonal antibodies linked with cytokines have been developed. A humanized GD2 antibody fused to IL2 (Neal etÂ€al. 2004; Yu etÂ€al. 1998; King etÂ€al. 2004) has been investigated in a COG Phase II trial, and future trials are being developed to combine these antibodies with anti-angiogenic drugs. Limiting the broad application of this strategy are the difficulties with antibody production and the development of neutralizing antibodies, even to the chimeric molecules (Neal etÂ€al. 2004; Yu etÂ€al. 1998; King etÂ€al. 2004). Neuropathic pain is also a significant immediate toxicity and can be dose-limiting (Yu etÂ€ al. 1998) (hu14.18-IL2) (Neal etÂ€ al. 2004; King etÂ€al. 2004). Additional immunotherapeutic strategies early in the development include DNA (Bolesta etÂ€al. 2005), cellular (Rousseau etÂ€al. 2003), and anti-idiotypic vaccination strategies (Yu etÂ€al. 2004) as well as using engineered cytolytic T lymphocytes for cellular immunotherapy (Gonzalez etÂ€al. 2004).

Retinoids Based on the results from the randomized trial of 13-cis-retinoic acid following myeloablative chemotherapy (Matthay etÂ€al. 1999), most investigators consider this agent standard of care for high-risk neuroblastoma patients who have a complete or very good partial response following myeloablative chemotherapy and stem cell rescue. Current investigation is focused on finding the retinoid compound that gives optimal systemic exposure allowing for maximal antitumor effect without excess toxicity (Reynolds 2004). Fenretinide is currently the lead retinoid in the development because it has been shown to achieve multilog cell kill in multiple neuroblastoma cell lines, even those resistant to other retinoic acids, at least in part through ceramide upregulation (Maurer etÂ€ al. 1999, 2000). Phase I studies done in the United States and Italy have shown that the drug is generally well-tolerated and a Phase II trial recently completed enrollment within the COG (Garaventa etÂ€al. 2003;

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Reynolds etÂ€al. 2003). The current Fenretinide formulation, however, has low oral bioavailability requiring young children to take large numbers of capsules. Liquid and intravenous formulations are currently in phase I trials through the NANT to circumvent this issue.

Angiogenesis Inhibitors Tumor vascularity is correlated with an aggressive phenotype in neuroblastoma making angiogenesis inhibitors an attractive therapeutic option. High-risk tumors are highly vascular, and the tumor vascular index is strongly correlated with adverse prognostic features, including MYCN amplification and poor survival probability (Meitar etÂ€al. 1996). In addition, several pro-angiogenic factors, including VEGF, have been shown to be differentially expressed in primary neuroblastomas in a pattern suggesting promotion of an angiogenic phenotype in high-risk tumors (Eggert etÂ€al. 2000; Fotsis etÂ€al. 1999), while low-risk neuroblastomas are characterized by a rich stromal component that produces angiogenesis inhibitors (Chlenski etÂ€ al. 2002; Huang etÂ€ al. 2000). Preclinical therapeutic studies, however, have shown variable efficacy in neuroblastoma (Erdreich-Epstein etÂ€al. 2000; Katzenstein etÂ€al. 1999; Morowitz etÂ€al. 2005; Shusterman etÂ€al. 2001) that is not always predicted by the activity seen in adult and other pediatric tumors, making the translation to the clinic somewhat slow. Strategies focused on neutralization of circulating VEGF are currently in early clinical trials with specific development for neuroblastoma underway. The challenge of providing neovascular inhibition strategies in young children with a developing cardiovascular system need to be considered during preclinical and clinical development.

Targeting MYCN The cancer genes most commonly altered in adult carcinogenesis (e.g., TP53, CDKN2A, RAS) are rarely aberrant in neuroblastoma. TP53 inactivating mutations are infrequent in primary tumors, although they have been documented in cell lines at relapse (Carr etÂ€al. 2006; Keshelava etÂ€al. 2001; Tweddle etÂ€al. 2001). Homozygous deletion of CDKN2A (INK4A/p16) has been identified in a subset of neuroblastoma cell lines (Thompson etÂ€al. 2001), but there is no consistent evidence for inactivation of this locus in primary tumors. Finally, though persuasive evidence supports RAS and MYC gene cooperation in tumorigenesis (Sears etÂ€al. 2000; Yaari etÂ€al. 2005), activation of RAS does not appear to constitute a preferred secondary pathway for neuroblastomas, even those with MYCN amplification (Dam etÂ€ al. 2006). Thus, major oncogenic pathways governing human neoplasia do not appear deregulated in neuroblastoma with the exception of MYCN in a subset. The MYCN protein provides an obvious potential therapeutic target, but it is so massively dysregulated in neuroblastoma cells with amplification that this has been

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difficult to date. Constitutive activation of the BDNF:TrkB signaling pathway in high-risk neuroblastoma provides another rationale target that might be exploited. Evans and colleagues showed that small molecule inhibition of the Trk tyrosine kinases causes significant growth inhibition of neuroblastoma xenografts (Evans etÂ€ al. 1999, 2001), providing the rationale for an ongoing Phase I trial. Another strategy currently being explored is the use of demethylating agents such as Decitabine. The rationale for this approach is that methylation of genes critical for apoptosis such as caspase 8 appear to occur frequently in neuroblastomas, particularly in association with MYCN amplification (Teitz etÂ€al. 2000). Histone deacetylase inhibitors have demonstrated preclinical activity against neuroblastoma (Coffey etÂ€al. 2001; Jaboin etÂ€al. 2002b), and there are several in clinical trials for patients with refractory solid tumors. With the relatively small number of pediatric patients available for experimental therapeutic trials and the growing number of potential drugs to be tested, it is becoming increasingly important to have firm biological rationale and evidence of efficacy in appropriate preclinical models to help prioritize drug development. Many other strategies, some of which are listed in TableÂ€1 are under development in the laboratory or in early phase clinical trials.

Mitotic Spindle Inhibition Among several potential mechanisms for chromosome instability in cancer cells, much attention has recently been given to functional abnormality of centrosomes because of its prevalence in the majority of solid tumors as well as leukemias and lymphomas, and a strong association between centrosome abnormality and aneuploidy in cancers (D’Assoro etÂ€al. 2002; Fukasawa 2005, 2007). The Aurora family of serine/threonine protein kinases plays a critical role in the regulation of chromosomal segregation and cytokinesis during mitotic progression. The Aurora A kinase gene is amplified and/or overexpressed in many tumors, including colon, breast, pancreatic, and bladder cancers, and its overexpression results in the transformation of normal cells, thus supporting its role as an oncogene. Preliminary data suggest that the Aurora A kinase gene is overexpressed in neuroblastoma cells and that selective small molecule inhibition of this kinase has potential application in this disease.

Other Strategies Epigenetic silencing of genes that are critical for induction of programmed cell death, such as caspase 8, appears to occur frequently in neuroblastoma (Teitz etÂ€al. 2000). Therefore, demethylating agents such as decitabine are currently being studied. Histone deacetylase inhibitors have also demonstrated preclinical activity against neuroblastoma (Coffey etÂ€al. 2001; Jaboin etÂ€al. 2002b). At least three histone deacetylase inhibitors are now in clinical trials for patients with refractory solid

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tumors. Heat shock protein 90 inhibitors are also of interest because these agents alter the function of molecules associated with neuroblastoma cell growth and proliferation, including the Type I insulin-like growth factor receptor AKT, and TrkB. With an expanding portfolio of potential drugs to be tested in pediatric Phase I clinical trials, it is becoming increasingly important to have a firm biological rationale and evidence of efficacy in preclinical models to help prioritize drug development.

Future Directions and Challenges Despite extensive literature correlating common genomic alterations with disease outcome, no bona fide target genes have been identified in neuroblastoma with the exception of MYCN. Hemizygous deletions are typically dozens of megabases without demonstrable biallelic inactivation of regional candidate genes, suggesting that complex multiple gene repression and/or haploinsufficiency may be involved. Whole genome copy number alterations (CNAs) detected with array-based CGH have confirmed the true complexity of chromosomal aberrations in neuroblastoma (Mosse etÂ€ al. 2005b), identified distinct genetic subgroups (Vandesompele etÂ€ al. 2001; Wang etÂ€al. 2006) and inferred models for progression (Bilke etÂ€al. 2005). It is likely that refined platforms will replace region-specific methods to detect crucial CNAs used in risk-stratification schemas, and enhance gene discovery efforts (Beheshti etÂ€ al. 2003; Chen etÂ€ al. 2004; Mosse etÂ€ al. 2005a). Pathway discovery may be further facilitated by integrating genomic DNA alterations with mRNA expression profiles. Transcriptional profiles have been used to define prognostic signatures (Schramm etÂ€ al. 2005) and regional CNAs have been correlated with transcriptional alterations (Wang etÂ€al. 2006). Biological pathways engaged or suppressed as a result of recurring CNAs are being sought. To date, these include upregulation of genes involved in host immune response and antigen processing in high-risk neuroblastomas without MYCN amplification, as well as enrichment for sympathoadrenal developmental genes in low-risk neuroblastomas (Wang etÂ€ al. 2006). Ultimately, improvements in survival will likely result from innovative treatment approaches based upon a better understanding of the critical biological pathways responsible for neuroblastoma initiation and progression.

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Ewing’s Sarcoma Family of Tumors: Molecular Targets Need Arrows Jeffrey A. Toretsky and Aykut Üren

Ewing’s Sarcoma Patients Require Improved Therapy Ewing’s Sarcoma Family of Tumors (ESFT) are highly malignant tumors of bone and soft tissue that occur in children, adolescents, and young adults (Arndt and Crist 1999; Toretsky 2003). Currently, the standard therapy for ESFT patients is a five-drug regimen that consists of alternating cycles of doxorubicin/vincristine/ cyclophosphamide and etoposide/ifosfamide over the course of approximately 9 months. Side effects include nausea, vomiting, and severe hematologic cytopenias, and patients often develop life-threatening infections while receiving chemotherapy. Patients who present with localized ESFT have approximately 70% disease-free survival. Patients who present with metastatic ESFT have a poor prognosis, reporting only 20% disease-free survival despite receiving intensive therapy (Grier etÂ€ al. 2003). These clinical response rates have persisted for the past decade, even after patients received dose-intensifying chemotherapy and bone-marrow transplantation. Current treatment-related morbidity includes cardiac, musculoskeletal, and second malignancies (Fuchs etÂ€al. 2003). We need to discover novel therapeutic approaches to reduce treatment-related morbidity as well as improve overall survival. Novel therapies should exploit tumor vulnerability based on ESFT ontogeny, oncogenesis, and tumor-maintenance pathways.

Ontogeny of ESFT In 1921, James Ewing, M.D., suggested an endothelial cell origin for Ewing’s Sarcoma (Ewing 1921), and there has been a debate over the matter ever since. Morphologically, Ewing’s cells appear as small round cells with a high nuclear to

J.A. Toretskyâ•›() Departments of Oncology and Pediatrics, Lombardi Comprehensive Cancer Center, Georgetown University, 3970 Reservoir Rd. N.W., Washington, DC 20057-1469, USA e-mail: [email protected] P.J. Houghton and R.J. Arceci (eds.), Molecularly Targeted Therapy for Childhood Cancer, DOI 10.1007/978-0-387-69062-9_18, © Springer Science+Business Media, LLC 2010

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cytoplasm ratio, and Ewing’s tumors contain a very limited amount of stromal tissue. These features indicate an undifferentiated malignancy and make it difficult to determine the cell or tissue of origin. Ewing’s sarcoma and peripheral primitive neuroepithelioma cells were found to share a chromosomal translocation (Turc-Carel etÂ€al. 1984; Whang-Peng etÂ€al. 1984) and a similar pattern of oncogene expression (McKeon etÂ€ al. 1988). The ESFT-specific chromosomal translocations t(11;22), t(21;22), and others result in the expression of pathognomonic fusion proteins. The most common of these chimeric proteins, EWS-FLI1, is critical for ESFT survival. The cellular origins of ESFT have been investigated through different experimental approaches, including analysis of gene expression patterns, forced induction of differentiation, immunohistochemical profiles, and inÂ€vitro transformation followed by tumor xenograft models. Studies using cDNA microarray and other related technologies found thousands of gene expression signatures for ESFT cells, including a prevalence of neuronal markers (Baer etÂ€al. 2004; Khan etÂ€al. 2001; Prieur etÂ€al. 2004; Staege etÂ€al. 2004). This discovery is consistent with the expression of neuronal and neuroectodermal markers in ESFT as detected by immunohistochemistry (Cavazzana 1994; Lipinski etÂ€al. 1987; Noguera etÂ€al. 1992). The potential neuroectodermal origin of ESFT is further supported by the finding that modulation of Notch signaling in ESFT cell lines induces neural differentiation (Baliko etÂ€al. 2007). Expression of EWS-FLI1 in a variety of non-ESFT models demonstrates profound effects on cell fate through altered cellular differentiation (Eliazer etÂ€al. 2003; Rorie etÂ€al. 2004; Torchia etÂ€al. 2003). Most recently, GLI1 was identified as a direct target of EWS-FLI1, and its expression likely suppresses ESFT differentiation (Beauchamp etÂ€al. 2009; Zwerner etÂ€ al. 2008). On the other hand, epithelial markers are expressed in a fraction of ESFT samples (Collini etÂ€al. 2001; Gu etÂ€al. 2000; Schuetz etÂ€al. 2005), suggesting a more pluripotent cell of origin. Another characteristic feature of ESFT is the very common expression of the cell surface molecule CD99MIC2 (Ambros etÂ€ al. 1991; Fellinger etÂ€ al. 1991; Hamilton etÂ€ al. 1989a), which was identified as an androgen inducible gene in prostate cancer cells. CD99MIC2 is expressed in normal prostate cells only during embryogenesis; however, expression of CD99MIC2 in adult cells can be observed in undifferentiated carcinomas and some hyperplasias. CD99MIC2 is also expressed in CD34+ hematopoietic precursor cells (Dworzak etÂ€al. 1994). Therefore, CD99MIC2 expression in ESFT suggests an undifferentiated precursor cell of origin for Ewing’s Sarcoma. Expression of EWS-FLI1 in pluripotent bone marrow stromal cells was found to inhibit their ability to differentiate osteogenic and adipogenic lineages (Torchia etÂ€al. 2003). The effects of EWS-FLI1 modulation on gene expression were investigated with the hypothesis that loss of EWS-FLI1 expression might remove the inhibition of differentiation in ESFT and revert the tumor cells back to their cell of origin. The study found that inhibition of EWS-FLI1 expression in three ESFT cell lines resulted in cDNA microarray profiles resembling those of mesenchymal stem cells (Tirode etÂ€al. 2007).

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EWS-FLI1 can transform only a subpopulation of cells inÂ€ vitro, a result suggesting that the ESFT cell of origin is particularly susceptible to EWS-FLI1induced transformation. EWS-FLI1 can transform NIH3T3 cells, but it has failed to transform other fibroblast cell lines (May etÂ€ al. 1993). In primary mouse embryo fibroblasts (Deneen and Denny 2001) or human foreskin fibroblasts (Lessnick etÂ€al. 2002), EWS-FLI1 expression was shown to induce growth arrest and apoptosis. More successful models of expression were obtained when primary murine bone marrow-derived cells were transfected with EWS-FLI1, supporting a mesenchymal stem cell origin for ESFT (Castillero-Trejo etÂ€al. 2005). When bone marrow-derived cells were enriched for mesenchymal progenitor cells and transfected with EWS-FLI1, the results were malignant transformations resembling ESFT phenotypes (Riggi etÂ€al. 2005). These studies provide evidence to support a pluripotential mesenchymal stem cell as the potential cell of origin for ESFT. Cancer stem cells, which represent a small population of therapy-resistant cancer cells that are capable of inducing a new tumor, have been described in many types of cancer (Reya etÂ€al. 2001). These cells are considered the primary source of tumor recurrence following a patient’s good clinical response to initial therapy. The clinical course of ESFT is consistent with this hypothesis, but no cancer stem cells have been described for ESFT. The identification, purification, and propagation of a cell origin for ESFT would help advance models of the disease, but even in the absence of this knowledge, several models have been developed to provide clues to its pathogenesis.

Models of ESFT Knowledge of ESFT has been accrued by studying cell lines established in culture from patient tumors as well as by studying these tumors directly. Spheroid models of ESFT cell lines demonstrate different gene expression and enhanced resistance to anticancer compounds compared to monolayer cultures (Kang etÂ€ al. 2007; Lawlor etÂ€ al. 2002). These spheroid models are postulated to closely resemble actual ESFT growth in patients; however, this conclusion requires additional supportive evidence. To achieve a xenograft model, ESFT cell lines are injected into nude or athymic mice, creating tumors that are close replicates of human tumors (Vormoor etÂ€ al. 2001). Recently, one model was developed by injecting a human cell line into the gastrocnemius to replicate an orthotopic setting found in humans. When the orthotopic tumor grows to a certain size, the primary tumor is resected and virtually all the mice develop metastases, mostly to the lung (Merchant etÂ€al. 2004). An additional metastatic model was developed from the cyclic re-injection of tumor cells into the tail vein of a nude mouse, followed by the re-injection of those cells that grew as pulmonary metastases (Jia etÂ€al. 2003).

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Additional models have been created based upon the expression of EWS-FLI1 in various cell types, and some of these derived cell lines have been used to initiate xenografts. It does not appear that all of these derived cell lines closely approximate human tumor xenografts (Braunreiter etÂ€al. 2006), but these models could still be used to analyze the tumorigenicity of EWS-FLI1. Unlike other diseases driven by chromosomal translocations, such as myeloid leukemia (Dobson etÂ€ al. 1999), alveolar rhabdomyosarcoma (Keller etÂ€ al. 2004), and synovial sarcoma (Haldar etÂ€al. 2007), there are no transgenic models of ESFT. Since the cell origin of ESFT is unknown, choosing a promoter that appropriately expresses the fusion protein EWS-FLI1 in the correct tissue and developmental stage has been challenging. In one murine model, expression of EWS-ERG, a translocation occurring in about 10% of ESFT patients and a very close homolog to EWS-FLI1, led to the development of myeloid leukemia rather than Ewing’s Sarcoma (Codrington etÂ€ al. 2005). Recent advances in molecular biology have made it possible to generate transgenic animals with the EWS-ERG and EWS-FLI1 fusion proteins in hematopoietic stem cells (Forster etÂ€al. 2005; Torchia etÂ€al. 2007). These transgenic animals showed a leukemia phenotype similar to that observed in human cases, which is a promising development. A further attempt to create an EWS-FLI1 transgenic mouse, using a Prx1 promoter, designed to express in mesenchymal progenitors, resulted in animals with disrupted skeletons and poorly differentiated sarcomas (Lin etÂ€ al. 2008). At the present time, promising therapies should be evaluated in existing models until future advances lead to a better endogenous model of ESFT.

An Ideal Molecular Target While the EWS-FLI1 fusion protein has presented challenges in modeling, it is an ideal opportunity for targeted therapy because it only exists in ESFT cells. EWS-FLI1 arises from the balanced chromosomal translocation t(11;22) (q24;q12), which occurs in >95% of ESFT. This translocation joins the Ewing’s Sarcoma (EWS) gene located on chromosome 22 to an ets family gene, most commonly either the Friend Leukemia Insertion (FLI1) gene located on chromosome 11, t(11;22), or the ERG gene located on chromosome 21, t(21;22). Unfortunately, despite the knowledge that inactivation of EWS-FLI1 causes ESFT cell death, strategies to inactivate this ideal target have not yet been brought to the clinic. Chromosomal translocations involving EWSR1 and FLI1 in ESFT may take place at different exon–intron boundaries. Exons 7, 9, or 10 from EWS are combined with exons 4, 5, 6, 7, or 8 from FLI1, resulting in a family of EWS-FLI1 translocations. The most common combination is the EWS exon 7 fused to FLI1 exon 6; this is known as a Type-1 translocation and occurs in approximately 50 to 64% of patients with ESFT. Initial retrospective analyses identified that patients who have localized tumors with the 7/6 fusion have a 70% 4-year survival rate, while

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patients with the other variants have a 20% 4-year survival (de Alava etÂ€al. 1998; Zoubek etÂ€ al. 1996); however, follow-up investigations have not confirmed these earlier findings (Huang etÂ€al. 2005). The finding that EWS-FLI1 is expressed only in tumor cells makes it a very promising molecular target. Therapies directed toward inactivating EWS-FLI1 might address the significant problem of recurrent disease, and eliminate nonspecific side effects for patients.

EWS-FLI1 Modulates Transcription and Splicing EWS-FLI1 has been found to modulate transcription, and some of the protein partners of EWS-FLI1 indicate a role in RNA synthesis and splicing (Fig.Â€1, upper). Unfortunately, the entire EWS-FLI1-containing transcriptosome has not been described. There is enough evidence, however, to conclude that EWS-FLI1 functions differently from both untranslocated EWS and untranslocated FLI1 (May etÂ€al. 1993). One known protein partner of EWS-FLI1 is hsRPB7, the seventh largest subunit of human RNA polymerase II. In Ewing’s sarcoma cell lines, EWS-FLI1 and hsRPB7 were found in a complex by immunoprecipitation (Petermann etÂ€al. 1998). Similarly, inÂ€vitro translated EWS-FLI1 and hsRPB7 were found in an immunoprecipitated complex, suggesting a direct interaction between these proteins. In the same study, researchers did not detect association of hsRPB7 with either full-length endogenous

Fig.Â€1â•… EWS-FLI1 regulates transcription and RNA metabolism. EWS-FLI1 is a protein partner in complexes that regulate transcription and translation. The proteins shown are those that have been identified as binding to EWS-FLI1 either directly or indirectly. Proteins that are directly regulated by EWS-FLI1 are shown in the lower half of the figure

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EWS or full-length endogenous FLI1, which implies that hsRPB7interacts with EWS-FLI1 only in its chimeric state. The functional interaction between these two proteins is further supported by the finding that hsRPB7 augmented EWS-FLI1 transcriptional activity on a reporter construct. EWS-FLI1 binds to DNA through the conserved ets binding domain. Many studies have identified transcripts that are increased or decreased as a result of EWS-FLI1 expression (Fig.Â€1, lower). These studies investigated changes in gene expression based on EWS-FLI1 expression in different cell lines (Braun etÂ€al. 1995) and expression profiles of ESFT patients compared to those of related childhood tumors that lack EWS-FLI1 (Baer etÂ€ al. 2004; Khan etÂ€ al. 2001). The observed changes in gene expression could be a result of direct interaction of EWS-FLI1 with cell-specific coregulatory expression or a secondary effect. Genes that appear to be directly regulated by EWS-FLI1 include PTPL1 (Abaan etÂ€ al. 2005), Id2 (Fukuma etÂ€al. 2003; Nishimori etÂ€al. 2002), p21 (Nakatani etÂ€al. 2003), MK-STYX (Siligan etÂ€al. 2005), IGFBP-3 (Prieur etÂ€al. 2004), TGFßRII (Hahm etÂ€al. 1999), NROB1 (Kinsey etÂ€ al. 2006; Mendiola etÂ€ al. 2006), NKX2.2 (Smith etÂ€ al. 2006), Uridine Phosphorylase (Deneen etÂ€al. 2003), PLD2 (Kikuchi etÂ€al. 2007), caveolin-1 (Tirado etÂ€al. 2006), and thrombospondin (Potikyan etÂ€al. 2007). Although it is interesting to note that these are all genes whose protein products contribute to cell growth or transformation, a clear picture of how EWS-FLI1 regulates these genes has yet to emerge. Identification and evaluation of proteins that interact with EWS-FLI1 might clarify the mechanisms of transformation, and these mechanisms might also help to explain the differences in patient survival based on the breakpoint of the fusion protein. There is also evidence to suggest that EWS-FLI1 is directly involved in RNA splicing. The link between EWS-FLI1 and RNA splicing is supported by the interaction between EWS-FLI1 and a small nuclear ribonucleoprotein (snRNP), which is part of the spliceosome complex involved in mRNA splicing. U1C, a member of the U1 snRNP-specific protein family, was identified as an EWS-FLI1 protein partner, using a yeast two-hybrid system with the EWS domain of EWSFLI1 as bait (Knoop and Baker 2000). In vitro, translated U1C and EWS-FLI1 expressed in bacteria were found to coprecipitate in GST pull-down experiments. This interaction was mapped to the amino terminus of EWS, revealing that both EWS-FLI1 and full-length EWS can bind to U1C. Transcription by an EWSFLI1-dependent reporter construct was inhibited when the cells were transfected with U1C expression plasmids. As promising as these inÂ€vitro results may be, a direct interaction between endogenous EWS-FLI1 and U1C in ESFT, as well as the involvement of EWS-FLI1 in U1C driven RNA splicing, remains to be investigated. Functionally, EWS-FLI1 can alter mRNA by alternatively splicing an E1A transcript (Knoop and Baker 2001). There are no literature reports of altered mRNA splicing in ESFT cells, which would manifest as functional consequences such as loss of growth regulation or transformation, but it is possible that some of the genes thought to be modulated by EWS-FLI1 are, in fact, regulated by mRNA splicing

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rather than direct transcriptional regulation. Additional evidence suggests that EWS-FLI1 inhibits splicing modulated by RNA polymerase and YB-1 (Chansky etÂ€al. 2001; Yang etÂ€al. 2000).

Elimination of EWS-FLI1 Reduces ESFT Cell and Tumor Growth EWS-FLI1 is required to maintain the growth of ESFT cell lines. When EWS-FLI1 expression is reduced, ESFT cell lines die and tumors in nude mice regress. Reduction of EWS-FLI1 by antisense oligodeoxynucleotides (ODN) (Tanaka etÂ€al. 1997; Toretsky etÂ€ al. 1997a) and by antisense RNA expressed from a vector (Kovar etÂ€al. 1996; Maksimenko etÂ€al. 2003a; Ouchida etÂ€al. 1995) inhibited not only the proliferation of ESFT cell lines, but also the growth of xenografted tumors in nude mice. Encouraging results were also obtained when EWS-FLI1 was inhibited by siRNA, a type of RNA interference that serves as a sequence-specific posttranscriptional silencing mechanism. This evolutionarily conserved mechanism defends the cell from viruses by degrading double-stranded RNA fragments. siRNA uses 18 to 21 nucleotide double-stranded RNA to induce sequence-specific gene silencing. Targeting the junction point of EWS-FLI1 fusion protein by siRNA significantly diminished the expression of EWS-FLI1 in ESFT cell lines (Chansky etÂ€al. 2004; Dohjima etÂ€al. 2003; Kovar etÂ€al. 2003). Like the antisense oligonucleotides, siRNA inhibited the proliferation of ESFT cell lines inÂ€vitro and stopped xenografted tumor growth in nude mice (Hu-Lieskovan etÂ€al. 2005). The ODN, antisense RNA, and siRNA studies have each established that EWSFLI1 is critical to ESFT cells, but effective strategies of treatment for ESFT patients remain to be developed. The pharmacokinetics and pharmacodynamics of siRNA and antisense ODN have been less than satisfactory in humans. Recent advances in nanotechnology have improved delivery and controlled the release of siRNA and antisense ODN in animal models (Lambert etÂ€al. 2000; Maksimenko etÂ€al. 2003b), but even with current technologies, reduction of EWS-FLI1 with antisense ODN and siRNA remains far from possible in humans. An alternative approach to reducing EWS-FLI1 involves blocking EWS-FLI1 function with a dominant negative protein, the Kruppel-associated box (KRAB) (Kovar etÂ€al. 1996). KRAB is a 75 amino acid protein domain that can function as a transcriptional repressor. When linked to the DNA-binding domains of different transcription factors, KRAB inhibited promoter–reporter constructs. Similarly, a KRAB-FLI1 fusion protein was found to inhibit EWS-FLI1 oncogenic function, most likely by competing for the same DNA-binding sites in the cells (Chan etÂ€al. 2003). Based on multiple models of biologic intervention, EWS-FLI1 is critical for ESFT; therefore, inhibition of EWS-FLI1 should be evaluated as a specific cancer therapy for ESFT.

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Molecular Targeting of ESFT The development of targeted molecular agents for patients with ESFT is necessary in order to improve patient survival. The most obvious target for these agents is to disable or eliminate EWS-FLI1. As a regulator of transcription, EWS-FLI1 serves as an Achilles’ heel to the tumor when it is inhibited. As discussed earlier, developments in nanotechnology may improve the delivery of siRNA and antisense ODN to eliminate EWS-FLI1 in tumor cells; however, the technology has not been tested in humans. Alternatives to eliminating EWS-FLI1 are the inhibition of its function by disrupting the binding of EWS-FLI1 to key functional protein partners, finding small molecules to mimic EWS-FLI1 reduction, using single chain antibodies, and inhibiting specific key transcriptional targets of EWS-FLI1. Additional molecular targeting could be directed toward key growth factor pathways or other aspects of tumor biology that present a reasonable therapeutic window. The overall goal of targeted therapy is to eliminate the tumor and spare normal function as much as possible in terms of both acute and long-term effects. Knowledge gained by studying and targeting EWS-FLI1 would have broad benefits beyond ESFT. EWS and FLI1 homologs are partners in translocations that occur in a wide range of sarcomas and leukemias (Helman and Meltzer 2003). EWS and its homolog TLS are involved in chromosomal translocations of clear cell sarcoma, mixoid liposarcoma, desmoplastic small round cell tumors, chondrosarcoma, and acute myeloid leukemia. The FLI1 homolog ERG is translocated in acute myeloid leukemia. The importance of EWS and FLI1 homologs in so many forms of cancer suggests that EWS-FLI1 may serve as a model system, possibly shedding further light on a family of diseases that are related by translocation partners.

EWS-FLI1: The Perfect Target Small-Molecule Protein–Protein Interaction Inhibitors EWS-FLI1 is clearly established as a molecular target for ESFT, but strategies to reduce protein levels are not ready for clinical use. Small-molecule inhibitors of EWS-FLI1 function may lead to targeted therapeutics; however, since EWS-FLI1 lacks intrinsic enzymatic function, targeted inhibition represents a significant challenge. Disruption of key EWS-FLI1 protein–protein interactions, a strategy that has been suggested to regulate other ets family proteins (Li etÂ€al. 2000a), might lead to novel therapeutic agents. Computer-assisted design of small molecules that fit into functional pockets has been shown to prevent max from binding to myc, and has thereby inhibited myc-modulated transcription (Yin etÂ€ al. 2003). While threedimensional structures have been critical to the optimization of targeted smallmolecule inhibitors, EWS-FLI1 presents a challenge based upon its biochemical characteristics, described below (Uren etÂ€al. 2004a).

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The novelty of EWS-FLI1 may cause it to interact with other proteins, such as basal transcriptional regulators, in unique three-dimensional protein–protein interactions. The separation of EWS-FLI1 from the key proteins required for EWS-FLI1 oncogenesis might cause ESFT cell death. Structural information about EWS-FLI1 as a fusion protein is limited. EWS-FLI1 has the potential to both homodimerize and heterodimerize with EWS; however, it is not clear that this happens in ESFT cells (Spahn etÂ€al. 2003; Watson etÂ€al. 1997). While crystallographic and NMR structures of ets DNA-binding domains are available, no data exist for EWS-FLI1. Furthermore, due to changes in the overall amino acid composition and charge distribution in the fusion protein, EWS and FLI1 domains in EWS-FLI1 may have a unique three-dimensional structure that is different from both wild-type protein partners (Uren etÂ€ al. 2004a). An analysis of the primary amino acid sequence of EWS-FLI1 suggests that the secondary structure of EWS-FLI1 is largely disordered under native conditions due to low overall hydrophobicity. This hypothesis was supported by the discovery that EWS-FLI1 lost function following mutation of its aromatic residues (Ng etÂ€al. 2007). These analyses suggest that EWS-FLI1 will be in considerable motion in solution and, as a result, may not be crystallized easily. The overall size of EWS-FLI1 and its poor solubility make it difficult to study with current magnetic resonance techniques (Uren etÂ€al. 2004a). Strategies used to identify EWS-FLI1 protein partners include yeast two-hybrid, column chromatography, promoter analysis, and phage display. Yeast two-hybrid and column chromatography have identified partners that are components of transcription, such as RNA Pol II (Petermann etÂ€al. 1998) and TFIID (Bertolotti etÂ€al. 1998). Scrutiny of the uridine phosphorylase promoter, an EWS-FLI1-regulated transcript, revealed AP-1 as a critical EWS-FLI1 cofactor (Kim etÂ€al. 2006). Recently, we generated a functional recombinant EWS-FLI1, which was then used to screen bacterial phage libraries to identify peptide sequences that bind to EWS-FLI1. These peptides led to the identification of proteins that directly bind to EWS-FLI1 as well as small molecules whose homologous structure might prevent EWS-FLI1 from binding to key proteins. We identified RNA helicase A (RHA) as a significant protein partner of EWS-FLI1, whose interaction with EWS-FLI1 represents a therapeutic target (Toretsky etÂ€al. 2006).

YK-4-279 is a Novel Small-Molecule Inhibitor of EWS-FLI1 Disrupting the interaction between EWS-FLI1 and its key protein partner RHA holds particular promise as a targeted therapy because of the specific protein interaction of the two partners. The region of RHA (aa 647-1075) that interacts with EWS-FLI1 uniquely binds to EWS-FLI1; it has yet to be identified as a binding partner with the basal transcription apparatus (Anderson etÂ€al. 1998; Aratani etÂ€al. 2001; Nakajima etÂ€al. 1997; Tang and Wong-Staal 2000). A ten amino acid peptide that mimics the interaction of RHA blocks the interaction with EWS-FLI1 (Erkizan etÂ€al. 2009). A library of small molecules led to the discovery of a lead compound,

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YK-4-279, that also binds to EWS-FLI1 and, in addition, prevents RHA binding (Erkizan etÂ€ al. 2009). Moreover, this novel compound inhibits EWS-FLI1 transcriptional activity, reduces ESFT cell-line viability, induces apoptosis, and inhibits xenograft growth (Erkizan etÂ€al. 2009). While still early in the development process, YK-4-279 demonstrates a novel approach to the inhibition of tumor-specific fusion proteins and promises to be a future therapeutic for patients with ESFT.

Alternate Approaches to Small Molecule Identification Recently, a novel approach to the inactivation of EWS-FLI1 was developed through cDNA expression arrays that established a signature for EWS-FLI1-expressing cells (Lessnick etÂ€al. 2002). An ESFT cell line was treated with a panel of compounds, most of which are already in clinical use. cDNA signatures were established for each of the compounds used on ESFT cells, and these signatures were compared with those that resembled the siRNA reduction of EWS-FLI1. The results of this comparison identify cytosine arabinoside (Ara-C) as a molecule that caused ESFT cells to have an expression profile most similar to that of EWS-FLI1 reduction (Stegmaier etÂ€al. 2007). Unfortunately, a recent Children’s Oncology Group phase II study for patients with recurrent ESFT demonstrated that Ara-C did not have clinical activity (DuBois etÂ€al. 2009).

Single-Chain Antibodies The use of single-chain antibodies to inactivate proteins was developed to better study the antigen–antibody interaction (Denzin etÂ€al. 1991) and, in part, to develop recombinant therapies (Begent etÂ€al. 1996). The use of a specific antibody to target EWS-FLI1 has been explored, but the most recent result was an antibody that recognizes wild-type EWS but not the fusion protein in its native form (Aryee etÂ€al. 2006).

Targeting the Targets Knowing that EWS-FLI1 regulates many transcripts that result in ESFT, one clear challenge is to decide which of these is a worthy therapeutic target, in terms of both specificity and drug ability. Since the targets of EWS-FLI1 include genes that might be important for nontumor cells, including hematopoietic cells, the specificity gained by targeted therapy may be lost. Alternatively, there may be a key target of EWS-FLI1 that is not important for nontumor physiology and would thus make a good anticancer target. The ideal targets would have unique biology, from kinases

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to phosphatases to nonenzymatic proteins. In each case, the biology of the target needs to be considered and validated as critical to the growth of ESFT tumors. This validation would include reducing the level of the target gene to demonstrate reduced ESFT growth (Kinsey etÂ€ al. 2006; Potikyan etÂ€ al. 2007). For example, caveolin-1 is a putative EWS-FLI1 target (Tirado etÂ€al. 2006), but a general targeting of caveolin-1 might disrupt signaling in all cells, thus reducing the specificity of a potential caveolin-1-directed therapy. Given that many of the current anticancer therapies are not targeted and that agents like proteosome inhibitors or anti-heat shock agents appear to convey some sense of therapeutic index, the approach of inhibiting the targets of EWS-FLI1 may be fruitful. However, targeting less ESFTspecific molecules may lead to significant and intolerable toxicity.

Disruption of EWS-FLI1 DNA Binding Another targeted approach for disabling EWS-FLI1 might stem from the disruption of DNA binding. There are two challenges to this approach. The first is that DNA binding may not be necessary for EWS-FLI1 transformation (Welford etÂ€al. 2001), although DNA binding may still be necessary for tumor maintenance. Second, the DNA sequence that EWS-FLI1 binds is a well-recognized ets-binding site and demonstrates significant promiscuity in transcription factor binding (Watson etÂ€al. 1997). While specificity of novel compounds may overcome these challenges, disrupting EWS-FLI1 and DNA binding is not a feasible therapeutic strategy at this point.

Non-EWS-FLI1 ESFT Targets In order to improve survival for ESFT patients, many agents are being used in phase I and phase II studies (TableÂ€1). These agents are supported by varying levels of rationale. Those with specific evidence in ESFT are described here.

Modulation of Apoptotic Tendency Apoptosis signaling pathways can be categorized into two classes: the intrinsic pathway involving the mitochondria and the extrinsic pathway involving cell surface receptors. These death receptors are members of the tumor necrosis factor (TNF) receptor family, which can induce apoptosis independently of p53 (Ashkenazi 2002). One subgroup of this family includes TNF receptor 1 (TNFR1), FAS, and death receptors 4 and 5 (DR4 and DR5). These receptors contain an intracellular death domain that, upon ligand binding, can recruit the FAS-associated death domain (FADD) and activate caspase 8. The ligand for DR4 and DR5 is

TableÂ€1â•… Current clinical trials using targeted therapies in ESFT Agent Other names Function Phase Manufacturer 17-AAG 17-AAG Geldanamycin analog, HSP-90 inhibitor Phase I CP-751,871 CP-751,871 IGF-IR inhibitor monoclonal antibody Phase I Pfizer R-1507 IGF-IR inhibitor monoclonal antibody Phase II Roche Imatinib Gleevec, STI-571 TX inhibitor BCR-ABL, c-KIT, PDGFRb Phase II Novartis Depsipeptide FK-228, NSC-630176 HDAC inhibitor Phase I/Il Reolysin Reolysin Wild-type human reovirus with anti-ras Phase II Oncolytics Biotech Inc. activity Bevacizumab Avastin Monoclonal antibody against VEGF Phase II Genentech Topotecan Hycamtin Topoisomerase 1 inhibitor Phase III GlaxoSmithKline Irininotecan Camptosar Topoisomerase 1 inhibitor Phase II Pfizer Cytarabine Ara-C, Cytosar-U DNA polymerase inhibitor Phase II Pfizer Trabectedin ET-743, Yondelis Directly binds to DNA, inhibits replication Phase II PharmaMar, Johnson & Johnson and repair Desatinib BMS-354825, Sprycel TK inhibitor BCR-ABL, SRC, c-KIT, EPHA2, Phase II Bristol-Myers Squibb and PDGFRb Phase II Bayer Sorafenib Nexavar TK inhibitor Raf, VEGFR, PDGFR, Fit-3, c-Kit, RET Sunitinib Sutent TK inhibitor VEGFR, PDGFR, Fit-3, c-Kit Phase II Pfizer Key to abbreviations: 17-AAG 17-(Allylamino)-17-demethoxygeldanamycin, IGF-IR insulin-like growth factor-I receptor, PDGFRb platelet derived growth factor receptor-b, HDAC histonedeacetylase, VEGF vascular endothelial cell growth factor, VEGFR vascular endothelial cell growth factor receptor, TK tyrosine kinase, FLT Fms-like tyrosine kinase

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TNF-related, apoptosis-inducing ligand (TRAIL); DR4 and DR5 are also called TRAIL receptor 1 and TRAIL receptor 2, respectively. TRAIL receptors function as homotrimers that can activate the extrinsic apoptosis pathway by converting inactive procaspase 8 to active caspase 8. Once activated, caspase 8 can activate caspase 3 and other downstream caspases to initiate apoptosis. Caspase 8 can also cleave BID and activate intrinsic apoptosis pathway through the mitochondria (Duiker etÂ€al. 2006). Since TRAIL receptors are not expressed in normal cells but are highly expressed in many tumor cells, they have been evaluated as a potential therapeutic target for cancer (Fesik 2005; Takeda etÂ€al. 2007). A common approach has been to activate these death receptors either by agonistic antibodies or recombinant soluble TRAIL proteins. Both approaches activate the TRAIL receptors and induce apoptosis in tumor cells with little to no toxic effects on normal cells. However, some tumor cells are able to resist TRAIL-induced apoptosis by expressing decoy receptors, which can bind TRAIL but cannot propagate the signal due to their lack of a cytoplasmic death domain. In these cases, use of the antibody approach may have benefits over using recombinant ligands. Agonistic antibodies not only activate the death receptor to initiate apoptosis, but they can also initiate an immune response through their Fc domain. An antibody attached to the surface of a tumor cell can activate both complement-dependent cytotoxicity and antibody-dependent cellular cytotoxicity. ESFT cell lines express both FAS and TRAIL receptors (Kontny etÂ€ al. 2001; Kontny etÂ€al. 1998). Furthermore, application of ligands to Ewing’s cells induces apoptosis in most cell lines in both inÂ€ vitro and xenograft models. Interestingly, resistant cells become sensitive when they are pretreated with interferon-g (IFN-g) (Abadie and Wietzerbin 2003; Merchant etÂ€ al. 2004). This cooperation between IFN-g and TRAIL or TNF-a appears to stem from the stimulation of ESFT cells with IFN-g as well as TNF-a-induced expression of TRAIL (Abadie etÂ€al. 2004). IFN-g also induces expression of caspase 8. Induction of caspase 8 by either IFN-g or an expression vector was enough to convert a TRAIL-resistant ESFT cell line into a sensitive one (Lissat etÂ€al. 2007). These findings provide accumulating evidence toward a rationale for using TRAIL and TNF-a in combination with IFN-g.

Cytokine-Regulated Growth Pathways The insulin-like growth factor type 1 receptor (IGF-IR) pathway, which leads to transformation, cell division, and survival, is activated by the ligands IGF-I or IGF-II. The receptor is a heterotetrameric membrane-bound tyrosine kinase that, upon autophosphorylation, directly binds a number of molecules to advance signaling, including the docking protein insulin-receptor substrate-1 (IRS-1), thus initiating multiple growth and survival pathways (Butler etÂ€al. 1998; Singleton etÂ€al. 1996; Tanaka etÂ€al. 1996; Valentinis and Baserga 1996). The IGF-IR pathway is required for transformation by many oncogenes; therefore, it is thought to be a critical pathway for cells to become malignant. (Baserga 1995).

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IGF-IR was first recognized as important in ESFT based on its modulation of the antigen HBA-71 (CD99, p30/32MIC2), effects upon cell-line growth, and IGF-I ligand expression (Hamilton etÂ€al. 1989b, 1991; van Valen etÂ€al. 1992; Yee etÂ€al. 1990). An antibody bound to the IGF-IR reduced ESFT cell-line growth and enhanced the effects of chemotherapy (Hofbauer etÂ€al. 1993), suggesting that IGF-I acts as an autocrine factor for the growth of ESFT. Whether autocrine, paracrine, or endocrine, IGF-I appears to activate a significant pathway in ESFT. The expression of the IGF-IR was found to be necessary for the EWS-FLI1 oncogene to transform fibroblasts (Toretsky etÂ€al. 1997b). Xenograft studies that blocked the IGF-IR with antisense RNA and antibodies further demonstrated the importance of IGF-IR signaling for ESFT growth (Benini etÂ€al. 2001; Scotlandi etÂ€al. 1998). In addition, the IGF-I ligand has been shown to enhance tumor cell survival, and a pilot study suggested that a high ratio of IGF-I to IGFBP-3 led to reduced overall survival in ESFT patients (Toretsky etÂ€al. 1999, 2001). Recently, the IGF-binding protein-3 (IGFBP3) was identified as a transcriptional target suppressed by EWS-FLI1 (Prieur etÂ€al. 2004), which is an interesting finding because IGFBP-3 may directly or indirectly enhance apoptosis (Gill etÂ€al. 1997; Rajah etÂ€al. 1997). The inhibition of the IGF-IR pathway in patients with ESFT has been a research goal for almost 20 years. The first small-molecule inhibitors of the IGF-IR kinase activity were effective at reducing the growth of ESFT cell lines and xenografts (Scotlandi etÂ€al. 2005), but there are currently no clinical trials for these inhibitors in ESFT patients. The experience with the EGFR suggests that the small-molecule kinase inhibitors are most useful when patients have constitutively activated mutations of the EGFR (Lynch etÂ€al. 2004; Pao etÂ€al. 2004). ESFT have not yet been systematically screened for IGF-IR-activating mutations. In fact, no IGF-IRactivating mutations have been described in any naturally occurring tumor. Tumors that appear to rely on IGF-IR signaling generally have a significant overexpression of the receptor, leading to a more constitutive activation. The EWS-WT1 fusion protein, which results from a t(11;22) with a different fusion partner and is found in desmoplastic small round blue cell tumors, directly activates expression of the IGF-IR, while EWS-FLI1 does not directly increase IGF-IR expression in ESFT (Karnieli etÂ€al. 1996). Despite these caveats, there is significant interest in targeting the IGF-IR pathway in ESFT. A number of commercially developed antibodies are currently available in phase I and II trials. These clinical trials will answer whether IGF-IR is both targetable and a worthwhile target in ESFT. Another potential therapeutic target for ESFT is the platelet-derived growth factor (PDGF), a secreted glycoprotein with important mitogenic and chemoattractant functions (Heldin etÂ€ al. 1986). There are two well-characterized PDGF ligands: PDGF-A and PDGF-B, which form heterodimers as PDGF-AB and homodimers as PDGF-AA and PDGF-BB (Williams 1989). Recently, two new members of this family have been discovered: PDGF-C and PDGF-D (LaRochelle etÂ€al. 1993; Li etÂ€al. 2000b). PDGF-A and PDGF-B chains are secreted from the cell as mature ligands, while PDGF-C and PDGF-D require proteolytic cleavage outside the cell in order to become active. PDGF ligands exert their effect on target cells through specific dimeric receptors. The two different subunits of PDGF receptors (PDGFRs)

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have different ligand-binding specificity; a-PDGFR binds PDGF-A, B, and C chains, but b-PDGFR binds only PDGF-B and D chains. PDGF-C has affinity toward a-PDGFR similar to PDGF-A, and PDGF-D has affinity toward b-PDGFR similar to PDGF-B. Ligand binding initiates dimerization and autophosphorylation of the specific receptors. PDGF is important in physiologic processes, including embryonic development, wound healing, angiogenesis, hemostasis, and platelet aggregation. Over-activity of PDGF signaling contributes to the pathogenesis of many diseases, including lung fibrosis, glomerulonephritis, atherosclerosis, and cancer (Ostman and Heldin 2001). The PDGF-B homolog, v-sis, was one of the earliest discovered oncogenes (Doolittle 1983, Waterfield 1983). Overexpression of PDGF ligands and receptors has been reported for many solid tumors, including glioblastoma, meningioma, melanoma, ovarian cancer, prostate cancer, lung cancer, pancreatic cancer, and gastric carcinoma (Ostman and Heldin 2001). Analysis of ESFT cell lines and tumor samples provided evidence that PDGF signaling could be used as a molecular target. ESFT cell lines express b-PDGFR, which has a significantly higher transforming capacity than a-PDGFR (Merchant etÂ€ al. 2002; Uren etÂ€ al. 2003). Expression of b-PDGFR was also detected in a majority of tumor samples from ESFT patients (Bozzi etÂ€al. 2007; Uren etÂ€al. 2003). Activation of b-PDGFR in ESFT cell lines induces proliferation and chemotaxis, so it may contribute to both tumor development and metastasis. Screening of a cDNA library from EWS-FLI1-transformed fibroblasts revealed PDGF-C as an EWS-FLI1-induced gene (Zwerner and May 2001). Later, PDGF-C expression was found to have a strong correlation with EWS-FLI1’s ability to transform different fibroblast cell lines (Zwerner etÂ€ al. 2003). The ability of EWS-FLI1 to induce PDGF-C expression suggests a potential autocrine signaling pathway in ESFT cells. However, unlike fibroblasts that express both PDGF receptor isoforms, ESFT cells mainly express b-PDGFR, which cannot bind PDGF-C (Merchant etÂ€al. 2002; Uren etÂ€al. 2003). A dominant negative PDGF-C inhibited the growth of ESFT cell lines (Zwerner and May 2002), but in this study, a potential cross reactivity of dominant negative PDGF-C was not ruled out. Since PDGF ligands can form heterodimers, a dominant negative PDGF-C may inhibit PDGF-B function as well. Additionally, clinical samples also showed strong phosphorylation of b-PDGFR. This paradoxical finding may be explained by a paracrine signaling mechanism. PDGF-BB from serum and stromal cells can activate b-PDGFR in ESFT cells and induce their growth and motility. PDGF-C secreted from ESFT cells can act on tumor stroma to provide vascularity. KIT is a cell surface receptor glycoprotein found in the Type III class of receptor tyrosine kinases, the same class as PDGFRs (Fantl etÂ€al. 1993). This class of receptor tyrosine kinases is characterized by five extracellular immunoglobulin-like domains and an intracellular tyrosine kinase domain that is interrupted by a large insert sequence termed the “kinase insert” region. The ligand for KIT is stem cell factor, which induces dimerization and autophosphorylation of the receptor. KIT plays an important role in erythropoiesis, lymphopoiesis, mast cell development, megakaryopoiesis, gametogenesis, and melanogenesis (Ronnstrand 2004). KIT

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gain-of-function mutations have been described in gastrointestinal stromal tumors (GIST), mastocytomas, sinonasal T-cell lymphomas, dysgerminomas, and acute myelogenous leukemias (Heinrich etÂ€al. 2002). Autocrine and paracrine activation of wild-type KIT has been implemented in several types of tumors as well. Expression of KIT in ESFT cell lines and tumor samples has been shown in multiple studies (Bozzi etÂ€al. 2007; Merchant etÂ€al. 2002; Tamborini etÂ€al. 2003), but expression of KIT in ESFT did not correlate with patient survival and there were no gain-of-function mutations of KIT. There is still some evidence, however, of autocrine or paracrine activation of KIT in ESFT tumor samples (Bozzi etÂ€al. 2007; Tamborini etÂ€al. 2003). Imatinib (STI-571, Gleevec®) is a specific protein tyrosine kinase inhibitor that inhibits Abl, KIT, and PDGFR kinases with high specificity. Imatinib found clinical use for treating chronic myelogenous leukemia (CML), which expresses the BcrAbl chimeric oncoprotein as the result of a specific chromosomal translocation (Philadelphia chromosome). Another successful clinical application of Imatinib came in GIST, which contain KIT gain-of-function mutations in over 70% of patients. Clinical trials of Imatinib in ESFT patients did not demonstrate activity as a single agent in a Phase II trial (Bond etÂ€al. 2007). Other cytokine-activated pathways found to be important in cell-based models of ESFT include bFGF (Girnita etÂ€ al. 2000; Schweigerer etÂ€ al. 1987; Westwood etÂ€al. 2002; Williamson etÂ€al. 2004), VEGF-R (Fuchs etÂ€al. 2004; Guan etÂ€al. 2005; Lee etÂ€al. 2006), and wnt (Uren etÂ€al. 2004b). Although all these findings require tissue validation, the pathways identified may be critical for the growth of ESFT. The CD99MIC2, first identified as HBA-71 (Hamilton etÂ€al. 1989a), is relatively specific for ESFT (Weidner and Tjoe 1994), and crosslinking of this antigen is toxic to ESFT cells (Scotlandi etÂ€al. 2006). CD99MIC2 may be involved in metastatic growth of ESFT through downstream effects on the potassium channel KCMF1, leading to enhanced cell motility (Kreppel etÂ€ al. 2006). Although the function of CD99MIC2 remains cryptic in ESFT and its possible role in therapy is uncertain, it could potentially allow for reasonably targeted antibodies to ESFT cells.

Molecular Targets from Other Tumor Models Some therapeutic agents are active against ESFT with a broad mechanism; others are developed for mechanisms in non-ESFT cancers that might also apply to ESFT. Agents that inhibit topoisomerase, irinotecan, and topotecan are active in recurrent ESFT in combination with alkylating therapy, temozolomide, and cytoxan, respectively (Saylors etÂ€al. 2001; Wagner etÂ€al. 2007). By their nature, these combinations do not act toward specific molecular targets. Additional agents have undergone preclinical and early stage clinical development in adult cancers, and they may hold less-specific promise – but promise nonetheless – for patients with ESFT. Some agents, such as ET-743, demonstrate good activity against ESFT, but the mechanisms they affect remain cryptic (Scotlandi etÂ€al. 2002). Other targeted therapies for

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patients with ESFT include agents that affect protein binding to DNA, protein chaperoning, and protein degradation. Among these potentially promising agents for ESFT are the histone deacetylase (HDAC), heat shock protein 90 (HSP90), and proteosome inhibitors. These agents do not specifically act upon EWS-FLI1, but there is hope that they may provide ESFT patients more general therapy with tolerable side-effects. The HDAC family of proteins regulates the acetylation of histone proteins, thereby regulating transcription factor access to DNA. The recent discovery of nonhistone acetylation of proteins suggests novel posttranslational protein regulation mechanisms. This class of anticancer agents has yielded a long list of molecular classes and clinical trial results. For instance, cutanteous T-cell lymphoma has been one of the most responsive malignancies to HDAC inhibitors. The bicyclic peptide HDAC inhibitor FK-228 (depsipeptide, NSC-630176) induces apoptosis in a series of ESFT cell lines and reduces xenograft tumor growth (Sakimura etÂ€al. 2005). Phase I studies of depsipeptide have been completed in children, and phase II studies are currently underway. MS-275, a benzamide HDAC inhibitor, demonstrated reduced ESFT cell and xenograft growth (Jaboin etÂ€al. 2002). Both HDAC studies showed that ESFT cells re-expressed transcripts that are putatively suppressed by EWSFLI1, suggesting a potential mechanism of compound action. FK-228 also appeared to reduce the expression of EWS-FLI1. Heat shock proteins (HSP) are both specific and general targets of anticancer therapeutics. HSP are specifically inactivated by benzoquinonoid ansamycins, suggesting that they are specific molecular targets of HSP90. Significant reduction of HSP90 leads to a dramatic reduction of many client proteins, including Akt, IGF-IR, telomerase, and other key kinases. ESFT cells are very sensitive to reduction of xenograft growth and treatment with geldanamycin (IC50â•›=â•›5 to 8Â€nM) in a 3 H-thymidine incorporation assay (Whitesell etÂ€al. 1992). The key client proteins of HSP90 have not been determined in ESFT, but IGF-IR or Akt are logical possibilities. Two pediatric phase I studies have been performed with 17AAG, a more water soluble form of geldanamycin. Phase II studies are pending the development of more potent compounds with better “drug-like” qualities. HSP90 inhibitors may be helpful to further elucidate a series of key protein targets in ESFT, given the cell lines’ significant sensitivity to HSP90 depletion. The potential role for HSP inhibitors as targeted therapy in the clinical practice is still under investigation. Protein synthesis and degradation are balanced modulators of protein levels. They are important because they lead to regulation of cell functions via the presence or absence of key proteins. In cancer cells, proteins required for cell division are synthesized rapidly while proteins that slow the cell cycle are degraded at a high rate in order to maintain tumor growth. Both of these processes can be the molecular targets of cancer therapy. For example, Rapamycin has been shown to bind to FKBP and block the binding of raptor to the mammalian target of Rapamycin (mTOR) (Hara etÂ€ al. 1998, 2002). This binding prevents the activation of key proteins needed to enhance protein translation. Used in isolation, Rapamycin moderately decreases ESFT cell growth, but in combination with EWS-FLI1-directed antisense oligonucleotides, it

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significantly reduces xenograft growth (Mateo-Lozano etÂ€ al. 2006). Rapamycin reduces protein levels of EWS-FLI1, suggesting that a high level of protein synthesis is necessary to maintain EWS-FLI1 in the tumor cells. This implies that Rapamycin may be a compound synergistic to agents that disrupt EWS-FLI1 function by causing dissociation of key protein partners. Protein degradation is regulated by ubiquitination followed by proteosomemediated catabolism. While the standard proteosome inhibitors have not yet been tested in ESFT cell lines, blocking proteosomal activity causes an increase in levels of the cell cycle inhibitor p27, leading to ESFT cell senescence (Matsunobu etÂ€al. 2004). In combination with antisense therapy directed to EWS-FLI1, these results suggest a cooperative mechanism of potential novel therapeutics. There are no published phase II studies of agents that regulate protein levels with useful numbers of ESFT patients.

Conclusion New and improved therapies are needed for more effective treatment of ESFT, especially in patients with metastatic disease. The discovery of the importance of EWS-FLI1 in ESFT has sparked a significant amount of research in the past two decades, but the translation of this basic science to the bedside has been slow. Although ESFT contains a perfect molecular target in EWS-FLI1, therapeutic development has not yet yielded a successful drug. Still, there has been some progress regarding ESFT. Advances in modern molecular biology techniques have led to a long list of genes that are modulated, at some level, by EWS-FLI1. The identification of EWS-FLI1 protein–protein interactions, as well as the role of EWS-FLI1 in RNA processing, has broadened the understanding of EWS-FLI1-mediated oncogenesis. However, improved resolution of the molecular mechanisms of EWS-FLI1 oncogenesis is required. The recently developed small-molecule and peptide probes will clearly help to advance understanding and therapy of EWS-FLI1. Additional challenges to effective targeted therapeutics include the lack of a definitive cell of origin and a clinically relevant animal model of ESFT. More recent knowledge points toward a mesenchymal cell; however, earlier data supported a neural crest origin cell of origin. This cell of origin might be clarified by identification of a promoter that will allow the proper timing and tissue expression of EWS-FLI1 to recapitulate ESFT in a mouse. An experimental transgenic animal model will be of great value both for studying ESFT biology and for testing new chemotherapeutic agents. The progression toward clinical EWS-FLI1 inhibitors has been challenged by a general scientific belief that such inhibitors were not possible, and if they were possible, pharmaceutical development would not occur due to the potentially small numbers of affected patients. The recent demonstration of a small-molecule inhibitor of EWS-FLI1 represents a breakthrough for a new discovery method as well as a class of compounds as therapeutic agents for ESFT. Novel approaches to therapeutic

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development for rare diseases, such as ESFT, will clearly require partnership from philanthropy, industry, academia, and NIH resources. If we can join these forces, we can make the translation of molecular biology to bedside practice much more imminent. Acknowledgmentsâ•… The authors would like to thank Audrey Kubetin for her skillful assistance with this manuscript.

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Molecular Targeted Therapy for Wilms’ Tumor James I. Geller and Jeffrey S. Dome

Introduction Wilms’ tumor (nephroblastoma, WT) is the most common pediatric primary renal malignancy, originating from aberrant differentiation of a pluripotent renal stem cell derived from embryogenic metanephric blastema (Beckwith etÂ€al. 1990). In the United States, WT has an annual incidence of 7.6 cases per million children, with approximately 500 new cases diagnosed each year, accounting for 6% of all childhood cancers (Bernstein etÂ€al. 1999). The incidence rate is slightly higher in girls (female:male is 1.09), is slightly higher in black children, and significantly lower in Asian children (Dome etÂ€ al. 2006a). Over 77% of WT patients are diagnosed prior to age 5, with girls and boys presenting at a median age of 3 and 2Â€ years, respectively (Pastore etÂ€al. 2006).

Current Treatment Strategies Treatment for WT utilizes a combination of surgery, chemotherapy, and radiation. Traditionally, the National Wilms Tumor Study Group (NWTSG) has favored upfront surgical resection followed by adjuvant chemotherapy and radiation as needed, whereas the International Society of Pediatric Oncology (SIOP) has favored neoadjuvant chemotherapy followed by resection and additional chemotherapy and radiation as needed. Both approaches have yielded good results. Antimitotic agents, anthracyclines, topoisomerase II inhibitors, oxazophosphorines, platinum analogs, and now camptothecins have all shown clinical activity in WT patients and form the basis of current therapeutic strategies, which aim to maximize cure and simultaneously minimize toxicity.

J.S. Domeâ•›(*) Division of Oncology, Center for Cancer and Blood Disorders, Children’s National Medical Center, 111 Michigan Avenue NW, Washington, DC 20010, USA e-mail: [email protected] P.J. Houghton and R.J. Arceci (eds.), Molecularly Targeted Therapy for Childhood Cancer, DOI 10.1007/978-0-387-69062-9_19, © Springer Science+Business Media, LLC 2010

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The current SIOP-2001 protocol employs a treatment algorithm built upon (1) randomization of stage II and stage III patients to upfront vincristine/dactinomycin versus vincristine/dactinomycin/doxorubicin; and (2) postoperative chemotherapy dependent on histologic features assessed at the time of postneoadjuvant chemotherapy nephrectomy (Metzger and Dome 2005). The COG Renal Tumor Committee (RTC), which has replaced the NWTSG, has recently analyzed the results of prior NWTSG and SIOP studies and designed new biologic and clinical pediatric renal tumor trials. At the helm of this new design is a risk stratification protocol termed AREN03B2, whereby pathology slides, tumor cytogenetics (loss of heterozygosity at chromosomes 1p and16q), operative reports, and radiology studies are centrally reviewed by national experts. Based on a central risk assignment, patients are enrolled in one of four therapeutic studies: AREN0532 (Very low and standard-risk favorable histology (FH) Wilms tumor), AREN0533 (Higher-risk favorable histology Wilms tumor), AREN0321 (high-risk renal tumors), and AREN0534 (Bilateral Wilms tumor or bilaterally-predisposed). AREN03B2 also serves to maintain a biological samples bank for future research and monitors outcomes for patients not eligible for a COG therapeutic study. Ninety percent of WT patients have favorable histology, of which 10 to 15% relapse, 10% have anaplastic histology, of which 40% relapse, and 7% present with or develop bilateral disease, of which 30 to 40% relapse (Green 2004; Dome etÂ€al. 2006b; Pastore etÂ€ al. 2006). Thus, approximately 15% of WT patients develop recurrent disease and are at risk for significant morbidity and mortality. These patients require more effective and tolerable treatment approaches.

Molecular Pathways in Wilms Tumor Although Wilms tumor was one of the original models for Knudson’s two-hit hypothesis, (Knudson and Strong 1972) the genetically heterogeneous nature of WT pathogenesis is now undeniable. Over the last two decades, preclinical and clinical investigation have led to significant advances in our understanding of WT histologic differentiation and pathogenesis, WT predisposition, genetics and epigenetics, putative WT tumor suppressor genes and oncogenes, and of WT signal transduction pathway integrity and function. From such research, clinically meaningful prognostic variables have emerged enabling the application of risk-stratified therapy based on WT histology and genetic features. In addition, such advances provide a preliminary understanding of the cancer’s heterogeneous molecular profile, forming the basis for the development and application of new molecular targeted therapies. The complexity of Wilms tumor genetics is reflected by the number of Wilms tumor susceptibility genes and loci that have been identified (TableÂ€1). Rather than focusing on individual genes, this review discusses Wilms tumor biology in the context of molecular pathways. We first discuss pathways that are defective in Wilms tumor and then review preclinical efforts to test novel therapeutic agents.

20% 9%

Deletion Mutation 3p21 CTNNB1 (b-Catenin) Mutation 17q12-q21 FWT1 Mutation-germline 19q13.3-q13.4 FWT2 Mutation-germline LOH 7p14-15 POU6F2 Mutation-germline LOH 1p13 DCLRE1B LOH 1q25.3 CACNA1E Amplification 1q25-q32 HPRT (Parafibromin) LOH 5q21-q22 APC LOH MSI 6q21 HACE Hypermethylation 9q P16INK4a LOH Methylation 11q23.2 SKCG-1 Loss (PCR); LOH? 15q IGFR1 Gain 16q E-Cadherin LOH; mutation

DCC

18q21.1

LOH MSI

21.6% 7.8% 15% Unknown Unknown Unknown 2/24 16% Unknown 8% 4/10 30% 16% 4/5 11.8% 23.7% 38% 12% 2/20; 8/35

WTX

Xq11.1

Perotti etÂ€al. (2004)

Reference Gessler etÂ€al. (1994) Baudry etÂ€al. (2000) Diller etÂ€al. (1998) Grundy etÂ€al. (1994, 1996) Ravenel etÂ€al. (2001, 2002) Pritchard-Jones and Vujanic (2006) Grundy etÂ€al. (1994, 1996) Rivera etÂ€al. (2007) Major etÂ€al. (2007) Koesters etÂ€al. (1996) Rahman etÂ€al. (1996, 1998) McDonald etÂ€al. 1998

Tumor suppressor; apoptosis

Tumor suppressor? Oncogene; growth, Anti-apoptosis Adhesion; b-catenin

Ubiquitination CDK inhibition

Fernandeaz etÂ€al. (2001) Natrajan etÂ€al. (2007b) Arcellana-Panlilio etÂ€al. (2000) Singh and Roy (2006) Natrajan etÂ€al. (2007c) Schulz etÂ€al. (2000) Safford etÂ€al. (2005) Ramburan etÂ€al. (2004)

DNA repair; telomere Natrajan etÂ€al. (2007a) Cav2.3 – PKC; MEK/ERK; EGR; b-Catenin Natrajan etÂ€al. (2006a) RNA processing; b-catenin Zhao etÂ€al. (2007) b-Catenin Ramburan etÂ€al. (2005)

Unknown

Oncogene; b-catenin Unknown Tumor suppressor?

Tumor suppressor; b-catenin

TableÂ€1â•… Putative genetic loci/genes involved in WT pathogenesis Locus Gene Genetic event Frequency Function/pathway 11p13 WT1 Mutation 10% Tumor suppressor Sporadic 10% Germline 4% LOH 30% 11p15.5 WT2 (IGF2/H19) LOI 25–40% Oncogene – growth; Anti-apoptosis LOH 35%

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IGF Signaling The role of IGF signaling in cancer has received significant attention concurrent with the evolution of IGFR1 inhibitors that are now in early phase clinical trials. In general, via presumed pleiotropic mechanisms, IGF signaling provides an antiapoptotic signal, mediates proliferation, and promotes tumorigenesis (Werner and Le Roith 1997; Kurmasheva and Houghton 2006; Reidemann and Macaulay 2006). The net effect of alterations in the IGF signaling pathway is challenging to predict, (Samani etÂ€ al. 2007) however, and growth factor receptor cross talk, particularly with the EGFR pathway, is adding complexity and possibly opportunity. IGF2, a well characterized fetal mitogen, has long been implicated in the pathogenesis of Wilms tumor. IGF2 is an imprinted gene that resides at chromosome 11p15.5, the locus for Beckwith-Wiedemann Syndrome (BWS), a cancer predisposition syndrome associated with a 5% risk of WT. (Koufos etÂ€al. 1989; Ping etÂ€al. 1989) Because 11p15.5 contains several imprinted genes that are implicated in BWS, the precise gene that incites WT is not completely elucidated, though IGF2 has emerged as a leading candidate (Cooper etÂ€al. 2005; Prawitt etÂ€al. 2005; Alger etÂ€al. 2007). IGF2 is expressed at increased levels in WT and in embryonic kidney compared with adjacent normal kidney or adult kidney, respectively (Scott etÂ€ al. 1985; Reeve etÂ€al. 1985). A common molecular defect in WT and BWS is loss of imprinting (LOI) of IGF2, which involves reactivation of a normally silent maternally-derived allele (Ogawa etÂ€al. 1993). Increased IGF2 protein expression mediated through LOI is found concurrent with downregulation of H19, another gene at the 11p15 locus, via parental-origin specific DNA methylation of the H19 promoter (Steeman etÂ€al. 1994; Moulton etÂ€al. 1994). Hypermethylation of H19 blocks binding of the insulator protein CTCF, permitting IGF2 activation via an enhancer shared by IGF2 and H19 (Mummert etÂ€al. 2005; Hancock etÂ€al. 2007). The frequency of IGF2 LOI in sporadic WT is reported to be between 25% and 40%, indicating that defects in IGF2 signaling are not restricted to BWS-related WT (Ravenel etÂ€al. 2001; Pritchard-Jones and Vujanic 2006). Another mechanism reported to increase the effective gene dosage or protein product of IGF2 is maternal allele specific loss of heterozygosity (LOH) at 11p15. LOH of 11p15, present in approximately 35% of WTs (Grundy etÂ€al. 1996), is characterized by selective inactivation of the maternal allele and duplication of the paternal allele, yielding a relative increase in the IGF2 “oncogene” (Dome etÂ€al. 2006a). Aberrant imprinting of the IGF2 receptor gene (IGFR2) also can increase the IGF2 dosage because the IGFR2 receptor targets IGF2 for degradation (Xu etÂ€al. 1997). Decreased expression of paternal IGFR2 was noted in 7/16 (44%) of kidneys and associated WTs. IGF2 released by WT cells can send a growth-promoting autocrine signal via its receptor, IGFR1 (Zumkeller etÂ€ al. 1993; Qing etÂ€ al. 1996; Schmitt etÂ€ al. 1997). Interestingly, the protein product of WT1, the first Wilms tumor suppressor gene, has been demonstrated to suppress IGF2 transcription, possibly important in negatively regulating blastemal cell proliferation at various developmental stages (Drummond etÂ€al. 1992).

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IGFR1, a transmembrane heterotetramer tyrosine kinase that is structurally and functionally related to the insulin receptor, transmits a growth-promoting signal via the PI3kinase/AKT/mTOR and RAS/RAF/MAPK pathways when bound to IGF2 (Reidemann and Macaulay 2006). Like IGF2, IGF1R has been found to be increased in WT at the transcript, as well as protein levels, (Gansler etÂ€al. 1988) and it is similarly subject to transcriptional repression by WT1 (Werner etÂ€ al. 1993). Recent investigation has documented gain of IGFR1 gene copy number and increased IGFR1 protein expression in WT, a finding associated with 15q gain and with relapse (Natrajan etÂ€al. 2006b, 2007c). In summary, data supporting IGF2/IGFR1 as a key pathway to target in WT include its frequent upregulation via various genetic and epigenetic events, its known growth promoting effects, its ability to stimulate autocrine WT growth, and its association with relapse. IGF signaling also activates numerous downstream targets amenable to therapeutic targeting (Fig.â•›1) and activates additional oncogenic pathways such as the b-catenin pathway, the latter via multiple signaling cascades including RAS/RAF activation and GSK-3b inhibition (Desbois-Mouthon etÂ€al. 2001).

IGF and mTOR Signaling Pathways IGF1 IGFR

RAS

IGFR PI3K Akt

IRS1

Raf GSK-3b

Raptor 4EBP1

MEKK

mTOR

Foxo

eIF2B ERK

eIF4E

p70S6K

Protein Synthesis

Fig.Â€ 1â•… IGF and mTOR signaling pathways: upon binding its receptor IGFR1, IGF transmits a signal via IRS1 downstream through the RAS and AKT/mTOR pathways. IGFR and its downstream pathway components subject to pharmaceutical targeting are shaded

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WNT-b-catenin Pathway The WNT-b-catenin signaling pathway plays an important role during embyrogenesis, tissue regeneration, and tumorigenesis. b-catenin-driven transcription regulates multiple cellular processes including survival/apoptosis, differentiation, proliferation, and motility. Overactive WNT signaling has been linked to both tumorigenesis and cancer progression in numerous cancer models, making this pathway a desirable target for molecular therapeutic intervention (Takahashi-Yanaga and Sasaguri 2007; Herbst and Koligs 2007). WNT-4 is known to autoinduce mesenchymal to epithelial transition of normal nephrons in utero. Dysregulation of this pathway has been postulated to be involved in WT transformation. Mutations in b-catenin, downstream of WNT-4, and other WNT receptors, have been found in approximately 15% of WTs, and such mutations lead to a loss of critical b-catenin phosphorlyation sites leading to overactive b-catenin signaling (Koesters etÂ€ al. 1996). There exists a striking association between b-catenin mutation and WT1 mutation, whereby approximately 75% of WTs with WT1 mutations also harbor b-catenin mutations (Maiti etÂ€al. 2000; Li etÂ€al. 2004). The b-catenin pathway was further implicated in WT with the recent discovery of the WTX gene. Approximately 30% of WT have deletions or point mutations of WTX, which encodes a protein that forms a complex with b-catenin and other proteins, ultimately promoting ubiquitination and degradation of b-catenin, thereby attenuating TCF-mediated transcription (Major etÂ€al. 2007). A defective WNT-b-catenin pathway (overactivated), due to either b-catenin or WTX mutation, is found in up to 45% of WTs (Major etÂ€al. 2007; Nusse 2007) (Fig.â•›2). Other proteins found to be dysregulated in WT have been linked to b-catenin signaling. APC protein, a binding partner that targets b-catenin for proteasomal degradation, has been found to be subject to LOH and microsatellite instability in 30% and 16% of WT tested, respectively (Ramburan etÂ€al. 2005). These findings further widen the spectrum of WTs likely to be affected by b-catenin signaling. In WT, b-catenin expression has been demonstrated to concentrate in the membranous component (93 to 95% of WT), and less so in the cytoplasmic component (58% of WT), of epithelial, blastema, and stromal cells (Alami etÂ€ al. 2003a; Ramburan etÂ€al. 2005, 2006). In one large study, b-catenin and E-cadherin expression was noted in 133/140 (95%) and 75/140 (54%) of WTs tested, respectively. Given its high prevalence, b-catenin expression in WT, in general, lacks prognostic value (Ramburan etÂ€al. 2006). However, WT with a loss of membranous staining and shift toward cytoplasmic staining, demonstrated a statistically significant shorter survival (Ramburan etÂ€al. 2005). Nuclear accumulation is notably increased in metastatic WT (Alami etÂ€ al. 2003a) and in WT with associated activating b-catenin mutations (Koesters etÂ€ al. 2003). E-cadherin, a binding partner of b-catenin at the cellular membrane, was found to be increased in pretreated WT specimens (Ramburan etÂ€ al. 2006) and is expressed at low levels in metastatic tumors with nuclear b-catenin localization (Alami etÂ€al. 2003a). cDNA microarray studies of b-catenin-mutated WT versus nonmutated WT suggest that PITX2, APCDD1, and two endothelin-related proteins EDN3 and EDNRA are

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Wnt /Beta-Catenin Signaling Wnt

E-Cadherin

Frizzled b-Catenin Cytoplasm

GSK3

Axin

b-Catenin

APC

WTX

Ub b-Catenin Nucleus Proteasomal Degradation b-Catenin CBP Survivin, c-Myc, Cyclin D1, p21, MMP-7, Axin-2, CD44, PPAR-g TCF-1, etc

LEF/TCF

Fig.Â€2â•… WNT/b-Catenin signaling: upon binding its receptor FRIZZLED, WNT transmits a signal via b-catenin that leads to b-catenin/TCF dependent transcription. Pathway components subject to genetic or epigenetic modulation in WT have hatched lines. DiSHEVELED and its downstream pathway components subject to potential pharmaceutical targeting are shaded

activated by b-catenin in WT, as are several likely compensatory upstream inhibitors of WNT signaling such as WIF1 and PRDC. Muscle-related genes are highly upregulated in b-catenin-mutated tumors, perhaps explaining the common myogenic changes found histologically in such tumors (Zirn etÂ€al. 2006). There has been much attention by the pharmaceutical industry in efforts to find b-catenin pathway inhibitors (Barker and Clevers 2006; Takahashi-Yanaga and Sasaguri 2007; Herbst and Koligs 2007). Inhibitors have been developed that block WNT-b-catenin signaling at the level of the Disheveled (Dvl) protein, which transduces Wnt signals from the receptor Frizzled (Fz) (Shan etÂ€al. 2005; DeAlmeida etÂ€al. 2007), at the putative binding site for b-catenin and Tcf (Lepourcelet etÂ€al. 2004), and at the site of cAMP response element-binding protein (CREB) binding protein (CBP) (Emami etÂ€al. 2004; Eguchi etÂ€al. 2005; Ma etÂ€al. 2005). ICG-001,

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which downregulates b-catenin/Tcf signaling via this latter pathway, has been shown to downregulate survivin expression and induce selective apoptosis in transformed but not normal colon cells, as well as demonstrate inÂ€ vivo activity in the SW620 colon cancer xenograft model (Emami etÂ€ al. 2004; Barker and Clevers 2006). Additional compounds proposed to be b-catenin signal inhibitors include quercitin (Park etÂ€al. 2005), lithium, flavinoids, and curcumin (Takahashi-Yanaga and Sasaguri 2007; Ohori etÂ€al. 2006). Networking between the WNT-b-catenin signaling pathway with the FGF, Notch, BMP, and Hedgehog signaling pathways is important for stem cell and progenitor cell homeostasis, disruption of which can result in cancer (Katoh 2007). b-catenin signaling pathway overlap has also been demonstrated for the EGF and PDGF pathways via EGF or PDGF-dependent b-catenin phosphorylation (Takahashi-Yanaga and Sasaguri 2007). b-catenin downstream effectors such as VEGF, Cyclin D1, survivin, and COX-2 are also potential targets for molecular inhibition (Herbst and Koligs 2007). Thus, further preclinical investigation of inhibiting multiple signaling pathways such as b-catenin in combination with EGFR, PDGR, IGF, or VEGF in WT is of interest. Similarly, simultaneous inhibition of both b-catenin transcription as well as downstream effectors such as VEGF, survivin, Cox-2, or Cyclin D1 (CDKs) may prove clinically meaningful.

EGFR Pathway Limited ex vivo data are available on EGF signaling in WT. Downregulation of EGFR from WT1-mediated transcriptional activity has been demonstrated in PC-12 neuronal cells as well as in osteosarcoma lines, with WT1-mediated apoptosis in the latter mitigated by constitutive EGFR expression, suggesting a protective role of EGFR in these models. However, EGFR signaling is normal in patients with Denys Drash Syndrome with germline WT1 mutations. (Liu etÂ€ al. 2000; Englert etÂ€al. 1995; Vicanek etÂ€al. 1997) EGFR, and specifically HER2 expression, has been shown in 45% of blastema WT, 53% of WT with epithelial differentiation, and 14% of stromal predominant WT. (Ghanem etÂ€al. 2001a; Salem etÂ€al. 2006) Importantly, anti-EGFR monoclonal antibodies have shown effect inÂ€vivo in WT xenograft models, (Pinthus etÂ€al. 2004; Yokoi etÂ€al. 2003) and in the COG Phase I study of the small molecule EGFR inhibitor gefitinib, two WT patients derived clinical benefit in the form of stable disease for 8 to ³60Â€weeks (Daw etÂ€al. 2005).

Angiogenesis The role of angiogenesis, and specifically, the role of vascular endothelial growth factor (VEGF) has drawn much attention in the oncology arena, and VEGF inhibitors are now readily available for clinical use. VEGF is induced by many cellular stimuli including HIF-1a, and functions as an endothelial cell mitogen. VEGF and

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HIF-1a have been shown to be co-expressed in all WTs, with one study demonstrating equivalent distribution of VEGF expression between the blastemal and epithelial components (Karth etÂ€al. 2000). Elevated levels of the proangiogenic protein basic fibroblast growth factor (bFGF) in the urine of pediatric WT patients as well as elevated levels of VEGF in the sera of WT patients have each been shown to correlate with tumor stage, to decrease following surgery, and postoperative increasing levels suggest relapse or persistent disease (Lin etÂ€ al. 1995; Blann etÂ€ al. 2001; Skoldenberg etÂ€al. 2001). Similarly, high WT microvessel density (MVD), a marker of tumor vascularity, has been shown to correlate with a worse prognosis (Ozluk etÂ€al. 2006). WTs characterized to have a decreased WT1 exon 5+/− ratio are associated with downregulation of VEGF, demonstrating differential levels of VEGF expression based on WT1 isoform expression pattern (Baudry etÂ€al. 2002). VEGF and VEGFR isoforms and WT subcomponent analysis have also demonstrated that VEGF (VEGF-A) and its receptor VEGFR-1 (Flt-1) are expressed in 52% and 47% of blastemal cells, respectively, and positive expressing cells were associated with higher MVD and risk for clinical progression (Ghanem etÂ€ al. 2003). Similarly, VEGF-C expression is noted in 100% and 30% of WT blastema and stromal cells, respectively. VEGFR-2 is highly expressed in stroma and epithelial cells, but absent from WT blastema tested. While VEGF-C was shown to be associated with clinical progression by Nowicki etÂ€al. (2007), all WTs studied were pretreated and multivariate analysis was not conducted to rule out independence of this purported prognostic factor from the confounding factor of posttreatment residual blastema. The finding that blastema lacks VEGFR-2 is of clinical importance as some antiVEGF targeted agents are VEGF receptor-specific.

Apoptotic Pathways Conflicting data have emerged regarding the relative prognostic value of various apoptosis activators or inhibitors in WT. In pediatric renal tumors, a high survivin:Fas ratio demonstrated a positive predictive value for tumor recurrence of 85.7% and negative predictive value of 71.4% (Takamizawa etÂ€al. 2001). However, subsequent investigation in 92 primary WTs showed survivin mRNA increased in WT compared with expression levels in adjacent kidney, but neither survivin nor caspase 8, SMAC, Bid, and Fas (CD95) were independently, or in ratio, associated with stage or risk for tumor recurrence (Miller etÂ€ al. 2005). Both antiapoptotic BCL-2 and pro-apoptotic BAK have been demonstrated to be under WT1-mediated transcriptional control, and therefore suggest a possible role in WT tumorigenesis (Fukuzawa etÂ€al. 2004; Morrison etÂ€al. 2005). Additionally, BCL-2 expression has been found in nephrogenic rests (Wunsch etÂ€al. 2001), possibly providing protection from apoptosis during early tumor development. However, several investigations have not found any prognostic value of BCL family member expression, either antiapoptotic (BCL-2, BCL-X(L/S)) or proapoptotic (BAK), in WT (Re etÂ€al. 1999; Tanaka etÂ€al. 1999). One study demonstrated increased BCL-2 and decreased

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BAX in WT blastema of increasing pathologic stage, with an increasing BCL-2/ BAX ratio indicative for tumor progression (Ghanem etÂ€al. 2001b). Nonetheless, the compromised integrity of the apoptotic program via abnormal expression of proteins involved in the apoptotic pathway in WT is supported by activation of the anti-apoptotic effects of the IGF pathway, (Werner and Le Roith 1997; Kurmasheva and Houghton 2006; Reidemann and Macaulay 2006) and by TP53 mutation inducing attenuated apoptosis in anaplastic histology WT (Bardeesy etÂ€al. 1995). Additionally, WT blastema commonly overexpresses the MYCN oncogene, known to have both proliferative and pro-apoptotic effects, the latter typically inhibited in cancer cells via perturbation of other cellular proteins required for apoptosis execution (Shaw etÂ€al. 1988). Similar to MYCN amplified neuroblastoma and other tumor models, pro-apoptotic caspase-8 is silenced via promoter methylation in greater than 40% of WTs. (Morris etÂ€al. 2003) WT also commonly express DR5, a receptor for both TNF-related apoptosis inducing ligand (TRAIL) and agonist DR5 monoclonal antibodies, each of which transmit a pro-apoptotic signal by directly activating the apoptotic pathway and each currently in early phase clinical development (Takamizawa etÂ€al. 2000). A biomarker of active apoptosis, cytokeratin-18-related tissue polypeptide-specific (TPS) antigen, typically released by epithelial cells undergoing apoptosis, is elevated in nearly all epithelial and blastemal WT specimens tested, suggesting that it may be a reasonable biomarker of tumorspecific apoptosis, easily assessable in the serum of WT patients who undergo future therapies targeting the apoptotic program (Rebhandl etÂ€al. 2001).

Cell Cycle WT1 has been shown to induce G1 phase arrest, possibly via induction of p21CIP1, abrogated by overexpression of cyclin/cdk complexes (Kudoh etÂ€al. 1995; Englert etÂ€al. 1997). Suppressed p16 expression either via promoter methylation or alternative genetic or epigenetic events has also been well documented in WT (ArcellanaPanlilio etÂ€ al. 2000; Natrajan etÂ€ al. 2007b). Cyclin D1 is a well established transcriptional endpoint of overactive b-catenin signaling. Thus, WT1 mutation, p16 silencing, or activated b-catenin signaling all could lead to uninhibited cyclin/ cdk activity, enabling rapid cell cycle transitioning and proliferation. A preliminary association between CDK4 overexpression and relapse in WT has been suggested (Faussillon etÂ€al. 2005). These data support further exploration of cell cycle inhibition, perhaps CDK4 inhibition, in the treatment of WT.

HGF/c-MET Hepatocyte growth factor (HGF) and its receptor c-MET are involved in cell migration, differentiation, cell growth, angiogenesis, apoptosis inhibition, and increased cancer metastases (Christensen etÂ€al. 2005; Sattler and Salgia 2007). HGF binding

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to c-MET has also been shown to induce c-MET/b-catenin disassociation, leading to increased free b-catenin, analogous to the effects of EGF on E-cadherin/bcatenin. The complexity of such pathway cross-talk is exemplified by the findings that HGF treatment of cancer cells increases b-catenin/TCF transcription, b-catenin fosters HGF ligand-independent c-MET mediated cell scattering, and suppression of b-catenin inhibits the HGF-induced motile phenotype (Rasola etÂ€al. 2007). HGF has been shown to be expressed in the serum of WT patients at levels three times higher than controls (Skoldenberg etÂ€al. 2001). Additionally, HGF and c-MET colocalize in WT and are associated with an increased proliferative index (Alami etÂ€ al. 2002). Further investigation of HGF/c-met signaling and inhibition is warranted in WT. A number of therapeutics, both small molecule inhibitors (ARQ-197, SU11271, SU11274, SU11606, SGX523, PHA665752, and PF2341066), or antibodies are currently in early clinical development in adults. Both humanized (AV299) and fully human antibodies (AMG102) against HGF, as well as MET decoys (CGEN214) that prevent ligand binding, or receptor dimerization, and onearmed monoclonal antibodies against the MET receptor are in development.

Other Pathways Heat-shock proteins (HSP) 27 and 70 have been found to be co-expressed with one another as well as with glutathione peroxidase in WT, with HSP70 expression confined to blastema and epithelial components (Stammler and Volm 1996; Efferth etÂ€al. 2001a). High HSP70 expression, as well as HSP90a expression, was found in WT predominantly from children who survived (Efferth etÂ€al. 2001a; Yang etÂ€al. 2006). WT1 mediated inhibition of cellular proliferation has been shown to require association with HSP70 (Maheswaran etÂ€al. 1998). From these data, it is not likely that HSP inhibition will yield an overall positive effect on WT patients and could, in fact, be utilized to discourage such clinical investigation. Similarly, c-KIT has not been found to be strongly nor diffusely overexpressed in WT, with one study showing 0/6 WTs expressing c-KIT, diminishing enthusiasm for the employment of single agent c-kit inhibitors for WT (Smithey etÂ€al. 2002; Miliaras etÂ€ al. 2004). TRK A and B receptors are found predominantly in WT stromal compartments, whereas TRK C receptors are found within WT epithelial structures (Donovan etÂ€al. 1994). However, high levels of full-length TRKB transcripts in WT are associated with a worse outcome, suggesting a possible role for TRKB inhibition (Eggert etÂ€al. 2001). Epigenetic methylation of various genetic loci in WT, potentially amenable to demethylation via various clinically available demethylating agents, notably includes the tumor suppressor caspase-8 (methylated in 43% of WT). However, demethylating agents could have potentially undesirable effects at other loci, including the locus for O-6 methylguanine DNA methyl transferase (MGMT, methylated in 30% of WT) (Morris etÂ€al. 2003). Upregulation of MGMT expression may mediate cellular resistance to alkylator-based therapy. Interestingly, 60% of

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WTs are characterized by general hypomethylation, particularly in centromeric satellite alpha DNA, possibly contributing to karyotypic instability and WT oncogenesis or tumor progression (Ehrlich etÂ€al. 2002, 2003). The net effect of demethylating agents on WT awaits preclinical and clinical exploration. The multidrug-resistance protein (MRP) P-glycoprotein is expressed in the majority of WT at various levels, predominantly in blastemal and epithelial compartments. In WT, MRP levels have been associated with TP53, HSP70, and LRP/ MVP expression, but have not been associated with tumor grade, stage, or clinical course (Efferth etÂ€al. 2001b; Camassei etÂ€al. 2002).

Preclinical Identification of New Agents for Wilms Tumor Given the overall excellent prognosis for WT, few patients are candidates for phase I and II clinical trials. With a plethora of agents coming through the drug development pipeline, oncologists need a rational approach to prioritize agents or combinations of agents for clinical study. The identification of molecular pathways that are dysregulated in Wilms tumor is a starting point to identify drugs of interest. However, as illustrated in the sections above, these molecular pathways are complex and interactive, so it is difficult to predict the physiological effect of targeting a component of a particular pathway. To that end, investigators have developed preclinical WT models with which to test the efficacy of novel agents.

In Vitro Investigation of WT Attempts at establishing reliable WT cell lines have unfortunately met limited success. The T3/73 cell line was established from a WT resected from a 9Â€month old boy with aniridia. Initial reports demonstrated growth factor independent rapid growth of desmin positive fusiform cells, capable of undergoing over 100 passages (Kumar etÂ€al. 1987). Subsequent investigation showed this cell line to be dependent on co-exposure of IGF2 for inÂ€vitro proliferation (Zumkeller etÂ€al. 1993). Similarly, WTCL-1 – WTCL-4 cells established using human WT were used to demonstrate that IGF2 is released by WT cells and that WT-secreted IGF2 sends a growth-promoting autocrine signal via IGFR1 (Qing etÂ€al. 1996; Schmitt etÂ€al. 1997). Brown etÂ€al. (1989) established five cell lines produced from five Wilms’ tumors; however, all cultures were defined by a finite lifespan, no cells showed the expected characteristics of the putative WT stem cell, and all failed to show a “transformed” phenotype. Garvin etÂ€ al. (1985, 1987) report specialized media and conditions that permit the growth of blastemal or skeletal components of WT inÂ€vitro, though limited passage and restrictive conditions are somewhat prohibitive for general use. However, the blastemal WT cell line, W13, established in the Garvin laboratory,

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was used to demonstrate the growth suppressive effects of suramin and ATRA in WT, possibly due to inhibition of IGF2 mediated crosslinking of IGFR1 and IGFR2, and by modulation of MYCN, IGF2, and IGF-binding proteins, respectively (Vincent etÂ€ al. 1996a, b). The G401 and SKNEP-1 cell lines, utilized in numerous inÂ€vitro and inÂ€vivo published studies, have now been shown to represent rhabdoid tumor of the kidney and Ewing’s Sarcoma, respectively (Garvin etÂ€ al. 1993; Smith etÂ€al. 2006). In a similar manner, one must question the origin of the “sarcomatoid” WT model, WCCS-1 (Talts etÂ€al. 1993). Most recently, investigators at The Hospital for Sick Children in Toronto have established the WiT 49 cell line from an anaplastic histology (AH) WT lung metastasis. The cell line harbors a TP53 mutation, co-expresses HGF and c-MET, lacks E-cadherin, expresses high levels of b-catenin co-immunolocalized to membranous c-MET, and expresses moderate levels of g-catenin and ezrin. Constitutive activation of MMP9 and latent MMP2 suggest active b-catenin-driven transcription (Alami etÂ€al. 2003b). WiT 49 has subsequently been used by investigators to further demonstrate (1) podocalyxin (PODXL), an antagonist of cellular adhesion possibly via interaction with ezrin, is positively regulated by WT1 and transcriptionally repressed by TP53 in WT, (Stanhope-Baker etÂ€al. 2004), (2) that siRNA-mediated downregulation of C/EBPB, a transcription factor involved in proliferation and differentiation and implicated in lymphoma tumorigenesis (Piva etÂ€al. 2006) induces apoptosis in WT, (Li etÂ€al. 2005) and (3) that STAT1 is a prosurvival factor in WT pathogenesis (Timofeeva etÂ€al. 2006).

In Vivo Xenograft Testing in WT The dearth of Wilms tumor cell lines prompted the development of WT xenograft models, in which human Wilms tumors are transplanted into immunodeficient mice. Several groups have successfully employed WT xenografts to study novel agents. In 1989, investigators at the Medical University of South Carolina tested the effect of intraperitoneal injection of a human IGFR1 antibody (aIR-3) against nude mice bearing human WT heterotransplants. Models were assessed, indirectly, for IGFR1 function. Following 1Â€week of growth after subcutaneous inoculation, when greater than 2/3 of mice from each treatment cohort (7 to 10 mice) had developed measurable flank tumor growth, mice were treated with intraperitoneal injections of 0.5Â€ mg antibody three times weekly. Mice were sacrificed between 30 and 50Â€days. Overall, at 3Â€weeks into treatment, the tumor diameters of treated and control mice were significantly different, with approximately 80% growth inhibition. Mice crossed over from control to aIR-3 showed initial response, and mice crossed over from aIR-3 to control showed rapid growth of tumor (Gansler etÂ€al. 1989). In this elaborate study, which employed methods that parallel current techniques used for xenograft testing, two FH WT and one AH WT showed statistically significant response to anti-IGFR1 antibody but not to control IgG, providing additional rationale for the testing of anti-IGFR1 therapy in WT patients.

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Preliminary Anti-EGFR and Angiogenesis Testing Two groups of investigators have confirmed the in-vivo activity of anti-erbB2 monoclonal antibodies in human WT xenografts. Pinthus etÂ€ al. (2004) demonstrated that 3 intraperitoneal injections of N29 anti-erb2 monoclonal antibody could prevent WT growth inÂ€ vivo. ErbB2 expression was documented in 13/14 human WT (the one negative being AH WT). Yokoi etÂ€al. (2003) showed that anti-her2/neu monoclonal antibody could suppress tumor growth in her2/neu(+) WT xenografts, but not in her2/neu(−) WT xenografts. Suppression of angiogenesis in the responding WT xenograft was mechanistically important. Decreased antiangiogenesis as a physiologically important mechanism of anti-WT activity was also demonstrated in two xenograft models of FH WT treated with the angiogenesis and collagen type I synthesis inhibitor halofuginone. Decreased angiogenesis leading to tumor growth inhibition was accompanied by a reduction of collagen synthesis, reduced HGF receptor (c-MET), and increased levels of WT1 (Pinthus etÂ€al. 2005). Additional anti-angiogenic agents have been tested in xenograft models of SK-NEP-1. However, SK-NEP-1 has now been shown to be a Ewing’s sarcoma, not a WT (Huang etÂ€al. 2004; Frischer etÂ€al. 2004; Smith etÂ€al. 2006).

Pediatric Preclinical Testing Program WT Xenograft Testing The majority of preclinical inÂ€vivo drug testing in WT xenografts has come out of the lab of Peter Houghton at St. Jude Children’s Research Hospital, either via internal testing or via the National Cancer Institute-sponsored Pediatric Preclinical Testing Program (PPTP). Outside the auspices of the PPTP, the Houghton laboratory has published inÂ€ vivo activity in WT with the histone deacetylase inhibitor depsipeptide and with the antimitotic agents ABT-751 and ixabepilone (Peterson etÂ€al. 2005; Graham etÂ€al. 2006; Morton etÂ€al. 2007). Response criteria, in general, were complementary to that used formally by the PPTP (Houghton etÂ€al. 2007a). ABT-751, a novel oral antimitotic agent that binds tubulin at the colchicine binding site, demonstrated objective tumor regression in 1/6 WT models (Morton etÂ€ al. 2007). Depsipeptide induced tumor regression and tumor stabilization in 1 WT model each, accounting for 2/7 of the sensitive models out of 39 tested models (11 kidney) (Graham etÂ€al. 2006). Ixabepilone induced WT regressions in 5/6 models at 10Â€mg/kg (mouse MTD) and in 1/5 models at 4.4Â€mg/kg, given every fourth day, demonstrating a steep dose response. Accompanying pharmacokinetic studies showed that a dose of 10Â€mg/kg provided an AUC/dose of 5.8Â€mmol/L- h, approximating that achieved in adult patients receiving 40Â€ mg/m2/dose (Peterson etÂ€ al. 2005). Both ABT-751 and Ixabepilone are currently undergoing single agent Phase II study in WT through the COG. Two WT patients were treated on the COG Phase I trial of depsipeptide and like all other patients on study, neither responded (Fouladi etÂ€al. 2006). Given preclinical data suggesting WT susceptibility to HDAC inhibition inÂ€ vivo, it is worth considering more formal exploration of the more potent HDAC inhibitor, SAHA, in WT patients.

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The PPTP utilizes four WT xenografts (and four rhabdoid tumor xenografts) in its panel, all which were derived from human WTs obtained at diagnosis. Two tumors (KT-10 and KT-11) are favorable histology, one tumor has diffusely anaplastic histology (KT-13) and one tumor is characterized as a favorable histology WT with nuclear unrest (KT-5). The efforts and methods of the PPTP are designed to perform xenograft experiments in as controlled a fashion as possible, and all models have undergone microarray characterization to supplement inÂ€vivo activity with possible scientific insight. Thus far, the PPTP has investigated 13 agents for which data have been released. The inÂ€ vivo activity of cisplatin, vincristine, and cyclophosphamide was broad, including significant tumor regressions in WT models (Houghton etÂ€al. 2007a; Tajbakhsh etÂ€al. 2007). Other agents tested showed limited activity and include bortezomib (proteasome inhibitor), (Houghton etÂ€ al. 2008) dasatinib (src inhibitor), (Kolb etÂ€ al. 2007) 17-DMAG (Hsp 90 inhibitor), (Smith etÂ€ al. 2007) ispinesib (anti-mitotic), (Houghton etÂ€ al. 2006) vorinostat (HDAC inhibitor), (Houghton etÂ€ al. 2007b) rapamycin (mTOR inhibitor), (Houghton etÂ€ al. 2007c) AZD2171 and sunitinib (anti-angiogenic agents targeting VEGF), (Maris etÂ€ al. 2007; Houghton etÂ€ al. 2007d) lapatinib (anti-EGFR/AKT), (Houghton etÂ€al. 2007e) and SCH717454 (an anti-IGFR1 inhibitor) (Houghton etÂ€al. 2007f). Unfortunately, tested agents thus far have not provided a large “response signal” in the PPTP WT models. Tumor growth delay in WT models was noted from anti-VEGF treatment, antimitotic agents, and HDAC inhibition. The above data provide the preclinical rationale for continued exploration of novel antimitotic agents in WT. There is also a suggestion that antiangiogenic agents, histone deacetylase inhibitors, and anti-EGFR agents may be worth clinical exploration in WT. Early data with IGFR1 inhibition inÂ€ vivo combined with the molecular characterization of WT suggest that testing IGFR1 inhibitors, now in clinical development, is a worthwhile pursuit in WT patients. b-catenin pathway activation in a majority of WT samples strongly supports further investigation of this pathway as well. Additional ex vivo WT preclinical data suggest that demethylating agents, cell cycle modifiers (CDK 4 inhibitors), and apoptosis activating or modulating agents such as agonist DR5/TR2 or TRAIL analogs may have a role in the future treatment of WT patients. It is difficult to ignore the activity of mTOR inhibitors in other renal neoplasms, (Hudes etÂ€al. 2007) and either single agent or combination testing in WT, either with conventional chemotherapeutic agents or with inhibitors of upstream targets such as IGFR1 is justifiable.

Future Directions and Challenges Barriers to new agent development in WT exist at both the preclinical and clinical level. A major challenge is the lack of reliable, validated WT cell culture systems readily available to interested investigators. Several laboratories are actively working on the establishment of such WT cell lines, as it would both greatly facilitate

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more rapid signaling pathway interrogation as well as provide cost-effective drug activity screening. A second issue requiring refinement is the identification of more robust prognostic tools predictive of treatment-specific response and outcome. Current markers (LOH of 1p and 16q) while useful in general, are nonetheless, rudimentary (Grundy et al. 2005). In addition, in the era of molecular targeted therapy, the identification of biomarkers of disease responsiveness must also be addressed. The genetic and molecular heterogeneity of WT suggests that the application of novel molecular therapies will not be a “one size fits all” situation. The nonuniformity of IGFR1 expression, b-catenin activation, and heterogeneous makeup of additional signaling pathways of current and future interest presents the challenge of matching patient to drug. Thus, in this new era of targeted therapy, we not only continue to battle issues of under- or over-treatment due to poor markers of disease response to conventional therapy, but we must focus our awareness of individual patient tumor biology and consider this in molecular targeted therapy options. Therefore, inclusion of individual tumor biology characterization and pharmacodynamic endpoint monitoring, in conjunction with traditional clinical endpoints, becomes all the more critical. Remaining challenges include the solicitation of pharmaceutical industry support, acquisition of additional supportive preclinical data, establishment of clinical pathways for trial implementation, and patient enrollment. To achieve these ends, the COG RTC has developed tight collaboration with the PPTP and COG Developmental Therapeutics Committee (DVL) to channel both relevant WT models and targets of interest into the screening and testing pipeline provided by those two entities, respectively. The establishment of these collaborations holds much promise for more rapid transit of new agents into the clinical arena, with appropriate correlative investigation necessary to refine assessments of clinical utility based on specific tumor biology. It is noteworthy that the recently opened WT strata of ADVL0524 (Phase II study of Ixabepilone) completed accrual in under 1Â€ year, providing proof that “when built, the patients do come.” Via this pathway, the COG RTC has recently endorsed Phase I and II study of the monoclonal anti-IGFR1 antibody IMC-A12 (Imclone) as well as a Phase II study of the novel oral antimitotic, ABT-751 (Abbot Laboratories). Phase III investigation of new agents or new treatment strategies requires significant patient numbers, remedied by either prolongation of accrual time or by maximizing patient access. The latter has the advantage of facilitating more rapid progress, and can be achieved via international cooperation. Through improved model discovery and refinement of our understanding of relevant signaling pathways in WT, careful patient tumor biological profiling, design of trials with thorough pharmacodynamic and correlative studies, and international collaboration, molecular targeted agents hold promise of enabling maximization of cure with minimization of toxicity for children with Wilms’ tumor.

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Molecular Therapy for Rhabdomyosarcoma Raushan T. Kurmasheva, Hajime Hosoi, Ken Kikuchi, and Peter J. Houghton

Introduction Rhabdomyosarcoma (RMS) is the most common sarcoma of childhood representing about 23% of all sarcomas, and approximately 7% of all pediatric malignancies (Arndt and Crist 1999). Histologically, RMS presents as three major variants, embryonal (ERMS), representing about 60%, botryoid that are usually combined with embryonal tumors, and alveolar RMS (ARMS) that comprise 30% of RMS. The pleiomorphic variant is an unrelated high-grade sarcoma with various degrees of muscle differentiation and is rarely (if ever) diagnosed in children. Embryonal RMS is not associated with any chromosomal translocation, but loss of heterozygosity at 11p15.5 is a common feature (Scrable etÂ€ al. 1987; Scrable etÂ€ al. 1989) associated with loss of imprinting of the IGF-2 locus (Anderson etÂ€ al. 1999). Alveolar RMS is characterized by specific translocations. Approximately 60 to 70% of histologically diagnosed ARMSs involve translocations of t(2;13)(q35;q14) leading to PAX3-FKHR gene fusion (Galili etÂ€ al. 1993), whereas 10% have the t(1;13)(q36;q14) translocation that encodes the PAX7-FKHR fusion (Davis etÂ€ al. 1994). Both translocations generate in-frame fusion between the PAX gene DNA binding domain and the transactivation domain of FKHR. Interestingly, both ERMS and translocation-positive ARMS may have loss of imprinting at 11p15.5 (Anderson etÂ€al. 1999), suggesting that dysregulation of IGF2 may be common to both histologies. Recent studies using expression profiling suggest that histological ARMS that are translocation negative cluster with ERMS, hence the molecular classification of these tumors may differ from the histopathologic classification. Further, a small signature comprising as few as ten genes differentiates translocation-positive from translocation-negative RMS (Lae etÂ€al. 2007). Thus, the understanding of genetic events characteristic of RMS is at a point where these may be applied to differential diagnosis, and potentially to novel treatment strategies. It is well established that P.J. Houghtonâ•›(*) Department of Molecular Pharmacology, St. Jude Children’s Research Hospital, 332 N. Lauderdale Street, Memphis, TN 38105-2794, USA e-mail: [email protected]

P.J. Houghton and R.J. Arceci (eds.), Molecularly Targeted Therapy for Childhood Cancer, DOI 10.1007/978-0-387-69062-9_20, © Springer Science+Business Media, LLC 2010

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ARMSs are more aggressive than ERMS, and have a poorer prognosis (Qualman and Morotti 2002; Breneman etÂ€al. 2003). Further, the prognosis for patients with metastatic disease at diagnosis is significantly worse for ARMS having the t(2;13) (q35;q14) translocation compared to those having the t(1;13)(q36;q14) variant (Sorensen etÂ€ al. 2002). Thus, the genetic alterations impact on chemo- or radiosensitivity. However, it is also clear that any advanced stage RMS still presents a clinical challenge. Essentially, the cure rate for metastatic disease has not changed significantly in 30Â€years, despite intensification of cytotoxic therapy and introduction of novel cytotoxic agents (Pappo etÂ€al. 2007). Thus, while introduction of novel cytotoxic agents, such as the camptothecins that target topoisomerase 1 (Furman etÂ€al. 1999; Pappo etÂ€al. 2007), may ultimately improve outcome, it is probable that it will be at the expense of additional toxicity or necessitate reduced dose intensity of other agents used in the treatment of RMS. Alternative approaches that exploit the molecular characteristics of RMS conceptually appear to offer potential benefit with lower toxicity and with reduced sequellae. The obvious example is that of imatinib mesylate in the treatment of chronic myelogenous leukemia (Druker 2003; Druker etÂ€al. 2006) or gastrointestinal stromal tumors (Rubin etÂ€al. 2007; Siehl and Thiel 2007). However, the complexities of RMS biology suggest that a single “genetic driver” is unlikely, and that combinations of agents that target different molecular abnormalities will be necessary to eradicate these tumors using these rational approaches. Indeed, the development of therapeutic strategies that exploit synthetic lethal interactions that are consequential to the molecular aberrations in these tumors will be the major challenge in developing curative approaches to childhood RMS. Here, we review the reported molecular characteristics of RMS as they relate to the development of molecularly targeted therapy.

What Is the Cell of Origin? It has been proposed that tumor growth may be sustained by a rare population of cells, termed cancer stem cells (Hope etÂ€al. 2003, 2004). These cells, analogous to stem cells in normal self-renewing tissues, have the ability to self-perpetuate, and it has been proposed that they may give rise to recurrent tumor following surgery or chemo-radiation therapy. It is also postulated that failure of conventional treatments is because the stem cell is resistant, and that therapeutics should be targeted to this population. For example, in AML only a small proportion of cells (approximately 1â•›×â•›10−6) in human samples resulted in tumor growth when inoculated immune deficient mice. However, these studies with human AML cells have relied extensively on xenotransplantation techniques where human cancer cells have been inoculated into irradiated nonobese severe combined immunodeficient mice. In contrast, transplantation of syngeneic Em-myc or Em-N-RAS lymphomas, or PU.1−/− AML indicate that inoculation of as few as 10 cells results in lethal disease (Kelly etÂ€al. 2007). Thus, while the cancer stem cell hypothesis is of interest, it will be critical to demonstrate its role in RMS. Currently, there is considerable focus on

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identifying the putative RMS stem cell. Although the histogenesis of RMS is not certain, skeletal muscle characteristics of these tumors sugges t that they originate from fetal rhabdomyoblasts that fail to undergo normal differentiation. In fusion negative RMS, PAX7 is upregulated and may contribute to tumorigenesis in the absence of chimeric PAX transcription factors. In contrast, PAX3 expression is independent of translocations (Tiffin etÂ€ al. 2003). In normal myogenesis, expression of PAX7 is concurrent with PAX3 in the dermomyotome. Expression of PAX3 suppresses myogenic differentiation in migrating myoblasts, whereas PAX7 expression is downregulated in these cells. Thus, high expression of PAX7 but not PAX3 in translocation deficient RMS suggests that these tumors are not derived from proliferating cells in the dermomyotome. PAX7 expression is essential (Asakura etÂ€al. 2002) for specification and maintenance of myogenic satellite cells (Seale and Rudnicki 2000; Seale etÂ€al. 2000), and as myogenic satellite cells are known to express PAX7 (Asakura etÂ€al. 2002) it has been proposed that translocation-negative RMS may be derived from the myogenic satellite cell lineage (Tiffin etÂ€al. 2003). This is consistent with the finding of the hepatocyte growth factor (HGF) receptor, MET, expression in all translocation-negative RMS as MET is expressed in quiescent satellite cells (Cornelison and Wold 1997). That the type of tumor induced by altered pathways in RMS may be dependent on the stage of development of the cell of origin is demonstrated by the studies of Linardic etÂ€al. (2005). Less differentiated human skeletal muscle precursors gave rise to tumors with variable histologies when stably expressing T/t-Ag, hTERT and H-RAS, whereas committed human skeletal muscle myoblasts formed RMS-like tumors of embryonal morphology.

Whole Genome Expression Profiling As discussed briefly above, childhood RMS can be classified histologically as ERMS, ARMS or botryoid. More recently, there have been a series of reports using genome-wide expression that have sought to identify expression signatures that distinguish ERMS from ARMS, and potentially identify drug targets (Wachtel etÂ€al. 2004; Davicioni etÂ€al. 2006; De Pitta etÂ€al. 2006; Goldstein etÂ€al. 2006; Romualdi etÂ€al. 2006; Lae etÂ€al. 2007). Despite differences in approach (for example different platforms for assessing expression), a gene set found to be differentially expressed between fusion-positive and fusion-negative tumors has been identified. The overlap between the top 50 genes that distinguish between ARMS and ERMS in three independent studies (Wachtel etÂ€al. 2004; Davicioni etÂ€al. 2006; Lae etÂ€al. 2007) identified twelve genes that were common. The ranked list overlap analysis from these three studies is shown in Fig.Â€1. Interestingly, the canabinoid receptor (CNR1) is highly expressed in ARMS compared to ERMS, and may be a potential therapeutic target in several human cancers (Flygare etÂ€ al. 2005; McAllister etÂ€ al. 2005; Sarfaraz etÂ€ al. 2005, 2006). A small molecule agonist (WIN-55,212-2) of the canabinoid receptor has been shown to induce apoptosis. An alternative approach may be to develop antibodies that act as agonists, or that selectively deliver a cytotoxic

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Fig.Â€1â•… Ranked list overlap analysis. The overlap between the top 50 genes (probe sets) distinguishing between ARMS and ERMS in three independent studies. Lae etÂ€al. (2007), Wachtel etÂ€al. (2004) and Davicioni etÂ€al. (2006) study is shown graphically. (From reference Lae etÂ€al. 2007 with permission)

agent to tumor. The largest expression profiling study (Davicioni etÂ€ al. 2006) reported analysis of 139 RMS, and revealed a distinct common expression profile for PAX3-FKHR expressing tumors compared to those that were fusion negative, Fig.â•›2. Thus, by this analysis there is a common profile irrespective of the fusion (PAX3 or PAX7), and this differs from fusion-negative tumors, Fig.Â€ 2. In another cytogenetic and molecular analysis of primary tumors AURKA, the gene encoding aurora kinase A required for chromatid axial shortening (Mora-Bermudez etÂ€ al. 2007), was found to be overexpressed in all RMS studied. Whether or not overexpression signifies this mitotic kinase as a tumor-selective drug target needs to be established. Several aurora kinase A inhibitors are in clinical development (Hoar etÂ€al. 2007; Manfredi etÂ€al. 2007), and are currently being evaluated against childhood cancer preclinical models.

Chimeric Transcription Factors and Oncogenes PAX3-FKHR While expression profiling has been of some utility in identifying potential therapeutic targets, the development of novel anticancer agents to treat patients with tumors such as ARMS needs elucidation of the pathogenesis based on the abnormal gene and protein expression and aberrant signaling pathways in the tumor. Various

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Fig.Â€2â•… PAX-FKHR expression in ARMS is associated with a unique gene expression profile that is independent of tumor histology. (a) Multidimensional scaling analysis of 139 primary rhabdomyosarcoma tumors based on semisupervised analysis reveals tight clustering of most alveolar tumors, in contrast to the heterogeneous distribution of embryonal and spindle/botryoid tumors included in the main alveolar cluster are three mixed histology alveolar/embryonal tumors.The legend indicates the histologic diagnoses. (b) Replot of (a) based on normalized QRT-PCR expression levels of PAX3-FKHR and PAX7-FKHR for 59 tumors with alveolar or mixed alveolar/ embryonal histology. Relative PAX-FKHR mRNA levels are depicted by colored dots as indicated in the color scale. Note that the two tumors with low but detectable PAX-FKHR expression (blue dots) had median PAX-FKHR expression levels 1,000-fold less than alveolar tumors expressing high levels of PAX-FKHR (orange to pink dots). Rhabdomyosarcoma samples for which no QRTPCR data were available are also indicated (gray dots). Note that all nonARMS tumors were shown at diagnosis to be fusion negative by conventional RT-PCR. (From reference Davicioni etÂ€al. 2006 with permission)

genes have been proposed as the downstream candidates for transcriptional activation by the chimeric PAX3-FKHR gene. Earlier studies focused mainly on the observation that the PAX3-FKHR fusion protein exerts greater transcriptional activity than that of wild type PAX3 protein (Fredericks etÂ€al. 1995). Several studies, in particular, used microarrays and expression profiling to determine downstream consequences of the wild type PAX3 and PAX3-FKHR fusion proteins. In a cell culture-based approach to identify targets, the wild type gene or fusion gene was transfected and ectopically expressed in a cell line. A set of target genes, which were similarly regulated by both the wild type and fusion proteins, were identified. However, another set of target genes, which were regulated only by PAX3-FKHR, or were repressed by PAX3 but induced by PAX3-FKHR, were also identified. Of importance is that the specific downstream target genes identified were dependent on the cell line in which the fusion gene was ectopically expressed, Tableâ•›1. An example of this cell type specificity is the downregulation of BMP4 by PAX3-FKHR in SaOS-2 cells and the upregulation of BMP4 by PAX3-FKHR when expressed in RD ERMS cells. In addition, all these results were obtained from experiments using the method of forced expression, and using cells other than alveolar rhabdomyosarcoma cells, such as mouse fibroblast cells (Zhang and Wang 2003), osteosarcoma cells (Begum etÂ€al. 2005) or embryonal rhabdomyosarcoma

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TableÂ€1â•… Genes regulated by ectopic expression of PAX3 or PAX3-FKHR in nonARMS cells Gene PAX3 PAX3-FKHR Cell type References → ↑ Human osteosarcoma, SaOS-2 Begum etÂ€al. (2005) PLN CNR1 ↑ ↑ Human osteosarcoma, SaOS-2 CNR1 ↑ ↑ Human ERMS, RD cells PTHLH ↓ ↑ Human osteosarcoma, SaOS-2 SLIT2 → ↑ Human osteosarcoma, SaOS-2 SLIT2 → Human ERMS, RD cells EPHA4 ↑ Human osteosarcoma, SaOS-2 EFNB2 ↑ Human osteosarcoma, SaOS-2 MSX2 ↓ Human osteosarcoma, SaOS-2 BPM4 ↓ ↓ Human osteosarcoma, SaOS-2 BPM4 ↑ Human ERMS, RD cells PAX3 ↑ Human ERMS, RD cells Tomescu etÂ€al. (2004) CXCR4 ↑ Human ERMS, RD cells Tomescu etÂ€al. (2004), Begum etÂ€al. (2005) CXCR4 → Human osteosarcoma, SaOS-2 Begum etÂ€al. (2005) PAX7 Human ERMS, RD cells Tomescu etÂ€al. (2004) ↑ Human ERMS, RD cells, and Nabarro etÂ€al. (2005) H2-K 76-9 cells (MHC class 1) PAI1 ↓ Human ERMS, RD cells STAT1 ↓ Human ERMS, RD cells STAT3 ↓ Human ERMS, RD cells TAP1 ↓ Human ERMS, RD cells CXCL10 ↓ Human ERMS, RD cells CCL5 ↓ Human ERMS, RD cells IGF2 ↑ Human ERMS, 76-9 cells TRAM1 ↑ Human ERMS, 76-9 cells COLSA1 ↑ Human ERMS, 76-9 cells SKP2 ↑ Murine NIH3T3 fibroblast cells Zhang and Wang (2003)

cells (Tomescu etÂ€al. 2004; Begum etÂ€al. 2005; Nabarro etÂ€al. 2005). Therefore, the issue must be critically assessed as to the degree to which any given ectopic cell culture system provides a model of the actual ARMS tumor environment. In other words, in these studies, direct effects of PAX3-FKHR in alveolar rhabdomyosarcoma cells have not yet been conducted. Up- and downregulated genes by ectopic expression of PAX3 or PAX3-FKHR reported in the literature are summarized in TableÂ€1. To elucidate targeted genes downstream of PAX3-FKHR, targeted knockdown of PAX3-FKHR expression in ARMS cells by antisense oligonucleotides and siRNA have also been attempted (Bernasconi etÂ€ al. 1996; Ebauer etÂ€ al. 2007). However, again these studies have not addressed the specific effects of selectively downregulating PAX3-FKHR. For example, the antisense oligonucleotides and the siRNAs used were designed against the PAX3 gene and were not specific for PAX3-FKHR. Thus, while this approach suppressed PAX3-FKHR, it also suppressed the expression

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of PAX3 itself. Hence, the phenotypes reported may be derived from PAX3 gene suppression, and not necessarily from the suppression of PAX3-FKHR. Recently, we established an inÂ€ vitro system using several siRNAs designed against the PAX3FKHR fusion point where PAX3-FKHR fusion gene expression is specifically and efficiently depleted with no effects on expression of either PAX3 or FKHR. We have examined the effects of specific PAX3-FKHR suppression in several ARMS cell lines. Our results differ from previous and recent studies that used antisense oligonucletides and siRNA approaches to regulating PAX3-FKHR. Specifically, we found that the downregulating PAX3-FKHR causes inhibition of the rate of cellular proliferation, and an accumulation of cells in G1 phase of the cell cycle, but no increase in cell death through apoptosis. Downregulation of PAX3-FKHR led to reduced levels of the MET receptor, and reduced motility in response to stimulation with HGF, its ligand. Suppression of PAX3-FKHR induced the myogenic differentiation gene, myogenin (Fig.Â€ 3), and muscle differentiation (morphologic change and the expression of muscle specific proteins, desmin and myosin heavy chain). These results suggest that PAX3-FKHR has important functions in ARMS cells to promote malignant phenotypes such as proliferation, motility and to suppress differentiation. Candidate genes regulated by PAX3-FKHR that may be relevant to maintenance of the malignant phenotype, include SKP2, CXCR4, HDAC4, myogenin (myogenic factor 4), MRF5 (myogenic factor 5). Potential drug targets upregulated by PAX3FKHR include MET, the cytokine receptor CXCR4, fibroblast growth factor receptor 4 (FGFR4) and the cyclin-dependent kinase CDC2 (CDK1). PD173074, is a synthetic compound of the pyrido(2,3-d)pyrimidine class, that inhibits tyrosine kinase activities of FGF receptors including FGFR4 (Ezzat etÂ€al. 2006). Crystal structure elucidation has identified PD173074 in complex with the tyrosine kinase domain of FGFR1 with a high degree of surface complementarity with the hydrophobic, ATPbinding pocket of FGFR1 (Mohammadi etÂ€al. 1998). Systemic administration of this compound effectively blocks FGF-induced angiogenesis and neurotrophic actions (Skaper etÂ€al. 2000). The staurosporine derivative PKC412 that inhibits FGFR/FLT3/ PKC has entered clinical trials (Stone etÂ€al. 2004), and has shown some benefit in patients with cholangiocarcinoma. Nausea/vomiting and diarrhea were the most common toxicities in adults (Propper etÂ€al. 2001). CXCR4 antagonists are in clinical development as “entry fusion inhibitors” for the treatment of type 1 human immunodeficiency virus and include small peptide agents (ALX40-4C) (Doranz etÂ€ al. 2001) and small molecule inhibitors (AMD 3100; Plerixafor). Plerixafor is a bicyclam derivative that acts as a stem cell mobilizer by blocking the CXCR4 chemokine receptor, which triggers the rapid movement of stem cells out of the bone marrow and into circulating blood. Plerixafor is in phase III clinical trials in stem cell transplantation among cancer patients. Inhibition of CDK1 has been proposed as a potential therapy for tumors that overexpress MYC, based on the selective killing of MYC-overexpressing cells by purvanolol or RNAi targeting MYC (Goga etÂ€ al. 2007). This may be particularly of interest if the same synthetic lethal interaction occurs in RMS that overexpress MYCN independent of p53 functional status. Agents that target CDK1 are in preclinical testing (Lin etÂ€al. 2007), and agents such as roscovitine or R547 (DePinto etÂ€al. 2006) are in clinical trials.

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Fig.Â€3â•… Specificity of RNAi designed against the fusion point (siP-F) in suppressing PAX3-FKHR mRNA and the impact from PAX3-FKHR knockdown on MET, myogenin, and IGF-IRb mRNAs and proteins. (a) The relative amounts of PAX3-FKHR mRNA relative to GAPDH mRNA 24Â€hours after transfection. The levels of the PAX3-FKHR mRNA were not changed in the mediumonly control, mock transfection, and si-CON-transfected (scrambled control). (b) Western blot of PAX3-FKHR, MET, Myogenin, IGF-1Rb protein, and b-actin 48Â€hours after transfection in the medium-only control, mock transfection and si-CON-transfected (scrambled control). (c) The relative abundance of transcripts of PAX3-FKHR, PAX3, and FKHR in Rh30 cells transfected with si CON, and transfected with siP-F. In siCON-transfected cells, the mRNA levels of both PAX3 and FKHR were very low compared to that of PAX3-FKHR, being about 1/30 and 1/300 the level of PAX3-FKHR, respectively. Although siP-F decreased the mRNA level of PAX3-FKHR by 83%, but the mRNA levels of both PAX3 and FKHR were not largely affected by this siP-F (adapted from Kikuchi K, Tsuchiya K, Otabe O, Gotoh T, Tamura S, Katsumi Y, Yagyu S, TsubaiShimizu S, Miyachi M, Iehara T, Hosoi H. 2008 Effects of PAX3-FKHR on malignant phenotypes in alveolar rhabdomyosarcoma. Biochem Biophys Res Commun 365:568–74.)

MYCN Dysregulation Amplification of the MYCN oncogenic transcription factor locus is associated with poor prognosis in neuroblastoma. However, amplification and overexpression of MYCN has been reported to occur frequently in RMS, predominantly in ARMS

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(Dias etÂ€al. 1990; Driman etÂ€al. 1994; Hachitanda etÂ€al. 1998). Cumulatively, these relatively small studies observed amplification of MYCN in 16 of 28 ARMS and 0 of 27 ERMS. However, whether amplification correlated with outcome was controversial. Larger studies (Gordon etÂ€al. 2000; Lu etÂ€al. 2001) indicated that the 2p24 MYCN locus is frequently amplified, and even more frequently genes in this region are overexpressed in RMS. A report where expression (nâ•›=â•›113 cases) and copy number (nâ•›=â•›92) were determined concluded (Chesler etÂ€ al. 2006) that increased copy number of MYCN was a feature of both ERMS and ARMS. In ERMS high expression occurred even without increased copy number. Further, in patients with ARMS, overexpression, greater than the median, or gain of MYCN copy number were significantly associated with poor outcome (Williamson etÂ€ al. 2005). Thus, MYCN, or genes downstream of this transcription factor appear to be of importance in the genesis or progression of RMS and may reveal potential targets for therapy (Lu etÂ€ al. 2003; Pession and Tonelli 2005; Tonelli etÂ€ al. 2005; Morgenstern and Anderson 2006). However, whether dysregulation of MYCN is critically involved in the pathogenesis of RMS has yet to be demonstrated. Several approaches to downregulating MYCN have been proposed, including antisense oligonucleotides that retard the growth of the MYCN-driven transgenic model of neuroblastoma in mice (Burkhart etÂ€ al. 2003) and peptide nucleic acids (Tonelli etÂ€ al. 2005) that induce apoptosis in some cell lines. Whether such approaches, including therapeutic inhibitory RNA (siRNA) will ultimately have therapeutic utility remains a matter of conjecture at this time. Further, the effect of downregulating MYCN in RMS models has not been reported. MYC proteins form heterodimers with MAX through their HLHZip domains and the heterodimer activates transcription of multiple genes by binding the E-box motif (CACGTG). Berg etÂ€al (2002) have reported a small molecule inhibitor of MYC/MAX dimerization; however, this does not discriminate between MYC family members, hence may have pleiotropic effects against MYCdependent tissues. Of importance for developing therapeutic approaches is that MYC proteins have a relatively short half-life of about 20 to 30Â€min. MYCN is also expressed only in early development, hence may represent a tumor-specific target. In neuroblastoma cells, MYCN transcription is stimulated by IGF-1 (Misawa etÂ€al. 2000) through the mitogen activated protein kinase pathway. Inhibition of phosphoinositol 3¢-kinase (PI3K) reduces MYCN protein and decreases transcription of two downstream genes (mdm2 and mcm7). MYCN induction in response to IGF-1 is decreased by PI3K inhibitors putatively through a combination of effects on translation and MYCN protein stability (Chesler etÂ€al. 2006). The proposed mechanism for downregulation of MYCN protein is that inhibition of PI3K leads to the activation of glycogen synthase kinase 3b (GSK3b) a kinase that phosphorylates MYCN and causes its destabilization, and induces apoptosis in neuroblastoma cells. MYCN amplified neuroblastoma cell lines are also hypersensitive to a small molecule inhibitor of translation elongation (Radhakrishnan and Gartel 2006) that causes rapid downregulation of MYCN protein (Radhakrishnan etÂ€al. 2008). Thus, in addition to inducing selective cytotoxicity through targeting CDK1 in the context of MYC overexpression, there appear to be numerous approaches to regulate MYCN. Inhibitors of PI3K (BEZ235, SF1126), GSK3b (LiCl) and Akt (RX-0201, VQD-

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002, XL418, GSK609693) are in development, and several agents are in adult clinical trials. GSK609693 is currently undergoing the evaluation against pediatric solid tumors and acute lymphocytic leukemia models in the Pediatric Preclinical Testing Program (Houghton etÂ€al. 2007).

TP53 Dysregulation The tumor suppressor P53 (TP53) is mutated in approximately 50% of human cancer; however, its status in RMS is somewhat unclear. In part, this is because the function of TP53 can be attenuated by overexpression of HDM2, the human homolog of MDM2, which binds TP53 preventing its transcriptional activity (Bottger etÂ€al. 1997). HDM2 also has ubiquitin ligase activity, which targets TP53 for proteasomal degradation (Honda etÂ€al. 1997). The reported incidence of TP53 mutations in RMS is relatively low (approximately 5%) and similarly amplification of HDM2 is found in 10% of clinical samples (Taylor etÂ€ al. 2000). ImmunoÂ� histochemical staining for TP53 and HDM2 has been variable; clinical RMS samples showed both TP53 and HDM2 to be expressed at low levels in both ERMS and ARMS (Leuschner etÂ€ al. 2003), although overexpression of TP53 (interpreted as indicative of stabilized mutated TP53) was postulated to be a crucial step in metastatic disease for patients with ERMS. In contrast, the study of Takahashi etÂ€al. (2004) found overexpression of TP53 in 30% of cases, 22% had gene abnormalities, and HDM2 was amplified in 13%. However, determination of the functional status of TP53 is further complicated as TP53 is a member of a multigene family that includes TAP63 and TAP73 (Flores etÂ€al. 2002, 2005), which encode proteins that have overlapping function with TP53, for example induction of apoptosis in response to DNA damage (Flores etÂ€al. 2002; Melino etÂ€al. 2004; Flores 2007). Multiple isoforms of TAp73 can be produced by alternative use of intronic promoters, alternative splicing, or use of an internal ribosome entry site (IRES) (Melino etÂ€al. 2002; Stiewe and Putzer 2002; Sayan etÂ€al. 2007). Notably, N-terminal truncated forms (DNp73) act as a potent transdominant negative regulators of all p53-members (Grob etÂ€ al. 2001; Zaika etÂ€ al. 2002), and suppress multiple developmental programs including muscle differentiation (HuttingerKirchhof etÂ€al. 2006). Of importance is that DNp73 is expressed in many clinical samples of RMS and cell line models (Cam etÂ€al. 2006), and confers chemoresistance through suppressing TP53-induced CD95 and proapoptotic BCL2 members (Gressner etÂ€al. 2005; Muller etÂ€al. 2005). Thus, it is probable that TP53 function is attenuated through HDM2 and DNp73, as well as mutations within the TP53 gene itself. As p53-family members regulate stress-induced apoptosis (Vogelstein etÂ€al. 2000), and loss of function confers drug or radiation resistance, strategies to reactivate TP53 function appear to have some application for the treatment of RMS. The most developed approach is to attenuate the activity of HDM2, and several chemical classes of inhibitor that block HDM2-p53 interactions have been described (Vassilev 2007). This may be particularly effective in tumors where the

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HDM2 gene is amplified or overexpressed (Tovar etÂ€ al. 2006; Vassilev 2007), however, to date no studies with RMS cell lines have been reported. Nutlin 3a, a small molecule inhibitor of HDM2-p53 interactions may induce G1 cell cycle arrest and apoptosis (Logan etÂ€ al. 2007) consistent with recruitment of p53 to chromatin and increased expression of p53-responsive genes, CDKN1A (encoding p21CIP1), PUMA, GADD45, and HDM2. Nutlin 3a can also induce p53-dependent senescence (Efeyan etÂ€al. 2007). Interestingly, nutlin 3a can enhance chemotherapy-induced apoptosis in p53-mutant cell lines through an E2F1-dependent induction of the proapoptotic proteins TAp73a and NOXA (Ambrosini etÂ€al. 2007). An alternative strategy proposed (Kranz and Dobbelstein 2006) is to use the HDM2 inhibitor to selectively block cells with wild type TP53 in G1 phase. In contrast, cells mutant for TP53 progress into S-phase and are selectively killed by S-phase specific agents such as gemcitabine, a strategy equally applicable to topotecan or irinotecan used in treatment of RMS. HDM2 antagonists may also have antiangiogenic activity through disrupting the HDM2-hypoxia inducible factor 1a (HIF1a) interaction that enhances transcription of vascular endothelial growth factor (VEGF) (Bardos etÂ€al. 2004; LaRusch etÂ€al. 2007; Secchiero etÂ€al. 2007). However, the strategy for use of HDM2 inhibitors will be successful only if the apoptotic machinery downstream of TP53 is functional. For example, overexpression of antiapoptotic proteins of the BCL2 family or MCL1 could antagonize cell death induced by these agents. Similarly, overexpression of DNp73 would be anticipated to antagonize HDM2 inhibitors, and abrogate approaches such as introduction of wild type p53 using an adenoviral vector (Ganjavi etÂ€ al. 2005), shown to sensitize RMS cells to DNA damaging cytotoxic drugs.

Aberrant Growth Factor Signaling in RMS Insulin-Like Growth Factor Signaling RMSs show the presence of both active type-1insulin-like growth factor receptor (IGF-1R) and the autocrine production of its ligand IGF-2 (Minniti etÂ€ al. 1994). IGF-1 and -2 and IGF-1R regulate all aspects of the malignant phenotype with IGF-1R being activated by its ligands and also by steroid hormones (Kaleko etÂ€al. 1990; Stewart and Rotwein 1996; Sepp-Lorenzino 1998). The activated IGF-1R is capable of phosphorylating other tyrosine-containing substrates of which the insulin receptor substrates (IRS-I-4) link the receptor to a cascade of enzyme activations via PI3K-Akt-mTOR and RAF-MAPK systems (LeRoith etÂ€al. 1995). Activation of these pathways results in a variety of responses, such as cell proliferation, differentiation, migration, and in mouse models allows maintenance of the malignant phenotype (Bohula etÂ€ al. 2003; Baserga 2004). IGF-1R signaling transcriptionally upregulates antiapopototic genes and suppresses proapoptotic genes, through a multitude of pathways as shown in Fig.â•›4. IGF-1R signaling is

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Survivin

PAR4

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cIAP-2 XIAP

CREB p53

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AKT

p38 NF k B

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BIM

AIB1 JNK

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Fig.Â€4â•… IGF-IR-mediated survival through transcriptional regulation. IGF-IR signaling via IRS-1, AKT, or MAP kinases (ERK1/2, p38) positively regulates transcription of antiapoptotic genes (red ) and negatively regulates proapoptotic genes (green). IGF-IR signaling regulates transcription factors that themselves regulate antiapoptotic programs. (From reference Kurmasheva and Houghton 2006 with permission)

tightly regulated since high IGF-1 levels result in a decline in IGF-1R, and IGFs may also act as negative feedback signals to repress expression of IGF-1R (Yang etÂ€al. 1996; Hernandez-Sanchez etÂ€ al. 1997). It has been recently shown that IGF-1R mediates Akt activation in response to inhibition of mTOR through a feedback loop in which IRS-1 becomes stabilized in Rh-30 and RD cells (Petricoin etÂ€al. 2007). In contrast to the effect of IGFs, other growth factors, including bFGF, PDGF, and EGF, as well as estrogens, glucocorticoids, GH, FSH, luteinizing hormone, thyroid hormones stimulate IGF-1R expression (LeRoith etÂ€ al. 1995). IGF-2 is consistently overexpressed in both embryonal and alveolar RMS, where it also cooperates with PAX3-FKHR (El-Badry etÂ€ al. 1990; Minniti etÂ€ al. 1994; Wang etÂ€al. 1998). At the transcriptional level, PAX3-FKHR is able to transactivate the IGF-1R promoter in sarcoma-derived cell lines, whereas PAX3 exhibited a reduced potency in comparison to the fusion protein (Ayalon etÂ€ al. 2001). Low concentrations of IGF-1 protein in media can be detected in RMS cells inÂ€vitro by sensitive methods such as ELISA, although the molar ratio of IGF-2:IGF-1 is approximately 500 (RTK, unpublished data). Thus, constitutive activation of IGF-1R by its ligands (mainly IGF-2) can disrupt the delicate balance between proliferation and differentiation, creating the expanded pool of cycling myogenic progenitors that become targets of further mutagenesis and potential seeds of

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future RMS. In animal models, IGF-2 is essential for the development and malignant behavior of RMS (Wang etÂ€al. 1998; Hahn etÂ€al. 2000). Interestingly, wild type TP53 inhibits transcription of the IGF-2 gene (Zhang etÂ€al. 1996, 1998a). As the function of p53 appears to be suppressed in RMS cells through mutation, overexpression of HDM2 or expression of DNp73, an N-terminal truncated isoform of TAp73, this could help explain overexpression in RMS. In normal development, IGF-2 mRNA is most abundant during the fetal development and rapidly declines after birth. The activity of the IGF-2 gene in normal mouse and human muscle tissues is regulated by genomic imprinting, which is a form of nonMendelian inheritance in mammals, where the imprinted genes are expressed uniquely from one allele. The expressed allele, either paternal or maternal, is constant for each imprinted gene, unless a genetic or epigenetic alteration has occurred. Almost all imprinted genes identified to date can be classified as regulators of embryonic growth, placental growth, or adult metabolism (Morison etÂ€ al. 2005). The IGF-2 signaling pathway has implications in all three of these functions. Moreover, it has been documented that upregulation of IGF-2, a common outcome of loss of imprinting (LOI), occurs at high frequency in a large variety of human tumors including childhood cancers. In normal tissues, the IGF-2 gene is exclusively silent at the maternal allele. Zhan etÂ€ al. (1994) reported that normally imprinted allele of the IGF-2 gene was activated in alveolar RMS tumors as well as in Rh-28 cells. Embryonal RMS are usually characterized by LOI, overexpression of IGF-2 and paternal disomy of the IGF-2 locus. It appears that both alveolar and embryonal histologies of RMS have alterations of the normal IGF-2 locus. Loss of heterzygosity (LOH) that is caused by the loss of maternal chromosome has only been observed in tumors belonging to the embryonal subtype (Scrable etÂ€al. 1989). Zhan etÂ€al. (1994) have suggested that LOI represents the functional equivalent of LOH with paternal disomy in RMS, and possibly both events are related to the increased expression of IGF-2 seen in both histologic subtypes of RMS. In addition, uniparental paternal disomy (UPD) of the 11p15 locus have been described in predisposition to the development of tumors of embryonic origin (Weksberg etÂ€al. 1993). Most of the imprinted genes known so far are organized in clusters. Two genes with inÂ€vitro growth inhibitory capacity, namely H19 and p57KIP2 (Hao etÂ€al. 1993; Matsuoka etÂ€al. 1995), and growth promoting IGF-2 gene, are members of such a cluster with the first two genes being paternally imprinted. Low H19 mRNA was associated with the loss of the maternal allele or with relaxation of IGF-2 imprinting, although LOI of the IGF-2 gene was not necessarily coupled to low H19 expression in both ERMS and ARMS (Casola etÂ€al. 1997). Low H19 mRNA levels relative to normal tissues have been found in the great majority of Wilms’ tumor samples as well as in RMS, suggesting the role of a tumor suppressor for this gene (Steenman etÂ€al. 1994; Casola etÂ€al. 1997). It was shown that forced expression of the transfected H19 gene inhibited cell growth of a RMS cell line and suppressed tumorigenicity of Wilms’ tumor cells (Hao etÂ€al. 1993). The mechanism of regulation of H19/IGF-2 expression has become clearer with identification of a new component in the regulatory system. On the maternal chromo-

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some, an enhancer-blocking protein CCCTC-binding factor CTCF (recognizes the DNA sequence CCCTC) binds to the differentially methylated regions (DMR), preventing the enhancers from interacting with the promoter of IGF-2, and instead favoring H19 expression (Schoenherr etÂ€al. 2003). But on the paternal chromosome, the DNA of the boundary element is methylated, the blocking protein cannot bind, and IGF-2 can be expressed while H19 is not. Physiologically, CTCF repeats are required to maintain the hypomethylated state of the entire DMR during the period of active deÂ€novo methylation that occurs after implantation. Possibly, the presence of CTCF engenders a tight engagement between the DMR, the H19 promoter and the downstream enhancers, thus excluding access of the IGF-2 gene to the enhancers that allows maintenance of monoallelic IGF-2 expression (Engel etÂ€al. 2006). It is not presently known if CTCF is the only protein capable of this type of imprinted gene regulation. IGF-2 promoters play different roles throughout development and in different tissues: they are normally active during fetal growth, but silent in postnatal life, and become reactivated in cancer. The IGF-2 gene is transcribed from four different promoters (P1–P4). P2–P4 contains CpG islands, and transcription from these promoters is subject to imprinting while biallelic expression is observed from the other alternative promoter (P1) (Pavelic etÂ€al. 2007). The developmentally regulated transcription factor AP-2 is expressed at high expression levels in RMS and human fetal skeletal muscle cells but not in adult skeletal muscle cells. This suggests that AP-2 may also contribute to the high expression of IGF-2 in RMS cells (Zhang etÂ€al. 1998b). The role of IGF-1R signaling in the pathogenesis of RMS, and its role in preventing apoptosis induced by a multitude of cellular stresses including cytotoxic drugs, radiation, and hypoxia (Kurmasheva and Houghton 2006) indicate that targeting this pathway may have considerable utility for therapy of RMS. As dysregulated IGF-1 signaling is common to several adult malignancies, targeting IGF-1R has become a major focus for therapeutic development (Braczkowski etÂ€ al. 2002; Cohen etÂ€ al. 2005; Wang etÂ€ al. 2005; Guerreiro etÂ€ al. 2006; Kurmasheva and Houghton 2006; Sachdev and Yee 2006, 2007). Currently, there are both small molecule drugs and fully human or humanized antibodies directed at the IGF-1R. Several classes of small molecule inhibitors of IGF-1R have been described, including tyrphostins, pyrrolopyrimidines (Mitsiades etÂ€al. 2004; Scotlandi etÂ€al. 2005), substituted benzimidazole derivatives (Haluska etÂ€ al. 2006) and diarylureas (Gable etÂ€ al. 2006). Small molecule ATP-competitive IGF-1R inhibitors do not induce downregulation of the receptor, and in most cases do not discriminate between the insulin receptor and the IGF-1R (Haluska etÂ€al. 2006; Wittman etÂ€al. 2007). As a consequence, these inhibitors may have significant effects on glucose homeostasis (causing hyperglycemia and hyperinsulinemia). Several small molecule inhibitors of IGF-1R. Five fully human (CP751871, AMD479, R150 (OSI-906 and BMS-754807) are inclinical trials. 7, IMC-A12, SCH717454) or humanized antibodies (H7C10) are in adult phase-I or -II clinical trials. These agents show specificity for the IGF-1R although they may inhibit chimeric receptors formed through heterodimerization with the insulin receptor. In preclinical models of childhood cancers, the prototypical antiIGF-1R antibody, a-IR3 mediated downregulation of IGF-1R significantly retards theÂ€growth of many cell lines inÂ€vitro (El-Badry etÂ€al. 1990), and retards the growth of RMS xenografts (Kalebic etÂ€al. 1994). SCH717454 significantly inhibits the growth of

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RMS xenografts and induces regressions in several sarcoma histotypes notably osteosarcoma and Ewing sarcoma (Kolb etÂ€al. 2008). Molecular characterization of these sensitive models where IGF-1R signaling appears to be critical could identify subsets of tumors that have become “addicted” to this pathway (Weinstein and Joe 2006). In other preclinical models, blocking IGF-1R signaling results in significant retardation of tumor growth although in a clinical setting this response would be scored as progressive disease. In these models with intermediate sensitivity, such as RMS, combinations of signaling inhibitors would potentially be a more effective antitumor therapy. One strategy that is being evaluated in preclinical models is the combination of the mTOR inhibitor, rapamycin, with IGF-1R inhibitors. The basis for this combination is that inhibition of mTOR upregulates IGF-1R signaling through stabilization of IRS-1 (Easton etÂ€al. 2006), and IGF-1R signaling blocks rapamycin-induced apoptosis (Huang etÂ€al. 2003; Thimmaiah etÂ€al. 2003). Alternative approaches to inhibiting IGF-1R signaling include the development of ligand binding antibodies. Targeting receptor ligand rather than the receptor per se has proven a valuable approach for the antiangiogenic antibody, bevacizumab, and high affinity fully human antibodies have been developed against IGF-II (Feng etÂ€ al. 2006). Another approach to limiting IGF-1R signaling is to administer IGF-binding proteins as therapeutics. The activity of IGFs is regulated by a family of six structurally related high affinity IGF-binding proteins (IGFBPs) (Hwa etÂ€al. 1999). In the plasma, 99% of IGFs are complexed to IGFBPs that modulate the availability of free IGF-1 to the tissues. At the same time, binding of IGFBPs to IGFs protects IGFs from proteolytic degradation increasing their bioavailability. Normally, more than 90% of the circulating IGFs are bound to IGFBP-3. Of the IGFBPs, IGFBP-6 (approximately 30Â€ kDa) binds IGF-2 with the greatest affinity, having a 20- to 100-fold greater binding affinity for IGF-2 than for IGF-1 (Bach etÂ€al. 1995). High levels of IGFBP-6 expression in cells are associated with nonproliferative and differentiated states, both inÂ€vitro and inÂ€vivo. In particular, IGFBP-6 expression is correlated with quiescence in myoblast culture and IGFBP-6 protein was not identified in media conditioned by RMS cells, and expression is strongly downregulated in spontaneously metastatic RMS cells compared with nonmetastatic parental RMS cells (Scholl etÂ€ al. 2000). Additionally, IGFBP-6 inhibits IGF-2 mediated differentiation and proliferation of myoblasts (Bach etÂ€ al. 1994, 1995). Unlike in normal cells, lower IGFBP-6 expression in RMS than in myoblasts would therefore be expected to enhance the actions of IGF-2. Gallicchio etÂ€al. (2001) reported that IGFBP-6 significantly inhibited monolayer RD and Rh-30 cell proliferation in a dose-dependent manner. Further, overexpression of IGFBP-6 resulted in a 74 to 88% reduction in Rh-30 tumor size inÂ€vivo after 18Â€days, showing that IGFBP-6 can be a potent antitumor agent.

Hepatocyte Growth Factor/MET Autocrine Signaling Increased expression of the MET receptor or its ligand hepatocyte growth factor/ scatter factor (HGF) occurs frequently in human cancer, and increases with tumor progression in some malignancies (Longati etÂ€al. 2001). In myogenic precursors,

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PAX3 activates transcription of several genes, including MYOD and MET. As discussed above, MET is consistently downregulated when PAX3-FKHR is downregulated by antisense or siRNA, and is overexpressed in cells expressing PAX3-FKHR. MET encodes a cell-surface receptor transcriptionally activated by both PAX3 and PAX7 that mediates delamination of myoblast precursors from the epithelial dermomyotome and their migration in the limb bud in response to HGF. MET has been shown to be highly overexpressed in ARMS (Epstein etÂ€ al. 1996; Ferracini etÂ€ al. 1996; Ginsberg etÂ€al. 1998), but while the gene is not mutated or amplified (Chen etÂ€al. 2007) the expression level of MET was found to be significantly higher in patients who died of disease. MET is highly expressed in cell lines derived from ARMS (Epstein etÂ€ al. 1996) and HGF induces motility and confers drug resistance (Jankowski etÂ€al. 2003). Conditioned media from bone marrow derived fibroblasts chemoattracts RMS cells in an HGF-dependent manner and RMS cells with higher expression of MET increased homing/seeding to bone marrow of RMS cells inoculated intravenously into mice. In contrast to ARMS, ERMS have more variable expression of MET, but appear to express the ligand HGF at higher levels than in ARMS. Analysis of RMS xenografts indicates phosphorylation of MET (Y1234/1235), in both ARMS and some ERMS suggesting auto-activation. As murine HGF does not activate the human receptor, these data imply autocrine activation, at least under conditions of RMS growth in the mouse. Transcripts for human HGF are also detected in both ERMS (2/2) and ARMS (2/5) xenografts (unpublished data). Consistent with the model data, MET was detected in all of 68 RMS specimens and 62% of tumors coexpressed HGF, however, neither high MET nor HGF expression correlated with metastatic disease (Rees etÂ€ al. 2006). Somewhat unexpectedly, MET was a consistent feature of ERMS and not ARMs. MET appears to be an essential mediator of PAX3-FKHR-induced oncogenesis, as exogenous HGF rescued the ability of PAX3-FKHR-transduced cells to form colonies in soft agar under low serum-containing conditions. Further, PAX3-FKHR was capable of transforming wild type murine embryo fibroblasts but not those derived from mutant (inactive) MET (Taulli etÂ€al. 2006). Together with data showing the downregulation of MET inhibits proliferation (although induction of apoptosis has been variable between reports) and migration of RMS cells inÂ€ vitro and inÂ€ vivo, the evidence points to the MET receptor being a potential target for therapy of both ARMS and ERMS. Relatively specific inhibitors of MET (SU11271, SU11274, SU11606 (Wang etÂ€ al. 2003), SGX523, PHA665752 (Ma etÂ€ al. 2005), and PF2341066) as well as multitargeted kinase inhibitors that are potent against MET (XL880 and MP470) have been reported. Both humanized (AV299) and fully human antibodies (AMG102) against HGF, as well as MET decoys (CGEN214) that prevent ligand binding, or receptor dimerization, and one-armed monoclonal antibodies against the MET receptor are in development. Preliminary clinical results in adult trials with ARQ197, a small molecule MET inhibitor, suggest that this agent is well tolerated, with fatigue, diarrhea, and elevated ALP and AST as the most frequent toxicities (Garcia etÂ€al., 2007), and antitumor activity has been observed with oral dosing. XL880, a multitargeted kinase/MET inhibitor, has demonstrated reversible Grade 3 elevations in lipase, hepatic transaminases and proteinurea, with objective responses

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in four of 40 adult patients (Eder etÂ€al., 2007). Common adverse events included hypertension and fatigue with the loss of concentration. These data from early clinical trials in adults are encouraging, and suggest these agents should be evaluated against relevant models of RMS, and potentially advanced to clinical trials.

Hedgehog Signaling Pathway Gorlin syndrome, also known as nevoid basal cell carcinoma syndrome (NBCCS) is caused by mutations in the hedgehog receptor PTCH gene, and is characterized by the developmental defects and predisposition to the development of tumors including fetal rhabdomyomas and ERMS (Tostar etÂ€al. 2006). Binding of hedgehog (Hh) to PTCH suppresses smoothened (SMO) the direct activator of the transcription factor GLI1. GLI1 amplification occurs in some RMS, and overexpression of GLI1 and GLI2 suppresses skeletal muscle differentiation (Gerber etÂ€al. 2007) through binding to and suppressing MYOD heterodimerization with E12 proteins. Losses in the PTCH locus 9q22 have been identified in 12 to 30% of ERMS (Bridge etÂ€al. 2000; Calzada-Wack etÂ€al. 2002; Tostar etÂ€al. 2006). Genomic loss at 10q24 a locus encoding the GLI-suppressor SUFU occurs in 18% of ERMS (Bridge etÂ€al. 2002). Thus, cumulative loss of negative regulatory components in the Hh pathway approaches 50% in ERMS. Interestingly, a positive regulator of GLI transcription (Mao etÂ€al. 2002), the survival kinase DYRK1/MIRK is significantly expressed in most RMS (Mercer etÂ€ al. 2006). Several classes of small molecule inhibitors of SMO have been reported (Chen etÂ€ al. 2002; Williams etÂ€ al. 2003). CUR61414, showed quite impressive activity against transgenic models of basal cell carcinoma in PTCH+/− mice (Williams etÂ€ al. 2003) and a precursor molecule HhAntag691 prevented the development of medulloblastoma in PTCH+/− p53−/− mice (Romer etÂ€al. 2004). CUR61414 advanced to clinical trials for the treatment of basal cell carcinoma, but failed to downregulate pharmacodynamic markers essential for establishing activity in the phase 1 clinical trial. More recently GCD-0449, another Hedgehog inhibitor has shown robust activity in treatment of basal cell carcinoma.

Other Pathways (RAS/MAPK/c-KIT) Proliferating myoblasts express the myogenic regulatory factors MYOD and MYF5, whereas myogenin and MRF4 are expressed as terminal differentiation progresses. Despite expression of these myogenic regulatory factors in myoblasts, mitogenic stimulation or activated oncogenes repress the differentiation program (Perry etÂ€al. 2001). Activation of RAS genes has been reported in ERMS with varying frequency (3 to 30%) (Stratton etÂ€al. 1989; Chen etÂ€al. 2006). However, oncogenic RAS induced the formation of RMS in zebrafish, concordant with expression

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profiles consistent with an activated RAS pathway. Extension of these studies indicated that RAS pathway activation appears to occur frequently in human RMS (Langenau etÂ€al. 2007). Activated RAF and MEK1, downstream of RAS can also prevent myogenic differentiation (Dorman and Johnson 2000; Perry etÂ€ al. 2001), and an inhibitor of MEK1 induces growth arrest, myogenic differentiation (in some cell lines) and downregulation of c-MYC. Thus, targeting the RAS/MAPK signaling pathway may offer some therapeutic potential. Targeting activated RAS through the use of farnesyltransferase inhibitors has utility limited to the treatment of hematologic diseases (Santos etÂ€al. 2004). However, inhibitors of RAF (Sorefenib) have clinical utility in the treatment of adult carcinomas, and MEK1 inhibitors (CI-1040, PD325901, and AZD6244) are in clinical trials for adults, and may have utility for the treatment of RMS. However, testing of AZD6244 against the PPTP panel inÂ€vivo did not demonstrate robust antitumor activity as a single agent (unpublished data). Overexpression of the stem cell factor receptor c-KIT has been reported in 10% of spindle cell RMS (Komdeur etÂ€al. 2003), but is absent in ARMS and only 2 of 13 (15%) of ERMS demonstrated strong, diffuse staining similar to that in GIST. Consistent with these data, imatinib mesylate has not demonstrated significant antitumor activity against RMS models inÂ€vivo (unpublished results) or in the treatment of childhood solid tumors (Bond etÂ€al. 2008).

Differentiation As discussed previously, RMS is characterized by arrested myogenic differentiation. It has been a goal of many studies to understand this process in light of molecular events that characterize normal myogenesis, and to devise approaches to induce terminal differentiation in these tumors. Failure to differentiate may be a consequence of activating mutations in oncogenes such as RAS, FOS, and SRC (Alema and Tato 1994), or epigenic changes in gene promoter methylation and silencing, as for PAX3 in ERMS (Kurmasheva etÂ€ al. 2005), or silencing through altered histone acetylation, as for the CDKN1A gene that encodes the cyclin-cdk inhibitor p21CIP1 (Jaboin etÂ€al. 2002; Kutko etÂ€al. 2003). Approaches to induce differentiation include the use of DNA methyltransferase inhibitors, such as 5-azacytidine (Lollini etÂ€ al. 1989), and the use of histone deacetylase inhibitors (Kutko etÂ€ al. 2003) that induce reexpression of p21Cip1. Numerous clinical trials have been carried out with demethylating agents, but unfortunately these trials highlight the need to understand better the complete pathways leading to LOI, since global demethylation, resulting from these treatments generated additional complications (Issa etÂ€al. 2005; Yoo and Jones 2006). However, as molecular detail of myogenic differentiation has emerged, it has been possible to determine why differentiation in RMS is attenuated. Many hypotheses have been proposed to explain how mitogenic signals suppress muscle differentiation. Phosphorylation of myogenic regulatory proteins (Li etÂ€al. 1992), induction of dominant helix-loop-helix (HLH) proteins that block E protein heterodimerization (Benezra etÂ€ al. 1990), cyclin D-CDK4-mediated

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inhibition of the differentiation program (Rao etÂ€al. 1994; Rao and Kohtz 1995; Skapek etÂ€ al. 1995, 1996), and MEK1-mediated inactivation of the transactivation domain of the myogenic bHLH proteins (Perry etÂ€al. 2001) have been implicated in arrested differentiation. Cyclin D-CDK4 complexes associate with the C-terminus of MYOD attenuating its interaction with DNA (Zhang etÂ€al. 1999), can indirectly inhibit the activity of myogenin (Skapek etÂ€al. 1996), and suppress the activity of RB (Novitch etÂ€al. 1996) that is required to promote later stages of skeletal muscle differentiation. Cyclin D-CDK4 activity also blocks the association of MEF2C with the coactivator protein GRIP1, thereby inhibiting MEF2 activity (Lazaro etÂ€ al. 2002). Expression of PAX3-FKHR also increases expression of the transcription factor SIX1 (Khan etÂ€al. 1999) that in addition to activating the myogenic program, activates a set of protumorigenic genes, including those encoding cyclin D1, c-MYC, and Ezrin (Yu etÂ€al. 2006). As cyclin D-CDK complexes are upregulated in both ERMS and ARMS (Knudsen etÂ€al. 1998; Saab etÂ€al. 2006), drive proliferation and suppress differentiation, targeting these complexes appears to be a valid therapeutic strategy. Relatively selective small molecule inhibitors of cyclin D-CDK4/6 have entered clinical trials for the treatment of adults with cancer (Fry etÂ€al. 2004; DePinto etÂ€al. 2006). PD0331992 is a potent inhibitor of cyclin-dependent kinases 4 and 6 and induced regressions of Colo-205 human colon tumor xenografts, eliminating phospho-RB and the proliferative marker Ki67 (Fry etÂ€al. 2004). However, the effects of PD332991 against RMS cell lines were less robust. Although it induced G1 phase accumulation in most RMS cell lines, and some degree of myogenic differentiation, it did not promote terminal differentiation. In part, this may be a consequence of incomplete cyclin D-CDK inhibition, as residual phosphorylation of RB (S780) was detected in treated cells. PD331992 induced some growth inhibition of RMS xenografts inÂ€vivo, although the effects were not particularly impressive (Saab etÂ€ al. 2006). These studies support the contention that elevated CDK2/CDK4 contribute to the inability of RMS cells to growth arrest when cultured in low-mitogen medium, but that these activities alone are not sufficient to explain the failure of cells to differentiate (Knudsen etÂ€ al. 1998). As DNp73 is expressed in many RMS and suppresses the myogenic program, it suggests additional approaches that will be required to develop effective therapies designed to induce terminal differentiation.

Angiogenesis Neovascularization of solid tumors is one component of the solid tumor microenvironment that is essential for sustenance and metastasis. Numerous approaches to decreasing tumor angiogenesis or targeting tumor vasculature are currently in preclinical or clinical trials. These include antibodies and peptides targeting the VEGF, including bevacizumab, and VEGF-Trap, small molecule inhibitors of VEGF receptors, as well as multitargeted tyrosine kinase inhibitors that inhibit both growth factor receptors and pathways downstream of receptor signaling. RMS cell

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lines secrete VEGF (Gee etÂ€ al. 2005; Kurmasheva etÂ€ al. 2007) as well as other angiogenic factors such as basic fibroblastic growth factor (bFGF) and interleukin 8 (Pavlakovic etÂ€al. 2001) as well as other potential angiogenic factors (De Giovanni etÂ€al. 1995). In most RMS cell lines, VEGF stimulates proliferation or activates the PI3K/Akt pathway (Gee etÂ€al. 2005; Kurmasheva etÂ€al. 2007), hence acts both as an autocrine growth factor as well as a paracrine factor involved in angiogenesis. The activity of bFGF may also be enhanced by glypican-5, a gene amplified and overexpressed in RMS (Williamson etÂ€al. 2007). RMS cells also secrete predominantly IGF-2, which in turn signals to activate HIF1a, and upregulation of VEGF (Beckert etÂ€al. 2006). Thus, the use of agents that blocks either secretion of VEGF or its interaction with receptors on vascular endothelial cells to inhibit RMS-driven angiogenesis appears to offer therapeutic potential. VEGF has emerged as the key stimulatory molecule for promoting angiogenesis in a variety of human malignancies (Ferrara 2002). This was first translated into successful application of an antiangiogenic strategy when the anti-VEGF monoclonal antibody bevacizumab showed a survival benefit for patients with metastatic colorectal cancer when combined with fluorouracil-based combination chemotherapy (Hurwitz etÂ€ al. 2005). Clearly, antibodies that bind and neutralize VEGF have already defined a role in the treatment of adult cancer, and are in clinical evaluation for the treatment of pediatric cancer. In preclinical studies, a human VEGF-specific antibody inhibited the growth of numerous tumors including A673 rhabdomyosarcoma (actually a Ewing sarcoma cell line, Barlow etÂ€al. 2006), although inhibition of both tumorderived (human) as well as mouse derived VEGF was required for optimal inhibition of tumor growth (Gerber etÂ€ al. 2000). Thus, in a xenograft model both circulating mouse-derived VEGF as well as tumor-derived factor contributes to tumor progression. Small molecule inhibitors of the VEGF receptors such as AZD2171 are in adult and pediatric clinical trials. AZD2171 can not only inhibit VEGFR1, VEGFR2, and VEGFR3 kinase activity at low nanomolar concentrations but it also inhibits c-KIT and platelet derived growth factor receptor (PDGFR) (a and b) at similar concentrations (Wedge etÂ€al. 2005). In preclinical testing, this agent has shown some utility against RMS xenografts, significantly retarding growth in each of five RMS models (Maris etÂ€al. 2008a); however, all RMS xenografts demonstrated progressive disease. Other multitargeted kinase inhibitors such as sunitinib (VEGFR1-3, PDGF-a and -b, FLT-3, c-KIT, CSR-1) also inhibit angiogenesis, and inhibit the growth of RMS xenografts to a similar extent as AZD2171 (Maris etÂ€al. 2008b). An alternative approach is to inhibit production of VEGF by tumor cells. Rapamycin, a selective inhibitor of mTOR downstream of PI3K/Akt regulates VEGF transcription and translation leading to decreased tumor angiogenesis. The antiangiogenic activities of rapamycin have been linked both to a decrease in VEGF production and to inhibited the response of vascular endothelial cells to stimulation by VEGF (Guba etÂ€al. 2002, 2004, 2005). Rapamycin also targets vascular mesenchymal cells (pericytes, smooth muscle cells, adventitial fibroblasts) inhibiting sustained VEGF and hepatocyte growth factor (HGF) expression, via inhibition of the PDGFRa-p70S6K pathway (Tsutsumi etÂ€al. 2004). Data that support a role for

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mTOR signaling in VEGF production include regulation of HIF-1a by mTOR signaling and increased VEGF in cells deficient in the tuberous sclerosis complex (TSC1/2) that negatively regulates mTOR via Rheb (Hudson etÂ€al. 2002; Brugarolas etÂ€al. 2003, 2004). In growth factor-deprived neuroblastoma cells inÂ€vitro, serum or IGF-1 induced increases in HIF-1a protein that temporally paralleled increases in VEGF mRNA. VEGF and HIF-1a levels were blocked by inhibitors of PI3K and mTOR, and to a lesser extent by the MEK1 inhibitor PD98059. In these cells, it was estimated that HIF-1a mediates approximately 40% of the growth factor activity stimulating VEGF protein expression (Beppu etÂ€ al. 2005). Further, inhibition of signaling via the type 1 insulin-like growth factor receptor (IGF-1R) resulted in decreased VEGF inÂ€vitro, and inÂ€vivo and decreased tumor volume and weight, vessel density, tumor cell proliferation and increased tumor cell apoptosis (Stoeltzing etÂ€ al. 2003). Rapamycin analogs (temsirolimus, everolimus) have recently completed phase 1 trials in children, and phase 2 trials are ongoing. In preclinical models, rapamycin significantly inhibited the growth of most solid tumors and induced regression of sarcomas including RMS and acute lymphocytic leukemia models (Houghton etÂ€al. 2008). Although mTOR signaling is implicated in the regulation of VEGF, there are data to suggest that VEGF may be regulated through PI3K/Akt signaling independent of mTOR (Arsham etÂ€al. 2002). Further, inhibition of mTOR in many cancer cell lines results in the anecdotal activation of Akt (Shi etÂ€al. 2005; O’Reilly etÂ€ al. 2006), as a consequence of inhibiting p70S6K1 phosphorylation, thus suppressing S6K1-induced phosphorylation of IRS-1 that leads to its proteasomal degradation (Haruta etÂ€al. 2000). In vitro an Akt inhibitor (A443654) was significantly more effective in suppressing RMS-derived VEGF than rapamycin, but the combination of these agents essentially blocked increases in VEGF induced by hypoxia (Kurmasheva etÂ€al. 2007). Whether inhibitors of Akt can be combined with rapamycin with acceptable toxicity remains to be determined. However, it is probable that to adequately suppress tumor angiogenesis, a combination of approaches will be required.

Future Directions and Challenges Childhood RMS is a genetically complex disease, with mutations and activation of multiple genes and signaling pathways. Consequently, it seems unlikely that inhibition of a single aberrant gene product, as in BCR-ABL “driven” CML will have such a dramatic effect on RMS proliferation and survival. However, novel targets, including MET, IGF-1R, VEGF, and indirectly MYCN, are now amenable to pharmacological intervention. Perhaps with the exception of MYCN inhibition, the effects of inhibiting the other signaling pathways would be anticipated to be largely cytostatic rather than cytotoxic. While the next step will be integrating these agents with conventional cytotoxic therapy, the real challenge will be in developing combinations of molecularly targeted agents that induce selective tumor cell death based on genetic changes that lead to tumor-specific dysregulation of signaling

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pathways. Such synthetic lethal interactions, first described in yeast systems, can be identified in mammalian cells by a variety of new techniques. Foremost would be whole genome approaches using RNAi to identify genes that encode products required for survival only in the context of the oncogenic change (PAX3-FKHR expression, or MYCN overexpression). Development of drugs that inhibit these gene products would potentially be tumor-selective. Pharmacologic inhibition of CDK1 resulting in death only of cells overexpressing MYC serves as a paradigm. Alternatively, identifying genes essential for survival in the presence of inhibitors of specific signaling pathways only in the context of a transforming gene may offer the opportunity to develop novel agents that in combination induce cell death selectively in RMS cells. Inhibition of IGF-1R signaling in combination with inhibition of mTOR in cells with attenuated p53 function, for example, results in shifting the cellular response to rapamycin from cytostasis to cell death, and shows quite promising inÂ€vivo preclinical results, particularly in sarcoma models (Kurmasheva et al. 2009). Pediatric trials of IGF-1R-directed antibodies alone and in combination with rapamycin are in the planning stage, and many of the agents discussed in this chapter are being evaluated against preclinical models of RMS. It is anticipated that many of these agents will enter clinical trials for the treatment of childhood RMS in the next few years. Acknowledgmentsâ•… Original work reported here was supported by PHS awards CA23099, CA96696, CA77776, and CA21675 (Cancer Center Support Grant), and by ALSAC.

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Molecularly Targeted Therapy for Osteosarcoma: Where Do We Go from Here? Rosanna Ricafort and Richard Gorlick

Introduction/Epidemiology Osteosarcoma at the molecular level is among the most complex pediatric Â�malignancies. Despite its relatively high incidence in the pediatric patient population many aspects of this disease continue to defy understanding. Osteosarcoma is the most common primary malignant bone tumor in children and adolescents (Arndt and Crist 1999). Approximately 400 children and adolescents are diagnosed each year in the United States (Meyers and Gorlick 1997). In the adolescent age range, it is the second most common malignancy, following only lymphoma in incidence. Osteosarcoma continues to be diagnosed solely based upon its histologic appearance. Despite having tremendously variable appearance, characterized descriptively by its histologic subtype, the presence of a malignant spindle cell which produces osteoid is the basis for diagnosing osteosarcoma. Molecular analyses of gene copy number, translocations, and gene expression do not contribute to making the diagnosis. In osteosarcoma numerous genetic abnormalities are present, each of which individually could lead to the development of a malignancy. This redundancy makes it difficult to decipher the relative timing and importance of each of these events. Quite similar to the molecular abnormalities, the epidemiology suggests multiple etiologic factors that may play a role in osteosarcoma development. Some of these factors may have differing levels of importance in different patient populations with the overall contribution of each of these factors unknown. Multiple studies have linked periods of rapid growth to the development of osteosarcoma, and not surprisingly incidence for this disease peaks during the second decade of life. Osteosarcoma has a predilection for the rapidly growing long bones of the extremities, with the distal femur and proximal tibia accounting for approximately 50% of all cases (Meyers and Gorlick 1997). Other factors suggesting R. Gorlickâ•›() Department of Pediatrics, Pediatric Hematology/Oncology, The Children’s Hospital at Montefiore and the Albert Einstein College of Medicine, 3415 Bainbridge Avenue, Rosenthal 3rd Floor, Bronx, NY 10467, USA e-mail: [email protected] P.J. Houghton and R.J. Arceci (eds.), Molecularly Targeted Therapy for Childhood Cancer, DOI 10.1007/978-0-387-69062-9_21, © Springer Science+Business Media, LLC 2010

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an association with growth include its increased incidence in taller individuals in the majority of reported studies, as well as its earlier peak incidence in females concordant with their earlier growth spurt. Perhaps among the more frequently cited data in making this assertion, is the high incidence of osteosarcoma among large breed dogs and its almost complete absence among smaller dogs. More recent studies have suggested that the incidence of osteosarcoma does not vary within canine breeds suggesting the determining factor may be their underlying genetics rather than height as a phenotype. More recent explanations of canine breed height variability based on a finite number of polymorphisms in the insulin-like growth factor axis may give further credence to that view. Independent of growth, accelerated bone turnover likely plays a role in the etiology of osteosarcoma, given its association with disorders of bone metabolism (i.e., Paget’s disease of the bone, and fibrous dysplasia) (Meyers and Gorlick 1997; Gelberg etÂ€ al. 1997). Other clear predisposing factors include exposure to nonspecific DNA damaging agents such as ionizing radiation. Radiation exposure clearly increases the incidence of osteosarcoma, but the vast majority of patients diagnosed have no known exposures and are too young to permit the latency period that typically occurs between the exposure and tumor development. The mechanism by which these etiologic factors predispose to osteosarcoma remains unclear. Multiple molecular alterations resulting in inactivation of tumor suppressor genes and overexpression of oncogenes are observed with considerable variability in every osteosarcoma. With the multitude of genetic lesions in osteosarcoma, it is difficult to discern which of these events is fundamental in tumorigenesis. Despite the many advances in the surgical and medical management of osteosarcoma in the past three decades, outcome of this disease more recently has not changed, with longterm survival rates remaining 70% in patients with localized disease; and the outcome for those with advanced or relapsed disease is poor (Chou etÂ€al. 2005b). Defining the pathogenesis of osteosarcoma likely will have an impact on the prognosis and therapy of this tumor through defining relevant treatment approaches. The following review will explore the molecular alterations of osteosarcoma in attempts to elucidate its pathogenesis, substantiate pathways as therapeutic targets, and contemplate current strategies and future challenges in the treatment of this tumor.

Molecular Features of Tumorigenesis: Alterations in Osteosarcoma Osteosarcoma Cell of Origin Tumors may be defined by both the cells they originate from as well as the genetic events that lead to tumor formation. Identifying the type of cell in which osteosarcoma arises is critical to understand the molecular mechanisms that drive its growth and differentiation. Given its characteristic production of osteoid, traditionally

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osteosarcoma is thought to be derived from osteoblasts, though evidence suggests that the cell of origin of osteosarcoma may be a more primitive precursor (Gibbs etÂ€al. 2005). Osteosarcoma can have varying histologic appearance with descriptions, including osteoblastic, chondroblastic, fibroblastic, telangiectatic, and small cell subtypes (Bertoni et al. 1989). Many osteosarcomas contain multiple histologic subtypes. These histologic subtypes, with limited clinical relevance, may represent an osteosarcoma’s capacity to differentiate into multiple lineages suggesting a more pluripotent cellular potential than is associated with a mature osteoblast. The formation of the various mesenchymal derivatives from a stem cell is analogous to hematopoiesis. Bone, cartilage, fibrous stroma, muscle, tendon, and adipose are all derived from a pluripotent mesenchymal stem cell. Similar to oncogenesis in the context of the hematopoietic system, sarcomas may in theory differentiate or dedifferentiate during the tumor formation further complicating defining the cell of origin. Several other recent murine findings have also implicated mesenchymal stem cells as a possible progenitor (Tolar etÂ€al. 2007); however, all of these data are far from definitive. Whatever the cell of origin in osteosarcoma, its pathogenesis involves complex molecular disruptions in proliferation and cell cycle control, apoptosis, and DNA damage response, along with disruptions in differentiation. All sarcomas can be divided into those that have recurrent characteristic chromosomal translocations such as the translocations of chromosome 11 and 22 in Ewing sarcoma and PAX gene translocations in alveolar rhabdomyosarcoma. The translocation-associated sarcomas generally have simple karyotypes. Osteosarcoma is characteristic of those sarcomas which do not have a consistent, recurrent chromosomal translocation. Osteosarcoma, typical of this group, has a complex karyotype with multiple alterations in various genetic pathways, including p53. Heterogeneity in osteosarcoma is manifest in many manners, including less robust clustering in comprehensive gene expression profiles.

Cancer Predisposition Syndromes Cancer predisposition syndromes associated with a high incidence of osteosarcoma provide clues as to potential mechanisms for its tumorigenesis. The cancer predisposition syndromes associated with osteosarcoma are summarized in Tableâ•›1. Among those with sporadic osteosarcoma few will be found to be associated with an un-suspected cancer predisposition syndrome or their related germline genetic alteration. The incidence of osteosarcoma is high in those with defined genetic predispositions due to several familial cancer syndromes as well as it is one of the most common tumors associated with other malignancies. Patients with hereditary retinoblastoma have a heterozygous germline retinoblastoma (RB) gene mutation and develop retinoblastoma with 90% penetrance (Aue etÂ€al. 1998). The cumulative incidence of a second malignancy by 50 years after the initial retinoblastoma diagnosis is 51%, with approximately half of these tumors being osteosarcoma

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TableÂ€1â•… Hereditary cancer syndromes that predispose to osteosarcoma Hereditary cancer syndrome Retinoblastoma

Chromosome location 13q14.2

Gene RB1

Li-Fraumeni

17p13.1

P53

Paget’s disease

18q21-q22 5q31 5q35 18q24.3

LOH18CR1 SQSTM1 MAPK8 RTS (ReQL4)

8p12-p11.2

WRN (RecQL2)

15q26.1

15q26.1

Rothmund Thomson syndrome Werner syndrome

Bloom syndrome

Function Cell cycle regulation DNA damage response IL-1/TNF signaling RANK signaling DNA helicase

DNA helicase Exonuclease activity DNA helicase

% of malignancies that are OS 50% 10% Not applicable

30%

<10%

<10%

(Wong etÂ€ al. 1997; Draper etÂ€ al. 1986). Both osteosarcomas and retinoblastomas occurring in patients with hereditary retinoblastoma have somatic loss of the normal RB allele and resultant complete absence of a functional RB protein (Hansen etÂ€al. 1985). Individuals with Li Fraumeni syndrome have germline mutations in p53 predisposing them to multiple malignancies, 10% of which are osteosarcoma (Li etÂ€al. 1988). Most tumors in these patients have loss of the normal p53 allele (Malkin etÂ€al. 1990). Familial cancer syndromes caused by mutations in RECQ DNA helicases include Rothmund-Thomson syndrome, Werner’s syndrome, and Bloom syndrome. RECQ DNA helicases are conserved proteins that separate the complementary strands of DNA duplexes; the three RECQ DNA helicases involved in cancer predisposition syndromes are RECQL4 in Rothmund-Thomson syndrome, RECQL2 in Werner syndrome, and RECQL3 in Bloom syndrome; they have related but not identical functions in maintaining genomic integrity, which may account for their differential tendency for developing osteosarcoma (Wang etÂ€ al. 2003; Goto etÂ€al. 1996; Ellis etÂ€al. 1995). Patients with Rothmund-Thomson syndrome have a 30% incidence of osteosarcoma, compared to less than 10% of patients with either Werner syndrome or Bloom syndrome (Wang etÂ€al. 2001; Wang etÂ€al. 2003; Goto etÂ€al. 1996; Ellis etÂ€al. 1995). Unlike in Li Fraumeni and hereditary retinoblastoma, in which the p53 and RB regulated pathways are also frequent targets for somatic mutations in sporadic osteosarcoma unrelated to cancer predisposition syndromes, mutations in the RECQ DNA helicases giving rise to osteosarcoma seem to be restricted to their specific familial cancer syndromes (Nishijo etÂ€ al. 2004). Nevertheless, these cancer predisposition syndromes, summarized in TableÂ€1, offer clues as to genetic instability and complex molecular alterations involved in the pathogenesis of osteosarcoma. The genetic pathways with among the most

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compellingÂ� data for their involvement in osteosarcoma tumorigenesis include p53 and RB based upon the hereditary predisposition syndromes, data from murine models and their frequent alteration in sporadic tumors.

Viral Targeting of Tumor Antigens Simian Virus 40 (SV40) is a monkey polyomavirus that has been shown to be oncogenic when injected into animal models, inducing various tumors including sarcomas and osteosarcoma (Carbone etÂ€al. 1996). SV40 large T-antigen protein affects cellular processes via regulation of p53-mediated transcriptional activation and apoptotic pathways (Mendoza etÂ€al. 1998) as well as cell cycle regulation by the RB tumor suppressor pathway (Ahuja etÂ€ al. 2005). In some studies, SV40 DNA sequences have been demonstrated in up to 50% of osteosarcoma tumor samples (Carbone etÂ€al. 1996) suggesting that contamination of poliovirus by SV40 which occurred in North America between 1955 and 1963 and further spontaneous transmission of the virus may be an etiologic factor in osteosarcoma. However, another study failed to find a correlation with integration of SV40 DNA and p53 or RB mutation status (Mendoza etÂ€ al. 1998). The most definitive study demonstrating SV40 is not a major epidemiologic factor in the development of osteosarcoma was a negative serologic study for antibodies in a large cohort of osteosarcoma patients (Carter etÂ€al. 2003).

Murine Models Although genetic alterations in humans, which are associated with osteosarcoma, have a more directly established relevance, murine models offer additional evidence as to genetic alterations and exposures which can predispose to osteosarcoma. Transgenic mice engineered to overexpress SV40 develop osteosarcoma, with the clinical pattern dependent upon the promoter driving expression (Knowles etÂ€al. 1990). This is supportive of the viewpoint that the p53 and RB tumor suppressor genes have a role in osteosarcoma development as the SV40 large T antigen abrogates the functions of both of these pathways. Knock out mice with mutant or absent p53 develop osteosarcoma along with a variety of other tumors. Depending upon the particular p53 alteration, osteosarcoma develops in between 5 and 50% of the mice (Donehower etÂ€ al. 1992; Jacks etÂ€ al. 1994; Liu etÂ€ al. 2000; Olive etÂ€ al. 2004). Knocking out the RB gene is lethal at embryonic stages in mice not permitting an assessment of whether or not these mice are prone to developing osteosarcoma (Khidr and Chen 2006). Transgenic overexpression of several oncogenes can also produce osteosarcoma. Almost 100% of transgenic mice which ubiquitously overexpress Fos develop osteosarcoma (Grigoriadis etÂ€al. 1993; Wang etÂ€al. 1995). Transgenic mice which conditionally overexpress Myc in fibroblasts/osteoblasts

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develop osteosarcoma (Jain etÂ€al. 2002). Radiating mice leads to the development of osteosarcoma (Erfle etÂ€ al. 1986) as does chronic exposure to parathyroid Â�hormone (Vahle etÂ€al. 2002).

Cell Cycle Regulation Molecular alterations in cell cycle regulation are an almost constant feature in the pathogenesis of osteosarcoma. Dysregulation of cell cycle transition leading to malignant proliferation may be the result of many different cellular processes, including inhibition of apoptosis, amplification of DNA replication, loss of DNA repair mechanisms, and loss of checkpoint control. Molecular lesions resulting in RB and p53 pathway inactivation account for the majority of these genetic alterations in osteosarcoma. The inactivation of these pathways is accomplished through any one of a variety of genetic alterations. These alterations typically occur in a nonoverlapping manner so that only one mechanism of RB inactivation and one mechanism of p53 inactivation are present in an osteosarcoma. The RB gene, mapped to 13q14, has been implicated to play a role in these various cellular functions (Classon and Harlow 2002) and genetic lesions in the RB gene itself have been shown to be present in approximately 70% of primary osteosarcoma tumor samples (Wadayama etÂ€ al. 1994; Benassi etÂ€ al. 1999; Hansen etÂ€ al. 1985). Once phosphorylated, RB regulates cell cycle progression from G1 to S phase by interacting with the transcription factor E2F (Classon and Harlow 2002). Abnormalities in either RB or in the various proteins responsible for its phosphorylation can result in loss of its checkpoint function (Nevins 1992). Germline RB mutations, either sporadic or inherited, result in a high incidence of osteosarcoma; half of the second malignancies developing in patients with hereditary retinoblastoma are osteosarcomas (Wong etÂ€al. 1997). Approximately 60% of osteosarcoma tumors will have the loss of heterozygosity at 13q, the site of RB (Wadayama etÂ€ al. 1994; Toguchida etÂ€al. 1988; Feugas etÂ€al. 1996). Gross structural rearrangements of the RB gene are present in approximately 30% of osteosarcoma tumors, but mutations are rare, occurring in less than 10% (Wadayama etÂ€ al. 1994). Cyclin dependent kinase-4 (CDK4) in complex with cyclin D1 (CCND1) is responsible for the phosphorylation of RB, and therefore amplification or overexpression of these genes results in functional inactivation of the RB signaling pathway and subsequent cell cycle progression. Amplification of the 12q13-15 chromosomal region which encompasses the CDK4 locus is observed in approximately 5 to 10% of osteosarcomas, and when measured directly by Southern blotting, CDK4 is amplified in approximately 9% of osteosarcoma tumor samples (Wei etÂ€ al. 1999a). The INK4A gene, localized to 9p21, encodes p16INK4A, a tumor suppressor that inhibits the CDK4-CCND1 complex, thereby halting cell cycle transition. CDK4 amplification seems to occur independently from INK4A alterations, suggesting mutually exclusive molecular aberrations being sufficient for RB pathway disruption (Nielsen etÂ€ al. 1998; Wei etÂ€ al. 1999a). Amplification of the INK4A region is observed in approximately

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5 to 10% of osteosarcomas (Wei etÂ€ al. 1999a, b). Interestingly, by virtue of an Â� alternative reading frame, INK4A also encodes another tumor suppressor, p19INK4A ARF or p14 , which interacts with MDM2 in the p53 pathway (Quelle etÂ€al. 1995). Tumorigenesis in osteosarcoma, as in many other tumor systems, is affected by disruptions in DNA damage control. The tumor suppressor p53 gene product plays a major role in the cellular response to DNA damage (Bode and Dong 2004). Its alteration may allow cells to proliferate despite genetic errors. Once stabilized in the nucleus, p53 binds DNA and initiates cellular responses through transcriptional activation or suppression of distinct target genes that function to prevent proliferation of damaged cells via damage repair, cell-cycle arrest, or apoptosis (Bode and Dong 2004). By the transactivation of its major target, cyclin dependent kinase inhibitor p21WAF1 (p21), p53 inhibits the activities of cyclin D/CDK4 or CDK6, thereby halting cell cycle progression from the G1 to S phase by keeping RB in a hypophosphorylated state (Mulligan etÂ€al. 1990; El-Diery 1998). Mutations in the p53 gene, localized to 17p13, are the most frequently occurring genetic alteration found in human malignancies (El-Diery 1998; Lonardo etÂ€al. 1997). It is known that patients with Li-Fraumeni syndrome, heralded by heterozygous germline p53 mutations, have a higher incidence of osteosarcoma, as the second most common malignancy in these patients (Siddiqui etÂ€al. 2005). Approximately 3% of sporadically occurring osteosarcoma patients have a germline mutation in p53. A significant proportion of sporadic osteosarcoma samples have some molecular modification abrogating normal p53 function (McIntyre etÂ€ al. 1994). In osteosarcoma tumor samples, alterations in p53 consist of point mutations (occurs in 20 to 30%), gross gene rearrangements (occurs in 10 to 20%), and loss of one 17q allele (occurs in 75 to 80%) (Lonardo etÂ€ al. 1997; Toguchida etÂ€ al. 1998). Alterations in genes encoding cellular proteins that regulate p53 have also been implicated in the pathogenesis of osteosarcoma. MDM2 is a ubiquitin E3 ligase which negatively modulates p53 function by binding the protein, concealing its activation site and facilitating its proteasomal degradation (Oliner etÂ€ al. 1993; Oliner etÂ€ al. 1992; Kubbutat etÂ€al. 1997). Amplification leading to MDM2 overexpression functionally eliminates p53 even in the presence of wild-type protein (Oliner etÂ€al. 1993; Oliner etÂ€al. 1992; Kubbutat etÂ€al.; â•›1997). The human homolog of the MDM2 gene, that maps to chromosome 12q13-14, is amplified in about 10% of osteosarcomas (Oliner etÂ€al. 1993; Ladanyi etÂ€al. 1993a; Kubbutat etÂ€al. 1997; Lonardo etÂ€al. 1997). In most tumor samples, amplification of MDM2 gene is mutually exclusive to p53 overeexpression (considered indicative of a mutant protein with greater stability), confirming the reciprocal relationship of these abnormalities, and suggesting a model of differential pathways for p53 pathway inhibition in osteosarcoma tumorigenesis (Lonardo etÂ€al. 1997). Another negative regulator of p53 is COPS3 (constitutive photomorphogenic homolog subunit 3) of the COP9 proteasome. The COP9 proteasome binds and phosphorylates p53, targeting the tumor suppressor to degradation by the ubiquitin system (Bech-Otschir etÂ€al. 2001). The COPS3 gene maps to chromosome 17p11.2, part of a region amplified in about 25% of cases of osteosarcoma (Van Dartel and Hulsebos 2004). COPS3 was found to be consistently overexpressed after gene amplification (Van Dartel etÂ€al. 2004) and additionally via

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alternative mechanisms in some tumors lacking COPS3 gene amplification (Henriksen etÂ€ al. 2003). COPS3 amplification seems to be mutually exclusive to MDM2 amplification or p53 mutations, again suggesting that any of these alterations in the p53 tumor suppressor pathway is sufficient in regards to the contribution of this pathway to tumorigenesis in osteosarcoma (Henriksen etÂ€al. 2003).

Growth Factors/Signal Transduction Pathways Another aspect contributing to abnormal cell proliferation in the pathogenesis of osteosarcoma involves growth factor receptors and autocrine or paracrine stimulation, which leads to the loss of regulation of cell proliferation, motility, and angiogenesis. The higher incidence during the pubertal spurt, in anatomic sites of greater growth, and in taller individuals, suggests that growth factors play an important role in the initiation or potentially pathogenesis of osteosarcoma. Several oncogenes involved in cellular growth and differentiation may contribute to this pathogenesis. The coexpression of these growth factors along with their receptors on osteosarcoma tissue samples and cell lines suggests an autocrine or paracrine mechanism of action may be driving the proliferation of malignant cells in this tumor. Difficulty in understanding osteosarcoma lies in the redundancy with a multitude of growth factor receptors simultaneously overexpressed and potentially dysregulated. As mentioned previously, osteosarcoma has been associated with accelerated bone turnover (Meyers and Gorlick 1997). Therefore, it is not surprising that type-1 insulin-like growth factor (IGF-1), a major regulator of skeletal growth, has been found to be mitogenic in inÂ€ vitro models of osteosarcoma (Pollak etÂ€ al. 1990; Kappel etÂ€al. 1994; Bostedt etÂ€al. 2001; MacEwen etÂ€al. 2004). Survival of osteosarcoma cell lines inÂ€vitro has been shown to be dependent on exogenous IGF-I, and proliferation of these cell lines can be inhibited by blocking signaling through the IGF-I receptor (IGF-1R) via monoclonal antibodies or antisense oligonucleotides (Kappel etÂ€ al. 1994; Haluska et al. 2006). IGF-1R has been found to be abundantly expressed in osteosarcoma cells (Burrow etÂ€ al. 1998). IGF-1R has emerged as the predominant receptor in cancer biology that contributes not only to cellular proliferation, but perhaps also more importantly to malignant transformation and protection from apoptosis (Baserga etÂ€al. 2003). Overexpression of IGF-I and its receptor may therefore play a role in the pathogenesis of osteosarcoma by contributing to the oncogenic transformation, and providing a proliferative and survival advantage to malignant cells (Sekyi-Otu etÂ€al. 1995). Of the growth factor circuits involved in the pathogenesis of osteosarcoma, the contribution of IGF-I/ IGF-IR to the malignant potential of this tumor has been identified to be of major importance (Scotlandi etÂ€al. 2005). Overexpression of the c-MET proto-oncogene tyrosine kinase receptor (MET) and its ligand, hepatocyte growth factor (HGF) or scatter factor, in osteosarcoma cell lines suggests a role for the MET in the metastatic phenotype of osteosarcoma. Binding of HGF or scatter factor to the MET/HGF receptor stimulates both cell

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proliferation and motility, features associated with the malignant potential of tumor cells (Scotlandi etÂ€al. 1996; Rong etÂ€al. 1993). The predominant expression of the MET/HGF receptor on epithelial cells, along with its ligand being produced primarily by cells of mesenchymal origin, suggests a paracrine or autocrine signaling system in governing stromal-epithelial interactions (Scotlandi etÂ€al. 1996; Ferracini etÂ€al. 1995). In a study of 17 osteosarcoma tumor samples, 60% expressed the HGF receptor at high levels (Scotlandi etÂ€al. 1996). When primary and metastatic tumors from the same patient are compared, metastatic tumors have higher MET expression than the primary tumors, again indicating its role in potentiating the metastatic phenotype of osteosarcoma (Scotlandi etÂ€al. 1996; Ferracini etÂ€al. 1995; Oda etÂ€al. 2000). Several MET inhibitors are undergoing clinical trials, and both selective and multitargeted tyrosine kinase inhibitors are being developed. In addition, fully human antibodies targeting the ligand, and MET decoys are also being developed. Initial clinical trials with the small molecule MET inhibitor, ARQ197, suggest that this agent is well tolerated. Platelet derived growth factor (PDGF) is another potent mitogen for cells of mesenchymal origin (McGary etÂ€al. 2002). Coexpression of PDGF and its receptors has been observed in various human solid tumors and correlates with inferior prognosis and metastasis in some (Kawai etÂ€al. 1997; Henriksen etÂ€al. 1993; Sulzbacher etÂ€al. 2000, 2001), thereby suggesting an autocrine or paracrine mechanism driving cellular proliferation and differentiation, chemotaxis, and survival in these tumors. The role of PDGF/PDGF-R in osteosarcoma has been explored. In one immunohistochemical study, PDGF-AA and its receptor, PDGFR-a, was found in 34 and 27% of osteosarcoma tumor samples, respectively, with coexpression of both proteins common (Sulzbacher 2000). Another analysis demonstrated PDGF-A and PDGFR-a expression in 80% of osteosarcoma tumor samples, with coexpression found in 76% of the patients; the expression of either growth factor or receptor, or both, correlated significantly with a shorter event-free survival (Kubo etÂ€al. 2002). Other tyrosine kinase receptors have been implicated in the pathogenesis of osteosarcoma. The ERBB2 proto-oncogene (also called HER2/neu), located on 17q12, encodes a protein structurally homologous to the epidermal growth factor receptor, although its actual ligand has not yet been identified. The importance of expression of ERBB family of tyrosine kinase receptors for tumor growth or clinical outcome has been demonstrated in a variety of human cancers, most notably in invasive breast cancers and non-small cell lung cancer (Wen etÂ€ al. 2007). In these carcinomas, activation of intracellular mitogenic signal transduction pathways is implicated in contributing to tumorigenesis (Wen etÂ€al. 2007). The data in osteosarcoma are somewhat controversial, with some investigators reporting overexpression, associated with either favorable or unfavorable prognosis, and other investigators reporting lack of overexpression of ERBB2 (Zhou etÂ€al. 2003; Akatsuka etÂ€al. 2002; Gorlick etÂ€al. 1999; Hughes etÂ€al. 2006; Scotlandi etÂ€al. 2005; Somers etÂ€al. 2005; Thomas etÂ€al. 2002; Maitra etÂ€al. 2001; Kilpatrick etÂ€al. 2001; Anninga etÂ€al. 2004; Scotlandi et al. 2004). In a study of 26 osteosarcoma tumor samples, 42% expressed ERBB2; the expression of ERBB2 in this cohort was associated with a poor Â�prognosis (Onda etÂ€al. 1996).

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Conversely, another study found HER2 overexpressionÂ�associatedÂ�with increased survival and less metastases (Akatsuka etÂ€al. 2002). A more recent article, utilizing immunohistochemistry and tissue microarray data, failed to find significant overexpression of the HER2 protein or gene amplification at the ERBB2 locus (Somers etÂ€ al. 2005). Various explanations may account for the discrepancies between studies, including the methodology and interpretation of immunohistochemical staining, the antibody clones utilized, and the cross-reactivity with other membrane antigens, along with inherent biologic variability among osteosarcoma samples tested in various institutions representing different geographical areas. TableÂ€2 summarizes key studies investigating the expression of ERBB2 and its clinicopathologic correlates. ERBB4 is another member of the epidermal growth factor receptor family that has been implicated in the pathogenesis of osteosarcoma. Hughes etÂ€al. evaluated the expression pattern of ERBB family members in primary osteosarcoma tumor samples, and found cell surface expression of EGFR and ERBB2, along with nuclear expression of the p80 isoform of ERBB4 (Hughes etÂ€al. 2004). In a followup study, the authors demonstrated constitutive phosphorylation of these ERBB family of receptor tyrosine kinases in osteosarcoma cells, and suggested their activated role in tumorigenesis (Hughes etÂ€al. 2006).

Signal Transduction Identification of growth factors that may play a role in the pathogenesis of osteosarcoma leads one to consider the downstream signal transduction pathways resulting from activation of their receptors. The majority of signaling by the aforementioned receptors are transduced by three inter-related parallel pathways; RAS/RAF/Â� mitogen activated protein kinases (MAPK), phosphatidylinositol 3¢-kinase/Akt (PI3K/AKT), and mammalian target of rapamycin (mTOR). Each of these pathways is comprised of a series of kinases, where signal is transmitted through phosphorylation of amino acids, tyrosine, serine or threonine. Transmission of signal in this manner permits it to occur rapidly as no RNA or protein synthesis is required. This transmission also permits marked signal amplification. RAS/RAF/MAPK – The MAPK pathways play pivotal roles in cell proliferation, differentiation and survival. The closely related MAPK pathways are regulated through a series of phosphorylation steps in a three component module: MAPKs are activated by MAPK kinases (MAPKKs) on dual residues of threonine and tyrosine, and MAPKKs are in turn phosphorylated by MAPK kinase kinases (MAPKKKs) on dual residues of serine/threonine. This unusual feature ensures the tight control on the signals transduced by MAPK pathways in terms of specificity, intensity, and duration. The ERK1 and ERK2 MAPKs (ERK1/2), often activated by growth factors, are widely considered as prosurvival and oncogenic. The constitutiveÂ� active form of the MAPK/ERK kinase (MAPKK of ERK, MEK1/2) is sufficientÂ�to transform murine fibroblast 3T3 cells (Mansour etÂ€al. 1994; Cowley etÂ€al. 1994).

TableÂ€2â•… Overview of clinicopathological studies of erb-B2 (HER-2/neu) expression in osteosarcoma Protein overexpression by IHC Pattern/scoring Reference Samples Antibody system %â•›+â•›samples Gene amplification Onda etÂ€al. 26 PT CB-11 Mem/qualitative 42% (11/26) Southern Blot (1996) CBE1 (cDNA probe) – no amplification or rearrangement in IHCâ•›+â•›samples PCR No activating mutation FISH (Her-2/neu 44% PT 11/25) Zhou etÂ€al. 25 PT Ab-3 Mem and/or Cy probe) 58% PulM (7/12) (2003) 12 PulM + >/=25% 6/7 IHC + *majority Cy+; positive stain 2/5 IHC − rarely Mem+ 5B5 Mem 42% (20/47) PT 47 PT Gorlick etÂ€al. Herceptest +>25% 50% (3/6) PulM (1999) 6 PulM 76% (10/13) Rec 13 Rec 62% (51/81) (either 81 PT, all L CB-11 Mem and/or Cy Akatsuka Mem etÂ€al. (2002) + >30% positive and/or Cy) stain 21 PT; 13 L, A0485 Mem 0% FISH: Ratio of Maitra etÂ€al. (2001) 8M HER-2/neu oncogene and ch17 centromere >/=2.0â•›=â•›amplified in none (0/21) NA

(continued)

Poor HR (pâ•›<â•›0.03), decreased EFS (localized at dx:47 vs 79% 5 years, pâ•›=â•›0.05) Increased EFS (72 vs 46% 5 years, pâ•›=â•›0.03)

Shorter overall DFS (pâ•›<â•›0.04); no correlation with HR

Clinical correlation IHCâ•›+â•›of primary tumor associated with: Decreased DFS (pâ•›<â•›0.01), Early pulmonary mets (pâ•›<â•›0.05), Poor HR (pâ•›<â•›0.01)

Molecularly Targeted Therapy for Osteosarcoma: Where Do We Go from Here? 469

40 PT 1 Rec parosteal OS

25 PT 9 PulM 84 PT, all L

15 PT 18 Res

Kilpatrick etÂ€al. (2001)

Thomas etÂ€al. (2002) Scotlandi etÂ€al. (2005)

Anninga etÂ€al. (2004)

Mem + if 2+ stain

Mem and/or Cy + if >/=25% positive stain

3B5 CB-11 Herceptest

Herceptest

Mem and/or Cy

CB-11 monoclonal antibody, and Oncor polyclonal) A0485

Mem and/or Cy

Protein overexpression by IHC Pattern/scoring Antibody system

0% Mem (47% Cy) 32% (27/84; 78% concordance between the 2 antibodies) 28% by Herceptest (12/43) 3% (1/27)

0% Mem (Cyâ•›+â•›in 98% by CB-11, 83% by Oncor)

%â•›+â•›samples

NA

IHCâ•›+â•›associated with inferior EFS (pâ•›=â•›0.03)

FISH: HER2/neu probe 13 IHCâ•›+â•›tested – none showed amplification FISH: no HER-2 gene amplification in the IHCâ•›+â•›case RT-PCR: no overexpression of HER2 mRNA

NA

Cy IHCâ•›+â•›did not correlate with HR, metastasis, or EFS

RT-PCR: 0/19 OS

Gene amplification

Clinical correlation IHCâ•›+â•›of primary tumor associated with:

PT primary tumor; PulM pulmonary metastases; L localized; M metastatic; Res resection; Rec recurrent; Mem membrane staining; Cy cytoplasmic staining; IHC immunohistochemistry; EFS event-free survival; HR histologic response to preoperative chemotherapy; NA not applicable

Samples

Reference

TableÂ€2â•… (continued)

470 R. Ricafort and R. Gorlick

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Activating mutations are frequently found in BRAF (a MAPKKK), or its upstream activator, RAS, in colorectal cancer and melanoma (Davies etÂ€ al. 2002). The ERK1/2 MAPK is among the most common dysregulated pathways identified in tumors, as indicated by activating mutations frequently occurring in RAS or BRAF in human cancer, particularly in colon cancer and malignant melanoma (Davies etÂ€al. 2002). Some evidence suggests that the ERK MAPK pathway may mediate differentiation in PC12 cells and osteoblasts (Cowley etÂ€al. 1994; Salasznyk etÂ€al. 2004). To date, little information is available regarding the role of this pathway in osteosarcoma. Several sources of data that suggest this pathway may be constitutively active in osteosarcoma (Kubo etÂ€al. 2002). PI3K/AKT and mTOR – The PI3K/AKT and mTOR signal transduction pathways play an important role in transducing a variety of receptor signals. mTOR, perhaps most importantly, serves as one of the intracellular pathways through which IGF signaling is transduced (Thimmaiah etÂ€al. 2003). Signal transduction via PDGF receptors occurs predominantly through the PI3K/AKT pathway (Thomas etÂ€ al. 1997). Ezrin, which will be discussed further subsequently, mediates its growth and survival signal via both PI3K/AKT and mTOR but not MAPK (Krishnan etÂ€al. 2006). These signaling pathways have a variety of components and downstream targets, a discussion of which is beyond the scope of this chapter. In the context of osteosarcoma, little is known about these pathways although several publications demonstrate activation of these pathways with a variety of ligands and with associated biological affects. Analogous to the MAPK pathway, both PI3K/ AKT and mTOR have been suggested to be involved in the differentiation of mesenchymal stem cells, in this case toward the adipogenic lineage (Yu etÂ€al. 2007). The pathways have been suggested as being regulators of osteoblast growth and apoptosis. The mTOR pathway plays a major role in cell growth and differentiation in a variety of tumors, including osteosarcoma (Bjornsti and Houghton 2004; Easton and Houghton 2006). PI3K/AKT has been implicated in osteosarcoma’s metastatic process (Fukaya etÂ€al. 2005) as well as chemotherapy sensitivity.

Other Oncogenes The contribution of alterations of other cellular proto-oncogenes to the molecular pathogenesis of osteosarcoma has been investigated. The MYC proto-oncogene, localized to 8q24, functions in the regulation of cell proliferation. c-Myc has the potential to homo and heterodimerize. When complexed with MAX, it activates the transcription of a large number of genes with many involved in the regulation of proliferation, cell growth, inhibition of differentiation and apoptosis. It has been found to be overexpressed in a subset of osteosarcoma tumor samples (Ladanyi etÂ€al. 1993a, b; Pompetti etÂ€al. 1996; Ikeda etÂ€al. 1989; Gamberi etÂ€al. 1998). The MYC gene is amplified in 44% of osteosarcomas (Kochevar etÂ€al. 1990; Ladanyi etÂ€al. 1993b; Gamberi etÂ€al. 1998; Stock etÂ€al. 2000). c-MYC expression has been suggested to be related with metastasis (Gamberi etÂ€al. 1998).

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Fos was initially identified as being related to osteosarcoma through recognition that mice inoculated with FBJ murine virus developed these tumors (Finkel etÂ€al. 1966; Van etÂ€al. 1983; Miller etÂ€al. 1984). The viral protein which induces osteosarcoma is v-fos, which led to the identification of the structurally related c-Fos protein as an oncogene (Curran etÂ€al. 1983). Fos heterodimerizes with Jun to regulate the AP1 transcription factor. The AP1 transcription factor is known to be regulated by Vitamin D, transforming growth factor-bTGF-b and parathyroid hormone (PTH) – all known to be involved in the regulation of bone growth, indicating a possible basis for its association with osteosarcoma development. Overexpression of FOS oncoprotein is found in the majority of osteosarcomas, and potentially associated with metastasis or recurrence (Ruther et al. 1989; Wu etÂ€ al. 1990; Pompetti etÂ€al. 1996; Franchi etÂ€al. 1998; Gamberi etÂ€al. 1998; David et al. 2005).

Genetic Complexity In addition to the known loss of tumor suppressor genes and gain of oncogenes, osteosarcoma has tremendous chromosomal complexity with numerous other genes affected by gross genetic changes. Osteosarcoma DNA ploidy ranges from diploid to hexaploid with the numbers of each chromosome quite variable. As one evidence of the genetic complexity, spectral karyotyping reveals an average of 39 chromosomal rearrangements per tumor (Bayani etÂ€al. 2003). Some of the genetic alterations appear to be random but recurrent regions of chromosomal gain and loss are present. In many of these cases, the target of the genetic amplification and loss are known and in many cases they are not. The most common genetic losses and regions with losses of heterozygosity involve chromosomes 2, 3, 6, 9, 10, 13, 17, and 18 with the RB gene located on chromosome 13 and P53 located on chromosome 17. Loss of heterozygosity on chromosome 18 is also seen in Paget’s disease and chromosome 3 in Brachmann-de Lange Syndrome, a bone dysmorphology syndrome (Nellissery etÂ€ al. 1998). The most common regions of amplification include regions within chromosomes 1, 5, 6, 8, 12, 16, and 17 with MYC located on chromosome 8 and MDM2 on chromosome 12. Candidate genes for some of the amplified regions include PRDM16, CDC5L, NFKBIE, IFNG, MGRN1, PMP22, MYCD, TOP3A, MAPK7, and COPS3 (Man etÂ€al. 2004).

Immortalization Immortalization heralded by telomere length stabilization has been explored as contributing to the pathogenesis of osteosarcoma. Telomeres are nucleoprotein structures that cap chromosome ends and serve to potentiate the replicative process in cells by compensating for the loss of genetic material secondary to the inability to replicate end DNA. Maintaining telomere length is regarded as a fundamental

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necessity for overcoming cellular senescence in tumor cells. Some osteosarcoma tumor models attribute the complex unbalanced karyotypes that characterize osteosarcoma to telomere dysfunction. Although this model has not been disproven, the patterns of genetic rearrangements observed in osteosarcoma are not typical of tumors undergoing chromosomal fusion-bridge-breakage cycles secondary to telomere length erosion. Maintaining telomere length is accomplished by most cancers via activation of an enzyme, telomerase (TERT), a ribonuclease protein. In contrast, TERT activity has been shown in a minority of osteosarcoma tumors, and does not seem to be essential to tumorigenesis. When present in osteosarcoma tumor samples, TERT activity has been shown to be low in comparison to HeLa cell positive controls (Aue etÂ€ al. 1998). The clinicopathological correlation of telomerase has varied: an inverse relationship was reported between TERT activity and pulmonary metastases in patients treated with chemotherapy (Sangiorgi etÂ€ al. 2001), while another study found decreased survival associated with TERT expression in primary osteosarcoma tumor samples (Sanders etÂ€ al. 2004). Alternative mechanisms of telomere lengthening (ALT), a recombination-based method characterized by heterogeneous and elongated telomeres, has been the mechanism of maintaining telomere length that is seen more often in osteosarcoma. About 60% of osteosarcoma tumor samples demonstrated elongated telomeres with ALT (Ulaner etÂ€al. 2004). Aggressiveness of osteosarcoma tumors has been associated with telomere integrity: a subset of osteosarcoma patients that lacked both TERT activity and evidence of ALT demonstrated a more favorable prognosis (Ulaner etÂ€al. 2003).

Bone Differentiation The complex molecular alterations in osteosarcoma may not only involve dysregulation of cellular pathways involved in proliferation and immortalization, but also disruptions in osteoblast differentiation. Osteosarcoma can be regarded as evolving from a primitive pluripotent progenitor cell that retains its proliferative capacity while undergoing partial differentiation, or alternatively, a more differentiated precursor cell that de-differentiates and gains a propensity to proliferate, or loses its nonproliferative commitment. Understanding the molecular mechanisms underlying osteogenic differentiation and discovering genetic alterations within these pathways in osteosarcomas, contributes to our understanding of the molecular pathogenesis of osteosarcoma. The differentiation status of osteosarcoma tumors is variable (Haydon etÂ€al. 2007). Transforming growth factor-beta (TGF-b) isoforms play an important role in regulating bone formation. An immunohistochemical analysis demonstrated expression of one or more TGF-b isoforms, particularly TGF-b3, in all of the 25 high grade osteosarcoma tumor samples studied (Kloen etÂ€ al. 1997). TGF-b3 was correlated with disease progression, and the pattern of staining in the endothelial and perivascular layers in the tumor stroma suggested an angiogenic activity of this growth factor (Kloen etÂ€ al. 1997). Another study

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demonstrated increased expression of TGF-b2 and VEGF correlated with Â�osteosarcoma grade (Jung etÂ€al. 2005). Bone morphogenetic proteins (BMPs), part of the TGF-b superfamily, are multifunctional cytokines that regulate bone and skeletal development. BMPs are involved in the differentiation of mesenchymal cells to cells of osteoblastic lineage, and in the differentiation of immature osteoblasts into mature osteoblasts (Wei etÂ€ al. 1999b; Ohta etÂ€ al. 1992). In vitro data suggest that BMPs stimulate the growth of osteosarcoma cells (Ohta etÂ€al. 1992). Numerous BMPs and/or their receptors are highly expressed in osteosarcoma tumors, implicating their importance in autocrine or paracrine growth stimulation of osteosarcoma (Wei etÂ€al. 1999b). Overexpression of BMP Receptor II has been associated with poor prognosis and metastatic potential in osteosarcoma (Wei etÂ€al. 1999b; Weiss etÂ€al. 2006; Yoshikawa etÂ€al. 1988). BMPs have been demonstrated to induce the expression of c-fos mRNA in animal models of osteoblast formation, suggesting that the sustained expression of c-fos has a role in the pathogenesis of osteosarcoma (Ohta etÂ€al. 1992). The process of osteoblast differentiation is driven by wingless (WNT) signaling within the mesenchymal stem cell. WNTs, encompassing the wingless and int genes, encode a family of highly conserved, secreted ligands that play an important role in osteoblast development and tumorigenesis (Hoang etÂ€ al. 2004b). WNT binds to the cell surface receptor frizzled (FRZ) and the coreceptor lipoprotein related proteins 5 and 6 (LRP-5/6), leading to the stabilization and accumulation of b-catenin that in turn elicits a variety of effects, including the induction of differentiation and proliferation (Hoang etÂ€ al. 2004a, b). Aberrant activation of WNT signaling is associated with many human cancers (Polakis 2000). The role of Wnt pathway signaling in the pathogenesis of osteosarcoma purports that canonical Wnt signaling functions in maintaining an undifferentiated, proliferating progenitor population (Boland etÂ€al. 2004). Cytoplasmic or nuclear accumulation of b-catenin, owing to b-catenin deregulation by WNT pathway signaling, has been found in a majority of osteosarcomas (Haydon etÂ€al. 2002a), and has been correlated with its metastatic potential (Iwaya etÂ€ al. 2003). Expression of LRP5 seen in 50% of osteosarcomas is associated with a less-differentiated phenotype and metastasis (Hoang etÂ€al. 2004a, b). Overexpression of dickkopf-3, DKK-3, a WNT agonist, and inhibition of LRP5 by using a dominant-negative LRP5 receptor in osteosarcoma cell lines was shown to inhibit tumor cell motility and invasion, providing evidence linking WNT pathway signaling to osteosarcoma tumorigenesis (Hoang etÂ€ al. 2004a). DKK-1, a secreted inhibitor of canonical WNT signaling, inhibits osteogenesis, and has been found to be elevated in pediatric osteosarcoma samples when compared to unaffected individuals (Lee etÂ€al. 2007). The WNT signaling pathway, along with other signaling pathways that are involved in osteoblast differentiation, can be induced, perhaps in parallel, by PTH (Kulkarni etÂ€al. 2005; Carpio etÂ€al. 2001). PTH plays a central role in the regulation of bone and mineral metabolism, and has been implicated in the pathogenesis of osteosarcoma. In response to parathyroid hormone-related protein (PTHrP), osteoclastic resorption of bone is stimulated, and as a result, growth factors such as TGF-b and IGF-1 are released from the surrounding extracellular matrix, further

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stimulating PTHrP secretion and additional tumor growth. This positive-feedback loop culminates in osteosarcoma tumor growth and metastasis (O’Keefe and Guise 2003; Yang etÂ€al. 2007a).

Metastasis Chemokine stromal cell-derived factor 1 (SDF-1) is a cytokine-like protein expressed on the surface of vascular endothelial cells that, through binding to its chemokine receptor, CXCR4, plays a role in cytoskeleton rearrangement, adhesion to endothelial cells, and chemotaxis (Bleul etÂ€ al. 1996; Gupta etÂ€ al. 1998). Involvement of the CXCR4/SDF-1 pathway has been implicated in the metastatic potential of many cancers, including rhabdomyosarcoma and lymphoma (Taichman etÂ€al. 2002; Libura etÂ€al. 2002; Corcione etÂ€al. 2000). CXR4 mRNA has been shown to be expressed in osteosarcomas and associated with the presence of metastases at the time of diagnosis (Laverdiere etÂ€al. 2005). In vitro assays showed that migration of osteosarcoma cells expressing CXCR4 follows an SDF-1 gradient and that their adhesion to endothelial and bone marrow stromal cells is promoted by SDF-1 treatment (Perissinotto etÂ€al. 2005). This study also provided a rationale for the propensity of osteosarcoma to metastasize to the lung, where SDF-1 concentration is high, and demonstrated the prevention of pulmonary metastasis in a murine model by the administration of a CXCR4 inhibitor (T134 peptide), suggesting molecular strategies inhibiting this axis as a therapeutic target (Perissinotto etÂ€al. 2005). Ezrin is a membrane-cytoskeleton linker protein that allows direct cellular interactions with the microenvironment, facilitating signal transduction through growth factor receptors and adhesion molecules, thereby regulating cell migration and metastasis (Khanna etÂ€al. 2004). The role of ezrin in potentiating the metastatic potential of osteosarcoma has been investigated. In an orthotopic model of murine osteosarcoma, ezrin expression was threefold higher in the more aggressive K7M2 cell line and correlated with the metastatic potential of the aggressive osteosarcoma when compared to the less aggressive K12 cell line (Khanna etÂ€al. 2000, 2001). In follow-up experiments, ezrin expression was found to provide an early survival advantage for pulmonary metastatic osteosarcoma, mediated partly by MAPK but not AKT (Khanna etÂ€ al. 2004). A significant correlation between high ezrin expression and poor outcome has been shown, with shorter disease-free survival and higher risk of metastatic relapse, thereby corroborating the animal model data (Khanna etÂ€al. 2004).

Angiogenesis In addition to cell proliferation and motility, another feature contributing to the Â�metastatic potential of tumors is neovascularization. Angiogenesis elicits Â�proliferation and migration of endothelial cells to allow the formation of new

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Â� capillaries (Kaya etÂ€al. 2000). Vascular endothelial growth factor, VEGF, is a Â�peptide that acts as a mitogen for endothelial cells, directing new vessel formation and vascular maintenance. The role of neoangiogenesis in the pathogenesis of osteosarcoma has been explored via expression analysis of VEGF in tumor samples. In one such study, VEGF expression was seen by immunostaining in 62% of 17 primary osteosarcoma tumor samples; VEGF positivity defined by expression in greater than 30% of tumor cells, correlated with a poor prognosis amid a significantly higher metastatic rate (Kaya etÂ€al. 2000). This association as a negative predictive factor persists following neoadjuvant chemotherapy, as another study illustrated the presence of VEGF expression in greater than 25% of tumor cells at the time of definitive surgery was associated with a poor outcome (Charity etÂ€al. 2006). This finding supports the concept that the degree of necrosis at the time of definitive surgery reflects the innate biology of osteosarcoma rather than treatment specific factors (Davis 1994, Gorlick and Meyers 2003a). Recent research involving VEGFR1-positive hematopoietic bone marrow progenitor cells initiating a premetastatic niche (Kaplan etÂ€al. 2005) exposes the possible role of VEGF in fostering the metastatic potential of osteosarcoma tumors via expression on osteoprogenitor cells.

Drug Resistance The utility of adjuvant and neoadjuvant chemotherapy for osteosarcoma has long been established (Meyers and Gorlick 1997; Carli 2003); however, its efficacy can be hindered by the presence of intrinsic and/or acquired drug resistance. Possible mechanisms of drug resistance in osteosarcoma include alterations in the expression of p-glycoprotein(P-GP), multidrug resistance protein expression, topoisomerase II, glutathione S-transferases, DNA repair enzymes, drug metabolism (inactivation), and reduced intracellular delivery (Gorlick etÂ€ al. 2003). Of these mechanisms, P-GP expression has been the most extensively studied in osteosarcoma. P-GP is a transmembrane ATP-dependent efflux pump protein encoded by the multidrug resistance (MDR1) gene, which is responsible for the efflux of numerous chemotherapeutic agents, including doxorubicin (Hanahan and Weinberg 2000). Immunohistochemistry or RT-PCR quantification studies have explored P-GP expression in osteosarcoma, and demonstrated overexpression of P-GP in 23 to 45%, with decreased survival reported for patients with P-GP-positive tumors (Baldini etÂ€ al. 1995; Chan etÂ€ al. 1997; Hornicek etÂ€ al. 2000; Yamamoto etÂ€al. 2000; Park etÂ€al. 2001; Serra etÂ€al. 2003). A meta-analysis also proposes that P-GP is associated with an increased risk of disease progression (Pakos and Ioannidis 2003). Though the literature suggests that P-GP may be a marker of drug resistance and aggressiveness in osteosarcoma (Baldini etÂ€ al. 1995; Chan etÂ€ al. 1997; Hornicek etÂ€al. 2000; Yamamoto etÂ€al. 2000; Park etÂ€al. 2001; Serra etÂ€al. 2003), a prospective clinicopathological national intergroup study concluded that there was no correlation between P-GP expression and percentage of osteosarcoma tumor necrosis after induction chemotherapy or event-free survival in localized osteosarcoma (Schwartz etÂ€ al. 2000, 2007). Overexpression of MDR1, the gene

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encoding P-GP, also has been explored in regards to its role in the progression of osteosarcoma and its prognostic relevance. Though a small pilot study showed a trend toward a worse outcome in patients exhibiting high levels of MDR1 expression (Wunder etÂ€al. 1993), the larger, prospective investigation did not delineate any correlation between MDR1 expression and disease progression in patients with osteosarcoma, with patients with either very low or very high levels of MDR1 fairing poorly (Wunder etÂ€al. 2000). High dose methotrexate (MTX) with leucovorin rescue is a major component of current protocols for the treatment of osteosarcoma (Meyers and Gorlick 1997; Rosen etÂ€al. 1982; Meyers etÂ€al. 2005). High-dose MTX is vastly more effective than conventional dose methotrexate in the treatment of osteosarcoma – a finding that is not observed in other malignancies (Gorlick etÂ€al. 1996), implying a mechanism of intrinsic MTX resistance within osteosarcoma tumor cells (Guo etÂ€ al. 1999). MTX is a potent inhibitor of dihydrofolate reductase, a key enzyme for intracellular folate metabolism. In experimental systems, resistance to methotrexate can occur through a variety of mechanisms, including impaired intracellular transport of the drug via the reduced folate carrier, upregulation of dihydrofolate reductase, and diminished intracellular retention secondary to polyglutamylation (Betino 1993; Banerjee et al. 1995; Yang et al. 2003). Studies have demonstrated that impairments of drug influx secondary to decreased expression and mutations in the reduced folate carrier gene is the major basis of intrinsic resistance: 65% of osteosarcoma tumor samples were found to have decreased reduced folate carrier expression at the time of initial biopsy (Guo etÂ€al. 1999). In contrast, dihydrofolate reductase overexpression was seen relatively infrequently at initial biopsy – in only 10% of osteosarcoma tumor samples, as opposed to in 62% of the tumors at the time of definitive surgery or relapse, suggesting this means as the major mechanism of acquired MTX resistance in osteosarcoma (Guo etÂ€al. 1999).

Rationale for Targeting Specific Pathways Tumorigenesis in osteosarcoma, as in all human cancers, is a multistep process reflecting genetic alterations that drive the progressive transformation of normal osteoprogenitor cells into highly malignant osteosarcoma. The molecular pathways presented thus far represent the molecular hallmarks of cancer development set forth by Hanahan and Weinberg (2000) – self-sufficiency in growth signals, insensitivity to antigrowth signals, evasion of apoptosis, strong replicative potential, sustained angiogenesis, and tissue invasion and metastasis. The multiplicity of genetic alterations described in osteosarcoma, and the redundancy of these pathways makes it difficult to discern which, if any, of these pathways is central in tumor initiation and progression, and therefore which pathways to target for anticancer drug development. Similar to many pediatric malignancies, the lack of a premalignant lesion and even a clearly defined cell of origin preclude direct investigations of the multiple steps in osteosarcomas pathogenesis. Although tumors that arise in patients with Li Fraumeni syndrome and hereditary retinoblastoma have

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defined inciting molecular aberrations in the p53 and RB pathways, and though these pathways encompass the majority of genetic alterations in osteosarcoma patients, they are not known to be the primary events in sporadic osteosarcoma. A summary of the genetic alterations are presented in TableÂ€ 3 and a schema in Fig.Â€ 1. Given the need for improved therapeutic options for patients with highgrade osteosarcoma, targeted therapies based on molecular knowledge of critical genetic events may offer attractive adjuncts to our current strategies for osteosarcoma. Factors central in the pathogenesis of osteosarcoma are likely to be prognostic and/or relevant therapeutic targets. In the context of other malignancies, therapeutically targeting signaling pathways that the tumor is addicted to, have generally met with the most success. Defining these addictions in osteosarcoma has been problematic with the most central alterations being loss of the p53 and RB tumor suppressor genes. Correcting these alterations, which would require a gain of function, has been difficult to achieve therapeutically. Most clinical trials have been conducted empirically focusing on conventional cyototoxic chemotherapy. Prioritizing clinical trials of chemotherapy directed to specific pathways will be the challenge for the future. Given the redundancy of the genetic alterations in osteosarcoma, clinical success may ultimately require inhibition of multiple pathways simultaneously. Current and future comprehensive expression profiling studies may further shed light as to the genes central in the molecular pathogenesis of osteosarcoma and provide new insight in guiding targeted therapeutic strategies.

Current Strategies The standard approach to the treatment of osteosarcoma employs multimodality therapy with surgery in addition to systemic neoadjuvant and adjuvant chemotherapy most standardly comprises high-dose methotrexate, cisplatin, and doxorubicin. Despite improvements in supportive care, surgical techniques, and aggressive chemotherapy approaches for osteosarcoma, 30 to 40% of all patients with high-grade osteosarcomas die of metastatic or recurrent disease (Kempf-Bielack etÂ€ al. 2005; Bacci et al. 2000). Therapeutic options for these high-risk patients are therefore needed. The conventional cytotoxic agents currently utilized in the treatment of osteosarcoma were discovered more than 20 years ago. These agents are characterized by their narrow therapeutic index and thus limited by associated toxicities. It appears that the doses of conventional cytotoxic agents have been maximally applied in osteosarcoma (Chou and Gorlick 2006); Accordingly, emerging therapeutic strategies have shifted from dose intensification of current cytotoxics to the development of novel therapies (Casas-Ganem and Healey 2005; Anderson and Pearson 2006). Radiation therapy has not been widely utilized in the upfront treatment of osteosarcoma as conventional teaching dictates that it is not sensitive to radiotherapy at doses that would allow the safe administration of systemic combination chemotherapy. However, radiation therapy is becoming a potential option for patients whose primary tumors are in sites where appropriate surgery is impossible, and

Location 13q14

9p21

12q13-15

17p13

12q13-14

17p11

Gene RB1

INK4A

CDK4

P53

MDM2

COPS3

Amplification

LOH – 75% Mutations – 20–30% Structural changes – 10–20% Amplification

Amplification

Genetic alteration LOH – 60% Structural changes – 30% Mutations – <10% Deletion

TableÂ€3â•… Overview of genetic alterations in osteosarcoma

Ligase than functionally inhibits p53 Phosphorylates p53; functionally inhibits p53

Tumor suppressor; encodes for p16INK4A inhibits CDK4-CCDN1 and p19ink4a inhibits MDM2 Phosphorylates of RB; functionally inactivates RB Tumor suppressor; DNA damage control

Function Tumor suppressor; cell cycle control

25%

(continued)

Insufficient

Good

Good 40–60%

Advanced presentation

Good

5–10%

10%

Good

Negative prognostic factor

Prognostic significance Negative prognostic factor

5–10%

Estimated frequency (%) of alteration in OS 70%

Strength of data regarding relative contribution to osteosarcoma tumorigenesis Good

Molecularly Targeted Therapy for Osteosarcoma: Where Do We Go from Here? 479

8q24

14q21-31

7q31

15q25-26

7p22

17q12

2q34

5p15.33

MYC

FOS

MET

IGF-1R

PDGF-AA/ PDGFALPHA ERBB2/HER2

ERBB4

TERT

b2: 1q41b3:14q24

BMP2: 20p12 BMP3: 14p22 BMP4: 14q22-q23 BMP6: 6p12.1 BMP7: 20q13

TGF-BETA3

BMP

ALT

Location

Gene

TableÂ€3â•… (continued)

Amplification

Amplification

Amplification

Genetic alteration

TGF-beta superfamily of cytokines; regulates skeletal development

Oncogene; cell proliferation; motility Mitogenic stimulus; protection from apoptosis Cell proliferation, differentiation, chemotaxis Intracellular mitogenic stimulus Intracellular mitogenic stimulus Maintenance of telomere length Maintenance of telomere length Autocrine growth factor

Oncogene

Oncogene

Function

Up to 94%

Up to 100%

60%

42%

Advanced presentation Negative prognostic factor

Insufficient

Insufficient

Insufficient

Insufficient

Insufficient

Controversial

Poor Negative prognostic factor

34%/27% up to 80%

Insufficient

Good

Insufficient

Good

Advanced presentation Advanced presentation Advanced presentation

Prognostic significance

40–50%

60%

61%

7–11%

Estimated frequency (%) of alteration in OS

Strength of data regarding relative contribution to osteosarcoma tumorigenesis

480 R. Ricafort and R. Gorlick

11q12–13

2q21

6q25.3

6p12

7q21

21q22.3

5q11.2-13.2

17p11

WNT LRP5

CXCR4

EZRIN

VEGF

MDR1

RFC

DHFR

MAPK7

Encodes p-gp; maintains Amplification; genomic integrity rearrangement Deletion or mutation Transport protein for methotrexate Dihydrofolate reductase; Amplification or mutation target of methotrexate

Angiogenesis

Osteoblast differentiation Co-receptor for Wnt; induction of differentiation and proliferation Chemokine receptor; binds SDF-1 Cell migration

10% (initial bx) 65% (post chemo)

35% (p-gp overexpression in 23–45%) 65%

62%

80%

Cell lines

50%

Advanced presentation Advanced presentation Negative prognostic factor Negative prognostic factor

Advanced presentation

Insufficient

Insufficient

Insufficient

Controversial

Poor

Good

Insufficient

Insufficient Insufficient

Molecularly Targeted Therapy for Osteosarcoma: Where Do We Go from Here? 481

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R. Ricafort and R. Gorlick

ERB2 ERB4

IGF / IGF-1R MET/HGF

PTH-R

PDGF-AA / PDGF-α

Pluripotent Mesenchymal Stem Cell

DIFFERENTIATION MDM2

? Precursor Cell

EARLY OSTEOSARCOMA

p14

ARF

COPS3 p16INK4A CDK4-CCND1 p21 p53 c-fos TERT ALT c-myc Rb mTOR wnt MAPK-RAS-AKT pathway

TGF-β BMP

Drug Resistance MDR1

ANGIOGENESIS VEG-F

METASTASES Ezrin SDF-1/CXCR-4

Osteoblast

ADVANCED OSTEOSARCOMA

Fig.Â€1â•… Potential mechanisms of pathogenesis in osteosarcoma

for palliation (DeLaney 2005a, 2005b). In addition to traditional external beam irradiation, bone-seeking Â�radiopharmaceuticals have also been employed in the treatment of osteosarcoma to provide therapeutic irradiation to osteoblastic bone metastasis. The most widely studied is samarium (Sm)-153 ethylene diamine tetramethylene phosphonate (153Sm-EDTMP). This compound has been shown to have high bone specificity and may be effectively used in the palliative setting (Anderson etÂ€al. 2002). The synergistic use of gemcitabine as a radiation sensitizer after the Sm-153 dose may increase its radiobiological effectiveness (Lawrence etÂ€al. 1999) and offer improved palliation, though a pilot study failed to demonstrate a durable response with this combination (Anderson etÂ€al. 2005). The biologic agent muramyl tripeptide phosphatidylethanolamine [MTP-PE] is a synthetic lipophilic analog of a component of the cell wall of Bacillus CalmetteGuerin, capable of stimulating macrophage cytotoxicity against osteosarcoma in animal models and sporadic canine osteosarcoma (MacEwen etÂ€al. 1989; Kleinerman 1995), ultimately leading to the development of a phase III clinical trial in pediatrics. However, its development, and hence its role in osteosarcoma therapy, has been hampered by the difficult to interpret initial results of the Children’s Oncology Group phase III clinical trial in pediatrics in which addition of MTP-PE trended toward having a favorable impact on event-free survival only when combined with the addition of ifosfamide to the standard 3-drug chemotherapy regimen (Meyers etÂ€ al. 2005). This interaction is not evident in later survival curves which more clearly demonstrate an advantage with the inclusion of MTP-PE but further development and use will depend on additional supplies of the drug becoming clinically

Molecularly Targeted Therapy for Osteosarcoma: Where Do We Go from Here?

483

available. Other standard agents considered for use in osteosarcoma include the bisphosphonates. Via their direct inhibitory effects on osteoclast-mediated bone resorption, effects on osteoblast activity, and possible antitumor properties, bisphosphonates may have a potential role in the prevention of bone tumor metastases and primary tumor progression (Diel etÂ€al. 2000). In vitro osteosarcoma models have demonstrated the ability of bisphosphonates to inhibit tumor growth and to disrupt tumor cell interactions with the surrounding microenvironment (Ashton etÂ€al. 2005; Cheng etÂ€al. 2004; Ory etÂ€al. 2005). A recent Phase II study conducted at Memorial Sloan-Kettering Cancer Center assessing the role of pamidronate in the treatment of osteosarcoma along with standard chemotherapy has closed to accrual; early indications are that bisphosphonates can be safely given in the context of osteosarcoma therapy. However, whether the addition of bisphosphonates to classical threedrug chemotherapy affords survival benefit remains to be proven in a larger subsequent study. Another avenue of research has been the development of novel delivery mechanisms to deliver focused high doses of agents to sites of disease, while avoiding systemic toxicity. Innovations in drug formulation may improve the delivery efficiency of current cytotoxic agents and improve the therapeutic index. Liposomally encapsulated doxorubicin has been shown to be an effective mode of delivery of doxorubicin, and has been shown to be safe in a limited number of studies (Chidiac etÂ€al. 2000; Judson etÂ€al. 2001; Muggia 2001; Skubitz 2003). Its activity in relapsed sarcomas suggests liposomal doxorubicin that may be able to overcome tumor resistance mechanisms (Alberts etÂ€al. 2004). Aerosolized granulocyte macrophage stimulating factor is being tested in a Children’s Oncology Group Phase II trial for relapsed osteosarcoma patients (Anderson 1999). An aerosolized liposomal formulation of cisplatin (Sustained release Lipid Inhalation Targeting [SLIT™] cisplatin) recently has been tested in a Phase Ib/ IIa setting for osteosarcoma with promising early results (Chou etÂ€al. 2007). Though targeting nonspecific therapies to sites of disease is one alternative in developing novel strategies against osteosarcoma, targeted therapies based on molecular knowledge of critical genetic events may offer new insight in guiding treatment. Identifying the genes and signal transduction pathways responsible for the malignant behavior in osteosarcoma may lead to the development of agents with better tumor specificity and therapeutic efficacy. Hence, there has been intense focus on the efficacy of pathway specific agents in osteosarcoma. Pathway selective agents, in the form of small molecule inhibitors and monoclonal antibodies, can function to antagonize ligand/receptor interactions, inhibit the anabolic activity of key tyrosine kinases, and intercept intracellular downstream functions. Though the majority of molecular alterations involve inactivation of the retinoblastoma or p53 pathways, attempts to target these genes have not been successful. Targeted therapies aimed at inhibiting growth factor and signal transduction pathways involved in osteosarcoma tumorigenesis have been more recently explored. Heat shock protein 90 (Hsp90) inhibition has been shown to simultaneously disrupt multiple signal transduction pathways associated with various cancer models, including osteosarcoma (Bagatell et al. 2005). However, a Phase I trial of a novel Hsp90 inhibitor, 17-N-Allylamino-17-demethoxygeldanamycin (17-AAG), failed to

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show benefit in this tumor (Bagatell et al. 2007). PDGF-AA and its receptor, PDGF-a, have been shown to be coexpressed on osteosarcoma tumor cells, correlating with an inferior prognosis (Kawai etÂ€al. 1997; Henriksen etÂ€al. 1993; Sulzbacher etÂ€al. 2001; Kubo etÂ€ al. 2002), making this loop an attractive target for novel therapeutic approaches in osteosarcoma. Imatinib mesylate (also known at ST1571, Gleevec), is a tyrosine kinase inhibitor developed as an inhibitor of the ABL protein tyrosine kinase (Druker etÂ€al. 1996), and in Â�addition also has been noted to inhibit the PDGF receptor (Buchdunger etÂ€ al. 1995, 2000). A Children’s Oncology Group Phase II study of imatinib in children with refractory or relapsed solid tumors, including osteosarcoma, demonstrated that this tyrosine kinase inhibitor had no activity against osteosarcoma as a single agent at conventional doses (Bond etÂ€al. 2008). Several investigators have demonstrated that overexpression of ERBB2, has prognostic significance in osteosarcoma and that it may play a role in maintaining the malignant phenotype in osteosarcoma, although these data are controversial (Zhou etÂ€al. 2003; Akatsuka etÂ€al. 2002; Gorlick etÂ€al. 1999; Hughes etÂ€al. 2006; Scotlandi etÂ€al. 2004, 2005; Somers etÂ€al. 2005; Thomas etÂ€al. 2002). Preclinical studies of trastuzumab, the humanized monoclonal antibody targeting the HER2 receptor, have suggested its ability not only to antagonize the function of growthsignaling properties of the HER2 system, but also to recruit immune effectors against the target (Weiner and Adams 2000), and to augment chemotherapy-induced cytotoxicity (Hancock etÂ€ al. 1991; Baselga etÂ€ al. 1998). Promising early clinical data of trastuzumab in HER2 overexpressing breast cancer (reviewed by Hudis 2007) have led to a Phase II study of trastuzumab in patients with metastatic osteosarcoma whose tumors overexpress HER2. The results of this study, which closed in October 2007, are eagerly awaited. Small molecule inhibitors of ERBB2 (Lapatinib) or pan-ERBB inhibitors (ARRY334543) are approved or in clinical development, and may have utility in treating osteosarcoma. The VEGF pathway is another growth factor signaling system recognized as a potential target of therapy in pediatric osteosarcoma, as studies have shown that VEGF expression correlates with metastasis and poor outcome (Charity etÂ€al. 2006; DuBois and Demetri 2007; Kaya etÂ€al. 2000; Kaya etÂ€al. 2002; Lee etÂ€al. 1999; Lee etÂ€al. 2007a, b). A number of VEGF pathway inhibitors are available now and are being tested in the preclinical arena. Encouraging results for AZD2171, a specific VEGF-receptor inhibitor, demonstrated growth inhibition in solid tumor xenograft models including osteosarcoma (Maris etÂ€al. 2008). Trials of bevacizumab (Avastin) an antibody that binds VEGF, added to conventional chemotherapy, are planned. Preclinical studies have suggested that the mTOR pathway may be a potentially effective target in osteosarcoma (Viella-Bach 1999; Wan etÂ€al. 2005; Houghton etÂ€al. 2007b). Rapamycin is widely available and several new rapamycin analogs (generally with less toxicity than rapamycin itself) have completed early stage clinical trials in sarcomas. A large phase III trial using the rapamycin analog (AP23573) is underway as a multi-institutional consortium study for the treatment of various sarcomas. Dysregulated insulin-like growth factor signaling is emerging as an important molecular target for cancer therapeutics development. Blockade of IGF-1 has been investigated using the sustained release sandostatin (OncoLar), which can reduce

Molecularly Targeted Therapy for Osteosarcoma: Where Do We Go from Here?

485

IGF-1 levels. A Phase I study of OncoLar with and without tamoxifen in relapsed osteosarcoma patients demonstrated a sustained 40 to 50% decrease in IGF-1 levels; however, there was no measurable tumor response (Mansky etÂ€al. 2002). The level of suppression of IGF-1 achieved in this trial may have been insufficient to produce a biologically relevant effect. Furthermore, the presence of redundant signal Â�transduction pathways along with autocrine and paracrine secretion of growth Â�factors may have compensated for the suppression of serum IGF-1 levels, rendering this therapy ineffective (Benini etÂ€al. 1999). Alternatively, targeting the receptor itself may result in improved inhibition of the IGF-1 signal transduction pathway and thus lead to better cytotoxic effects. In preclinical cancer models, antibody mediated down regulation of IGF-1R significantly inhibits the growth of neoplastic cells (Kappel etÂ€ al. 1994; Sachdev etÂ€al. 2003; Hailey etÂ€al. 2002; Quelle etÂ€al. 1995; Scotlandi etÂ€al. 2005), and induces regressions alone (Kolb etÂ€al. 2008) or when combined with cytotoxic agents (Khanna et al. 2002; Cohen etÂ€ al. 2005; Goetsch etÂ€ al. 2005). Many new IGF-1R antagonistic monoclonal antibodies are being introduced into Phase I and II studies for sarcomas and specifically in osteosarcoma. As an example, a phase II trial of R1507, a recombinant monoclonal antibody to IGF-1R, which includes a stratum for patients with osteosarcoma has already been initiated by the Sarcoma Alliance for Research through Collaboration (SARC). Multiple competing trials of IGF-1R antibodies are being performed simultaneously with slightly differing eligibility and response criteria using different monoclonal antibodies. As each of the antibodies has different epitope specificity, antibody subclass and toxicity, relative efficacy between these agents may ultimately need to be clarified. Further testing of these biological agents alone and in combination will need to be done in order to fully assess their role in osteosarcoma treatment. Particularly attractive are combinations with rapamycin or rapamycin analogs as IGF-1R signals via mTOR with combined inhibition potentially being more efficacious. Other targets of potential interest include the receptor activator for nuclear factor (RANK)/RANK-Ligand (RANKL)/Osteoprotegrin (OPG) system. The RANK/RANKL)/OPG system has been shown to be important in maintaining normal bone metabolism (Itoh etÂ€al. 2002; Wuyts etÂ€al. 2001). Recent studies suggest that this system may play a role in osteosarcoma pathogenesis (Miyamoto etÂ€al. 2002; Wittrant etÂ€al. 2006) and thus interference with this system represents another potential therapeutic strategy. RANKL antagonists (OPG, anti-RANKL antibodies and a soluble nonsignaling form of RANK, [RANK-Fc]) that bind ligand and prevent its interaction with the RANK receptor, have been used successfully in various animal models of bone metastases (Akatsu etÂ€al. 1998).

Targeting Bone Differentiation Pathways Given the indication that the complex molecular alterations in osteosarcoma may involve disruptions in osteoblast differentiation, agents promoting bone differentiation and/or circumventing differentiation defects may play a role in the therapy of

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osteosarcoma. In vitro studies suggest that targeting the WNT signaling pathway via immunodepletion of DKK1 or GSK3b inhibitors, may prevent inappropriate differentiation in osteosarcoma cell lines (Lee etÂ€al. 2007a, b). In addition, Â�strategies aimed at disrupting the PTHrP-based stimulation of growth factors such as TGF-b and IGF-1, leading to differentiation and tumor growth, may be exploited in Â�osteosarcoma therapy (O’Keefe and Guise 2003; Yang etÂ€ al. 2007a). Promoting terminal differentiation with nuclear receptors may represent another means of therapy in osteosarcoma. In vitro data suggest the treatment with PPARg agonists or 9-cis retinoic acid that was able to reduce proliferation rate and cell viability and induce differentiation (Haydon etÂ€al. 2002a, b).

The Pediatric Preclinical Testing Program With the bewildering number of agents under development, there needs to be a focused effort to systematically and rigorously evaluate their relevance to osteosarcoma therapy. The Pediatric Preclinical Testing Program (PPTP) is one such effort underway to facilitate the introduction of new, active agents into clinical trials for all childhood cancers. The number of children who are eligible for Phase I clinical trials are few, underscoring the importance of identifying potentially effective agents quickly and moving these agents into clinical trials in a timely manner. With a consortium of laboratories in the United States and abroad, the PPTP is able to quickly screen a large number of agents using inÂ€ vitro and inÂ€ vivo models. Preclinical testing potentially may predict the activity of new agents in patients with childhood cancers rationally designed clinical trials utilizing new agents can be formulated and completed quickly. The PPTP has evaluated several standard and novel agents, including cyclophosphamide, vincristine, topotecan, SCH717454 (anti-IGF-1R antibody) (Kolb etÂ€ al. 2008), AZD2171 (a specific inhibitor of VEGF-receptor 2) (Maris etÂ€al. 2007), AZD6244 (MEK1/2 inhibitor), bortezomib (proteosome inhibitor) (Houghton etÂ€al. 2007a, b, 2008), ABT-263 (a Bcl-2 inhibitor) (Lock etÂ€al. 2008), MLN8237 (aurora A kinase inhibitor), rapamycin (Houghton etÂ€ al. 2007a, b), vorinostat (histone deacetylase inhibitor), lapitinib (EGFR and ERBB2 inhibitor), and sunitinib (Houghton etÂ€al. 2007a, b, 2008). Thus far, of all the novel agents tested; SCH717454, AZD2171, and rapamycin have been among the most effective for the treatment of osteosarcoma. These data may help prioritize further development of these agents in pediatric clinical trials. The success of the PPTP will not be measured by the rapidity of the screening process but rather by the rapid identification of an active agent in osteosarcoma clinical trials. Other appealing model systems exist, most notably in domestic dogs which develop osteosarcoma spontaneously. The osteosarcomas in canines are quite similar to those that occur in humans and have the advantage of arising spontaneously, and therefore of being heterogeneous. The high incidence of osteosarcoma in dogs and the more accelerated time course for the progression of their disease facilitates the conduct of clinical trials in dogs analogous to those in humans.

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Linkages have increasingly been developed between the veterinary oncology consortiums and the oncology cooperative groups. Most notably, the National Cancer Institute and the Children’s Oncology Group are involved in comparative oncology programs in which osteosarcoma is a major interest.

Summary Osteosarcoma is a genetically complex cancer making the identification of Â�pathways which can be therapeutically targeted extremely difficult. Recent focus has been an increasing reliance on the use of preclinical model systems. Although selection of agents to test in these models remains empiric, the increased throughput possible in these systems may facilitate the identification of active chemotherapy. Perhaps recognition of effective therapies ultimately may inform the identification of pathways that are truly central in osteosarcomas pathogenesis or at a minimum which are worthy of further analysis.

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Nonrhabdomyosarcoma Soft Tissue Sarcoma in Children: Developing New Treatments Based on a Better Understanding of Disease Biology Stephen X. Skapek

Introduction Nonrhabdomyosarcoma soft tissue sarcomas (NRSTS) are a large and very Â�heterogeneous group of cancers in children. Although approximately 550 NRSTS diagnoses in the US each year represent only approximately 4% of cases of childhood cancer, NRSTS comprise nearly 60% of all soft tissue sarcomas in this age group [reviewed in Spunt etÂ€ al. (2006)]. The incidence is bimodally distributed, with relatively high incidence in the first year of life and a second peak during later childhood and adolescence. While NRSTS is typically a sporadic disease, some types of NRSTS are associated with cancer susceptibility syndromes, such as the Li–Fraumeni familial cancer syndrome (associated with heritable p53 mutations) and neurofibromatosis type I (associated with heritable mutations in NF1 tumor suppressor genes) (Li etÂ€al. 1988; Malkin etÂ€al. 1990; Sorensen etÂ€al. 1986); with certain environmental exposures (therapeutic ionizing radiation and HHV8, HIV, or EBV virus infection); and with certain chemical carcinogens (Spunt etÂ€al. 2006). Historically, therapy for NRSTS has comprised surgery and radiation for local tumor control and systemic cytotoxic chemotherapy to eradicate disseminated disease. Important prognostic factors include the histological grade of the tumor and the extent of disease. A sarcoma grading system validated for children was developed by the Pediatric Oncology Group (POG) (Parham etÂ€ al. 1995) (TableÂ€1). In many cases, pathological grade is reflected in the histological subtype; in others, grade is determined by morphologic criteria, such as necrosis of more than 15% of the tumor surface or a mitotic count greater than 5 per 10

S.X. Skapekâ•›(*) Pediatric Hematology/Oncology, The University of Chicago Comer Children’s Hospital, 900 E. 57th Street, Chicago, IL 60637, USA e-mail: [email protected] P.J. Houghton and R.J. Arceci (eds.), Molecularly Targeted Therapy for Childhood Cancer, DOI 10.1007/978-0-387-69062-9_22, © Springer Science+Business Media, LLC 2010

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TableÂ€ 1â•… Pediatric Oncology Group (POG) nonrhabdomyosarcoma soft tissue sarcoma grading system Grade I Myxoid and well-differentiated liposarcoma Deep-seated dermatofibrosarcoma protuberans Well-differentiated or infantile (£4 years old) fibrosarcoma Well-differentiated or infantile (£4 years old) hemangiopericytoma Well-differentiated malignant peripheral nerve sheath tumor Extraskeletal myxoid chondrosarcoma Angiomatoid malignant fibrous histiocytoma Grade II Sarcomas not specifically included in Grades I and III, and in which <15% of the surface area shows necrosis, and the mitotic count is £5/10 high power fields (hpf) using a 40× objective. As secondary criteria, nuclear atypia is not marked and the tumor is not markedly cellular Grade III Pleomorphic or round cell liposarcoma Mesenchymal chondrosarcoma Extraskeletal osteosarcoma Malignant triton tumor Alveolar soft part sarcoma Sarcomas not included in Grade I and with >15% of surface area with necrosis, or with ³5 mitoses/10 hpf using a 40× objective Marked atypia or cellularity are less predictive but may assist in placing tumors in this category Reference: Parham etÂ€al. (1995)

high-power fields. Perhaps the most important parameter for determiningÂ�Â�survival is the presence or absence of metastasis: large retrospective studies showed overall 5-year survival to be 89% in children with localized, resected NRSTS (Ferrari etÂ€al. 2005; Spunt etÂ€al. 1999) but less than 20% for those with Â�metastatic disease (Pappo etÂ€al. 1999). However, overall 5-year survival even in children with localized NRSTS is only approximately 50% unless the primary tumor is initially resected (Ferrari etÂ€ al. 2005; Spunt etÂ€ al. 2002). Among patients with resected tumors, survival is poorer if the tumor is greater than 5Â€cm in size or is locally invasive (Ferrari etÂ€al. 2005; Spunt etÂ€al. 1999). Systemic chemotherapy for children with NRSTS has typically included alkylating agents and anthracyclines. Standard chemotherapy has failed to improve disease control in most retrospective studies [e.g., Spunt etÂ€ al. (1999)] and in prospective POG trials that (1) compared adjuvant vincristine, actinomycin D, cyclophosphamide, and doxorubicin (VACA) to observation alone (Pratt etÂ€al. 1999) and (2) assessed the addition of dacarbazine to VACA chemotherapy (Pratt etÂ€al. 1998). These particular clinical trials are limited by their inclusion of a variety of NRSTS. There is hope that the next therapeutic breakthrough for high-risk disease will come from better understanding of NRSTS biology and the development of therapeutic agents that specifically block molecular events crucial to the malignant features of the tumor.

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NRSTS Biology Two Fundamental Groups of NRSTS There are many histological subtypes of NRSTS in children; synovial sarcoma and malignant peripheral nerve sheath tumor (MPNST) are the most common [e.g., Ferrari etÂ€al. (2005)]. The clinical behavior of most NRSTS depends on the histological grade and is similar in children and adults. Infantile fibrosarcoma and infantile hemangiopericytoma (a very rare childhood NRSTS) are exceptions to this rule. They are defined as occurring in children younger than 4 years and are considered low grade tumors despite their histological appearance, due to their relatively good outcome (Parham etÂ€al. 1995). Despite the daunting complexity of the numerous histological subtypes, childhood NRSTS can be divided into two fundamentally different groups: tumors with complex genomic abnormalities (e.g., aneuploidy, chromosomal breaks and translocations, and end-to-end chromosome fusions) and tumors with specific chromosomal rearrangements but relatively few cytogenetically evident genomic abnormalities [reviewed in Helman and Meltzer (2003)]. The mechanisms underlying generalized genomic instability have not been precisely defined, but some insight has been gleaned from mouse models. For example, p53 plays a critical role in preserving genomic integrity. Sarcomas frequently develop in mice with inactivated p53 or Arf (which activates p53) genes (Donehower etÂ€al. 1992; Jacks etÂ€al. 1994a; Kamijo etÂ€al. 1999). In mice lacking the Ink4a/Arf locus, loss of one allele of the gene encoding DNA ligase IV, an enzyme involved in nonhomologous DNA end joining, causes a wide range of soft tissue sarcomas with chromosomal translocations, amplifications, and deletions similar to those observed in human NRSTS (Sharpless etÂ€al. 2001). When bred into a line of mice with short telomeres because of telomerase deficiency, mice that lack functional p53 and Atm, which integrates DNA-damage sensing and repair mechanisms (Bakkenist and Kastan 2004), develop lymphomas with complex karyotypic abnormalities, some of which influence genes known to be involved in human cancer (Maser etÂ€al. 2007). Similar mechanisms may underlie the development of NRSTS with complex karyotypes, but specific studies are needed to identify potential therapeutic targets. Development of NRSTS in the presence vs. absence of global genomic derangement is likely to be fundamentally different. Most of these sarcomas have specific chromosomal defects initially identified by routine karyotypic analysis. In many cases, specific defects are now detectable by sensitive molecular techniques [such as fluorescence in situ hybridization (FISH)] even when the karyotype appears grossly normal. Many of the translocations generate novel fusion proteins (often transcriptional regulators) that presumably drive sarcomagenesis. The striking correlation of specific chromosomal rearrangements with histological subtype suggests their key role in driving the biology of particular sarcomas. Nonetheless, a single genetic event is not sufficient to cause malignancy. Therefore, even in cases of NRSTS with

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specific chromosomal translocations, additional genetic or epigenetic events are required to confer all of the “hallmarks” of cancer (Hanahan and Weinberg 2000).

What Is the Cellular Origin of NRSTS? NRSTS, like other sarcomas, are derived from mesenchymal cells. In some cases, for example, MPNST arising from a preexisting benign, plexiform neurofibroma, the cellular origin of the sarcoma is easy to explain. In most cases, however, the exact cell of origin is not known. In principle, different histological subtypes of NRSTS may originate from different types of mesenchymal cells. As in lymphoblastic leukemia, the relative degree of cellular differentiation within a class of tumors may indicate transformation at an earlier or later stage of development. It is also plausible that a common mesenchymal progenitor cell may give rise to different types of NRSTS. Just as a single transcription factor (MyoD) can determine the skeletal muscle differentiation of mesenchymal progenitors, NRSTS histology could be driven by abnormal transcription factors, such as EWS(or TLS)-CHOP, EWS-ATF1, and EWS-WT1, generated by tumor-restricted balanced chromosomal translocations. These fusion proteins are expressed exclusively in myxoid liposarcoma, clear cell sarcoma of soft parts, and desmoplastic small round cell tumor [reviewed in Skapek and Chui (2000), Spunt etÂ€al. (2006)].

Coupling Molecular Biology to Targeted Therapy for Childhood NRSTS Although there are many histological subtypes of NRSTS, current treatment strategies are largely independent of subtype and are instead based on pathological stage and extent of disease. If the molecular basis of individual NRSTS subtypes is clarified, it may be possible to direct subtype-specific therapy at essential molecular targets. The following sections review how understanding of the biology of the more common childhood NRSTS subtypes has led or may lead to targeted therapies. They also provide examples of less common tumors for which targeted therapies have already been identified.

Synovial Sarcoma and the SYT-SSX Gene Fusions Synovial sarcoma is the most common NRSTS in children, accounting for Â�approximately 27 to 30% of cases (Ferrari etÂ€al. 2005; Parham etÂ€al. 1995). Although it Â�commonly arises near joints, the cell of origin and its specific relationship to the

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joint synovium is not clear. Histologically, synovial sarcoma is composed either entirely of spindle-shaped mesenchymal cells or an admixture of spindle and Â�epithelial cells; these subtypes are termed monophasic and biphasic synovial sarcoma, respectively (Spunt etÂ€al. 2006). As in most types of NRSTS, surgical resection with or without ionizing radiation is the standard of care for localized disease. Important insight into the biology of synovial sarcoma came from the identification of nonrandom translocations between chromosomes X and 18 [t(X;18)(p11.2; q11.2)] in some cases (Turc-Carel etÂ€ al. 1987). Cloning and sequencing of the translocation breakpoints showed that the translocation fuses most of the SYT gene (encoding all but the last eight amino acids) on chromosome 18 to DNA encoding the carboxyl end of either SSX1 or SSX2 [(Clark etÂ€al. 1994; Ladanyi 2001)(review)]. Alternative translocations generating fusion proteins with similar motifs have occasionally been reported [e.g., Storlazzi etÂ€al. (2003)]. Molecular diagnostic studies have established that the SYT-SSX1 or SYT-SSX2 transcript (detected by reversetranscriptase polymerase chain reaction) is highly specific for synovial sarcoma and is present in almost all cases. The presence of SYT-SSX1 vs. SYT-SSX2 is correlated with histological subtype: the SYT-SSX1 fusion is found in both monophasic and biphasic tumors, in both histologic “phases” (Birdsall etÂ€al. 1999; Kawai etÂ€al. 1998). In contrast, SYT-SSX2 is found mostly in monophasic tumors. This finding implies that SYT-SSX2 may influence the expression of genes required for mesenchymal-to-epithelial cell transition. While some studies show that the SYT-SSX1 fusion portends a somewhat poorer outcome (Kawai etÂ€ al. 1998; Nilsson etÂ€ al. 1999), this conclusion is debated (Guillou etÂ€al. 2004). The molecular biology of the SYT-SSX1 and SYT-SSX2 fusion proteins is not fully understood [reviewed in Ladanyi (2001)]. Functional motifs in the SYT protein that are preserved in the fusion include interaction domains for (1) the transcriptional coactivator p300; (2) Brg1 and BRM (components of the SWI/SNF chromatin remodeling machinery); and (3) a QPGY motif acting as a transactivation domain. Normal SSX proteins include an amino-terminal region similar to a Kruppelassociated box (KRAB) transcriptional repression domain (which is not present in the fusion protein) and a carboxy-terminal repressor domain (RD) that mediates colocalization with Polycomb group proteins (Pgps), which also remodel chromatin. The predominantly nuclear localization of SYT-SSX and the aforementioned interaction motifs imply that SYT-SSX acts as a transcriptional regulator. Unlike most other fusion transcription factors (see below), it has no sequence-specific DNA binding motif but likely influences transcription by interacting with other proteins. In this regard, SYT-SSX has been shown to repress Candidate of Metastasis 1 (COM1) (Ishida etÂ€al. 2007) and E-cadherin (Saito etÂ€al. 2006); to colocalize with b-catenin in the nucleus (Pretto etÂ€al. 2006); to stabilize cyclin D1 (Xie etÂ€al. 2002); and to induce insulin-like growth factor-2 (Igf2) (Sun etÂ€al. 2006). Gene expression profiling and analyses of candidate proteins have also been used to identify molecular mechanisms operating downstream of SYT-SSX fusion (Allander etÂ€ al. 2002; Lee etÂ€ al. 2003; Nagayama etÂ€ al. 2002). Gene expression clustering observed in synovial sarcoma cases provides insight into both its Â�possible neuroectodermal lineage and its occasional epithelial differentiation. Protein studies

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confirm a number of candidate gene products that could contribute to tumor biology: synovial sarcomas frequently express detectable levels of the insulin-like growth factor1 receptor 1 (IGF-1R) (Xie etÂ€al. 1999); the epidermal growth factor receptor (EGFR) (Nielsen etÂ€al. 2003); the antiapoptotic protein Bcl2 (Nielsen etÂ€al. 2003); HER2/neu (Nielsen etÂ€al. 2003; Nuciforo etÂ€al. 2003); and platelet-derived growth factor receptor (PDGFR)-b (Tamborini etÂ€ al. 2004). However, there are conflicting data on the frequency of c-KIT expression (Nielsen etÂ€al. 2003; Smithey etÂ€ al. 2002; Tamborini etÂ€ al. 2004). More compelling evidence that a particular protein may drive tumor biology can come from genetic studies. For example, an activating mutation in the gene encoding PDGFR-a, which was found in one in a series of 12 cases, implies a causative role (Lopez-Guerrero etÂ€al. 2005). Similarly, frequent deletion of CDKN2A, which encodes the Cdk4/6-specific inhibitor p16Ink4a, suggests that Cdk4/6 inhibition may impede synovial sarcoma development or progression (Subramaniam etÂ€al. 2006). The importance of some of the aforementioned candidate therapeutic targets has been validated in experimental models. The SYT-SSX protein associates with chromatin remodeling proteins to repress gene expression; the histone deacetylase inhibitor FK228 (whose net effect can block gene repression) inhibits synovial sarcoma cell growth inÂ€vitro and in xenograft models (Ito etÂ€al. 2005). The antiapoptotic protein Bcl-2 is expressed in many synovial sarcomas; the Bcl-2-directed antisense oligonucleotide G3139 enhances doxorubicin-mediated apoptosis in two synovial sarcoma cell lines (Joyner etÂ€al. 2006). SYT-SSX1 can induce insulin-like growth factor (IGF)-2; blocking this signaling pathway promotes apoptosis and impedes tumor formation in cells expressing the fusion protein (Sun etÂ€al. 2006). Members of the Capecchi laboratory recently reported the generation of a mouse in which the conditional expression of human SYT-SSX2 causes a synovial sarcomalike tumor (Haldar etÂ€ al. 2007); this elegant model should be valuable in further clarifying the genetic and biochemical events that cooperate with SYT-SSX expression to drive synovial sarcomagenesis and in testing novel therapeutic strategies.

Malignant Peripheral Nerve Sheath Tumor and NF1 Mutation MPNST is the second most common NRSTS in children [e.g., Ferrari etÂ€al. (2005)]. It can occur in children with neurofibromatosis type 1 or sporadically in children with no known predisposition. In either case, the tumor often arises near major peripheral nerves, nerve roots, or the brachial plexus or from a preexisting plexiform neurofibroma [reviewed in Webber and Parham (1996)]. Although MPNST is commonly accepted as arising from Schwann cells, it must be emphasized that both plexiform neurofibromas and MPNST also contain fibroblasts, perineurial cells, and mast cells that can contribute to their biology (discussed more below). Surgical resection with or without ionizing radiation is central to the effective treatment. The use of intensive ifosfamide and doxorubicin-based therapy has not greatly influenced survival.

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Cytogenetic and molecular genetic analyses have not provided major insight into MPNST biology, instead revealing a variety of complex genomic abnormalities involving chromosome arms 7p, 9p, 17q, and 22q (Skapek and Chui 2000). However, the close link of MPNST with plexiform neurofibromas and neurofibromatosis type 1 strongly implies that loss of the NF1 tumor suppressor gene plays a role. Children who are born with only one normal NF1 allele are at increased risk of certain brain tumors, pheochromocytoma, myeloid leukemia, and a variety of other disorders, including cutaneous and plexiform neurofibromas [reviewed in Cichowski and Jacks (2001)]. A relatively small subset of the latter can degenerate into MPNST. NF1, residing at human chromosome 17q, encodes neurofibromin, which acts as a GTPase activating protein (GAP) (Cichowski and Jacks 2001). In response to signaling from a receptor tyrosine kinase, such as EGFR, the RAS protein is activated by binding to GTP and transduces a variety of signals that contribute to tumor biology. RAS signaling is “turned off” as its enzymatic activity converts bound GTP to GDP. As a GAP, neurofibromin facilitates this transition to curb RAS signaling; while RAS-independent effects of neurofibromin may exist, their importance to neurofibromin’s tumor suppression is not clear. While NF1 haploinsufficiency can alter the biology of perineurial cells and fibroblasts within a neurofibroma, there is good evidence that the normal NF1 allele is lost in the Schwann cells within these tumors. Additional genetic or epigenetic changes, such as loss of p53, p16INK4a, and p14ARF, appear to foster the transition from neurofibroma to MPNST. Importantly, key aspects of MPNST biology have been verified by using elegant mouse models. First, mere haploinsufficiency of mouse Nf1 predisposes mice to pheochromocytoma and myeloid leukemia but not to peripheral neurofibroma (Jacks etÂ€al. 1994b). Because Nf1 knockout mice are not viable, members of the Jacks laboratory used mouse chimeras possessing both wild-type and Nf1−/− cells to show that the loss of the second Nf1 allele is essential for neurofibroma development (Cichowski etÂ€al. 1999). Second, the combined loss of the second Nf1 allele and the p53 tumor suppressor causes MPNST and other malignant tumors found in children with neurofibromatosis type 1 (Cichowski etÂ€al. 1999; Vogel etÂ€al. 1999). Third, by proving that neurofibromas occur in mice when Nf1 is specifically inactivated in Schwann cells, members of the Parada laboratory provided solid evidence that neurofibromas originate from these cells (Zhu etÂ€al. 2002). Interestingly, they also showed that Nf1 haploinsufficiency in non-Schwann cells greatly enhances the development of peripheral neurofibromas, underlining both the complexity of heterotypic cell–cell interactions in this disease and pointing out the potential for therapeutic avenues distinct from those directed at Schwann cells. One important purpose of better defining the molecular biology of neurofibroma and MPNST formation is the development of rational therapy. Some important progress has been made. One obvious strategy is to block farnesyl protein transferase, which promotes RAS-dependent signaling by altering its subcellular localization (reviewed in Dilworth etÂ€ al. (2006)). A number of so-called farnesyl transferase inhibitors (FTIs), such as lonafarnib, tipifarnib, BMS-214662, FTI-277, L-744,832, and BMS-186511, have been tested in preclinical models with some

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success. However, because N- and K-RAS can also be activated by geranylgeranyl transferases, FTIs alone may not be sufficient. On the basis of EGFR expression and signaling activity in Schwann cells and MPNSTs from Nf1-deficient mice (DeClue etÂ€al. 2000; Li etÂ€al. 2002), it was recently shown that the genetic loss of EGFR activity dramatically improves survival of Nf1+/−, p53+/− mice (Ling etÂ€ al. 2005). In mouse and human MPNST cell lines, the EGFR inhibitor AG1478 blocks inÂ€ vitro accumulation of MPNST cells (DeClue etÂ€ al. 2000; Li etÂ€ al. 2002). PD184352, an inhibitor of MEK (which is activated downstream of EGFR and RAS), promotes apoptosis of MPNST-derived cells inÂ€vitro (Mattingly etÂ€al. 2006). The combination of erlotinib with oncolytic herpes simplex virus infection inhibits MPNST growth in a xenograft model as well (Mahller etÂ€ al. 2007). Finally, the observation that mTOR is also deregulated in the absence of NF1 implies that rapamycin and related compounds may have selective activity against plexiform neurofibroma and MPNST (Johannessen etÂ€al. 2005).

Infantile Fibrosarcoma and the ETV6-NTRK3 Fusion Protein Infantile fibrosarcomas are composed of fibroblast-like cells and nearly always occur in children less than 4 years old (Spunt etÂ€ al. 2006). Although their histopathological features suggest aggressive behavior (frequent mitotic figures and areas of necrosis), as does fibrosarcoma histology in adults, metastasis is rare and the prognosis is usually good. Insight into this favorable biology initially came from the observation that these tumors have a recurrent translocation involving chromosomes 12 and 15 (Knezevich etÂ€al. 1998). This translocation generates a fusion protein containing the dimerization domain of the ETV6 transcription factor (also known as TEL) and the protein tyrosine kinase domain of the neurotrophin-3 receptor, NTRK3 (also known as TRKC) (Knezevich etÂ€al. 1998). Unlike fusion proteins that show marked specificity for a single tumor, ETV6-NTRK3 has been described in congenital mesoblastic nephroma (Rubin etÂ€ al. 1998), secretory breast cancer (Tognon etÂ€ al. 2002), and acute myelogenous leukemia (Eguchi etÂ€al. 1999) as well as infantile fibrosarcoma, although it is not detected in adult-type fibrosarcoma. The finding that ectopic expression of ETV6-NTRK3 in NIH 3T3 cells induces a morphological change to a “transformed” phenotype and allows growth in soft agar suggests that the expression of ETV6-NTRK3 is integral to infantile fibrosarcoma biology (Wai etÂ€ al. 2000). Hence, elucidation of the function of ETV6-NTRK3 may suggest how its activity can be blocked as a therapeutic strategy. Studies of ETV6-NTRK3 biology have provided tremendous insight into the mechanisms by which it can drive sarcomagenesis. As expected from sequencing data, the fusion protein acts as a kinase capable of auto- or transphosphorylation (Wai etÂ€al. 2000). Its kinase activity and transformation potential in 3T3 cells are blocked by the deletion of the helix-loop-helix dimerization motif in the ETV6

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domain and by point mutations disrupting the kinase activity of the NTRK3 domain. Expression of this fusion protein activates both the RAS/ERK and the PI3K/AKT signaling pathways, leading to induction of cyclin D1 in cultured cells (Tognon etÂ€al. 2001). Pharmacological inhibition of either pathway blocks cellular transformation and cyclin D1 induction. IGF-IR suppresses apoptosis induced by anchorage-independent growth conditions (known as anoikis) in fibroblasts expressing ETV6-NTRK3 (Martin etÂ€ al. 2006). Interestingly, however, it is not needed for cyclin D1 induction. The adaptor protein c-Src fosters an interaction between the fusion protein and the insulin receptor substrate 1 (IRS1); c-Src kinase activity is needed for AKT activation by ETV6-NTRK3 (Jin etÂ€al. 2007). Finally, ETV6-NTRK3 physically interacts with and phosphorylates the transforming growth factor (TGF) b type II receptor, thereby blocking TGFB-dependent signaling (Jin 2005). As TGFBs have well-described antimitogenic properties [reviewed in Bierie and Moses (2006)], this interaction demonstrates a novel mechanism by which a constitutively activated fusion protein kinase may drive tumor formation. It is not yet known whether selective inhibition of ETV6-NTRK3 kinase activity or any of these cooperating pathways will be clinically useful.

Malignant Fibrous Histiocytoma The biology of malignant fibrous histiocytoma (MFH) remains enigmatic, and even its nomenclature is in flux. The 2002 WHO Classification of soft tissue tumors conceded that what had previously been called pleomorphic MFH is not a distinct entity (Fletcher 2006). This conclusion is consistent with recent gene expression array patterns in MFHs; they do not form tight clusters as a single entity, as is the case of synovial sarcoma, for example (Baird etÂ€al. 2005; Lee etÂ€al. 2003; Nagayama etÂ€al. 2002; Nakayama etÂ€al. 2007). Currently, the WHO considers that what was previously called pleomorphic MFH is better classified as undifferentiated pleomorphic sarcoma (Fletcher 2006). Similarly, giant cell MFH is likely to represent a giant cellrich osteosarcoma or other giant cell-containing soft tissue tumors, and most inflammatory MFH would currently be recognized as dedifferentiated liposarcoma. Also, unifying genetic or cytogenetic abnormalities have not been described in MFH, which instead often has complex karyotypic abnormalities similar to those in osteosarcoma; this fact is consistent with the concept that “MFH” may represent a broader array of tumors [reviewed in Fletcher (2006), Skapek and Chui (2000)]. Effective therapy for MFH in children, like therapy for other NRSTS, relies heavily on surgical resection; without resection, survival is poor despite intensive chemotherapy and radiation (Daw etÂ€ al. 2003). Interestingly, three of four tested MFH-derived cell lines express both c-KIT and PDGFRb; treatment with imatinib mesylate slows accumulation of the cells inÂ€ vitro and in xenograft models (Irsan etÂ€ al. 2007). Whether inhibition of these receptor tyrosine kinases will also be a useful therapy is not yet known.

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Dermatofibrosarcoma Protuberans and the COL1A1-PDGFB Fusion Dermatofibrosarcoma protuberans (DFSP) is a relatively uncommon soft tissue sarcoma that can occur in young adults and children. It is typically a low grade tumor, but higher grade lesions and metastatic disease can occur. Treatment in both children and adults has focused on achieving excision with wide margins, after which the chance of recurrence is relatively low [reviewed in McArthur (2006)]. Higher grade fibrosarcomatous elements (likely indicating gene mutation) can be histologically detected within an otherwise low grade tumor; while this finding can portend a worse prognosis (Abbott etÂ€al. 2006), adequate resection may be protective (Goldblum etÂ€al. 2000). As with synovial sarcoma, initial insight into DFSP biology came from cytogenetic studies showing that a balanced translocation involving chromosomes 17 and 22 is common in DFSP and in giant cell fibroblastoma, which is likely related (McArthur 2006). Molecular characterization of the breakpoint shows that it creates a fusion transcript involving variable regions of collagen type 1a1 and always involving exon 2 of platelet-derived growth factor B (Maire etÂ€al. 2007; McArthur 2006). Expression of the COL1A1-PDGFB fusion transcript is therefore controlled by the COL1A1 promoter but the transcript is processed to generate a normal PDGF-B protein, which is mitogenic for fibroblasts and other mesenchymal cells. Although this lesion is presumed to drive cell proliferation and hence tumorigenesis in an autocrine or paracrine manner, relatively little phosphorylation (activation) of PDGF receptors a or b is evident in protein lysates (McArthur etÂ€al. 2005). Imatinib mesylate, developed as a selective tyrosine kinase inhibitor, is known to block signals stemming from PDGF receptors. Rubin and colleagues reported that this drug significantly reduced the DFSP tumor burden in a single patient with unresectable, metastatic disease (Rubin etÂ€al. 2002). This report led to the successful use of imatinib in a child with unresectable disease (Price etÂ€al. 2005) and a prospective clinical trial in adults (McArthur etÂ€ al. 2005). The latter showed partial or complete responses in 9 of 10 patients enrolled, allowing surgical resection in some cases and long-term disease control. Xenograft studies using a DFSP-derived cell line indicate that imatinib causes apoptosis of the tumor cells but has little effect on cell proliferation (Sjoblom etÂ€al. 2001). While results to date are promising, additional studies are needed to determine whether continuous therapy is needed to eradicate DFSP and to address potential drug resistance (see additional discussion under GIST below).

Gastrointestinal Stromal Tumor and Activation of cKIT and PDGFR Gastrointestinal stromal tumors (GISTs) are relatively uncommon in children. A recent retrospective study identified only seven cases among 276 children with

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NRSTS over a 40-year period at a single institution (Cypriano etÂ€al. 2004). Until recently, it was difficult to distinguish GISTs from leiomyosarcoma of the gastrointestinal tract and therapeutic options were largely limited to surgical resection and radiation. Despite its rarity, GIST in children provides an exceptional example of the clinical application of molecular insight into sarcoma biology. A major breakthrough came from the finding that nearly all GISTs express the cKIT receptor tyrosine kinase (Hirota etÂ€al. 1998); in two smaller series in children, focal or diffuse cKIT expression was detectable in all cases (Cypriano etÂ€al. 2004; Prakash etÂ€al. 2005). This finding alone provides a useful tool to discriminate GIST from leiomyosarcoma. However, it further suggested that GIST arises from interstitial cells of Cajal, which express this receptor. The gene encoding cKIT is now known to be mutated in approximately 78% of GISTs [reviewed in Rubin etÂ€al. (2007)]. In most cases, the mutation alters the amino acid sequence in the juxtamembrane domain of the protein, activating its carboxy-terminal tyrosine kinase domain in the absence of its natural ligand, stem cell factor. The gene encoding the related receptor tyrosine kinase PDGFRA is mutated in a subset of GISTs with normal cKIT, thereby revealing a common theme underlying GIST biology. Activation of cKIT was proved to play a principal role in tumor formation by disruption of the juxtamembrane region of mouse cKit (Sommer etÂ€ al. 2003); survival of cKitV558D mice is significantly decreased and nearly all have gastrointestinal pathology (myenteric plexus hyperplasia in the distal esophagus, stomach, proximal duodenum, and cecum). The morphological appearance and uniform expression of cKit in the neoplastic lesions resemble that in GISTs. It is not clear exactly how constitutively active cKIT drives tumorigenesis. Evidence from cultured cells suggests that it secondarily activates PI3-kinase/AKT prosurvival pathways (Bauer etÂ€al. 2007), but enhanced cell proliferation is also observed inÂ€vivo in mice with mutated cKit (Sommer etÂ€al. 2003). While it is striking that cKit mutation is sufficient to cause tumors in a mouse model, the presence of a wide range of other chromosomal abnormalities in human tumors suggests that cooperating genetic events are important in human tumor progression (Rubin etÂ€al. 2007). As the relationship between cKIT (or PRGFRA) mutations and GIST was becoming clear, imatinib mesylate (STI571) was being developed as a selective inhibitor of the ABL tyrosine kinase (Druker etÂ€al. 1996). Imatinib competes with ATP for binding to the tyrosine kinase domain and is now known to block wild-type and mutant KIT, PDGFRA, and several other receptor tyrosine kinases in addition to ABL (Rubin etÂ€ al. 2007). A striking case report showing essentially complete resolution of tumor cell metabolic activity in an adult with widespread GIST opened the door to a number of clinical trials (Joensuu etÂ€ al. 2001). Numerous phase II and III trials enrolling 27 to more than 400 patients showed objective responses to imatinib in 48 to 71% of patients and tumor control (objective response or stable disease) in 74 to 90% of patients [Rubin etÂ€ al. (2007) and references therein]. When used as an adjuvant, imatinib effectively prevented recurrence after surgical resection (Nilsson etÂ€ al. 2007). The fact that disease recurrence is more common in patients who electively discontinued the drug after 1 year implies that

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it acts, at least partially, as a cytostatic agent (Blay etÂ€al. 2007). In cultured GIST cells, its proapoptotic effects appear to be due to the induction of phosphorylated histone H2AX (Liu etÂ€al. 2007). While imatinib has been tested in Phase II clinical trials in the Children’s Oncology Group, its effectiveness against childhood GIST is not yet known. Given the efficacy of imatinib in adult patients, attention has now turned to understanding the mechanisms of resistance and defining “second-line” therapies [reviewed in Rubin etÂ€al. (2007), Joensuu (2007)]. Patients whose tumors do not respond to imatinib tend to have activating mutations in exon 9 affecting the extracellular domain of the protein or no detectable KIT mutations. Those whose tumors respond initially but then become resistant usually have acquired new mutations that are likely to limit the ability of imatinib to bind the ATP-binding site. Secondary resistance is highly relevant because GISTs usually recur despite continued therapy [e.g., Blay etÂ€al. (2007)]. Sunitinib, a more broadly acting tyrosine kinase inhibitor, appears to have the activity against imatinib-resistant tumors, including those with exon 9 mutations (Joensuu 2007). In vitro studies of vatalanib (targets multiple tyrosine kinases), N-benzoyl-staurosporine (inhibits PKC), nilotinib (inhibits KIT), dasatinib (inhibits SRC and ABL), and geldanamycin (inhibits HSP-90) in imatinib-resistant GIST have yielded promising results.

Inflammatory Myofibroblastic Tumor and ALK Activation Inflammatory myofibroblastic tumor (IMT) is an uncommon soft tissue tumor composed of fibroblasts and myofibroblasts intermingled with an inflammatory infiltrate consisting mostly of lymphocytes and plasma cells (Webber and Parham 1996). Its classification as a true neoplasm vs. a “pseudotumor” and its status as a single disease vs. a group of tumors were debated until relatively recent studies confirmed that IMTs often have a balanced translocation involving chromosome band 2p23 that disrupts the gene encoding the anaplastic lymphoma kinase (ALK) [reviewed in Dehner (2004)]. IMTs are usually low grade neoplasms treated with surgical resection; their expression of COX-2 provides a rationale for the use of nonsteroidal anti-inflammatory drugs, but the efficacy of this therapy is not established (Applebaum etÂ€al. 2005). More aggressive tumors that metastasize are reported to be generally refractory to cytotoxic chemotherapy [e.g., Dishop etÂ€al. (2003)]. ALK rearrangement was originally reported in anaplastic large cell lymphoma (Morris etÂ€al. 1994). Numerous translocations are now known to generate a fusion protein in which the amino terminus contains a dimerization motif and carboxyl terminus represents the same 563 amino acids containing the ALK kinase domain [reviewed in Duyster etÂ€al. (2001)]. In IMTs, the dimerization motifs include portions of tropomyosin 3, tropomyosin 4, clathrin heavy chain, and ran binding protein 2 (Duyster etÂ€ al. 2001). In all cases, the promoter driving expression of the fusion partner causes increased expression of ALK; hence, immunohistochemical

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detection of ALK supports the diagnosis of IMT, but it may also be observed in other tumors. The oligomerization domain in the fusion protein leads to constitutive activation of ALK, which has potent transforming potential. Downstream effectors of ALK include PLC-g, PI3-K, STAT3, and STAT5. Because of the central role of ALK in IMT, pharmacological inhibition of ALK is an attractive strategy. Chemical biology screens have begun to identify compounds, including WHI-P131 (4-(40-hydroxyphenyl)amino-6,7-dimethoxyquinazoline) and WHI-P154 (4-[(30-bromo40-hydroxyphenyl)amino]-6,7 dimethoxyquinazoline) (Marzec etÂ€ al. 2005), and test their activity in preclinical models. Because NPM-ALK requires HSP90 for normal protein folding, the geldanamycin analog 17-AAG can destabilize NPMALK (Bonvini etÂ€al. 2004). It remains to be seen whether ALK inhibition will be helpful for low grade and malignant IMTs.

Leiomyosarcoma Leiomyosarcoma is an uncommon type of NRSTS in children. In a retrospective series of 276 children with NRSTS at St. Jude Children’s Research Hospital, only 4 (1.4%) were shown to have leiomyosarcoma (Cypriano etÂ€al. 2004). However, it has become the second most common neoplasm in children with HIV infection (Granovsky etÂ€ al. 1998). In either case, it occurs in gastrointestinal sites, liver, spleen, uterus, and other soft tissue sites. As with other NRSTS, survival is relatively poor unless the tumor can be completely removed surgically (Cypriano etÂ€al. 2004). Relatively little is known about leiomyosarcoma biology. The tumor is presumed to arise from smooth muscle cells or their progenitors but rarely arises from benign leiomyomas [reviewed in Sandberg (2005)]. Most biology studies have been performed on tumors in adults. Cytogenetic and molecular genetic analyses (e.g., comparative genomic hybridization) show that these tumors have complex and variable gains and losses involving a large number of chromosomes. This variability has made it difficult to develop a unifying hypothesis for the origin of leiomyosarcoma. Some have taken a “candidate gene” approach and have made some progress by using preclinical models. For example, building on the fact that the Wnt signaling pathway is generally important in a range of cancers and that Cripto-1 (CR-1) is a growth factor that can be induced by Wnt signaling, Strizzi etÂ€al. (2007) tested whether the deregulated expression of CR-1 could cause leiomyosarcoma in transgenic mice. Indeed, 20% of mice expressing this transgene developed uterine leiomyosarcoma as they aged. These investigators subsequently showed by immunostaining that 9 of 13 cases of human leiomyosarcoma expressed CR-1. Using similar logic, members of the Cordon-Cardo laboratory tested whether the activation of the PI3-kinase/AKT pathway in smooth muscle cells is sufficient to cause leiomyosarcoma (Hernando etÂ€ al. 2007). They found that cell type-specific genetic ablation of the PTEN tumor suppressor, which functions to block PI3K/AKT signaling, caused generalized

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smooth muscle hyperplasia in the gastrointestinal and urinary tracts (but not the uterus) that progressed to leiomyosarcoma in 80% of mice more than 2 months of age. Interestingly, p53 gene mutations were not observed, but Mdm2, which acts as a negative regulator of p53 tumor suppressor function, was highly expressed. Inactivation of this important pathway by Mdm2 supports the potential usefulness of therapeutic compounds aimed at disrupting Mdm2–p53 interactions. These investigators also observed molecular evidence of mTOR activation as a downstream effector of PTEN loss in smooth muscle cells. Survival was significantly prolonged in mice treated with the mTOR inhibitor everolimus, suggesting that mTOR activation plays a causative role. In view of these findings, mTOR and PI3K/AKT are very attractive therapeutic targets for this disease, especially if functional genomics approaches largely pursued in adult leiomyosarcoma [e.g., Segal etÂ€al. (2003)] can be extended to children.

Current Clinical Trials Using Targeted Therapies for Childhood NRSTS Currently, there are relatively few histology-specific clinical trials of targeted therapy for NRSTS in the US. A search of ClinicalTrials.gov revealed the following trials to be open as of June 2007: NCT00029354:â•… A randomized phase II trial of the FTI R115777 (tipifarnib) in children (ages 3 to 25 years) with neurofibromatofsis type I and progressive plexiform neurofibromas; conducted by the National Cancer Institute. NCT00148109:â•… A nonrandomized Phase II trial of cetuximab, an antibody directed against the EGF receptor, in children (16 years and above) and adults; conducted by the University of Michigan Cancer Center in collaboration with Bristol-Myers Squibb and ImClone Systems. NCT00171912:â•… A nonrandomized Phase II trial of imatinib in children (16 years and above) and adults with a variety of cancers, including dermatofibrosarcoma; conducted by Novartis. NCT00187174:â•… A nonrandomized Phase I/II trial of the mTOR inhibitor RAD001C (everolimus) in children (ages 3 to 21 years) with recurrent, refractory sarcoma, including NRSTS; conducted by St. Jude Children’s Research Hospital. NCT00401388:â•… A nonrandomized Phase II trial of the AKT/PKB inhibitor perifosine in children and adults (ages 13 and above) with refractory alveolar soft part sarcoma, chondrosarcoma, or extraskeletal myxoid chondrosarcoma; conducted by the Sarcoma Alliance for Research through Collaboration (SARC) in collaboration with AOI Pharma, Inc. NCT00464620:â•… A nonrandomized Phase II trial of the second-generation tyrosine kinase inhibitor dasatinib in children and adults with any of a variety of unresectable,

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recurrent, or metastatic soft tissue or bone sarcomas; conducted by SARC in collaboration with Bristol-Myers Squibb.

Future Directions and Challenges Great insight into the biology of many types of NRSTS in children and adults has come from cytogenetics, molecular genetics, and molecular biology studies largely using tumor-derived cell lines, human tissue samples, and elegant mouse models of the human diseases. The remarkable initial successes of imatinib against certain previously untreatable NRSTS, such as GIST, suggests great promise for the future of targeted therapies for this class of tumors, which are minimally susceptible to intensive chemotherapy with alkylating agents and anthracyclines. Of course, numerous challenges remain. Perhaps foremost among them are the remarkable diversity of histological subtypes of NRSTS and the relative rarity of any particular subtype in children. The recent opening of a national collaborative trial for NRSTS in children and young adults by the Children’s Oncology Group is hoped to encourage the enrollment of children on research protocols. This trial (ARST0332) tests the usefulness of a risk-based application of standard therapies, and the companion biology study (ARST D9902) will increase the availability of tissue and tumorderived cells to further dissect the molecular mechanisms of NRSTS. The rarity of individual subtypes of NRSTS would necessitate national collaborative studies of any histology-specific targeted therapies. However, histologically dissimilar tumors may be driven by similar molecular processes and may respond to the same therapies; for example, the unrelated GIST and DFSP are both treatable with imatinib mesylate. Therefore, eligibility for trials of targeted therapies may be based on biological rather than histological subtype. It is already becoming clear that the initial successes of targeted therapy for NRSTS are likely to lead us to the next challenge – refractory disease. This challenge must be met by continued molecular biological and genetic studies of refractory tumors; the development of second and subsequent generations of novel agents; better understanding of downstream signaling pathways and their changes in response to targeted therapy; the potential synergism of combined targeted therapies; and better understanding of parallel signaling pathways or genetic or epigenetic events necessary for tumor progression. The increased development and use of elegant, inÂ€vivo models such as those developed for the studies of synovial sarcoma and GIST provide an opportunity to discover cooperating pathways that may serve as additional targets.

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Index

A Abelson, H.T., 140 Acute lymphoblastic leukemia (ALL). See also Lymphoblastic lymphoma BCR/ABL tyrosine kinase and Ph+ ALL dasatinib and nilotinib, 5–6 imatinib mesylate, 4–5 potential toxicity, 5 SCT, 4 cancer stem cells self-renewing, 12 xenograft mouse model, 13 DNA technology, 3 FLT-3 pathway, MLL event free survival (EFS), 6 juxtamembrane mutations, 7 lestuartinib and midostaurin, 7–8 monoclonal antibodies alemtuzumab, 11–12 epratuzumab, 11 gemtuzumab ozogamicin, 12 rituximab, 10–11 therapeutic, 10 mutated gene, 4 Notch pathway c-Myc, 10 domain, 8–9 GSIs, 9–10 intracellular Notch (ICN), 9 members, 8 Acute megakaryocytic leukemia (AMkL) chromosome 21-localized genes, 112–115 description, 109–110 differential gene expression, 116–117 non-Down syndrome, treatment results, 111 somatic mutations, 116 Acute myeloid leukemia (AML). See also Down syndrome animal models, 74–75

development cellular and molecular targets, 62 cytoreduction, 61 drug resistance mechanisms cyclosporine, 69 P-glycoprotein, 68–69 proteasome inhibition, 70 epigenetic and chromatin remodeling DNA methylation and histone deacetylation, 71 gene expression, 70–71 sequencing, 72 FLI1 homolog ERG translocation, 380 immunotherapy and immunostimulatory gemtuzumab ozogamicin, 72–73 graft vs. host disease, 74 IL-2, 73–74 MDR1 transporter, 73 leukemogenesis mechanisms, 60 MDS and, 157, 163 relevance, 61 risk-adapted therapy, 59 signaling pathways, TK receptors FLT3, 67 FLT-3, 67 mTOR serine/threonine, 68 RAS mutations, 66 toxicity, 74, 75 tyrosine kinases, leukemia cell prolifeÂ� ration and survival CEP-701/lestaurtinib, 63–64 c-KIT mutations, 64–65 cytokines, 62–63 FLT3/ITD mutations, 63 inhibitors, 66 VEGF, 65 Acute promyelocytic leukaemia (APL) alemtuzumab, 101

521

522 Acute promyelocytic leukaemia (APL) (cont.) anthracycline cumulative dose and cardiotoxicity, 96 arsenic trioxide (ATO) induction and consolidation, 97 telomerase, 96 demographic features, 83–84 diagnosis distribution pattern, 86, 88 M3v, 86 reverse-transcription PCR, 88 tools, 87 failure, treatment antileukemic efficacy, 98 extramedullary (EM) relapse, 99–100 GO, 98–99 HSCT, 99 relapse, 97 salvage therapy, 97–98 FLT-3 inhibitors, 100 molecular monitoring MRD detection, 94–95 real-time quantitative RT-PCR assays, 95–96 pathogenesis chimeric protein, 85 PML-RAR and ATRA, 84–85 retinoic acid receptor-a(RARA) gene, 84 RA-sensitive and resistant cells, 100–101 SCT, 96 treatment anthracycline-based, 88–89 consolidation therapy, 92–94 induction therapy, 89–92 maintenance, 94 Adesina, A.M., 272 Akatsuka, T., 469 Al-Ahmari, A., 110 Albritton, K., 341 ALCL. See Anaplastic large cell lymphoma Aldosari, N., 272 ALL. See Acute lymphoblastic leukemia All-trans retinoic acid (ATRA), APL anthracycline-based chemotherapy, 88–89 arsenic trioxide (ATO) and, 96–97 consolidation therapy, 92–94 extramedullary relapses, 99 functions, 85 induction therapy, 89–92 maintenance chemotherapy, 94 PML-RARA fusion gene, 84 synergistic effect, 93 Alveolar RMS (ARMS), 425 AMkL. See Acute megakaryocytic leukemia

Index Ammatuna, E., 95 Anaplastic large cell lymphoma (ALCL) ALK rearrangement, 510 anaplastic lymphoma kinase (ALK) CD30, 197–198 CD52, 198 expression/function, fusion protein, 194–195 fusion partners, 194 inhibitors, 195–196 nucleophosmin (NPM), 193 overexpression, 193 proteasome inhibitors, 197 signaling pathways, 196–197 cDNA microarray analysis, 198 NPM-ALK multiple signaling pathways, 195 subtypes, 192–193 Anaplastic lymphoma kinase (ALK) activation, IMT, 510–511 Anderson, C.P., 356 Angiogenesis inhibition, astrocytomas blockade, VEGF, 245 avb3 integrin inhibitor cilengitide, 246 molecularly targeted therapies, 235 thalidomide, 245–246 VEGF receptor EGFR-mediated signaling, 244 ligand/receptor interactions, 244–245 Anninga, J.K., 470 Antimetabolite therapy, 160 APL. See Acute promyelocytic leukaemia Apoptotic tendency modulation, non-EWS-FLI1 DR4 and DR5, 383, 385 intrinsic and extrinsic pathway, 383 TRAIL receptors function, 385 Arceci, R.J., 59 Arcellana-Panlilio, M.Y., 403 ARMS. See Alveolar RMS Astrocytomas angiogenesis inhibition avb3 integrin inhibitor cilengitide, 246 thalidomide, 245–246 VEGF blockade, 245 VEGFR, 244–245 downstream signaling inhibitors, 240 drug resistance phenotype alkylguanine DNA alkyltransferase inhibition, 247–249 base-excision repair inhibition, 249–250 cell cycle regulatory proteins, 251–252 HDACi, 250–251 HSP and NF-kB, 251

Index epidermal growth factor receptor (EGFR) description, 238 gefitinib, 239 GW572016, 240 molecular signaling profile, 239–240 ZD 1839, 238–239 growth factor receptor and survival signaling angiogenic, 247 cytotoxic drugs, 246–247 high-grade gliomas anaplastic and oligoastrocytoma, 232–233 deÂ€novo, 233 malignant astrocytoma, 233–234 immunological/ligand-based therapies IL13-PE38QQR, 253–254 receptors, 252–253 Tf-CRM107, 253 TP-38, 254 vaccine-based, 252 inhibitors, growth factor receptor, 236 low-grade gliomas juvenile pilocytic, 232 non-pilocytic low-grade, 232 mitogen-activated protein kinase (MAPK) cascade signaling pathway, 242–243 sorafenib, 243 PI3K/Akt pathway inhibitors description and PTEN, 243 glioblastoma multiforme, 244 platelet-derived growth factor (PDGF) receptor expression, 236–237 identification, 236 inhibitors, 237 proliferation, 237–238 STI571, 238 protein kinase C (PKC) inhibition, 242 RAS processing inhibition cell-surface receptors, 240–241 farnesylation, 241 farnesyl transferase inhibitors (FTIs), 241 Athale, U.H., 111 B Babovic-Vuksanovic, D., 340 Badiali, M., 272 Baeza, N., 272 Bagatell, R., 169, 356 Baker, M.J., 251 Bakhshi, S., 270 Banerjee, D., 356

523 Barel, D., 270 Barnard, D.R., 111 Basic fibroblastic growth factor (bFGF), 444 Basso, K., 190 Baudry, D., 403 Bayani, J., 272 Beckwith–Wiedemann syndrome (BWS), 404 Begum, S., 430 Berg, T., 433 Berthold, F., 356 Bevacizumab, 299 Bianco, R., 247 Biondi, A., 83 B lymphocyte-induced maturation protein 1 (BLIMP-1), 186–187 BMPs. See Bone morphogenetic proteins Boehrer, S., 168 Bone differentiation, osteosarcoma BMPs, 474 PTHrP, 474–475 TGF-b isoforms, 473–474 WNT signaling, 474, 486 Bone morphogenetic proteins (BMPs), 474 Bosutinib, 147 Brain tumor polyposis syndrome, 275 Braun, B.S., 123 Bredel, M., 241 Brignole, C., 356 Brown, K.W., 412 Brown, P.A., 31, 34 Burkitt lymphoma (BL) antisense oligodeoxynucleotides, 192 benzodiazepine, Bz-423, 190, 192 description, 188, 190 differential gene signatures, 189 MYC subnetwork, 191 NOTCH1 signaling, 192 1q+ abnormalities, 188 subnetwork, 190 C Cairo, M.S., 177 Calabrese, C., 296 cAMP response element-binding protein (CREB), 407 Cancer predisposition syndromes BWS, 404 medulloblastoma, 270 osteosarcoma hereditary, 461–462 incidence, 461 RECQ DNA helicases, 462 Candidate of metastasis 1 (COM1), 503

524 Carroll, W.L., 3 Castleberry, R., 341 Cell cycle regulation, osteosarcoma INK4A gene, 464–465 malignant proliferation, 464 MDM2 and COPS3 gene amplification, 465–466 p53 gene, 465 Chakraborty, S., 160 Cheung, N.K., 356 Chimeric transcription factors and oncogenes, RMS MYCN amplification and overexpression, 432–433 downregulation, 433 GSK609693, 434 PAX3-FKHR antisense oligonucleotides and siRNA, 430–431 CXCR4, entry fusion inhibitors, 431 downstream consequences, 429 ectopic expression, 429–430 elucidation, pathogenesis, 428 expression, ARMS, 428, 429 specificity, RNAi, 431, 432 TP53 dysregulation, 434–435 Cho, Y.-J., 267 Chronic myelogenous/myeloid leukemia (CML) Bcr-Abl pathophysiology breakpoints, 140 hematopoietic stem cells, 139 kinase cellular proliferation, 140 murine retroviral transplantation, 140–141 structure and signaling function, 141–142 cancer stem cells, 12 chronic phase (CP) accelerated phase (AP), 143 blasts count, 142 description, 139 genetic lesion, 27 imatinib, 64, 145, 388 kinase inhibitors resistance Bcr-Abl domain mutations, 147 bosutinib, 148 dasatinib (See Sprycel™) imatinib therapy, 147 nilotinib (See Tisigna™) second generation tyrosine, response, 148

Index T315I mutation, 148–149 monitoring PCR, 146–147 Philadelphia chromosome, 145–146 pediatric, 142 targeted therapies diuretics, 144–145 imatinib, 144 interferon-a, 143 response criteria, 143–144 transplantation allogeneic stem cell, 149 HLA-matched sibling donor, 149–150 Clifford, S.C., 272 CML. See Chronic myelogenous/myeloid leukemia Coffey, D.C., 356 COM1. See Candidate of metastasis 1 Consolidation therapy, APL daunorubicin and cytarabine, 93 molecular evidence, 92 Corey, S.J., 139 Cortes, J., 139 Cox, C.V., 13 CR-1. See Cripto-1 Creutzig, U., 110 Cripto-1 (CR-1), 511 Cyclin D1 (CDKs), 408 Cytochrome P450 inhibitors, 92, 144 Cytokine-regulated growth pathways, non-EWS-FLI1 IGF-IR, 385–386 imatinib, 388 KIT, 387–388 PDGF, 386–387 Cytotoxic agents, neuroblastoma, 356–357 Czibere, A., 170 D Dahmen, R.P., 272 Dave, S.S., 188, 189 Davicioni, E., 428 De Bortoli, M., 272 de Chadarevian, J.P., 270 Delta-serrate-lag2 (DSL) family, 8 Dermal neurofibromas, 333 Dermatofibrosarcoma protuberans (DFSP), 508 Diamanti, P., 13 Diarrhea, 144 Dickson, P.V., 356

Index Diffuse large B-cell lymphoma (DLBCL) BCL-6 gene CD80, 182 chromatin remodeling, 183 expression deregulation, 181 germinal center-derived DLBCL (GC DLBCL), 180–181 germinal center (GC) phenotype, 180 peptide interference peptides, 183 protein interaction, 182 transcriptional regulation, 181–182 BLIMP-1, 186–187 B-NHL, 178–179 FAB/LMB96 international B-NHL trial, 183–184 genetic subtypes, 179 molecular targeting, 184 NF-kB pathway differential expression, 187 gene expression and transcriptional activation, 184–186 IkB proteins polyubiquitination, 186 rituximab, 186 PMBL children and adolescents, 180 differential expression, 184 gene expression, 180–181 IKK inhibitors, 187–188 Diller, L., 403 Distel, L., 270 DNA methyltransferase (DNMT) inhibitors gene transcription, 164, 166 HDACi, 166 p21Cip1 reexpression, 442 Doepfner, K.T., 67 Dome, J.S., 401 Douer, D., 83 Down syndrome (DS) AAML041, 117 acute myeloid leukemia DS ALL, 112 event-free survival (EFS) rate, 110, 111 GATA1, 115–116 infectious complications, 112 chemotherapy sensitivity anthracycline, 112 GATA1, 115–116 megakaryoblasts, 111 toxicity, 111–112 chromosome 21-localized genes allosteric regulation, 113 cystathionine-b-synthase (CBS), 112–113 GATA1, role of, 114–115 differential gene expression bone marrow stromal cell antigen 2 (BST2), 116–117

525 megakaryocytic blast maturation, 117 microarrays, 116 leukemia, 109 non-Down syndrome AMkL cases, 111 resistance, chemotherapy, 118 treatment outcome, 110 Drug resistance, astrocytomas alkylguanine DNA alkyltransferase inhibition Gliadel wafer implantation, 248–249 mechanism, 247–248 O6-BG, 249 O6-methylguanine, 248 base-excision repair inhibition cell cycle regulatory proteins, 251–252 DNA mismatch deficiency, 249–250 HDACi, 250–251 HSP and NF-kB, 251 INO-1001, 250 DS. See Down syndrome Duchayne, E., 111 Dyspnea, 144–145 E Eberhart, C.G., 272 Ebinger, M., 272 E-cadherin, 503 EGFR. See Epidermal growth factor receptor Ellison, D.W., 272 Ependymoma cancer stem cells and origins gene expression, 295–296 identification, 295 neural progenitor, 296 epidemiology and histopathology, 292 genetics, 294 genomics amplicons, 295 molecular alterations, 294–295 molecular targeted therapies, 297 stem cell niches, 296 therapy conventional chemotherapy, 293–294 surgery and radiation, 292 total tumor resection, 292–293 treatment improvement cancer stem cells, 299 disease-risk stratification system, 298 gene mutations, 298–299 JAGGED ligands binding, 297–298 minimal conventional therapy, 298 subgroups, genetic alterations, 297 Epidermal growth factor receptor (EGFR), 504 Epstein–Barr virus (EBV) apoptotic resistance, 220–221

526 EBV-PTLD molecular targets Epstein–Barr virus (EBV) (cont.) anti-viral medications, 221–222 immunosuppression, 222 monoclonal antibodies, 222–223 T cell adoptive immunotherapy, 223 vaccines, 221 host immune response cellular, 218–220 cytokine modulation and viral IL-10, 220 IgM and IgG titers, 218 infection, human B cell differentiation, 217–218 description, 217 ERBB2 proto-oncogene, 467–468 Erez, A., 272 ETV6-NTRK3 fusion protein, 506–507 Evans, A.E., 356 Ewing’s sarcoma family of tumors (ESFT) CD99MIC2 expression, 374 description, 373 discovery, 374 EWS-FLI1 approaches, small molecule identifiÂ�cation, 382 chromosomal translocations, 376–377 DNA binding, disruption, 383 elimination of, 379 expression, 374–375 single-chain antibodies, 382 small-molecule inhibitors, 380–381 targets of, 382–383 transcription and splicing, 377–379 YK-4-279, 381–382 features, 373–374 models murine, 376 spheroid, 375 xenograft, 375–376 non-EWS-FLI1 apoptotic tendency modulation, 383–385 current clinical trials, 383–384 cytokine-regulated growth pathways, 385–388 molecular targets, 388–390 targeted molecular agents, 380 Ewing’s sarcoma (EWS)-Friend leukemia insertion (FLI1) approaches, small molecule identification, 382 chromosomal translocations, 376–377 DNA binding, disruption, 383 elimination of, 379

Index ESFT molecular targeting, 380 expression, 374–375 RNA splicing mRNA, 377–378 U1C identification, 377 single-chain antibodies, 382 small-molecule inhibitors peptide sequences, 381 protein–protein interactions, 380–381 targets of, 382–383 transcriptional regulation hsRPB7, 377–378 and RNA metabolism, 377 YK-4-279 novel compound, 381–382 RHA region, 381 Extramedullary (EM) relapse, APL, 99–100

F Faderl, S., 5 Farnesyl transferase inhibitors (FTIs) AML, 66 antiproliferative effects, 241 NF1 related tumors, 339 RAS-dependent signaling, 442, 505 Fate-mapping experiments, 269 Feldkamp, M.M., 241 Felix, C.A., 31 Fernandeaz, C.V., 403 Feuerhake, F., 187 FMS-like tyrosine kinase-3 (FLT-3) childhood leukemia, 7 description, 6–7 inhibitors, APL, 100 midostaurin inhibitor, 8 receptor, 6 Fox, E., 356 Frappart, P.O., 273 Friedman, H.S., 249 FTIs. See Farnesyl transferase inhibitors

G Gallicchio, M.A., 439 Gamis, A.S., 110 Garvin, A.J., 412 Gastrointestinal stromal tumors (GISTs) description, 508–509 gene encoding cKIT, 509 phase II and III clinical trials, 509–510 second-line therapies, 510 Geller, J.I., 401

Index Genasense™ BCL-2 mRNA expression, 39–40 refractory/relapsed adult acute leukemia, 39 Gessler, M., 403 Ge, Y., 109 Gibson, B.E.S., 83 Gilbert, J., 251 Gilbertson, R.J., 291 GISTs. See Gastrointestinal stromal tumors Gleevec. See Imatinib Goodrich, L.V., 273 Gore, L., 155 Gorlick, R., 459, 469 Grignani, F., 84 Grimwade, D., 86, 88, 94, 95 Gross, T.G., 215 Grundy, P., 403 Gupta, A., 340 Guran, S., 270 Gutierrez, A., 19 H Hahn, H., 270 Hallahan, A.R., 273 Hamilton, S.R., 270 Hanahan, D., 477 Heat shock proteins (HSP). See also Astrocytomas HSP90, 46–48 anticancer therapeutics, 389 NPM-ALK, 511 Phase I trial, 483 PPTP, 415 HSP 27 and 70, 411 Hedgehog signaling pathway, 441 Hematopoietic stem cell transplantation (HSCT) allogeneic, 98, 123 AML graft vs. host disease, 74 therapy, 59 frontline therapy, 88 modality, 99 molecular disease, 96 PTLD, 215–316 rapamycin, 132 veno-occlusive disease (VOD), 72 Hepatocyte growth factor (HGF) HGF/c-MET, 410–411 ligand binding prevention, 440–441 MET, 439–440 PAX3-FKHR, 440 HER2/neu. See ERBB2 proto-oncogene Hirsch, B., 270

527 Histone deacetylase inhibitors (HDACi) cell cycle arrest, 72 histone deacetylases (HDAC) and, 166 as investigational agent, 356 neuroblastoma, 362–363 p21Cip1, 442 potential role, 100–101 vivo activity, WT, 414 Holcomb, V.B., 273 Ho, R., 356 Hosoi, H., 425 Houghton, P.J., 356, 425 Huang, H., 272 I IGF-2. See Insulin-like growth factor-2 (IGF)-2 IGF1R. See Insulin-like growth factor-1 receptor IGF signaling, WT IGF2/IGFR1, 405 IGFR1, IGFR2 and IGF2, 404–405 and mTOR, 405 IL13-PE38QQR domains, 253–254 resection, 254 Imatinib and chemotherapy, 5 children, 145 CML, 27, 64 description, 143 mesylate Abl, KIT, and PDGFR inhibition, 388 vs. ATP, 509–510 block signals stemming, PDGF receptors, 508 inhibitor, ABL, 484 neuroblastoma, 358 RMS, 426 resistance, 5–6 131 I-metaiodobenzylguanidine (131I-MIBG), 359 Immunomodulatory drugs (IMiDs), 167 IMT. See Inflammatory myofibroblastic tumor Induction therapy, APL ATRA and anthracycline, 89 complications APL differentation syndrome, 91–92 coagulation parameters, 91 headache and pseudotumor cerebri, 92 hemorrhage, 90–91 sequential treatment, 90 Infant ALL amino and carboxyl terminal fragments, 32 anti-apoptotic BCL-2 family members ABT-737, 43–44

528 Infant ALL (cont.) ABT-869, 44 ADVL0816, 42–43 basal expression levels, 42 classes, 39 drug resistance, 43 Genasense™, 39–40 homo-and heterotypic dimers, 40 neuroblastoma, 41 obatoclax, 40–41 silencing, 38 CD33 cell surface antigen, 49 epigenetic strategies, 48 FLT-3 tyrosine kinase inhibition CEP-701, 34–35, 37–38 chemotherapy regimens, 36 LSC, 36–37 microarray studies, 33 mutations, 33–34 plasma inhibitory activity (PIA), 37 signaling, 34 glycogen synthase kinase 3 small molecule inhibitors, 46 transduction, 46–47 HSP90, potential therapeutic target description, 47 inhibition, 47–48 MLL fusion transcripts, 45 mTOR inhibition glucocorticoid resistance, 48–49 serine/threonine kinase, 48 oncoprotein transcriptional deregulation, 33 partner genes, 31 PFWT, MLL partner protein interactions AF4-AF9 complexes, 45 synergistic, 46 treatment options, 31–32 Infantile fibrosarcomas kinase activity, 507 recurrent translocation, 506 Inflammatory myofibroblastic tumor (IMT) ALK rearrangement, 510–511 description, 510 INI1/hSNF5 tumor suppressor, RTs. See also Rhabdoid tumors differentiation cell of origin, 315 nonRT and normal cells, 314 interferon signaling, 314 loss cell characterization, 312–313 G0/G1 arrest, 313 mitotic spindle checkpoint activation, 313

Index mechanisms chromatin remodeling complexes, 310 SWI/SNF complexes, 310–311 Insulin-like growth factor-2 (IGF)-2, 503, 504 Insulin-like growth factor-1 receptor (IGF1R) characteristics, 385 inhibition, ESFT patients, 386 medulloblastomas description, 276–277 gene expression, 277 inhibitors, 277–278 Phase I and II studies, sarcomas, 485 predominant receptor, cancer biology, 466 RMS, 435–436, 438–439 synovial sarcomas, 504 VEGF production, 445 Insulin-like growth factor signaling, RMS CTCF engenders, 438 H19/IGF-2 expression, 437–438 IGFBPs, 439 IGF-2 mRNA, 437 IGF-1R signaling, 435–436, 438 specificity, 438–439 loss of heterzygosity (LOH), 437 International Prognosis Scoring System (IPSS), 158–159 J Jakacki, R., 340 Johnson, R.L., 270 Juvenile myelomonocytic leukemia (JMML), RAS signaling pathways description, 123 GM-CSF and deregulated RAS signaling effector cascades, 125 intracellular phosphoprotein analysis, 126–127 neurofibromin, 124–125 PTPN11 mutations, 125–126 quantitative assays, 126 RAS proteins, 124 JAK2-STAT5, 133–134 molecular genetics, 123–124 mouse models advantages, 128 conditional alleles, 127–128 Cre recombinase protein, 127 PI3K/AKT/mTOR AML, 132–133 rapamycin, 132 PTPN11/SHP-2, 133

Index RAF/MEK/ERK activation, 130–131 CI-1040, MEK inhibitor, 131–132 colony formation, 131 Nf1 mutant, 132 sorafenib, 131 targeted therapeutics activated RAS, inhibition, 128 CAAX protein, 128–129 oncogene addiction, 130 prenylation, 129 Juvenile pilocytic astrocytomas, 232 K Kalpana, G.V., 305 Karp, J.E., 65 Keshelava, N., 356 Kikuchi, K., 425 Kilpatrick, S.E., 470 Knudson’s two-hit hypothesis. See Wilms’ tumor Koch, A., 272 Koesters, R., 403 Kohler, 72 Kruppel-associated box (KRAB) repression domain, 503 transcriptional repressor, 379 Kudo, K., 110 Kurmasheva, R.T., 425 Kushner, B.H., 356 L Lae, M., 428 Lam, C.W., 272 Lam, L.T., 187 Lauchle, J.O., 123 Lee, Y., 273 Leiomyosarcoma, 511–512 Lenalidomide, 167 Leukemia initiating cells (LICs). See Leukemic stem cells Leukemic stem cells (LSCs) self renewal, 12 xenograft model, 36 Lie, S.O., 111 Lim, M.S., 177 Linardic, C.M., 427 Lo-Coco, F., 95 Lode, H.N., 356 Look, A.T., 19 Lorand-Metze, I., 157 Lymphoblastic lymphoma (LLs) ALCL molecular targets, 201 vs. ALL, 199

529 B-precursor (B-LLs) cytogenetic abnormalities, 205 and T-LLs, 199 cytogenetic and molecular changes, 200 description, 198–199 molecular abnormalities, 201–202 TAL1, 202–203 T-ALLs cryptic aberrations, 199–200 gene expression, 202 NOTCH1 mutations, 203–204 T-cells (T-LL) chromosomal translocations, 199 vs. T-ALL, 20, 200–201 M Maitra, A., 469 Major, M.B., 403 Maki, R.G., 341 Malignant fibrous histiocytoma (MFH), 507 Malignant gliomas genetic alterations, 234 molecular feature, 234 oligodendroglial features, 233–234 Malignant peripheral nerve sheath tumors (MPNSTs) description, 504 features, 505 molecular features, 338 and neurofibromas, 505–506 Malignant rhabdoid tumors (MRTs). See Rhabdoid tumors (RTs) Mammalian target of rapamcyin (mTOR) inhibitors, 279 NF1, 337–338 PI3K, 278–279 Marino, S., 273 Maris, J.M., 351, 356 Marks, P.A., 356 Marshall, J.L., 356 Matthay, K.K., 356 Maurer, B.J., 356 McDonald, J.M., 403 McKinnon, P.J., 273 Medulloblastomas cancer predisposition syndromes, 270 cellular origins cancer stem cells, 269, 271 pathogenesis pathways, 271 precursors, 269 classic, 267–268 molecular features CXCR4, 278

530 Medulloblastomas (cont.) EGFR family, 279–280 HDAC inhibitors and retinoic acid, 280–281 IGF1R (See Insulin-like growth factor-1 receptor) NOTCH, 276 PDGFR and TRKC, 280 PI3K/AKT/mTOR, 278–279 SHH (See Sonic hedgehog) WNT/b-catenin, 275–276 molecular pathways and targeted therapies, 271 mouse models, 273 nodular architecture, 268 risk stratification desmoplastic tumors, 268 diagnosis, 268–269 sporadic, mutations, 272 subtype classifications, 267 Methotrexate (MTX) DS patients, 112 human ALL xenografts, 48 osteosarcoma, 477 T-LL, 199 MFH. See Malignant fibrous histiocytoma Michiels, E.M., 272 Miller, B.S., 356 Milstein, 72 Minimal residual disease (MRD) detection, APL fusion transcripts, 95 leucocyte count, 94 relapse risk, 94–95 Mistry, A.R., 84 Mitotic spindle inhibition, neuroblastoma, 362 Mixed lineage leukemia (MLL) BCL2 family members, 38 FLT-3 pathway, 6–8 fusion transcripts, 45 native protein, 32 partner protein interactions, 45–46 rearrangements, 33–34, 36, 38, 42, 71 translocations, 31, 32 Modak, S., 356 Molecular targets, non-EWS-FLI1 ET-743, 388–389 HDACi, 389 HSP, 389 protein synthesis and degradation, 389–390 Morris, E.S., 11 Mossé, Y.P., 351 MPNSTs. See Malignant peripheral nerve sheath tumors MTP-PE. See Muramyl tripeptide phosphatidylethanolamine

Index MTX. See Methotrexate Multidrug resistance (MDR1) gene, 476–477 Multidrug-resistance protein (MRP), WT, 412 Muramyl tripeptide phosphatidylethanolamine (MTP-PE), 482 Myelodysplastic syndromes (MDS) vs. AML, 163 bone marrow angiogenesis, 65 chromatin-based transcriptional therapy DNMT inhibitors, 164–166 epigenetic and chromatin changes, 164 HDACs and HDACi, 166 classification adult and pediatric, 158–159 FAB and WHO, 157 IPSS, 156–157 myelofibrosis/myeloproliferative syndromes, 156 and DS, AML, 110 etiology chemotherapy/toxin exposure, 160 development, 157 genetic abnormalities, 160–162 hematopoietic disorders, 160, 163 predisposing genetic syndromes, 160 5q31 deletions, 163 IMiDs and anti-angiogenic agents, 167 incidence, 156 maturation-directed therapies, 167 SCT adults, 170 pediatric patients, 170 regimens, adults, 169 timing, 169–170 TKIs, 168 Myelosuppression gemtuzumab ozogamicin, 98–99 occurance, 145 N Nabarro, S., 430 Natrajan, R., 403 Neuroblastoma description, 351 locoregional tumors, 353 metastatic tumors common agents, relapse setting, 354 retinoids, 354–355 overall survival probability, high-risk patients, 351–352 relapse setting, high-risk disease angiogenesis inhibitors, 361 cytotoxic agents, 356–357 histone deacetylase inhibitors, 362–363

Index immunotherapy, 359–360 investigational agents, 355–356 mitotic spindle inhibition, 362 retinoids, 360–361 stem cell conundrum, 358–359 targeted delivery, radionucleotides, 359 targeting MYCN, 361–362 tyrosine kinase inhibitors, 357–358 risk classification system chromosomal aberrations, 353 MYCN oncogene amplification, 352 strategies, molecular targets identification, 355 Neurofibromas dermal, 333 and MPNST, 505–506 plexiform, 333–334 Neurofibromatosis type 1 (NF1) age stratification, 344 combination therapy, 345 description, 331 development, targeted treatments completed, ongoing and planned clinical trials, 339–341 phase II trial, TTP, 342–343 refractory cancer trial, 339, 342 diagnosis, 333 infrastructure, clinical trials, 343 molecular features, tumorigenesis angiogenesis, 336–337 growth factor receptors, 337 mTOR, 337–338 RAS pathway, 335–336 rationale, pathways, 339 tumor origin and environment, 336 MPNSTs, 338 pathway structure, 332 pilocytic astrocytomas, 232 preclinical evaluation, agents, 343 RAS activity, 332 target validation, 344 tumor manifestations dermal neurofibromas, 333 JMML, 335 malignant peripheral nerve sheath, 334 optic pathway gliomas, 334–335 PN, 333–334 NHL. See Non-Hodgkin lymphoma Nitta, T., 236 Non-Hodgkin lymphoma (NHL) BL (See Burkitt lymphoma) DLBCL ABC-DLBCL and GC-DLBCL, 183–184 BCL-6 gene, 180–183

531 BLIMP-1, 186–187 CARD11, 184 childhood and adolescent, 178–179 CHOP therapy, 179 gene expression profiles, 179–180 molecular targeting, 184 NF-kB pathway, 184–186 PMBL, 187–188 epratuzumab, 11 LLs (See Lymphoblastic lymphoma) Non-pilocytic low-grade astrocytomas, 232 Nonrhabdomyosarcoma soft tissue sarcomas (NRSTS) adjuvant VACA, 500 cellular origin, 502 clinical trials, 512–513 description, 499 DFSP, 508 fundamental groups chromosomal defects, 501–502 development mechanisms, 501 GISTs, 508–510 IMT, 510–511 infantile fibrosarcomas and ETV6-NTRK3 fusion protein, 506–507 leiomyosarcoma, 511–512 MFH, 507 MPNST and NF1 mutation, 504–506 POG, 499–500 synovial sarcoma, 502–503 SYT-SSX gene fusions, 503–504 NOTCH1 inhibition gastrointestinal toxicity, 23–24 g-secretase complex, 23 pathway description, 22 identification, 21 mutations, 22–23 resistance FBW7 tumor suppressor, 24 PTEN inactivation, 24–26 Nowicki, M., 409 NRSTS. See Nonrhabdomyosarcoma soft tissue sarcomas O Occhipinti, E., 157 Offit, K., 270 Ohgaki, H., 272 Oki, Y., 111 Onda, M., 469 Osenga, K.L., 356

532 Osteosarcoma description, 459 etiologic factors, 459–460 molecular alterations angiogenesis, 475–476 bone differentiation, 473–475, 485–486 cancer predisposition syndromes, 461–463 cell cycle regulation, 464–466 cell of origin, 460–461 drug resistance, 476–477 genetic complexity, 472 growth factors, 466–468 immortalization, 472–473 metastasis, 475 murine models, 463–464 oncogenes, 471–472 signal transduction, 468–471 SV40, 463 murine models, 463–464 pathogenesis genetic alterations, 479–481 mechanisms, 482 pediatric malignancies, 477–478 PPTP, 486–487 signal transduction, 468–471 SV40, 463 treatment strategies cytotoxic agents, 478 HER2 expression, 484 Hsp90 inhibition, 483–484 IGF-1, 484–485 liposomal doxorubicin, 483 MTP-PE, 482 pamidronate role, 483 radiation therapy, 478, 482 RANK/RANKL/OPG system, 485 rapamycin and VEGF pathway, 484 P Pagano, L., 111 Palomero, T., 24 Parathyroid hormone-related protein (PTHrP), 474–475 Parham, D.M., 500 Pasqualucci, L., 187 PDGF. See Platelet-derived growth factor PDGFR-b. See Platelet-derived growth factor receptor-b Pearson, A.D., 270 Pediatric preclinical testing program (PPTP) ABT-751 and Ixabepilone, 414 novel agents, tested, 486 osteosarcoma incidence, 486–487

Index tested agents, 415 xenografts utilization, 415 Perotti, D., 403 Pgps. See Polycomb group proteins Pieters, R., 3 Pietsch, T., 272 Pinkerton, R., 178 Pinthus, J.H., 414 Piva, R., 194 Platelet-derived growth factor (PDGF) and fibroblast growth factor (FGF), 234 isoforms, 236 neutralizing antibody, 236 overexpression, 387 PDGF-C and PDGF-D families, 386–387 potent mitogen, 467 Platelet-derived growth factor receptor (PDGFR) inhibition and inhibitors, 237 kinase activity, 247 PDGF isoforms, 236–237 PDGFRb signaling, 280 pediatric glial neoplasia, 231 signaling, 237–238 Platelet-derived growth factor receptor-b (PDGFR)-b, 504 Plexiform neurofibromas (PN), 333–334 PMBL. See Primary mediastinal B-cell lymphomas Pollack, I.F., 231 Polo, J.M., 183 Polycomb group proteins (Pgps), 503 Pomalidomid, 167 Pomeroy, S.L., 267 Post-transplant lymphoproliferative disorders (PTLD) classification, 215–216 EBV anti-viral medications, 221–222 apoptotic resistance, 220–221 cytotoxic T cells (CTLs), 216 host immune response, 218–220 human, 217–218 immunosuppression, 222 monoclonal antibodies, 222–223 T cell adoptive immunotherapy, 223 treatment, 217 vaccines, 221 occurance, 215 signs and symptoms, 216 PPTP. See Pediatric preclinical testing program Primary mediastinal B-cell lymphomas (PMBL) DLBCL and, 186–188, 205 gene expression, 180–181 prognosis, children and adolescents, 180

Index Pritchard-Jones, K., 403 PTEN Akt activation, 243 signaling, 247 high-grade gliomas, 234 inactivation, NOTCH1 inhibition, 24–26 preservation, 240 T-ALL, 26 transcriptional repression, 23 PTHrP. See Parathyroid hormone-related protein PTLD. See Post-transplant lymphoproliferative disorders R Raetz, E.A., 200 Raffel, C., 272 Rahman, N., 403 Ramburan, A., 403 Ranked list overlap analysis, RMS, 427–428 Rao, A., 110 Rao, G., 273 Rapamycin analogs, 484 medulloblastoma, 279 mTOR pathway, 204 VEGF, 444–445 Ravenel, J., 403 Ravindranath, Y., 109–111 Real-time quantitative RT-PCR (RQ-PCR) assays, APL, 95–96 Reardon, D.A., 238 Reifenberger, J., 272 Reinhardt, D., 111 Retinoids, neuroblastoma, 360–361 Reynolds, C.P., 356 Rhabdoid tumors (RTs) aggressiveness, 323 cell of origin cellular differentiation pathways, 315 INI1/hSNF5 reintroduction, 314 mesenchymal, 315 clinical trials molecular mechanism, 309–310 oxaliplatin, 309 peripheral blood stem cell rescue, 308 cyclin/cdk axis G0–G1 arrest and apoptosis, 318 phosphorylation, 318–319 cyclin D1 mouse model, 318 over-expression, 317–318

533 development, targeted therapy gene expression profile analysis, 321 interferon signaling pathway, 322–323 mitotic spindle checkpoint, 322 diagnosis cells, 306–307 description, 306 molecular genetics, 307 drug action, 323–324 flavopiridol, 321 4HPR apoptosis, 320 description, 319–320 ifosfamide, 308 INI1 loss characterization, 312–313 G0/G1 arrest, induction, 313 interferon signaling, 314 mitotic spindle checkpoint activation, 313 pathology, 306 radiotherapy, 307–308 suppression mechanisms, INI1-mediated core subunits, 311 hypothesis, 316–317 SWI/SNF complex, 310 transcription regulation, 311–312 surgical intervention, 307 Rhabdomyosarcoma (RMS) angiogenesis mTOR signaling, 445 neovascularization, solid tumors, 443 rapamycin, 444–445 VEGF stimulates proliferation, 443–444 ARMS, 425 cell of origin AML, 426 PAX7 and PAX3, 427 chimeric transcription factors and oncogenes MYCN dysregulation, 432–434 PAX3-FKHR, 428–432 TP53 dysregulation, 434–435 description, 425 ERMS, 425–426 genome expression profiling ERMS and ARMS, 427 PAX-FKHR expression, ARMS, 428, 429 ranked list overlap analysis, 427–428 growth factor signaling hedgehog, 441 HGF, 439–441 IGF-1R, 435–439 RAS/MAPK/c-KIT, 441–442 molecular characteristics, 426

534 Rhabdomyosarcoma (RMS) (cont.) myogenic differentiation cyclin D-CDK4 activity, 443 E protein heterodimerization blockage, 442–443 p21Cip1 reexpression, 442 pharmacologic inhibition, CDK1, 446 Ricafort, R., 459 Rivera, M., 403 RMS. See Rhabdomyosarcoma Rosenwald, A., 179, 180 Rossi, M.R., 272 Roy, D., 403 RTs. See Rhabdoid tumors Ruiz-Argüelles, A., 111 Ruud, E., 270 S Safford, S.D., 403 Sato, K., 236 Savasan, S., 117 Saylors, R.L., 272 Schulz, S., 403 Scotlandi, K., 470 g-Secretase inhibitors (GSIs) NOTCH1 activation, 23 T-ALL, 9–10 Shah, N.P., 356 SHH. See Sonic hedgehog Shipp, M.A., 180 Siapati, K.E., 356 Signal transduction pathways, osteosarcoma c-MET overexpression, 466–467 ERBB family genes clinicopathological studies, 469–470 ERBB4, 468 ERBB2 proto-oncogene, 467 IGF-IR, 466 PDGF, 467 PI3K/AKT and mTOR, 471 RAS/RAF/MAPK, 468, 471 Simian virus 40 (SV40), 463 Singh, K.P., 403 Skapek, S.X., 499 Slack, C., 356 Small, D., 59 Small hairpin RNAs (shRNAs), 183 Smith, M.E., 305 Sohara, Y., 356 Solid organ transplantation (SOT), 215 Sonic hedgehog (SHH) medulloblastoma cyclopamine and HhAntag, 274–275

Index pathway, 273 signaling, 273–274 stem cell determination and renewal, 358 Sprycel™, 147–148 Stark-Vance, V., 245 Stem cell transplantation (SCT) imatinib post transplant, 5 myelodysplastic syndromes (See Myelodysplastic syndromes) veno-occlusive disease (VOD), 72 Strizzi, L., 511 Sundberg, T.B., 190 SV40. See Simian virus 40 Synovial sarcoma description, 502–503 and SYT-SSX gene fusions, 503–504 SYT-SSX gene fusions IGF-2 induction, 504 and synovial sarcoma, 503–504 SYT-SSX1 vs. SYT-SSX2, 503 T Takahashi, Y., 434 T-ALL. See T cell acute lymphoblastic leukemia Tallman, M.S., 111 Taub, J.W., 109 Taylor, M.D., 272, 295 T cell acute lymphoblastic leukemia (T-ALL) gene expression, 199–202 mutations, oncogenes, 203–204 NOTCH1 inhibition, 23–24 pathway, 21–23 resistance, 24–26 oncogenic transcription factors chromosomal translocations, 20 protein–protein interactions, 21 PTEN, PI3K-AKT, and mTOR, 26 RAS, 27 surface antigen expression, 19–20 TCR gene rearrangements, 19 tyrosine kinase genes, 26–27 Teitz, T., 356 Tentori, L., 250 Testi, A.M., 83 Tf-CRM107, 253 Thiopurine methyltransferase (TPMT) drug, 160 Thomas, D.A., 5, 11 Thomas, D.G., 470 Thompson, M.C., 272 Tipifarnib time to progression (TTP), 342 Tischkowitz, M.D., 270

Index Tisigna™, 147–148 TKIs. See Tyrosine kinase inhibitors TNF-related apoptosis inducing ligand (TRAIL) receptors, 385 WT, 410 Tomescu, O., 430 Tong, W.M., 273 Toretsky, J.A., 373 TP-38, 255 Tumor suppressor P53 (TP53) dysregulation HDM2-p53 interactions, 434–435 nutlin 3a, 435 TAp73 isoforms production, 434 Turcot’s syndrome, 294 Tyrosine kinase inhibitors (TKIs) CML theraphy, 168 dasatinib and nilotinib, 5–6 neuroblastoma, 357–358 U Üren, A., 373 Uziel, T., 273 V Van Waes, C., 185 Vascular endothelial growth factor (VEGF) AML, 65 angiogenesis, 408–409 astrocytomas blockade, 245 receptor inhibition, 244–245 HDM2 gene, 435 PI3K/Akt pathway, 444 VEGF. See Vascular endothelial growth factor Vincristine, actinomycin D, cyclophosphamide and doxoru bicin (VACA), 500 von Recklinghausen disease. See Neurofibromatosis type 1 (NF1) Vora, A., 11 Vujanic, G., 403 W Wachtel, M., 428 Wang, C., 430 Wang, M., 215 Weinberg, R.A., 477 Wesenberg, F., 270 Wetmore, C., 273 Widemann, B.C., 331, 340 Wilms’ tumor (WT) description, 401

535 molecular pathways apoptosis activators, 409–410 cell cycle, 410 c-KIT, 411 demethylating agents, 411–412 EGFR, 408 HGF/c-MET, 410–411 HSP 27 and 70, 411 IGF signaling, 404–405 MRP, 412 putative genetic loci/genes involvement, 402–403 VGEF role, 408–409 WNT-b-catenin, 406–408 preclinical identification, novel agents anti-EGFR and angiogenesis testing, 414 PPTP, 414–415 inÂ€vitro investigation, 412–413 inÂ€vivo xenograft testing, 413 treatment strategies adjuvant and neoadjuvant chemotherapy, 401 SIOP-2001 and AREN03B2 protocol, 402 WNT-b-catenin, WT cDNA microarray studies, mutated vs. nonmutated WT, 406–407 E-cadherin, 406 inhibitors, 407–408 networking with FGF, Notch, BMP and Hedgehog, 408 WNT-4, 406 Wnt/beta-catenin pathway, medulloblastoma activation and mutations, 275 ZTM000990 and PKF118-310, 275–276 Wong, K.K., 232 WT. See Wilms’ tumor X Xenograft testing, inÂ€vivo, 413 Y Yan, C.T., 273 Yang, Q., 356 Yee, D., 356 Yokoi, A., 414 Yokota, N., 272 Yu, A., 356

536 Z Zeller, B., 110 Zhang, L., 430 Zhan, S., 437 Zhao, J., 403

Index Zhou, H., 356, 469 Zhou, L., 164 Zipursky, A., 110, 118 Zurawel, R.H., 272