Targeted Therapies in Oncology

Targeted Therapies in Oncology

Targeted Therapies in Oncology

Edited by

Giuseppe Giaccone

Free University Medical Center Amsterdam, The Netherlands

Jean-Charles Soria

Paris University XI, Institut Gustave Roussy Villejuif, France

Informa Healthcare USA, Inc. 52 Vanderbilt Avenue New York, NY 10017 ª 2007 by Informa Healthcare USA, Inc. Informa Healthcare is an Informa business No claim to original U.S. Government works Printed in the United States of America on acid-free paper 10 9 8 7 6 5 4 3 2 1 International Standard Book Number-10: 0-8493-9371-X (Hardcover) International Standard Book Number-13: 978-0-8493-9371-6 (Hardcover) This book contains information obtained from authentic and highly regarded sources. Reprinted material is quoted with permission, and sources are indicated. A wide variety of references are listed. Reasonable efforts have been made to publish reliable data and information, but the author and the publisher cannot assume responsibility for the validity of all materials or for the consequences of their use. No part of this book may be reprinted, reproduced, transmitted, or utilized in any form by any electronic, mechanical, or other means, now known or hereafter invented, including photocopying, microfilming, and recording, or in any information storage or retrieval system, without written permission from the publishers. For permission to photocopy or use material electronically from this work, please access www. copyright.com (http://www.copyright.com/) or contact the Copyright Clearance Center, Inc. (CCC) 222 Rosewood Drive, Danvers, MA 01923, 978-750-8400. CCC is a not-for-profit organization that provides licenses and registration for a variety of users. For organizations that have been granted a photocopy license by the CCC, a separate system of payment has been arranged. Trademark Notice: Product or corporate names may be trademarks or registered trademarks, and are used only for identification and explanation without intent to infringe.

Library of Congress Cataloging-in-Publication Data Targeted therapies in oncology / edited by Giuseppe Giaccone, Jean-Charles Soria. p. ; cm. Includes bibliographical references. ISBN-13: 978-0-8493-9371-6 (hardcover : alk. paper) ISBN-10: 0-8493-9371-X (hardcover : alk. paper) 1. Cancer–Treatment. I. Giaccone, Giuseppe. II. Soria, Jean-Charles. [DNLM: 1. Neoplasms–therapy. 2. Gene Therapy. 3. Immunotherapy. QZ 266 T1847 2007] RC270.8.T37 2007 616.990 406–dc22 Visit the Informa Web site at www.informa.com and the Informa Healthcare Web site at www.informahealthcare.com

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Preface

Rapid advances in tumor biology have led to the identification of the molecular circuitry that governs cancer cell proliferation. The better understanding of the key pathways that control tumor progression has enabled the pharmaceutical industry and academia to develop new anti-cancer agents targeting specific molecular events involved in the oncogenic process. The term “targeted therapies” refers to treatment strategies directed against molecular targets considered to be involved in the process of neoplastic transformation. This is not a totally new concept in oncology, since hormonal manipulations have long been applied for the treatment of advanced and local disease in breast, prostate, and thyroid cancers. In the past 30 years, alterations characteristic of neoplastic cells have been described, such as specific translocations, activating mutations, or gene amplifications, which have brought real changes to the nosological classification of cancers. The molecular classification of certain cancers has contributed to the development of a new class of drugs that aims at blocking, with various degrees of specificity, the activity of proteins involved in neoplastic cell development and progression. This book provides a concise and up-to-date panorama of existing targeted therapies and those being developed into valuable anticancer treatments, with an emphasis on the “clinical achievements” obtained with such agents. The biology behind each target has also been discussed. The large number of chapters included in this book reflects the variety of targeted therapies aiming at blocking a wide array of “hallmarks of cancer.” These notably include: signal-transduction inhibitors, anti-angiogenic and vascular-disrupting agents, telomere-targeting compounds, apoptosis modulators, and targeted agents at transversal mechanisms; however, only targeted agents that have already entered the clinical arena have been included. The introduction of these agents has already had a large impact across different tumor-types and in early- as well as advanced-stage cancer. Giuseppe Giaccone Jean-Charles Soria

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Contents

Preface iii Contributors

vii

1. Overview of Existing Therapies

1

Giannis Mountzios and Jean-Charles Soria 2. Signal Transduction Inhibitors, HER Family, EGFR Inhibition, and Clinical Achievements 19

Giuseppe Giaccone and Paolo Zucali 3. HER2 Inhibition and Clinical Achievements

45

Toru Mukohara and Pasi A. Jänne 4. Pan-HER Inhibitors 55

Gérard Milano 5. Inhibiting the Phosphoinositide 3-Kinase/Akt/Mammalian Target of Rapamycin Pathway 65

Janet E. Dancey 6. Farnesyl Transferase Inhibitors in Cancer: Promise, but Limited Proof

85

Laura Fanucchi, Michael P. Fanucchi, and Fadlo R. Khuri 7. Protein Kinase C Inhibitors in the Treatment of Non–Small Cell Lung Cancer

103

Yun Oh, Michael Lahn, Asavari Wagle, and Roy Herbst 8. Signal Transduction Inhibitors: PDGFR and c-KIT Inhibitors

123

Jean-Yves Blay, Jérome Fayette, Laurent Alberti, Severine Tabone-Eglinger, Hiba El Sayadi, Philippe Cassie, Armelle Dufresne, Dominique Ranchère, and Isabelle Ray-Coquard 9. The Insulin-Like Growth Factor 1 Receptor: A Target for Cancer Treatment

Yungan Tao, Jean Bourhis, and Eric Deutsch 10. Aurora Kinase Inhibitors

157

Mitesh J. Borad, Steven L. Warner, and Daniel D. Von Hoff 11. Apoptosis Modulators: p53 Targeting 177

Sunil Chada, Dora Bocangel, Kerstin Menander, and Jack A. Roth 12. Survivin

197

Dario C. Altieri 13. TRAIL Modulators

207

C. H. Mom, I. A. Sloots, S. de Jong, J. A. Gietema, E. G. E. de Vries, and S. Sleijfer 14. VEGF Targeting 223

Lee M. Ellis v

141

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Contents

15. Targeting Angiogenesis with Oral Agents

241

Benjamin Besse and Jean-Pierre Armand 16. Endothelial Cell Propagation Targeting 257

Gordon C. Tucker 17. HIF-1 Inhibitors

283

Giovanni Melillo 18. Antivascular Agents

295

Jane Robertson 19. Matrix Metalloproteinases

315

Stéphane Vignot and Jean-Philippe Spano 20. Src Inhibitors

333

Francisco Cruzalegui 21. Telomerase and Telomere Interacting Agents

349

Jean-François Riou, Anne de Cian, Lionel Guittat, Dennis Gomez, Céline Douarre, Laurent Lacroix, Chantal Trentesaux, and Jean-Louis Mergny 22. Targeting Hsp90: The Cancer Super-Chaperone

Paul Workman and Swee Sharp Index 401

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Contributors

Laurent Alberti Lyon, France

Department of Médecine, Centre Leon Berard, Laennec,

Dario C. Altieri Department of Cancer Biology and the Cancer Center, University of Massachusetts Medical School, Worcester, Massachusetts, U.S.A. Jean-Pierre Armand Department of Medicine, Institut Gustave Roussy, Villejuif, France, and Department of Medicine, Institut Cladius Regaud, Toulouse, France Benjamin Besse Department of Médecine, Paris University XI, Institut Gustave Roussy, Villejuif, France Jean-Yves Blay Department of Médecine, Centre Leon Berard, Laennec, Lyon, France, and Unité de Jour d'Oncologie Médicale Multidisciplinaire Hôpital Edouard Herriot, Place d'Arsonval, Lyon, France Dora Bocangel Departments of Clinical Research and Development, Introgen Therapeutics, Inc., Houston, Texas, U.S.A. Mitesh J. Borad Clinical Translational Research Division, Translational Genomics Research Institute, Phoenix, Arizona, U.S.A. Jean Bourhis Department of Radiation Oncology, Institute Gustave Roussy, Villejuif, France Philippe Cassie Department of Médecine, Centre Leon Berard, Laennec, Lyon, France, and Unité de Jour d'Oncologie Médicale Multidisciplinaire Hôpital Edouard Herriot, Place d'Arsonval, Lyon, France Sunil Chada Departments of Clinical Research and Development, Introgen Therapeutics, Inc., Houston, Texas, U.S.A. Francisco Cruzalegui Division of Cancer Research and Drug Discovery, Institut de Recherches Servier, Croissy-sur-Seine, France Janet E. Dancey Investigational Drug Branch, Cancer Therapy Evaluation Program, Division of Cancer Treatment and Diagnosis, National Cancer Institute, Rockville, Maryland, U.S.A. Anne de Cian Laboratoire de Biophysique, Muséum National d’Histoire Naturelle, Paris, France S. de Jong Department of Medical Oncology, University Medical Center Groningen, Groningen, The Netherlands Eric Deutsch Department of Radiation Oncology, Institute Gustave Roussy, Villejuif, France E. G. E. de Vries Department of Medical Oncology, University Medical Center Groningen, Groningen, The Netherlands Céline Douarre Laboratoire d’Onco-Pharmacologie, UFR de Pharmacie, Université de Reims Champagne Ardenne, Reims, France

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Contributors

Armelle Dufresne Department of Médecine, Centre Leon Berard, Laennec, Lyon, France, and Unité de Jour d'Oncologie Médicale Multidisciplinaire Hôpital Edouard Herriot, Place d'Arsonval, Lyon, France Hiba El Sayadi

Department of Médecine, Centre Leon Berard, Laennec, Lyon, France

Lee M. Ellis Departments of Surgical Oncology and Cancer Biology, M. D. Anderson Cancer Center, University of Texas, Houston, Texas, U.S.A. Laura Fanucchi Department of Hematology and Medical Oncology, Winship Cancer Institute, Emory University, Atlanta, Georgia, U.S.A. Michael P. Fanucchi Department of Medical Oncology, St. Vincent's Comprehensive Cancer Center, New York Medical College, New York, New York, U.S.A. Jérome Fayette Department of Médecine, Centre Leon Berard, Laennec, Lyon, France, and Unité de Jour d'Oncologie Médicale Multidisciplinaire Hôpital Edouard Herriot, Place d'Arsonval, Lyon, France Giuseppe Giaccone Department of Medical Oncology, Vrije Universiteit Medical Center, Amsterdam, The Netherlands J. A. Gietema Department of Medical Oncology, University Medical Center Groningen, Groningen, The Netherlands Dennis Gomez Laboratoire d’Onco-Pharmacologie, UFR de Pharmacie, Université de Reims Champagne Ardenne, Reims, France Lionel Guittat Paris, France

Laboratoire de Biophysique, Muséum National d’Histoire Naturelle,

Roy Herbst Department of Thoracic/Head and Neck, Medical Oncology, M. D. Anderson Cancer Center, University of Texas, Houston, Texas, U.S.A. Pasi A. Jänne Lowe Center for Thoracic Oncology, Department of Medical Oncology, Dana Farber Cancer Institute, Boston, Massachusetts, U.S.A. Fadlo R. Khuri Department of Hematology and Medical Oncology, Winship Cancer Institute, Emory University, Atlanta, Georgia, U.S.A. Laurent Lacroix Paris, France

Laboratoire de Biophysique, Muséum National d’Histoire Naturelle,

Michael Lahn Oncology Product Development, Eli Lilly and Company, Indianapolis, Indiana, U.S.A. Giovanni Melillo Developmental Therapeutics Program, SAIC Frederick, Inc., National Cancer Institute at Frederick, Frederick, Maryland, U.S.A. Kerstin Menander Departments of Clinical Research and Development, Introgen Therapeutics, Inc., Houston, Texas, U.S.A. Jean-Louis Mergny Laboratoire de Biophysique, Muséum National d’Histoire Naturelle, Paris, France Gérard Milano Laboratoire d'Oncopharmacologie, du Centre Antoine Lacassagne, Nice, France C. H. Mom Department of Medical Oncology, University Medical Center Groningen, Groningen, The Netherlands Giannis Mountzios Department of Médecine, Institut Gustave Roussy, Villejuif, France

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Contributors

Toru Mukohara Division of Hematology and Oncology, Research Center for Innovative Oncology, National Cancer Center Hospital East, Kashiwa, Japan Yun Oh Department of Thoracic/Head and Neck, Medical Oncology, M. D. Anderson Cancer Center, University of Texas, Houston, Texas, U.S.A. Dominique Ranchère Department of Médecine, Centre Leon Berard, Laennec, Lyon, France Isabelle Ray-Coquard Lyon, France

Department of Médecine, Centre Leon Berard, Laennec,

Jean-François Riou Laboratoire d’Onco-Pharmacologie, UFR de Pharmacie, Université de Reims Champagne Ardenne, Reims, France Jane Robertson Global Oncology Research and Development, AstraZeneca Pharmaceuticals, Aderley Park, Macclesfield, U.K. Jack A. Roth Thoracic and Cardiovascular Surgery, University of Texas, M. D. Anderson Cancer Center, Houston, Texas, U.S.A. Swee Sharp Cancer Research U.K. Centre for Cancer Therapeutics, The Institute of Cancer Research, Haddow Laboratories, Sutton, Surrey, U.K. S. Sleijfer Department of Medical Oncology, Erasmus University Medical Center, Rotterdam, The Netherlands I. A. Sloots Department of Medical Oncology, University Medical Center Groningen, Groningen, The Netherlands Jean-Charles Soria Department of Médecine, Paris University XI, Institut Gustave Roussy, Villejuif, France Jean-Philippe Spano Service d’Oncologie Médicale, Groupe Hospitalier Pitié Salpétrière, Paris, France Severine Tabone-Eglinger Lyon, France

Department of Médecine, Centre Leon Berard, Laennec,

Yungan Tao Department of Radiation Oncology, Institute Gustave Roussy, Villejuif, France Chantal Trentesaux Laboratoire d’Onco-Pharmacologie, UFR de Pharmacie, Université de Reims Champagne Ardenne, Reims, France Gordon C. Tucker Cancer Drug Discovery Department, Institut de Recherches Servier, Croissy-sur-Seine, France Stéphane Vignot Service d’Oncologie Médicale, Groupe Hospitalier Diaconesses Croix Saint Simon, Paris, France Daniel D. Von Hoff Clinical Translational Research Division, Translational Genomics Research Institute, Phoenix, Arizona, U.S.A. Asavari Wagle Oncology Product Development, Eli Lilly and Company, Indianapolis, Indiana, U.S.A. Steven L. Warner Clinical Translational Research Division, Translational Genomics Research Institute, Phoenix, Arizona, U.S.A. Paul Workman Cancer Research U.K. Centre for Cancer Therapeutics, The Institute of Cancer Research, Haddow Laboratories, Sutton, Surrey, U.K. Paolo Zucali Department of Medical Oncology and Hematology, Instituto Clinico Humanitas, Milan, Italy

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Overview of Existing Therapies Giannis Mountzios Department of Médecine, Institut Gustave Roussy, Villejuif, France

Jean-Charles Soria Department of Médecine, Paris University XI, Institut Gustave Roussy, Villejuif, France

INTRODUCTION Definitions Systemic cancer treatment, which followed a continuous progression all along the second half of the twentieth century, seems to be entering a new and exciting era, characterized by a more “sophisticated” selection of targets for cancer therapy, leading to the development of more “intelligent” drugs directed against these specific targets. By definition, molecular targeted therapy refers to every specific treatment strategy directed against well-defined molecular targets considered to be involved in the process of neoplastic transformation. Consequently, every pharmaceutical molecule with specific and unique properties against (or for) a welldefined molecular target implicated in the process of carcinogenesis may be considered as a molecular targeted agent (MTA). The above-mentioned definition does not include within MTA the “classic” cytotoxic agents (alkylating agents, antimetabolites, anticancer antibiotics, vinca alkaloids) or the more recently discovered ones (topo-isomerase inhibitors and taxanes), despite the fact that such agents are also directed against a specific target (e.g., thymidilate synthase or tubulines). Indeed such targets, although specific, do not characterize the process of tumor progression and transformation but participate in physiological process such as DNA synthesis and mitosis. To be more accurate, one could suggest that modern advances in molecular biology have helped to identify specific molecular abnormalities that characterize a certain type of cancer in a unique and repeated manner, allowing thus the development of specialized molecules that can selectively target the specific cancer-cell population and minimize the hazardous effects to normal cells. From “Classic” Chemotherapy to Molecular Targeted Therapy The idea of “attacking” a specific molecular target by interfering with the corresponding pathogenetic mechanism is not a new concept in oncology. Hormonal manipulation for the treatment of local and advanced disease in breast, prostate, and thyroid cancer have long been studied for potential benefits, taking advantage of their unique hormone-depended biological behavior. During the decade 1980–1990, the National Cancer Institute (NCI) established a new research approach, making the hypothesis that developing a solid tumor model in vitro could allow the discovery of potential new therapeutic targets. The choice of an in vitro model, compared to an in vivo model (mouse), was made for reasons of cost, simplicity, rapidity, efficacy, and automatization. The new model was applied in a panel of 60 human cell lines of various histological origin in an 1

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effort to establish a new, disease orientated, therapeutic strategy (1). Although the distinct biological characteristics of cancer cells in vitro were not always applicable in vivo, limiting thus the exploitation of the acquired knowledge in the clinical setting, the study of the biological behavior of cell lines has allowed the discovery of new pathogenetic mechanisms of oncogenesis and the elucidation of the various molecular pathways of signaling cascades that trigger neoplastic transformation. More recently, the sequencing of the human genome has allowed the full identification of the protein kinase complement, or “kinome,” including more than 520 proteins with tyrosine kinase domains, and leading to the generation of MTAs against most tyrosine kinases relevant to oncogenesis (2). When the tyrosine kinase inhibitor imatinib (Gleevec
; Novartis Pharmaceuticals, East Hannover, New Jersey, U.S.A.) became the first MTA proved to possess profound antitumor activity against chronic myeloid leukemia (3) and obtained the U.S. Food and Drug Administration (FDA) approval in 2001 for this disease, this was the first result of this long but fruitful procedure, followed by numerous other molecules targeting a wide variety of human tumors. Classification In a seminal article in 2000, Weinberg and Hanahan described the key-alterations of cancer cells (4); this first description of the six hallmarks of oncogenesis can be used as a framework to classify molecularly targeted agents (Fig. 1). Consequently, one could attempt a first classification according to the molecular mechanism modified by the agent: & & & & & &

Self-sufficiency in growth signals ! Cell-cycle inhibitors and signal transduction modulators Insensitivity to antigrowth signals ! Cell-cycle inhibitors and signal transduction modulators Sustained angiogenesis ! Antiangiogenic and antivascular agents Tissue invasion and metastasis ! Anti-invasive agents Evading apoptosis ! Apoptosis modulators Limitless replicative potential ! Antitelomerase and telomere-interacting agents

Alteration of DNA repairing capacity, as a recognised factor provoking genomic instability, as well as DNA epigenetic modulation, should probably also be added to the above mentioned mechanisms. Moreover, the elucidation of many molecular pathways involved in neoplastic transformation has resulted in numerous new targeted agents affecting diverse and sometimes overlapping mechanisms (heat shock proteins, COX2 interactions, DNA methylation, proteasome function, etc.). OVERVIEW OF THE EXISTING THERAPIES Signal Transduction Inhibitors Background Signal transduction describes the general process by which cells perceive changes in their environment. The more important steps for cellular signal transduction are the following: & & &

Recognition/binding of signal molecules to the cell surface Internalization of the signal Transmission of the signal through the cytoplasm

Overview of Existing Therapies

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FIGURE 1 The six fundamental mechanisms of carcinogenesis and their corresponding pharmaceutical agents according to their pathogenetic mechanisms. Source: From Ref. 4. & &

Entrance of the signal into the nucleus Signal-dependent modulation of gene activation

Schematically, cell growth signaling can be separated in three distinct parts that are closely connected (Fig. 2). First come the upstream growth factors and their receptors at the cell membrane. Engagement of the receptor by the ligand, which is usually a growth factor, usually triggers the activation of the intracellular domain of the receptor that possesses protein kinase properties. Protein kinases are enzymes that covalently attach phosphates to the side chain of either serine, threonine, or tyrosine residues of specific proteins inside cells. From there, molecular mediators of signal transduction and cytoplasmic messengers form a relay cascade through a “cross talk” involving activation and/or inactivation of intermediate downstream effectors, informing thus the nucleus of the received

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FIGURE 2 Important steps of the cell signaling process (left) and corresponding levels of potential pharmaceutical intervention (right).

stimuli. Activation or inactivation of the intermediate messengers is usually mediated by subsequent phosphorylations or de-phosphorylations of these molecules by specific enzymes, called kinases and phosphatases, respectively. Finally, an effector pathway leads the cell to the eventual division and proliferation. This signal transduction system, comprising growth factors, transmembrane receptor proteins, and cytoplasmic secondary messengers, is often exploited to optimize tumor growth and metastasis in malignancies. Indeed, in cancer cells, key components of these pathways may be altered by oncogenes through overexpression or mutation, leading to dysregulated cell signaling, inhibition of apoptosis, metastasis and cell proliferation (5). The components of these abnormal signaling pathways represent potential selective targets for new anticancer therapies. These potential targets, the majority of which are protein kinases, can be divided into subcategories, according to the level of interaction on the above described process, as follows: ligands (typically growth factors), cellular receptors, intracellular second messengers, and nuclear transcription factors. Agents targeting cellular receptors can be further categorized according to the mechanism of

Overview of Existing Therapies

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interference into monoclonal antibodies and tyrosine kinase inhibitors of the intracellular domain of the receptor (Fig. 2). Categories Inhibition of the Receptor Function Conceptually, there are three potential approaches to “attack” a membrane receptor (Fig. 2): & & &

Neutralization of the ligand Competitive inhibition of ligand–receptor engagement Inhibition of transduction of the signal from the receptor to secondary cytoplasmic messengers

The first, obvious target for intervention in a signaling cascade is the neutralization of ligands before they can associate with their receptors; this approach has been successfully validated with bevacizumab (Avastin
; Genentech Corp., San Francisco, California, U.S.A.), a humanized monoclonal antibody targeting circulating vascular endothelial growth factor (VEGF) (6). As a second option, direct inhibition of ligand–receptor engagement can be achieved by preventing the binding of the growth factors to their receptors by mimicking the ligand’s structure and interfering thus in the ligand–receptor affinity. The successful example of cetuximab (Erbitux
; ImClone systems, Branchburg, New Jersey, U.S.A.), a chimeric antibody against the epidermal growth factor receptor (EGFR), is a proof of this concept (7). Trastuzumab (Herceptin
; Genentech Corp.) activity against the HER2 receptor is also a clear, successful approach (8). Finally, inhibition of downstream signal transduction by interfering with the kinase activity of the receptors with small-molecule inhibitors (currently registered), like erlotinib (Tarceva
; Genentech Corp.) and geftinib (Iressa
; AstraZeneca Pharmaceuticals, Wilmington, Delaware, U.S.A.) is another effective therapeutic strategy (9). The list of HER-interacting TKI is extremely large (10), with very promising panHER inhibitors, such as lapatinib (Tykerb
; GlaxoSmithKlein Pharmaceuticals, Research Triangle Park, North Carolina, U.S.A.) and HKI-272 (Wyeth Pharmaceuticals, Madison, New Jersey, U.S.A.). Apart from EGFR, various cellular receptors can serve as attractive therapeutic targets, including c-KIT receptor, insulin-growth factor receptor (IGFR), platelet-derived growth factor receptor (PDGFR), and fibroblast growth factor receptor (FGFR). For example, imatinib mesylate has been shown to selectively inhibit a number of protein kinases, including c-KIT and PDGFR. Inhibition of Cytoplasmic Signal Transduction The Ras-Raf/Mitogen-Activated Protein Kinase Pathway. The Ras superfamily of genes encodes small GTP-binding proteins that are responsible for regulation of many cellular processes, including differentiation, cytoskeletal organization, and protein trafficking (Fig. 3) (11). At least 20 members of the Ras protein family are known in mammalian cells and share 30% sequence identity. Ras proteins localize to the inner surface of the cell membrane and act as on/off (Ras-GTP/Ras-GDP) switches controlled by cell surface receptors and transduce signals to the cell nucleus (5). Signaling via the Ras pathway has been elucidated in the last few years. Activated Ras activates Raf, which is a serine-threonine kinase. Raf activates mitogen-activated protein kinase-kinase (MAPKK), also called MEK, which in turn activates mitogen-activated protein kinase (MAPK) or extracellular regulated kinase (ERK). Finally, MAPK activation results in phosphorylation and activation

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FIGURE 3 Simplified illustration of some of the most important signal transduction pathways involved in ongenesis.

of transcriptional factors, such as c-jun, c-myc, and c-fos, provoking cell proliferation (Fig. 3). Oncogenic Ras mutations have been identified in non–small cell, lung, colorectal, pancreatic, bladder, kidney, and thyroid carcinomas (12). Raf mutations have been described in melanoma (13). RAS AND RAF KINASE INHIBITORS. The current approaches for inhibition of Ras include: (a) the inhibition of Ras protein expression through antisense oligonucleotides; (b) the prevention of membrane localization of Ras (farnesyl transferases);

Overview of Existing Therapies

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(c) engineering viruses to kill Ras-transformed cells; and (d) the inhibition of Ras function through inhibition of downstream Ras effectors. Inhibition of the Raf family of serine/threonine kinases, which consists of A-Raf, B-Raf, and C-Raf, is an effective way of proliferative signaling inhibition. Sorafenib (Nexavar
; Bayer Corp., Leverkuzen, Germany) is an orally bioavailable bisarylurea derivative developed initially as an inhibitor of mutant B-Raf, which was found later to inhibit the protein kinase domain of numerous receptors, including VEGFR, PDGFR, KIT, fetal liver (or Fms-like) tyrosine kinase 3 (FLT3), and P38a (14). The clinical use of sorafenib has so far resulted in tumor shrinkage and disease stabilization in renal cell, hepatocellular, colorectal, ovarian, and breast cancers (15). FARNESYL TRANSFERASE INHIBITORS. The enzyme Farnesyltransferase is involved in the posttranslational modification of Ras proteins by linking covalently a farnesylgroup onto Ras. On the basis of the fact that the biochemical procedure of farnesylation is critical for Ras maturation and function, farnesyltransferase inhibitors (FTIs) were originally envisioned as specific and sensitive inhibitors of Ras-mediated neoplastic transformation (16). However, it has become evident in the recent years that FTIs, apart from clear inhibition of Ras farnesylation, exhibit important activity against other polypeptides in addition to Ras that possess the ability to be farnesylated. Three FTIs, tipifarnib, lonafarnib, and BMS-214662, have been extensively tested in the clinic. Tipifarnib seems to exert significant clinical activity, especially in hematological malignancies (17). MEK INHIBITORS. There are two MEK homologues, MEK1 and MEK2, that are ubiquitously expressed in mammals and sequentially phosphorylate ERK1 and ERK2. CI-1040 was the first MEK-targeted agent to enter clinical trials, being a highly potent and selective inhibitor of both MEK isoforms (5). Other inhibitors of MEK kinase [PD0325901 (Pfizer Pharmaceuticals, New York, New York, U.S.A.), AZD6244 (AstraZeneca), MKI-833 (Wyeth)] are currently being evaluated either in preclinical or phase I/II settings.

The PI3K–Akt–Mammalian Target of Rapamycin Pathway. The mammalian target of rapamycin (m-TOR) (Fig. 3) is a serine/threonine kinase that belongs to the family of phosphatidylo-3-inositol kinase-like kinases (PI3K) involved in the regulation of a wide range of growth-related cellular functions including transcription, translation, membrane trafficking, protein degradation, and reorganization of the actin cytoskeleton (18). The best-known function of m-TOR, in the context of cell proliferation, is the regulation of translation initiation, presumably mediated by the activation of the 40S ribosomal protein S6 kinase. m-TOR activity seems to be regulated by PI3K/protein kinase B (PKB or Akt). The PI3K-Akt complex is activated as a result of the ligand-dependent activation of tyrosine kinase receptors, G-protein-coupled receptors, or integrins. On the other hand, phosphatase and tensin homolog (PTEN) is a dephosphorylating agent acting as a negative regulator of PI3K signaling. This phosphorylation stimulates the catalytic activity of Akt, resulting in the phosphorylation of numerous other proteins that affect cell growth, cell-cycle entry, and cell survival (5). PI3K AND AKT INHIBITORS. The main inhibitors of the PI3K-Akt pathway that have entered clinical trials are illustrated in Table 1.

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TABLE 1 Summary of PI3K-AKT Inhibitors Having Entered Clinical Trials Agent UCN-01 PERIFOSINE 17-AAG

Class

Target

Stage of development

Staurosporine Alkyl lipid Geldanamycin

PDK1 AKT HSP90, PDK, AKT

Phase II Phase III Phase III

RAPAMYCIN AND m-TOR INHIBITORS. Rapamycin was the first agent historically described to interact with m-TOR and inhibit the initiation of the translational process leading to slowing or arrest of cells in the G1 phase of the cell cycle. Since then, a number of compounds structurally related to rapamycin have been clinically developed, including tirosel/temsirolimus (Wyeth), RAD001 (Novartis), and AP23573 (Ariad Pharmaceuticals, Inc., Cambridge, Massachusetts, U.S.A.). A summary of the existing clinical trials assessing clinical activity of the various mTOR inhibitors is presented in Table 2. PROTEIN-KINASE C (PKC) INHIBITORS. The PKC family contains a number of serine/threonine specific protein kinases that have been shown to play an important role in a variety of cellular events potentially important for cancer development, including cell growth, cell-cycle progression, differentiation, drug efflux, apoptosis, and tumor angiogenesis (19). Two staurosporine analogues, PKC412 [N-benzoyl-staurosporine] (Novartis) and UCN-01 [7-hydroxy-staurosporin, NCI], which compete for binding to the ATP site on PKCs, have been recently evaluated in clinical trials (20). ISIS-3521, an antisense oligodeoxynucleotide that inhibits PKC-a mRNA, failed to demonstrate activity in a large phase III randomized trial in NSCLC. Enzastaurin (LY317615) is a PKC-b inhibitor (and to a lesser extent a PKC-a, -d, and -« inhibitor, too) with promising activity both in glioblastoma and lung cancer.

Signal Transduction and Activation of Transcription Pathway. One of the most recently recognized oncogenic signaling pathways involves the signal transduction and activation of transcription (STAT) proteins. This family of proteins comprises seven members that have dual roles as cytoplasmic signaling proteins and as nuclear transcription factors that activate a diverse set of genes, including some that are implicated in malignant progression (especially the STATs 3 and 5) (21). Activation of STAT signaling by growth factor receptors is effectuated through a mechanism similar to that described above for cytokine receptors except from the following unique properties: (a) the intrinsic receptor kinase may co-operate with nonreceptor tyrosine kinases (JAK- and SRC-family kinases); (b) once activated, TABLE 2 Summary of the Main Reported Clinical Trials Involving m-TOR Inhibitors Agent Tirosel/temsirolimus Tirosel/temsirolimus Tirosel/temsirolimus Tirosel/temsirolimus AP23573 Tirosel/temsirolimus Tirosel/temsirolimus Source: From Ref. 38.

Tumor

n

ORR (%)

Breast MCL Clioma Melanoma Lung Renal cell Renal cell

109 18 41 33 5 106 209

10 44 5 3 20 7 9

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STAT proteins form activated homo- or hetero-dimers that enter the nucleus; and (c) they bind by themselves to the corresponding promoter gene region of DNA, triggering permanent alterations in the genetic program controlling fundamental biological processes (Fig. 3). STAT

INHIBITORS.

The various approaches of inhibiting STAT signaling

comprise: & & &

&

Blocking of the upstream tyrosine kinases that are responsible for their activation [small molecule inhibitors of JAK (AG490), SRC and EGFR]. Targeting STAT3 and STAT5 directly by antisense oligonucleotides, dominantnegative expression vectors, and small interfering RNA molecules (siRNA). Blocking the dimerization and DNA-binding activity of STATs by short peptides (peptidomimetics), which are able to disrupt STAT signaling and induce tumor-cell apoptosis. Targeting knowing physiological regulators of STAT signaling, such as the suppressor of cytokine signaling (SOCS) family of proteins.

c-KIT Inhibitors. Imatinib mesylate is the first approved anticancer drug that may be considered to be a multitargeted tyrosine kinase inhibitor. Its targets include KIT, PDGFR, and BCR-ABL gene product. BCR-ABL has been linked with chronic myeloid leukemia (CML), while KIT and PDGFR have been associated with gastrointestinal stromal tumors (GIST). Imatinib is currently approved for first-line treatment of CML and GIST. While highly effective for these cancers, a limitation of chronic imatinib therapy is the development of resistance to the drug in a relatively large proportion of patients, for the most part due to acquisition of secondary mutations in the RTK driving the particular tumor (22). Cell-Cycle Inhibitors The cell cycle includes a series of precise, well defined, and coordinated events that include, successively, the post-mitotic G1 phase, the DNA synthesis S phase, the G2 phase, and the mitotic M phase, comprising itself a series of morphological and biochemical steps. Most of the “classic” chemotherapeutic agents, including the antimetabolites, the alkylating agents, the topo-isomerase inhibitors, and the tubulin polymerization/depolymerization inhibitors, exert their antitumor effect by interfering and blocking the cell-cycle process on a unique or multiple levels. Cyclin-dependent kinases (CDKs) are responsible for the phosphorylations required for the activation of the proteins in charge of cell-cycle progression. CDK inhibitors (CDKIs) are CDK counterparts that serve as negative regulators of the cell cycle. CELL-CYCLE INHIBITORS. The development of small molecule inhibitors of CDKs has progressed rapidly, with all of them targeting the ATP-binding site of the kinases and competing with ATP for inhibition. Staurosporine, a metabolite from Streptomyces spp., is a natural ATP-competitive inhibitor of CDKs that was initially identified as a potent inhibitor of protein kinase C (23). One other class of compounds that has been extensively studied is the flavones. Flavopiridol and deschloroflavopiridol are naturally occurring alkaloids showing cytotoxic properties against tumor cell lines. Flavopiridol (Sanofi-Aventis Corp., Paris, France) was

10

Mountzios and Soria

the first CDK pan-inhibitor (CDK 1, 2, 4, 6, and 9) to enter clinical trials, showing promising results in combination therapy, and enhancing the activity of paclitaxel in human trials (24). Purine analogs, such as roscovitine (Cyclacel Pharmaceuticals, Berkeley Heights, New Jersey, U.S.A.), have also been developed in phase I/II trials. AURORA KINASE INHIBITORS. The aurora kinase family of serine/threonine kinases comprises three members called A, B, and C. Aurora kinases primarily function in early mitosis as they are required for proper centrosome separation and mitotic spindle assembly. Following their unique properties in mitotic spindle formation, they have been considered an attractive target for molecular therapy. A summary of the most important aurora kinase inhibitors is provided in Table 3.

Apoptosis Modulators Apoptosis, defined as programmed cell death, is characterized by cell shrinkage, nuclear condensation, and formation of apoptotic bodies, and may follow one of two alternative pathways: the extrinsic death receptor pathway or the intrinsic mitochondrial pathway (25). In the death receptor pathway, several death domain (DD)-containing members of the tumor necrosis factor (TNF) family of receptors, located at the cell surface, bind to their respective ligands and recruit adaptor proteins, forming a death-inducing signaling complex (DISC) that leads to caspase activation. The mitochondrial pathway is activated in response to stress-associated signals, such as radiation, chemotherapy, or viruses. This appealing idea of inducing apoptosis to “immortal” cancer cells has long-time been exploited in an effort to drive malignant cells to a programmed death pathway. p53 TARGETING. Apoptosis and senescence are the two cornerstones of the “surveillance system” that every organism possesses in order to limit potentially deleterious, uncontrolled cell proliferation. The tumor suppressor protein p53 is a key-factor of this surveillance procedure. Loss of p53 oncosuppressive functions represents a major event during neoplastic transformation, and several therapeutic approaches aiming at the restoration of the control of p53 function in tumor cells have been developed. Furthermore, several mutations of the “wild-type” p53 gene have been described and strongly correlated to oncogenesis in certain types of tumors. Pharmacological methods are under development to either stimulate wild-type p53 protein function, or help mutant p53 proteins to recover wild-type functions. These methods are based on small chemicals (CP-31388, PRIMA-1), peptides (CDB3), or single-chain antibody fragments corresponding to defined p53 domains (26). p53 replacement by means of viral vectors have been tested (RPR/ IGN Adp53), while exploiting p53-deficiency through viral cytolytic infection was also developed (ONYX-015). TABLE 3 The Main Aurora Kinase Inhibitors Having Entered Clinical Trials Drug MLN8054 ZM447439 Hesperadin VX-680

Target

Phase of development

Aurora kinase A Aurora kinase A, B Aurora kinase B Aurora kinase A, B

I I–II I I–II

Overview of Existing Therapies

11

SURVIVIN. Survivin is one of the members of the inhibitor of apoptosis protein (IAP) family of proteins and is differentially overexpressed in many types of human cancer cells, representing therefore an attractive anticancer target either through inactivating the survivin protein or by stopping the production of survivin through inhibition of survivin gene expression. Tetra-O-methyl nordihydroguaiaretic acid (M4N) is a selective inhibitor of survivin transcription that has shown promising results in preclinical trials, including several cell lines and different animal models (27). A methoxyethyl-modified phosphorothiate is currently being developed by Lilly Pharmaceuticals (Indianapolis, Indiana, U.S.A.) as an antisense survivin oligonucleotide (ASO). TUMOR NECROSIS FACTOR-RELATED APOPTOSIS-INDUCING LIGAND. The activation of cell surface “death” receptors by the tumor necrosis factor-related apoptosisinducing ligand (TRAIL) results in direct stimulation of apoptotic signaling pathways (extrinsic stimulation). Molecules that directly activate these receptors, such as agonistic monoclonal antibodies to the TRAIL receptors [HGS-ETR1, HGSETR2, and HGS-TR2J, (Human Genome Sciences, Rockville, Maryland, U.S.A.)] as well as recombinant/modified TRAIL (Apo2L/TRAIL, AMG951; Amgen Corp., Thousand Oaks, California, U.S.A.), are being developed as monotherapies and as part of combination therapies with existing chemotherapeutic drugs and other therapeutic modalities. B-CELL LYMPHOMA 2. The B-cell lymphoma 2 (Bcl-2) family of proteins play a central role in apoptosis by regulating the mitochondrial membrane permeability that mediates the intrinsic pathway of caspase activation. The Bcl-2 proto-oncogene, originally identified at the chromosomal breakpoint t (14;18) (q32;q21) in B-cell follicular lymphomas, is the first recognized and most extensively studied member of the Bcl-2 family. Oblimersen sodium (G3139) is a phosphorothioate complementary to bcl-2 (Genasens) that has been developed in melanoma and chronic lymphocytic leucemia (CLL) patients.

Antiangiogenic and Antivascular Agents When a tumor’s size reaches 2–3 mm of diameter, further tumor growth necessitates vascular supply via a procedure called neoangiogenesis and is induced by a number of factors, among which hypoxia plays a critical role. Angiogenesis is thus a critical step for tumor progression and systemic spread, representing thus an appealing target for effective approach against cancer. VEGF TARGETING. The best characterized and, thus far, the only clinically validated approach in modulating VEGF signaling pathway is the use of bevacizumab (28), which is a recombinant humanized VEGF monoclonal antibody against circulating VEGF-A. Bevacizumab has already been approved by the U.S. FDA as first-line treatment of metastatic colorectal cancer in combination with intravenous fluorouracil-based therapy and has also been shown to be potentially active and associated with better clinical outcomes in combination with paclitaxel and carboplatin as first-line therapy for locally advanced or metastatic nonsquamous non–small cell lung cancer (29). Bevacizumab activity has also been demonstrated in breast cancer patients.

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Using the same principle of ligand sequestration are decoy protein receptors and aptamers. VEGF-Trap is an engineered decoy receptor protein that possesses a 100-fold higher affinity for VEGF than that of the monoclonal antibody. It is currently in phase I trials. Aptamers, on the other hand, are fully synthetic, short nucleic acid sequences of DNA or RNA that are capable of binding with high affinity and target specificity to many diverse types of molecules, such as peptides and proteins, with a mechanism similar to that of the monoclonal antibodies (30). VEGFR TARGETING. An alternate approach to inhibit VEGF function is to interrupt signaling at the receptor level. Ribozymes are antisense RNAs that can bind in a sequence-specific, complementary fashion to their respective target messenger RNA (mRNA), like antisense nucleotides, but at the same time possess the unique property of directly cleaving the target mRNA due to their intrinsic catalytic activity. Angiozyme is a ribozyme directed against the mRNA of VEGFR1 and has been tested in phase I trials (28). In a way similar to EGFR, VEGFR-mediated signaling cascade can also be interrupted by adenosine triphosphate-competitive small-molecule inhibitors of the VEGFR tyrosine kinase activity. Several selected agents exhibiting this kind of activity have been tested in phase I to III clinical trials (Table 4). ENDOTHELIAL CELL PROPAGATION TARGETING. The endothelium is increasingly recognized as a target for biomedical intervention, not only for its accessibility to molecular agents coming from the blood-stream, but also for the active role played by endothelial cell proliferation and propagation to support diseases such as cancer. During neovascularization, endothelial cells need appropriate receptors to interact with the extracellular matrix for migration and survival. Such integrators do exist and constitute a promising target for endothelial propagation targeted therapy: the integrins. Integrins are cell-surface heterodimers of so-called a and b subunits whose association defines the nature of the ligands recognized. Table 5 summarizes the main agents targeting integrins or other endothelial receptors that have entered clinical trials. HYPOXIA INDUCING FACTOR INHIBITION. The most important regulator of the cellular response to reduced oxygen levels identified to date is the hypoxiainducible factor 1(HIF-1). Since overexpression of HIF-1 is a consequence of intratumoral hypoxia, it has been proposed as an appealing target for molecular anticancer therapy. A number of small molecules that inhibit HIF-1 have been

TABLE 4 Most Advanced Agents Exhibiting VEGFR Tyrosine Kinase Inhibitory Activity Drug BAY 439006 PTK/ZK SU11248 AG013706 ZD6474 AEE788 AZD2171

Target Raf, VEGFR, c-KIT, Flt-3, P38a VEGFR PDGFR, VEGFR, c-KIT, Flt-3 VEGFR, PDGFR EGFR, RET, VEGFR EGFR, HER2/neu, VEGFR VEGFR, PDGFR

Clinical phase III/registered III III/registered II II/III I–II II/III

13

Overview of Existing Therapies TABLE 5 Main Endothelial Propagation Targeting Agents Having Entered Clinical Trials Name(s) Vitaxin (MEDI-523) Abegrin (MEDI-522) CNTO 95 Volociximab Eos-200-4 M-200 Angiostatin Endostatin Endostar

Type Humanized monoclonal antibody Humanized monoclonal antibody Human monoclonal antibody Humanized monoclonal antibody Recombinant protein Recombinant protein Recombinant modified endostatin

Target

Clinical status

avb3 integrin

Phase I/II

avb3 integrin

Phase I/II

av integrins

Phase I/II

avb1 integrin

Phase I/II

Integrins? Angiomotin avb1, other receptors avb

Phase I/II Phase I/II Phase I Phase III in lung cancer

identified, some of them initially described as having different molecular targets (rapamycin, topo-isomerase inhibitors, histone deacetylase inhibitors, antimicrotubule agents, and redox pathway inhibitors). ANTIVASCULAR AGENTS. Unlike the inhibition of angiogenesis, which aims at preventing the growth of new blood vessels, vascular targeting aims at the rapid and selective shutdown and/or damage of the established tumor vasculature, leading to secondary tumor cell death. Thalidomide is a non-VEGF-based angiogenesis inhibitor, which, apart from the mechanism of action as an antiangiogenesis agent, acts as well as a modifier of the established tumor microvasculature in diseases such as multiple myeloma. The vascular-targeting compound ZD6126
(AstraZeneca) destabilizes microtubules and selectively disrupts immature tumor endothelial cells, which stops tumor blood flow and induces tumor cell death (31). Combretastatine derivative AVE8062 (Sanofi-Aventis) is another vascular disrupting agent in phase I trials.

Anti-Invasive Agents TARGETING METALLOPROTEINASE. The metalloproteinases (MMP) are a family of zinc-dependent neutral endopeptidases that are capable of degrading essentially all of the components of the extracellular matrix. These proteases, which are synthesized by connective tissue cells, are important for the remodeling of the extracellular matrix that accompanies physiological processes, but also tumor growth, invasion, and metastasis. Several therapeutic approaches concerning MMP inhibition have been developed, including zinc-binding agents, covalent inhibitors, exosite-binding, and allosteric inhibitors (32). Most trials with MMP inhibitors have lead to negative results, including trials with, among others, AG3340 (Agouron/Pfizer Corp., New York, New York, U.S.A.), BAY 129566 (Bayer), and BMS-275291 (Bristol Meyer Squibb, Jacksonville, Florida, U.S.A.). SRC INHIBITORS. The tyrosine kinase pp60src (Src) is the prototypical member of a family of proteins that participate in a broad array of cellular signal transduction processes, including cell growth, differentiation, survival, adhesion, and migration. Abnormal Src family kinase (SFK) signaling has been linked to several disease states, including cancer metastases. Src has thus emerged as a

14

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molecular target for the discovery of small-molecule inhibitors that regulate Src kinase activity by binding to the ATP pocket within the catalytic domain. AP23451 (Ariad Pharmaceuticals) is a small-molecule inhibitor designed to inhibit Srcdependent bone resorption, and AP23464 is a small-molecule inhibitor designed to inhibit the Src-dependent metastatic spread of cancer (33). Dasatinib (Bristol Meyer Squibb) is on fast-track for registration for imatinib-resistant CML, and AZD0530 (AstraZeneca) is in phase II trials. Bosutinib (Wyeth) is another emerging potent Src inhibitor. Telomerase and Telomere Interacting Agents Telomeres, the extremities of chromosomes, are shortened at each cell division, leading eventually to the senescence of the cell. In order to achieve a great number of cell divisions, cancer cells must acquire the capacity to maintain their telomeres. Therefore, telomeres and telomerase, the enzyme responsible for telomere elongation, represent interesting targets for molecular cancer therapy. A summary of the main agents targeting telomeres or telomerase action is presented in Table 6. Targeted Agents with Transversal Mechanisms CYCLO-OXYGENASE-2 INHIBITORS. The evidence that cyclo-oxygenase (COX) inhibitors and nonsteroidal anti-inflammatory drugs (NSAIDS) could be of benefit against the development and progression of malignancies was originally derived from large epidemiological studies. Nevertheless, recent data indicating that COX isoform 2 (COX2) can play a role in tumor progression in many solid tumors has reinforced the value of COX2 as a cancer therapeutic target. The initial promising results obtained by the first clinical trials with celecoxib and rofecoxib as chemopreventors in colon cancer were followed by an increased risk of severe cardiovascular accidents (34). PROTEASOME INHIBITION. The ubiquitin–proteasome pathway is an important protein degradation system affecting indirectly signal transduction pathways

TABLE 6 Main Therapeutic Strategies for Telomere and Telomerase Targeting Agent AZT SiRNA, ribozymes

NPS, GRN163, S-ODN, 2-5A 5-azacytidine, tricostatine A

Target Catalytic subunit of telomerase (hTERT) hTERT mRNA

Human telomerase RNA component (hTR) Chromatine promoter region, histones

SiRNA, mutated hTR

Telomere structure

Porphyrine and acridine derivatives

Telomeres (G-quartet)

Mechanism of action Inhibition of reverse transcription RNA silencing, block of mRNA by specific binding and degradation Block of mRNA by antisense oligonucleotides Change of chromatide structure that inhibits hTERT transcription Introduction of mutations in telomeric DNA Telomere interacting agents

Overview of Existing Therapies

15

through regulation of cell-cycle proteins. Many of the protein products that can provoke or inhibit neoplastic transformation and growth are temporarily degraded during the cell cycle by the ubiquitin–proteasome pathway, including the tumor suppressor p53, several cyclins and cyclin-dependent kinase inhibitors (CDKIs), and the nuclear factor kB (NF-kB), which is a key factor regulating transcription, angiogenesis, cell adhesion, and apoptosis (5). Taken altogether, it is clear that proteasome inhibitors can act through multiple mechanisms to arrest tumor growth, spread, and angiogenesis. Bortezomib is a proteasome inhibitor that has entered clinical trials with a registration in multiple myeloma and promising results in several solid tumors (35). HEAT SCHOCK PROTEINS. A large body of evidence collected through the past decade has identified the molecular chaperone heat shock protein 90 (Hsp90) as a critical modulator of an extensive network of cellular signaling pathways. Many of the processes overseen by Hsp90 are deregulated in tumor cells, including cellcycle control, gene transcription, and apoptotic signaling. Therefore, Hsp90 inhibition offers the potential of simultaneous disruption of multiple signaling events critical to tumor cell growth and survival. Indeed, small molecule inhibitors of Hsp90 function are actively being evaluated in the clinic as anticancer agents. 17-allylamino-17-demthoxygeldanamycin (17-AAG) was the first pharmacological agent to reach phase I clinical trials, exhibiting prolonged disease stabilization, notably in malignant melanoma (36). HISTONE DEACETYLASE INHIBITORS. Epigenetic silencing of tumor suppressor genes by aberrant DNA methylation and chromatin deacetylation provides interesting targets for chemotherapeutic intervention by inhibitors of these events. 5-Aza-2’-deoxycytidine (decitabine, 5AZA-CdR) is a potent demethylating agent, which can reactivate tumor suppressor genes silenced by excessive DNA methylation. LAQ824 (LAQ) is a novel inhibitor of histone deacetylase (HDAC) that shows antineoplastic activity and can activate genes that produce cell-cycle arrest. Both 5AZA-CdR and LAQ as single agents are currently under clinical investigation in patients with cancer (37).

CONCLUSION This chapter aimed at clarifying the concept of molecular targeted therapy and providing a general overview of MTAs currently under clinical development. Important considerations to be kept in mind regarding their future development are the following: (a) the vast number of MTAs currently under pre-clinical evaluation that could potentially enter the clinical arena in the coming months and years; (b) the remarkable clinical efficacy of some of these agents (HER inhibition, antiangiogenic agents) against different tumor types, including their ability to reverse chemo-resistance to conventional chemotherapy; and (c) the huge impact that MTAs could have in every-day clinical practice of oncology if integrated in earlier stages (locally advanced and resectable disease) or in an earlier setting (first-line therapy). Another exciting and rapidly evolving new area of active research is the integration of MTAs within the established anticancer armamentarium (surgery, radiotherapy, and conventional chemotherapy), in order to optimize their combined action and therapeutic efficacy.

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Overview of Existing Therapies

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Vassilev LT, Vu BT, Graves B, et al. In vivo activation of the p53 pathway by smallmolecule antagonists of MDM2. Science 2004; 303(5659):844–8. Lambert JD, Meyers RO, Timmermann BN, and Dorr RT. Tetra-O-methylnordihydroguaiaretic acid inhibits melanoma in vivo. Cancer Lett 2001; 171(1):47–56. Dy GK and Adjei AA. Angiogenesis inhibitors in lung cancer: a promise fulfilled. Clin Lung Cancer 2006; 7(Suppl 4):S145–9. Sandler AB. Advanced non-small-cell lung cancer: new data, therapy choices, and challenging decisions. Oncology (Williston Park) 2006; 20(6):626–8. Rimmele M. Nucleic acid aptamers as tools and drugs: recent developments. Chembiochem 2003; 4(10):963–71. Bozec A, Lassalle S, Gugenheim J, et al. Enhanced tumour antiangiogenic effects when combining gefitinib with the antivascular agent ZD6126. Br J Cancer 2006; 95(6):722–8. Overall CM and Kleifeld O. Towards third generation matrix metalloproteinase inhibitors for cancer therapy. Br J Cancer 2006; 94(7):941–6. Dalgarno D, Stehle T, Narula S, et al. Structural basis of Src tyrosine kinase inhibition with a new class of potent and selective trisubstituted purine-based compounds. Chem Biol Drug Des 2006; 67(1):46–57. Bresalier RS, Sandler RS, Quan H, et al. Cardiovascular events associated with rofecoxib in a colorectal adenoma chemoprevention trial. N Engl J Med 2005; 352(11):1092–102. Rajkumar SV, Richardson PG, Hideshima T, and Anderson KC. Proteasome inhibition as a novel therapeutic target in human cancer. J Clin Oncol 2005; 23(3):630–9. Banerji U, O’Donnell A, Scurr M, et al. Phase I pharmacokinetic and pharmacodynamic study of 17-allylamino, 17-demethoxygeldanamycin in patients with advanced malignancies. J Clin Oncol 2005; 23(18):4152–61. Hurtubise A and Momparler RL. Effect of histone deacetylase inhibitor LAQ824 on antineoplastic action of 5-Aza-2’-deoxycytidine (decitabine) on human breast carcinoma cells. Cancer Chemother Pharmacol 2006; 58(5):618–25. Dancey JE. Therapeutic targets: MTOR and related pathways. Cancer Biol Ther 2006; 5(9):1065–73.

2

Signal Transduction Inhibitors, HER Family, EGFR Inhibition, and Clinical Achievements Giuseppe Giaccone Department of Medical Oncology, Vrije Universiteit Medical Center, Amsterdam, The Netherlands

Paolo Zucali Department of Medical Oncology and Hematology, Instituto Clinico Humanitas, Milan, Italy

INTRODUCTION The epidermal growth factor receptor (EGFR) has been extensively investigated as a target for anti-cancer therapy. EGFR is overexpressed in a large number of tumors, including head and neck, colorectal, lung, breast, ovary, prostate, kidney, brain, pancreas, and bladder carcinomas (1). The overexpression of EGFR correlates with poor prognosis and decreased survival in several of these solid tumors (2–4). Moreover, the overexpressing EGFR tumors often produce their own ligands, such as transforming growth factor alpha (TGF-a), leading to the activation of survival pathways via autocrine loops. Signaling through the EGFR axis has been implicated in mediating multiple processes involved in tumor progression and metastasis, including invasion, angiogenesis, proliferation, and inhibition of apoptosis (5). The efficacy of conventional chemotherapy was improved by the anti-EGFR-targeted therapies in both preclinical and clinical studies (1). Although such therapies may lead to partial response or disease stabilization in some patients, the majority of unselected patients do not benefit from anti-EGFR therapy, and those who do eventually develop resistance.

EGFR SIGNALING PATHWAYS EGFR is a member of the ErbB family of trans-membrane tyrosine kinase receptors, which includes ErbB1 (or HER1, or EGFR), ErbB2 (or HER2/neu), ErbB3 (or HER3), and ErbB4 (or HER4). EGFR was the first ErbB family member to be described and remains the best characterized to date (6,7). Epithelial cells and malignant tumors of epithelial origin express EGFR, but EGFR is not expressed on mature hematopoietic cells (7,8). EGFR has six known ligands: epidermal growth factor (EGF), transforming growth factor alpha (TGF-a), amphiregulin, betacellulin, heparin binding EGF, and epiregulin (9). After dimerization and activation, the receptor is internalized, and its degradation or recycling can transiently downregulate signaling mediated by the receptor. Multiple signaling pathways related to cellular proliferation and survival are activated downstream of EGFR, including the ras pathway with the extra-cellular signal-regulated kinase (ERK)/mitogenactivated protein kinase (MAPK), the phosphatidylinositol 3-kinase/Akt (PI3K/ Akt) pathway, and signal transducer and activator of transcription (STAT) 19

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pathways. EGFR activation can also induce cell cycle progression via various mechanisms, including the up-regulation of cyclin D1 (10). EGFR stimulation can significantly increase the activity of c-Src, a signaling intermediate involved in cell cycle progression, motility, angiogenesis, and survival (11) (Fig. 1). EGFR INHIBITORS Small Molecules Tyrosine kinase inhibitors (TKIs) (Table 1) prevent auto-phosphorylation of the EGFR intracellular TK domain by competitive adenosine tri-phosphate (ATP) inhibition at the intracellular catalytic domain. EGFR TKIs fall into two broad classes: reversible inhibitors, such as gefitinib (Iressa
; AstraZeneca, London, U.K.) and erlotinib (Tarceva
; Genentech, South San Francisco, California, U.S.A.), and irreversible inhibitors. Irreversible inhibitors covalently bind specific cysteine residues in the ATP binding site of EGFR. The clinical significance of reversible versus irreversible inhibition is uncertain at this point. The ability to irreversibly bind the tyrosine kinase domain could theoretically produce more sustained antitumor activity. Furthermore, some irreversible inhibitors have been shown to preserve activity against the T790M resistant mutant of EGFR (12). Gefitinib and erlotinib are currently approved in many countries for clinical use in advanced non–small cell lung cancer (NSCLC) patients who failed at least one line of chemotherapy. Erlotinib is also approved for clinical use in pancreatic cancer patients. Mechanistically, gefitinib is able to block MAPK and PI-3K/Akt path-

Ligand

Ras

Raf

Grb/ SOS

R

R

K

K

EGFR

PI3K PTEN

Src

Akt

mTOR MEK

Erk

Proliferation

Bad

STAT

Apoptosis

p70S6K

Cell cycle progression

FIGURE 1 Schematic representation of major components of the EGF receptor pathway.

Small molecule

Small molecule

Monoclonal antibody

AEE788

BMS-599626

Cetuximab (Erbitux
)

Irreversible inhibitor Inhibitor of EGFR and VGFR2 Inhibitor of EGFR, HER2, VEGFR2 Pan-inhibitor of all ErbB members Chimeric

EGFRa and ErbB2 TKs EGFR and VEGFR2 TKs EGFR, ErbB2 and VEGFR2 TKs EGFR, ErbB2 and ErbB4 TKs EGFR extracellular domain

Small molecule Small molecule

HKI272 ZD6474 (Zactima
)

Irreversible, pan-inhibitor of all ErbB members Irreversible inhibitor

EGFRa, ErbB2 and ErbB4 TKs EGFRa and ErbB2 TKs

Small molecule

Small molecule

Dual reversible inhibitor

EGFR and ErbB2 TKs

Small molecule

Specific reversible inhibitor

EGFR TK

Characteristics Specific reversible inhibitor

Small molecule

Target EGFR TK

Class

Small molecule

Lapatinib (Tykerb
, GW572016) Canertinib (CI-1033) EKB 569

Gefitinib (Iressa
, ZD1839) Erlotinib (Tarceva
, OSI-774)

Agent

TABLE 1 EGFR Inhibitors Route

Drug company

Bristol-Myers Squibb (New York, New York, U.S.A.) Imclone (New York, New York, U.S.A.) Bristol-Myers Squibb Merck KgGA (Darmstadt, Germany)

Oral IV

(Continued)

Novartis (Basel, Switzerland)

Oral

Oral Oral

Oral

Pfizer (New York, New York, U.S.A.) Wyeth Pharmaceuticals (Collegeville, Pennsylvania, U.S.A.) Wyeth Pharmaceuticals Astra-Zeneca

OSIP (Melville, New York, U.S.A.) Genentech (South San Francisco, California, U.S.A.) Roche (Basel, Switzerland) GlaxoSmithKline (Brentford, U.K.)

Astra-Zeneca (London, U.K.)

Oral

Oral

Oral

Oral

EGFR Inhibition and Clinical Achievements

21

Monoclonal antibody

Monoclonal antibody

Monoclonal antibody

Monoclonal antibody

Monoclonal antibody

MDX214

EGF vaccine

2C4

TheraCIMhR3

HuMax-EGFr

EGFR extracellular domain EGFR extracellular domain

Erb2 heterodimerization

Fully human

Humanized

hrEGF bound to protein and alum

Fully human

Fully human

Humanized

Characteristics

IV

IV

IV

ID

IV

IV

IV

Route

EMD (San Diego, California, U.S.A.) Merck KgGA Abgenix (Fremont, California, U.S.A.) Amgen (Thousand Oaks, California, U.S.A.) Medarex (Princeton, New Jersey, U.S.A.) CIMAB (Havana, Cuba) Biocon (Bangalore, India) CancerVax (Carlsbad, California, U.S.A.) YM BioSciences (Mississauga, Ontario) Genentech Roche YM BioSciences (Mississauga, Ontario, Canada) Genmab (Copenhagen, Denmark)

Drug company

Also active on the T790M resistant mutant. Abbreviations: EGF, epidermal growth factor; EGFR, epidermal growth factor receptor; ID, intradermally; IV, intravenously; TK, tyrosine kinase; VEGFR2, vascular endothelial growth factor.

a

EGFR extracellular domain

Monoclonal antibody

Panitumumab (Vectibix
, ABX-EGF)

EGFR extracellular domain EGF

EGFR extracellular domain

Target

Monoclonal antibody

Class

Matuzumab (EMD72000)

Agent

TABLE 1 EGFR Inhibitors (Continued )

22 Giaccone and Zucali

EGFR Inhibition and Clinical Achievements

23

ways, and its treatment is associated with cell-cycle arrest at G1, involving increased expression of the cyclin-dependent kinase (CDK) inhibitor p27KIP1 and decreased expression of CDK2, CDK4, CDK6, cyclin D1, and cyclin D3. Increased characteristics of apoptosis, such as DNA fragmentation, increased Fas protein expression, activation of initiator and effector caspases, and a change in plasma membrane phospholipids packing, were also observed (13,14). However, these effects may be limited and dependent on the tumor type (15). Similar cell-cycle and apoptotic changes have been seen after erlotinib treatment (14,16). Monoclonal Antibodies Monoclonal antibodies (mAb) against EGFR (Table 1) were the first approach used in clinical studies to target EGFR signaling in malignant cells. The mAb therapies competitively inhibit the binding of activating ligands to the extracellular domain of EGFR, inhibiting receptor auto-phosphorylation and, in contrast to the TKIs, inducing its internalization and degradation. Subsequent downstream signaling events are similar to those described for the TKIs gefitinib and erlotinib. Among mAb, cetuximab (Erbitux
; ImClone systems, Inc., Branchburg, New Jersey, U.S.A., licensed to Merck KGaA, Darmstadt, Germany) has achieved approval for use in colorectal cancer (CRC) refractory to irinotecan (Campto
; Pfizer, Capelle aan den Ijssel, The Netherlands) and in locally advanced squamous cell carcinomas of the head and neck (SCCHN) in combination with radiotherapy.

Other EGFR Inhibitors Active immunization could be an attractive alternative to inhibit the EGFR because it would circumvent both the need for multiple infusions and the danger of inducing an immune response typical of the antibodies. Mimotopes are small peptides that mimic a given epitope structurally, but not necessarily by amino acid sequence. The only important prerequisite is that an antibody recognizes the mimotope; for example, antibodies with proven beneficial antitumor properties, such as cetuximab. Riemer and colleagues observed that the epitope-specific immunization is feasible for active anti-EGFR immunotherapy. The in vitro biologic features of mimotope induced antibodies are similar to those of the monoclonal antibody cetuximab (17). Bing Hu and collegues observed that an active antitumor immunity could be induced by dendritic cells pulsed with recombinant ectodomain of mouse EGFR (DC-edMER), which may involve both humoral and cellular immunity, and may provide insight into the treatment of EGFR-positive tumors through the induction of active immunity against EGFR (18). Epidermal growth factor could be another possible target. Ramos and colleagues developed an active specific immunotherapy based on EGF deprivation, observing a correlation between antibody titers, serum EGF levels, and patient survival in a Phase I trial of 43 patients with NSCLC receiving the EGF vaccine (19). The use of antisense oligonucleotides inhibiting EGFR synthesis and the use of antibody-based immunoconjugates represent other interesting strategies. In preclinical studies, both treatments showed a significant inhibition of growth in EGFR positive tumors (20,21).

24

Giaccone and Zucali

CLINICAL TRIALS WITH EGFR INHIBITORS Non–Small Cell Lung Cancer Phase II studies of unselected patients with NSCLC treated with gefitinib and erlotinib as single agents after chemotherapy failure produced response rates and median survivals ranging from 10% to 19% and 6.0 to 8.0 months, respectively (Table 2). On the basis of the encouraging response rate and symptomatic benefit TABLE 2 Randomized Trials of EGFR TKIs in Advanced NSCLC

Study IDEAL 1 2nd/3rd line (Phase II) IDEAL 2 (22) 2nd/3rd line (Phase II) BR.21 (24) 2nd/3rd line (Phase III) ISEL (25) 2nd/3rd line (Phase III) INTACT 1 (26) 1st line (Phase III)

No. of patients

Response rate (%)

Median timeto-progression (months)

Median survival (months)

1-year survival (%)

Gefitinib 250 mg Gefitinib 500 mg

103 105

18.4 19

2.7 2.8

7.6 8.0

35 29

Gefitinb 500 mg Gefitinib 250 mg

102 114

12 10

7.0 6.0

7.0 6.0

27 24

Erlotinib 150 mg placebo

488 243

Gefitinib 250 mg placebo

1129 563

Regimen

Cisplatingemcitabineplacebo Cisplatingemcitabinegefitinib 250 mg Cisplatingemcitabinegefitinib 500 mg INTACT 2 (27) Carboplatin1st line paclitaxel(Phase III) placebo Carboplatinpaclitaxelgefitinib 250 mg Carboplatinpaclitaxelgefitinib 500 mg TALENT (29) Cisplatin1st line gemcitabine(Phase III) placebo Cisplatingemcitabineerlotinib 150 mg TRIBUTE (28) Carboplatin1st line paclitaxel(Phase III) placebo Carboplatinpaclitaxelerlotinib 150 mg

9 <1 (p < 0.001) 8.0 1.3

2.2 1.8 (p < 0.001)
3.0 2.6

6.7 4.7 (p < 0.001) 5.6 5.1

31 21 27 21

363

47.2

6.0

10.9

44

365

51.2

5.8

9.9

41

365

50.3

5.5

9.9

43

345

28.7

5.0

9.9

42

345

30.4

5.3

9.8

41

347

30.0

4.6

8.7

37

586

29.9

5.6

10.1

42

586

31.5

5.4

9.9

41

540

19.3

4.9

10.5

43.8

539

21.5

5.1

10.6

46.9

Abbreviations: EGFR, epidermal growth factor receptor; TKI; tyrosine kinase inhibitors; NSCLC, non–small cell lung cancer.

EGFR Inhibition and Clinical Achievements

25

observed in the IDEAL 1 and 2 studies, gefitinib, at the oral dose of 250 mg/daily, was the first molecularly targeted agent to be approved by the U.S. Food and Drug Administration (FDA) for NSCLC patients progressing after platinum- and docetaxel-containing chemotherapy regimens (22). In the IDEAL 1 and 2 studies (23,24), the patients were randomized to receive gefitinib 250 or 500 mg/daily. There was no significant difference in response rate and survival between the two dosages. Moreover, there was a good correlation between clinical response and symptomatic improvement that was reached in approximately 40% of patients. Adverse effects were, in general, well tolerated, but were more severe with the 500 mg dose in both studies. In IDEAL 2 the 500 mg gefitinib induced more acnelike rash (p ¼ 0.04) and diarrhea (p ¼ 0.006). In a phase II study of advanced NSCLC patients with prior chemotherapy treated with erlotinib 150 mg daily (25) the response did not appear to be correlated to the extent of prior exposure to chemotherapy. Survival was correlated to occurrence and degree of skin toxicity, and there was no relation with EGFR expression. In two large phase III trials (BR21 and ISEL) evaluating the efficacy of erlotinib or gefitinib as second or third line therapy in unselected patients with NSCLC, a statistically significant survival advantage over placebo was demonstrated only for erlotinib (26,27) (Table 1). In the BR21 study, patients treated with erlotinib had a statistically significant increase in terms of progression-free survival (2.2 vs. 1.8 months), median survival (6.7 vs. 4.7 months), and 1-year survival (31% vs. 21%) compared with patients receiving placebo. Major symptoms (cough, dyspnea, and pain) were also significantly improved by erlotinib treatment. Toxicity was acceptable, consisting mainly of skin toxicity and diarrhea. The survival benefit observed in this study was the basis for the approval in many countries of erlotinib for patients with NSCLC who had progressed after one or two systemic chemotherapy regimens. Unfortunately, the phase III study comparing gefitinib to placebo in 1692 patients failed to show an improvement of survival with the EGFR TKI. There was no statistically significant improvement in survival for gefitinib compared with placebo in the whole population (median 5.6 vs. 5.1 months; p ¼ 0.11), although significant improvement was observed in Asians (n ¼ 342; 0.66 [0.48–0.91], p ¼ 0.01; median survival 9.5 vs. 5.5 months) and in patients who never smoked (n ¼ 375; 0.67 [0.49–0.92], p ¼ 0.012; median survival 8.9 vs. 6.1 months). Although gefitinib and erlotinib have some differences in pharmacological properties, there are very many similarities between the two compounds, and it seems plausible that the choice of dose may have played a role in determining the different outcome of these studies. The dose of erlotinib used was 150 mg/day, the maximum tolerated dose of this drug, which corresponds to about 600–700 mg/day of gefitinib. The fact that the IDEAL studies did not discern differences in outcome between the 250 and the 500 mg/day gefitinib, may have to do with the small sample size of these randomized phase II studies, which does preclude conclusions on survival. Gefitinib and erlotinib were also tested in combination with standard chemotherapy (Table 2). Two large, randomized, placebo-controlled, double-blind phase III trials (INTACT 1 and 2) investigated the efficacy of gefitinib at the dose of 250 or 500 mg/daily in combination with chemotherapy as first-line therapy for NSCLC. Chemotherapy was given for up to six cycles, and the EGFR tyrosine kinase inhibitors were continued in nonprogressing patients until progression. However, the addition of gefitinib to chemotherapy failed to improve survival, time-to-progression, or response rate compared with chemotherapy alone (28,29).

26

Giaccone and Zucali

A subset analysis of patients with adenocarcinoma who received 90 days’ chemotherapy or more in the INTACT 2 study, demonstrated statistically significant prolonged survival, suggesting a gefitinib maintenance effect. In general, treatment was well tolerated and the toxicity of chemotherapy was nonoverlapping with that of gefitinib (skin rash and diarrhea). However, as expected, gefitinib 500 mg was associated with a higher degree of toxicities, as observed in the IDEAL studies, which led to more dose reductions and treatment interruptions. Two other studies employed the same combination chemotherapy regimens with erlotinib at 150 mg versus placebo (TRIBUTE and TALENT studies). Erlotinib was continued after termination of chemotherapy like in the INTACT studies. These combinations were well tolerated, and there was a positive correlation of survival with the degree of skin rash in the TRIBUTE study. However, these two large studies failed to demonstrate superiority of the erlotinib combinations in terms of survival (30,31). The absence of patient selection may have caused the negative outcome in these studies. EGFR inhibitors act mainly by reducing proliferation in wild type EGFR tumor cells, and proliferating tumor cells are those most affected by chemotherapy, therefore an antagonistic effect between EGFR TKIs and chemotherapy is plausible. However, the landmark analysis and the time-to-progression and survival curves, showing that the maintenance arms do better than the placebo arms after discontinuation of chemotherapy, (29,30), suggest that sequential therapies be a reasonable approach to this disease for front-line therapy. Based on these considerations, sequential studies of EGFR TKIs (both gefitinib and erlotinib) following chemotherapy have been planned or are presently accruing; these studies investigate whether TKIs may improve outcome in patients who are not progressing on chemotherapy. The activation of uncontrolled, tumor-induced angiogenesis through an increase in vascular endothelial growth factor (VEGF) secretion by cancer cells is a mechanism linked to acquired resistance to EGFR-inhibitor treatment. Significant and sustained anti-tumor activity in this context can be obtained by combining selective anti-EGFR drugs with antiangiogenic agents. Advanced NSCLC patients pretreated with one prior chemotherapy regimen were given erlotinib and bevacizumab (Avastin
; Genentech), a recombinant anti-VEGF monoclonal antibody (32) in a phase I/II study. Both agents could be given at the full dose of 150 mg/day of erlotinib and 15 mg/kg bevacizumab every 3 weeks. In the 40 patients enrolled (34 of which were in the phase II part) the response rate was 17.5% and median survival was 9.3 months. These results appear promising and have stimulated further studies of this combination in first- and second-line treatment of advanced nonsquamous lung cancers. In a preliminary analysis of a phase II randomized trial evaluating the efficacy and safety of bevacizumab in combination with either chemotherapy [docetaxel (Taxotere
; Sanofi-Aventis, Paris, France) or pemetrexed (Alimta
; Eli-Lilly and Co., Indianapolis, Indiana, U.S.A.)] or erlotinib compared with chemotherapy alone for treatment of recurrent or refractory nonsquamous lung cancer, data favored the addition of bevacizumab to either chemotherapy or erlotinib over chemotherapy alone (33). The bevacizumab-erlotinib combination showed a favorable toxicity profile compared with either chemotherapy-containing group and may represent an alternative to chemotherapy-based treatment in relapsed NSCLC if the results can be confirmed in a phase III trial. Definitive phase III trials of this combination are ongoing in relapsed (BeTa trial) and in first-line non-squamous carcinomas of the lung (ATLAS). ZD6474 (Zactima
; AstraZeneca, Macclesfield, U.K.) is an orally bioavailable, aniliquinazoline derivative, with a potent activity against the VEGF receptor 2,

EGFR Inhibition and Clinical Achievements

27

EGFR, and Ret. Phase II evaluation of ZD6474 has included two randomized studies in patients with advanced NSCLC. In one trial the efficacy of ZD6474 monotherapy (300 mg) was compared with that of the EGFR inhibitor gefitinib in previously treated patients. The adverse event profile of ZD6474 included rash, diarrhea, and asymptomatic QTc prolongation. A statistically significant improvement in time-to-progression was observed for ZD6474 compared with gefitinib (11.0 vs. 8.1 weeks, p ¼ 0.025) (34). In a second trial, 127 patients with platinumrefractory NSCLC were randomly allocated to be treated with ZD6474 (100 vs. 300 mg) or placebo, in combination with docetaxel [75 mg/m2 by intravenous (IV) infusion every 21 days (24)]. Also in this study, preliminary results showed a significant improvement of progression-free survival. The median time-to-progression was 12.0 weeks for docetaxel alone, 19.0 weeks for ZD6474 100 mg plus docetaxel (p ¼ 0.074 for comparison with control arm), and 17.0 weeks for ZD6474 300 mg plus docetaxel (p ¼ 0.461) (35). A randomized trial of ZD6474 in combination with carboplatin (Paraplatin
; Bristol-Myers Squibb, New York, New York, U.S.A.) and paclitaxel (Taxol
; Bristol-Myers Squibb) is also ongoing (36). Interestingly, however, the progression-free survival prolongation did not translate into an overall survival advantage in both studies (32,33). Remarkably, the ZD6474 arms had worse survivals (6.1 months in ZD6474 arm vs. 7.4 months in gefitinib arm; 13.4 months in docetaxel þ placebo arm vs. 13.1 months in docetaxel þ ZD6474 100 mg arm vs. 7.9 months in docetaxel þ ZD6474 300 mg arm), though the difference was not significant. The relatively short follow-up or the cross-over effect could be an explanation for these results. The improvement of progression-free survival led Heymach and colleagues to initiate a phase III evaluation of ZD6474 þ docetaxel in 2nd-line NSCLC. A few clinical studies have been conducted with the EGFR-directed antibodies for patients with NSCLC. In a phase II trial of previously treated patients, singleagent cetuximab (400 mg/m2 loading dose followed by 250 mg/m2 weekly) produced an objective response in 3 of 66 eligible patients and a median survival of 8.1 months (37). Treatment was well tolerated, with skin rash being the principal toxicity. The vast majority of patients (60 of 66) expressed EGFR by immunohistochemistry. Two single-arm phase II trials testing cetuximab in combination with a platinum-based doublet in previously untreated patients showed responses in the range of 26% to 29%, with median survival times of 10–11 months (38,39). A European randomized phase II trial tested cisplatin (Platinol
; Bristol-Myers Squibb) 80 mg/m2 on day 1 and vinorelbine (Navelbine
; Pierre Fabre, Castres, France) 25 mg/m2 on day 1 and 8 with or without cetuximab given at 400 mg/m2on day 1 and at 250 mg/m2 weekly thereafter as first-line therapy in 86 patients with advanced non–small cell lung cancer expressing EGFR (40). Response rate was slightly higher in the cetuximab arm (35% vs. 28%). A large phase III trial with this combination (FLEX) has been recently completed. Patients with EGFR-expressing advanced NSCLC (stage IIIB with documented malignant pleural effusion and stage IV) were randomized 1:1 to Group A (cetuximab 400 mg/m2 initial dose then 250 mg/m2 weekly, cisplatin 80 mg/m2 on day 1, vinorelbine 30 mg/m2 on day 1 and 8) or to Group B (cisplatin and vinorelbine as before) for a maximum of 6 threeweekly cycles. Cetuximab was administered until progression or unacceptable toxicity. 1,037 patients have been randomized, 689 were still under treatment, and 348 discontinued the study as recently reported. (41). In a multicenter phase I/II study, cetuximab combined with paclitaxel and carboplatin was administered to 32 untreated stage IV NSCLC patients, achieving a median time-to-progression of

28

Giaccone and Zucali

5 months, a median survival of 11 months, and 1- and 2-year survival rates of 40% and 16%, respectively (42). U.S. investigators from the Southwest Oncology Group recently completed a randomized phase II study of cetuximab, given both concomitantly and after carboplatin and paclitaxel, or given only after chemotherapy, to address the best sequence of administration of cetuximab and chemotherapy (42). In a phase II study, 175 advanced NSCLC patients were treated with panitumumab (Vectibix
; Amgen, Thousand Oaks, California, U.S.A.) combined with paclitaxel and carboplatin versus paclitaxel and carboplatin alone as first line therapy. The patients expressing EGFR were randomized 2:1 to receive panitumumab 2.5 mg/kg weekly plus paclitaxel and carboplatin every three weeks (Arm 1) or paclitaxel and carboplatin alone every three weeks (Arm 2). Paclitaxel and carboplatin were continued until progressive disease or up to a maximum of 6 cycles, whereas panitumumab was continued until progressive disease or intolerability. The improvement in the primary end-point of this study, time-to-progression, was not reached (4.2 months in panitumumab plus chemotherapy arm vs. 5.3 months in chemotherapy alone arm; log-rank p ¼ 0.55) (43). Colorectal Cancer Cetuximab was recently approved for recurrent metastatic CRC. Preclinical studies have shown that therapeutic synergy exists between cetuximab and several chemotherapeutic agents, and also that such synergy can occur in tumor cells already resistant to drugs such as irinotecan (44). Although cetuximab has shown later benefit as a single agent in CRC, it was primarily developed for use in combination with chemotherapy (Table 3). In a phase II study, Saltz and colleagues treated 121 patients with advanced CRC unresponsive to irinotecan with a combination of cetuximab (400 mg/m2 loading dose followed by 250 mg/m2 weekly) and irinotecan (at the same dose and schedule that the patient had previously progressed on) observing a radiographically objective tumor shrinkage of 19% (45). To determine whether this antitumor effect was due to synergy between the two drugs or due to the independent activity of cetuximab, Saltz and colleagues conducted another phase II study, where 60 similar patients were treated with cetuximab alone and 10% of them had radiologically significant tumor shrinkage (46). These data were confirmed and extended in a randomized phase II study by Cunningham and colleagues: 329 patients with advanced CRC refractory to irinotecan were randomly assigned in a ratio of 2:1 to receive cetuximab with irinotecan or TABLE 3 Cetuximab Trials in Colorectal Cancer

Agents Cetuximabirinotecan Cetuximab Cetuximab Cetuximabirinotecan

No. of patients

Response rate (%)

Median time-toprogression (months)

Phase II

121

19

NR

NR

(43)

Phase II Phase II (Randomized)

57 111 218

11 10.8 22.9 (p ¼ 0.007)

1.4 1.5 4.1 (p < 0.001)

6.4 6.9 8.6 (p ¼ 0.48)

(44) (45)

Study

Abbreviation: NR, not reported.

Median survival time (months)

Refs.

EGFR Inhibition and Clinical Achievements

29

cetuximab alone (47). In cases of disease progression, the addition of irinotecan to cetuximab monotherapy was permitted in the cetuximab alone arm. In comparison with the monotherapy group, the combination-therapy group had a significantly higher improvement in terms of response rate (22.9% vs. 10.8%; p ¼ 0.007), timeto-progression (median 4.1 vs. 1.5 months; p < 0.001), but not survival (median 8.6 vs. 6.9 months; p ¼ 0.48). The side effects of cetuximab are relatively mild, with an acne-like rash and drying and fissuring of the skin being the most common. Toxic effects were more frequent in the combination-therapy group, but their severity and incidence were similar to those of irinotecan alone. A phase III trial (EPIC) comparing cetuximab plus irinotecan to irinotecan alone in second-line metastatic, EGFR-expressing CRC patients (target N ¼ 1,300 patients) is still ongoing. A recruitment of 1220 patients with EGFR-detectable tumors in a phase III trial (CRYSTAL) comparing cetuximab (400 mg/m2 loading dose followed by 250 mg/m2 weekly) added to FOLFIRI [irinotecan 180 mg/m2, FA 400 mg/m2, 5-FU (Adrucil
; SICOR Pharmaceuticals, Irvine, California, U.S.A.) bolus 400 mg/m2, 2400 mg/m2 infusional 5-FU] versus FOLFIRI alone was completed in December 2005; an accrual of 1080 patients was planned in order to be able to detect a difference of 2 months in PFS with 80% power. The results of this study will be soon available. Gefitinib and erlotinib do not appear to have activity against metastatic CRC as single agents (48,49). Nonetheless, oral EGFR TKIs may augment the activity of combination chemotherapy against metastatic CRC. Gefitinib combined with infusional FU, leucovorin, and oxaliplatin (Eloxatin
; Sanofi-Aventis) (IFOX) showed a promising activity (response rate, 33%; median event-free survival, 5.4 months) in patients previously treated for metastatic CRC (50). Similarly, erlotinib combined with capecitabine (Xeloda
; Roche, Basel, Switzerland) and oxaliplatin yielded a response rate of approximately 25% in two phase II studies (51,52). However, these studies with gefitinib and erlotinib have the limitation of small sample size. Also in CRC a combined approach with a combination of an anti-EGFR drug and an anti-angiogenesis agent is currently under evaluation. In a randomized phase II study (BOND-2 trial) patients with irinotecan-refractory, metastatic CRC have been randomized to receive cetuximab (400 mg/m2 loading dose followed by 250 mg/m2 weekly) plus bevacizumab (5 mg/kg weekly) versus the combination of cetuximab, irinotecan (at the same dose and schedule as last received prior to study), and bevacizumab. The preliminary results reported a 35% response rate with a time-to-progression of 5.8 months in the triple drug combination, whereas a 23% response rate and a time-to-progression of 4 months were observed in the patient population treated with cetuximab plus bevacizumab. No unexpected toxicities were encountered (53). Head and Neck Cancer Inhibition of EGFR signaling by small molecules, monoclonal antibodies directed against ligands or the receptor, immunotoxin conjugates, or antisense oligonucleotides have demonstrated robust activity in preclinical models of squamous cell carcinoma of the head and neck (SCCHN) (54). Currently, Cetuximab is approved for use in locally advanced SCCHN patients in combination with radiotherapy. In phase I trials in recurrent/metastatic (R/M) disease, cetuximab combined with platinum agents with or without fluorouracil was found to be safe and tolerable at full doses of the cytotoxic agents (55–57). ECOG performed a placebocontrolled, randomized phase III trial of cisplatin with or without cetuximab,

30

Giaccone and Zucali

enrolling 117 assessable patients who had not been treated previously for R/M disease (58). More than 95% of the participants had received prior therapy for locally advanced disease, and approximately two-thirds of patients in each arm had metastatic disease at study entry. Patients in the experimental arm were allowed to cross over and received cetuximab on progression. The cisplatincetuximab arm achieved an objective response rate of 26% versus 10% in the cisplatin alone group (p ¼ 0.03). However, the primary end point of progressionfree survival did not meet statistical significance for the cisplatin-cetuximab group (4.2 vs. 2.7 months; p ¼ 0.09), and nor did overall survival (9.2 vs. 8 months; p ¼ 0.21). Cetuximab also shows an activity in patients with platinum-refractory disease. In a phase II trial (59), 103 patients who had progressed while receiving a platinum-based regimen were treated with cetuximab alone (Table 4). On progression during treatment with cetuximab, patients were offered combination therapy TABLE 4 EGFR TKIs Trials in R/M SCCHN

Agent Gefitinib (phase II) Gefitinib (phase II) Gefitinib (phase II)

Erlotinib (phase II) Cetuximab (phase II)

Dose/ schedule

Prior therapy

Progression No. of Response free survival patients rate (%) (months)

Overall survival (months)

Refs.

250 mg daily

No limit

70

1

1.8

5.5

(63)

500 mg daily

1 prior

47

11

3.4

8.1

(61)

500 mg daily

Cohort A: none Cohort B: 1 prior 1 prior

20

15

3

6

(62)

12

0

NA

NA

115

4

3.8

6

(64)

103

13

2.3

5.9

(57)

96

10

2.2

5.2

(58)

76

10

2.8

6.1

(59)

213 211

64 74

12.4 17.1

29.3 49.0

(60)

(p ¼ 0.02)

(p ¼ 0.006)

(p ¼ 0.03)

150 mg daily

400 mg/m2 loading, 250 mg/m2 weekly Cetuximab þ 400 mg/m2 loading, platinum 250 mg/m2 (phase II) weekly 400 mg/m2 Cetuximab þ loading, platinuma (phase II) 250 mg/m2 weekly HDRT – Cetuximab þ 400 mg/m2 loading, HDRT 250 mg/m2 (phase III weekly randomized)

1 prior platinumcontaining regimen 1 prior platinumcontaining regimen 1 prior platinumcontaining regimen No prior treatment

No prior treatment

a After failure of a platinum-containing regimen, patients on these studies received cetuximab in combination with a platinum-containing chemotherapy regimen. Abbreviations: EGFR, epidermal growth factor receptor; HDRT, high dose radiotherapy; NA, not available; R/M SCCHN, recurrent/metastatic squamous cell carcinoma of the head and neck; TKI, tyrosine kinase inhibitor. Source: From Refs. 57–64.

EGFR Inhibition and Clinical Achievements

31

with cetuximab and a platinum agent. An overall response rate of 13% with five patients achieving a complete response and a median survival of 5.9 months was reported. In 53 patients who went on to receive cetuximab with cisplatin or carboplatin, no responses were seen. Two phase II studies have evaluated cetuximab in combination with either cisplatin or carboplatin in patients who progressed while receiving platinum-based regimens (Table 4) (60,61). The efficacy of cetuximab as a single agent in platinumrefractory patients suggests that the activity in these trials can be attributed mostly to the addition of cetuximab, and that the agent alone may achieve the same results. A phase III trial (EXTREME) randomizing R/M SCCHN patients to receive cetuximab (first dose 400 mg/m2 then 250 mg/m2 weekly) plus a maximum of 6 three-weekly cycles of cisplatin (100 mg/m2 IV on day 1) or carboplatin (AUC 5, day 1) and 5-FU (1000 mg/m2/day continuous infusion for the first 4 days of each cycle) versus cisplatin or carboplatin with 5-FU as before as first-line treatment has been completed and results are expected in 2007. Cetuximab has received U.S. FDA approval for the treatment of patients with locally advanced SCCHN on the basis of a recently published phase III trial where patients were randomly assigned to treatment with high-dose radiotherapy alone (213 patients) or high-dose radiotherapy plus weekly cetuximab (211 patients) given as loading dose of 400 mg/m2, followed by 250 mg/m2 weekly for the duration of radiotherapy (62). The median duration of loco-regional control was 24.4 months for patients treated with combined therapy versus 14.9 months for those treated with radiotherapy alone (hazard ratio for locoregional progression or death, 0.68; p ¼ 0.005). Radiotherapy plus cetuximab significantly prolonged progression-free survival (hazard ratio for disease progression or death, 0.70; p ¼ 0.006) and overall survival (hazard ratio for death, 0.74; p ¼ 0.03). With the exception of acneiform rash and infusion reactions, the incidence of grade 3 or greater toxic effects, including mucositis, did not differ significantly between the two groups. Gefitinib has been studied in R/M SCCHN as a single agent in single arm phase II trials (Table 4). In the first study, the authors concluded that gefitinib at the dose of 500 mg is active and well tolerated in R/M disease, with a response rate of 10.6% (1 complete response) and 1-year survival rate of 30%. They also observed a strong positive correlation between skin toxicity and clinical outcome (63). These results were supported by a similar trial where patients were prospectively separated into those who received prior systemic therapy and those who did not, with treatment of gefitinib 500 mg daily (64). Gefitinib appeared to be more active in the first-line setting, but this difference did not reach statistical significance. Gefinitib was also tested at a dose of 250 mg daily in a phase II trial, and the relatively low activity observed led the authors to conclude that gefitinib at the dose of 250 mg appears less effective than 500 mg (65). Erlotinib was also investigated in R/M disease (Table 4). In a phase II study, some activity was shown. Correlations between EGFR expression and survival or severity of skin rash were not observed. However, the presence and grade of skin toxicity was again correlated with a more favourable outcome (66). Gefitinib or erlotinib were also tested in combination with cytotoxic chemotherapy or other targeted agents. Belon et al. (67) treated 24 patients with a docetaxel, cisplatin, and gefitinib (250 mg daily) regimen and reported a 50% overall response rate, with seven patients achieving a complete response. A similar

32

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trial employing erlotinib with docetaxel and cisplatin yielded an overall response rate of 78%, with three complete responses in 27 patients (68). In a phase I/II trial, erlotinib was combined with bevacizumab in patients with R/M disease (69). This combination appeared well tolerated, without doselimiting toxicities observed in the phase I portion of the trial, and the recommended phase II dose of erlotinib 150 mg daily and bevacizumab 15 mg/kg every 3 weeks was administered to an expansion cohort of 48 patients achieving an overall response rate of 15% and a median survival of 6.8 months. Moreover, in patients who had not received prior therapy for R/M disease, the objective response rate, median progression-free survival, and median overall survival were 17%, 4.8 months, and 7.5 months, respectively, suggesting again that this group of patients might benefit most from these targeted strategies, comparing favourably with cytotoxic chemotherapy regimens. Several phase III trials are being conducted in this disease with EGFR inhibitors. A large, international phase III trial randomly assigning patients to one of three arms [methotrexate (Methotrexate
; Wyeth Lederle, Madison, New Jersey, U.S.A.) monotherapy, methotrexate plus gefitinib 250 mg, and gefitinib 500 mg alone] has completed accrual. Patients were required to have had progressive disease while receiving a platinum-containing chemotherapy regimen or have been considered poor candidates for platinum-based therapy. ECOG is enrolling subjects to a randomized trial of docetaxel with or without gefitinib as first- and second-line therapy in R/M SCCHN patients. Other Tumors Erlotinib in combination with chemotherapy has demonstrated benefit in patients with pancreatic cancer. In a Phase III trial that involved 569 patients with advanced pancreatic carcinoma, the addition of erlotinib to gemcitabine improved overall survival and progression-free survival compared with gemcitabine alone (70). The median overall survival (6.37 vs. 5.91 months; p ¼ 0.025) and median progression-free survival (3.75 vs. 3.55 months; p ¼ 0.003) were significantly longer for the erlotinib arm. Results from that trial are the first that demonstrate a clinical benefit from an EGFR TKI used in combination with chemotherapy. A phase II trial to investigate the efficacy, tolerability, and pharmacokinetics of gefitinib in pre-treated patients with metastatic gastric carcinoma included 75 subjects who were randomized to receive 250 or 500 mg/d gefitinib orally. Gefitinib monotherapy was generally well tolerated in pretreated patients with gastric metastatic adenocarcinoma, and disease control was achieved in 18.3% of cases. The most common drug-related adverse events were diarrhea, rash, and anorexia (71). Data on the use of erlotinib in gastric cancer is limited to a single study of 70 patients having either gastric cancer (n ¼ 26) or gastroesophageal junction cancer (GEJC) (n ¼ 44). No patient in the gastric cancer cohort presented an objective response, but five patients in the GEJC cohort did so, one being a complete response. The overall response rate was 11% (72). Gefinitib showed a modest activity also in second-line treatment of advanced esophageal cancer (73). In patients with glioblastoma, the most common primary malignant brain tumor of adults, erlotinib and gefitinib showed some activity in a small subgroup of patients (74,75). In recent studies, ABX-EGF and cetuximab reported obtained different results in renal cell carcinoma patients. In a phase II trial evaluating the

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safety and efficacy of ABX-EGF, 20 patients were treated at 1.0 mg/kg, 23 at 1.5 mg/kg, and 15 at 2.0 mg/kg. Grade 2/3 adverse events reported were skin rash, pruritus, dyspnea, fatigue, diarrhea, abdominal pain, and nausea and vomiting. Two of 31 patients evaluated who failed or were unable to receive IL-2 (Proleukin
)/IFN-a (Roferon
; Roche) achieved objective responses, and 58% had minor responses or stable disease (76). In contrast to ABX-EGF, in 55 patients treated with cetuximab (first dose 400 mg/m2 then 250 mg/m2 weekly), no patients achieved either a complete or partial response (77). Given these disappointing results and the lack of prolongation of time-to-progression compared with historical controls (IFN-a), the investigators concluded that further study of cetuximab was not recommended (77). In a phase II study in 21 patients treated with 500 mg gefitinib once daily as a 28-day cycle, stable disease was reported in eight patients (38%), all of whom had previously been progressing. Median progression-free survival was 2.7 months and median overall survival was 8.3 months: the difference in overall survival between stable disease and progressive disease was 16 þ and 6.1 months, respectively (p ¼ 0.0007) (78). In breast cancer, gefitinib monotherapy obtained a disease control between 10% and 38% in heavily pre-treated metastatic patients (79–81). Two trials have investigated the efficacy of single agent gefitinib in hormone-therapy resistant breast cancer in ER positive disease with acquired resistance and ER negative disease (82,83). One study was terminated prematurely due to low clinical benefit rates of 2.5 and 8% disease control in the two groups, respectively, while the second trial has observed one patient with partial response and two with stable disease of only three evaluable patients in the ER positive group. Dual blockade of HER2 and EGFR using concomitant trastuzumab (Herceptin
; Roche) and gefitinib has been attempted in 34 patients, three quarters of whom received the therapy as first line treatment (84). Disappointingly, there was no additive effect on response rates, and the observed time-to-progression was shorter than that of standard trastuzumab monotherapy. These results may suggest antagonism. Combined therapy against both the hormonal and EGFR pathways may hold more promise. In one study, anastrazole in combination with gefitinib induced disease stabilization in 20% of patients (85). In a larger study, patients with both ER and EGFR positive primary breast cancers were randomized to gefitinib and anastrazole (Arimidex
; AstraZeneca) versus gefitinib with placebo. Both groups experienced similar primary tumor size decrease after four to six weeks of treatment, and there was a greater reduction in the Ki-67 tumor proliferation index in the combined therapy group (86). In combination chemotherapy, gefitinib plus docetaxel was well tolerated and showed encouraging antitumor activity (87). However, when gefitinib was given with paclitaxel and carboplatin, efficacy was similar to that reported for the doublet chemotherapy alone (88). There are fewer published studies of erlotinib. A phase II study of erlotinib with gemcitabine (Gemzar
; Eli-Lilly and Co.) in pre-treated metastatic breast cancer of 59 patients showed a response rate of 14%, which was lower than the expected antitumor activity for this trial (89). A second study combining erlotinib with bevacizumab obtained stabilization in 33% of patients (90). In a phase I trial erlotinib, trastuzumab and paclitaxel were simultaneously employed and 4 of 14 (29%) patients with advanced breast cancer responded to this combination, which was well-tolerated (91).

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In a phase II study of first-line lapatinib (Tykerb
; GlaxoSmithKline, Brentford, U.K.) monotherapy in patients with advanced HER2-amplified breast cancer, 38% and 46% derived partial response and stable disease, respectively (92). This degree of disease control, although anticipated to be higher in the first line setting than in heavily pre-treated patients, is impressive, and more comprehensive data are eagerly awaited. Additional studies have focused on patients with metastatic disease refractory to trastuzumab; lapatinib monotherapy has a reported disease control rate of 22% (93), while lapatinib and capecitabine in combination significantly prolonged median time-to-progression (HR 0.51, 95% CI 0.35-0.74) when compared to capecitabine alone (94).

PREDICTORS OF RESPONSE Clinical Features Retrospective analyses of NSCLC phase II trials showed that never smoking history, female gender, adenocarcinoma histology, and Asian origin were significantly associated with a higher response to EGFR TKIs (23–25,95,96). Among clinical characteristics, smoking history is the most important predictor. In the TRIBUTE trial (30), median survival was significantly longer in never smokers treated with erlotinib than in never smokers treated with placebo. Similarly, in the BR21 and ISEL trials, it was shown that never smokers treated with EGFR TKIs had a significantly longer survival when compared to never smokers treated with placebo, with no survival difference in smokers irrespective of the treatment (26,27). Thus far, there appears to be no clinical subgroup of patients with SCCHN or CRC that is more likely to respond or benefit from EGFR-TKIs. Nevertheless, patients with SCCHN or CRC who develop skin rash appear to achieve greater benefit when treated with either TKIs or MAbs. Unfortunately, a clear association between signaling inhibition in skin and anti-tumor response has not been found and the mechanism underlying this correlation is currently unclear (97). Development of skin rash has been associated with sensitivity to erlotinib (25) and cetuximab (42) also in NSCLC. However, the role of rash as a marker of sensitivity to gefitinib in NSCLC is more controversial, as some trials show a correlation between rash and response (98,99), while others do not (23).

Biological Features In NSCLC, mutations within the EGFR tyrosine kinase domain (missense mutations in exons 18 and 21, deletions in exon 19, and insertions in exon 20) confer sensitivity to TKIs. These mutations were found to be significantly related to Asian ethnicity, female gender, adenocarcinoma histology, and never smoking history (100–102). Despite the initial success of gefitinib or erlotinib in the treatment of NSCLC with EGFR mutations, all patients will ultimately develop disease progression. Two studies examined tumor specimens from patients with EGFR mutations who had significant initial clinical benefit from either gefitinib or erlotinib but subsequently developed progressive disease (103,104). In four of seven patients analyzed, a common secondary mutation, a substitution of methionine for threonine at position 790 (T790M), was identified only in the recurrent tumor specimen. However, in the remaining patients, a secondary mutation was not found in the

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EGFR tyrosine kinase domain in the recurrent tumor specimens, suggesting that other mechanisms of resistance must also exist. NSCLC tumors expressing EGFR mutations generally do not present K-ras mutations typically occurring in smokers, and significantly associated with primary resistance to TKIs (105). Mutations in K-ras are found more frequently in patients who develop disease progression with either gefitinib or erlotinib therapy and are also associated with a worse outcome when patients are treated with chemotherapy in combination with erlotinib (106). Therefore, the presence of K-ras mutations has to be considered as negative selector for response and the fact that EGFR and K-ras mutations are mutually exclusive, suggests a different pathogenic mechanism in smokers from never smoker NSCLC patients (107). However, approximately 18% of patients responding to EGFR TKIs do not have identifiable EGFR mutations. Moreover, EGFR mutations are virtually absent in patients with stable disease (108), and in the BR-21 study it was demonstrated that disease stabilization contributed to the overall survival advantage seen with erlotinib (26). Although survival analyses of retrospective series showed a significant association of EGFR gene mutations and improved survival of TKIs treated patients, association with survival was not significant in subgroup analyses of large NSCLC phase II-III trials. Emerging data suggest that NSCLC patients with EGFR gene mutations have a better prognosis than individuals with EGFR wild-type regardless of the treatment (106,109), although in resected NSCLC no difference in prognosis was observed (110). NSCLC patients with a high tumoral EGFR gene copy number by fluorescence in situ hybridization (FISH) analysis show a trend to experience shorter survival (111). Recent data indicate that increased copy number of the EGFR gene is crucial for TKIs sensitivity in NSCLC. A significant association between increased EGFR gene copy number as measured by FISH and response to gefitinib, time-to-progression and survival was observed in NSCLC (108,112–114). All these data suggest that FISH of the EGFR gene is a good predictor of sensitivity to EGFR-inhibitors in NSCLC. In a preclinical report, NSCLC tumors with synchronous overexpression of EGFR and HER2 are particularly sensitive to the drug (115). In a retrospective analysis, NSCLC patients with an increased HER2 gene copy number at FISH who were also EGFR positive (either at IHC or FISH) had better response rate, disease control rate, time-to-progression, and survival (116). Recent studies also point out to p-Akt expression in NSCLC as a predictor of response to gefitinib (117) and for this it may be essential to have a high expression of EGFR. In comparison with NSCLC, the prevalence of EGFR tyrosine kinase mutations in CRC and SCCHN is very low and cannot account for the activity seen with EGFR inhibitors. In unselected SCCHN tumor specimens from Korean patients, an EGFR tyrosine kinase mutation was described in 7% of cases (118). Moreover, in a North American study of tissue samples from eight SCCHN patients who responded to either gefitinib or erlotinib, no mutations were detected by in the EGFR tyrosine kinase domain (119). The prognostic role of EGFR expression by immuno-histochemistry (IHC) is still controversial. Although the U.S. FDA approval states that cetuximab should be administered only to CRC patients with EGFR-positive tumors, the EGFR detection by IHC has little or no predictive value of response to cetuximab, either as a single agent or in combination with irinotecan (46,47). In the BOND study, the efficacy of cetuximab was not related to levels of EGFR expression. Morover,

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cetuximab has also shown activity in colorectal tumors that are negative for EGFR by IHC (120). In SCCHN, clinical trials with either TKIs or MAbs have not demonstrated a positive association between EGFR expression by IHC and tumor response (23). Paradoxically, in the ECOG randomized phase III trial, participants with low-to-moderate EGFR expression appeared to have a higher response rate to the combination of cisplatin and cetuximab compared with the high expression group (40% vs. 12%; p ¼ 0.03) (121). The high EGFR gene copy number by fluorescence in situ hybridization (FISH) analysis is associated with a poor prognosis in SCCHN (122). In CRC, the increased EGFR copy number detected by FISH is associated with response to the anti-EGFR monoclonal antibodies cetuximab or panitumumab (123). The polymorphism in intron 1, the CA-single sequence repeat (CA-SSR), modulates transcriptional efficiency of the EGFR gene and subsequent EGFR expression (124). Interestingly, shorter repeat lengths which are associated with higher EGFR expression, have been shown to be predictive of response to erlotinib in SCCHN cell lines (125). Prospective clinical trials of patients selected on the base of biological and/or characteristics are ongoing. FUTURE DIRECTIONS Further improvement to identify predictors of response will help to better select patients who are most likely to benefit from EGFR TKIs. On the other hand, the mechanisms of resistance also need to be elucidated. Emerging data suggest that EGFR-inhibition may not be effective in the presence of independently activated proteins (e.g., MAPK, K-ras, PI3K/Akt, STAT3), tumor cell dedifferentiation (e.g., epithelial-mesenchymal transition), or the expression of other cell surface receptors (e.g., insulin-like growth factor I receptor or an increase in vascular endothelial growth factor). If these mechanisms underlie resistance in the clinic, then the emerging therapeutics targeting these processes or proteins are expected to synergize and provide wider applicability of the EGFR inhibitors. There are already clinical trials underway combining an EGFR-inhibitor with different target-inhibitors, such as rapamycin analogs that inhibit a downstream activator of AKT, mTOR (mammalian target of rapamycin), or antiangiogenesis agents.

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41. Von Pawel J, Park K, Pereira JR, et al. Phase III study comparing cisplatin/vinorelbine plus cetuximab versus cisplatin/vinorelbine as first-line treatment for patients with epidermal growth factor (EGFR)-expressing advanced non-small cell lung cancer (NSCLC) (FLEX). Abstr 7109, 42nd Annual Meeting of American Society of Clinical Oncology, Atlanta, Georgia, June 2–6, 2006. Alexandria, VA: American Society of Clinical Oncology (ASCO), 1964. 42. Thienelt CD, Bunn PA, Jr, Hanna N, et al. Multicenter phase I/II study of cetuximab with paclitaxel and carboplatin in untreated patients with stage IV non-small-cell lung cancer. J Clin Oncol 2005; 23:8786–93. 43. Crawford J, Swanson P, Prager D, et al. Panitumumab, a fully human antibody, combined with paclitaxel and carboplatin versus paclitaxel and carboplatin alone for first line advanced non-small cell lung cancer (NSCLC): a primary analysis. Eur Cancer Conf (Abstr 1123) 2005. 44. Baselga J, Albanell J. Epithelial growth factor receptor interacting agents. Hematol Oncol Clin North Am 2002; 16:1041–63. 45. Saltz L, Rubin M, Hochster H, et al. Cetuximab (IMC-C225) Plus Irinotecan (CPT-11) is Active in CPT-11-Refractory Colorectal Cancer (CRC) that Expresses Epidermal Growth Factor Receptor (EGFR). Abstr 7, 37th Annual Meeting of American Society of Clinical Oncology, San Francisco, California, May 12–15, 2001. Alexandria, VA: American Society of Clinical Oncology (ASCO), 1964. 46. Saltz LB, Meropol NJ, Loehrer PJ, Sr, et al. Phase II trial of cetuximab in patients with refractory colorectal cancer that expresses the epidermal growth factor receptor. J Clin Oncol 2004; 22:1201–8. 47. Cunningham D, Humblet Y, Siena S, et al. Cetuximab monotherapy and cetuximab plus irinotecan in irinotecan-refractory metastatic colorectal cancer. N Engl J Med 2004; 351:337–45. 48. Hidalgo M, Siu LL, Nemunaitis J, et al. Phase I and pharmacologic study of OSI-774, an epidermal growth factor receptor tyrosine kinase inhibitor, in patients with advanced solid malignancies. J Clin Oncol 2001; 19:3267–79. 49. Dorligschaw O, Kegel T, Jordan K, et al. ZD 1839 (Iressa)-based treatment as lastline therapy in patients with advanced colorectal cancer (ACRC). Abstr 1494, 39th Annual Meeting of American Society of Clinical Oncology, Chicago, Illinois, May 31–June 3, 2003. Alexandria, VA: American Society of Clinical Oncology (ASCO), 1964. 50. Kuo T, Cho CD, Halsey J, et al. Phase II study of gefitinib, fluorouracil, leucovorin, and oxaliplatin therapy in previously treated patients with metastatic colorectal cancer. J Clin Oncol 2005; 23:5613–9. 51. Meyerhardt JA, Zhu AX, Enzinger PC, et al. Phase II study of capecitabine, oxaliplatin, and erlotinib in previously treated patients with metastastic colorectal cancer. J Clin Oncol 2006; 24:1892–7. 52. Delord JP, Beale P, Van Cutsem E, et al. A phase 1b dose-escalation trial of erlotinib, capecitabine and oxaliplatin in metastatic colorectal cancer (MCRC) patients. Abstr 3585, 40th Annual Meeting of American Society of Clinical Oncology, New Orleans, Louisiana, June 5–8, 2004. Alexandria, VA: American Society of Clinical Oncology (ASCO), 1964. 53. Saltz LB, Lenz H, Hochster H, et al. Randomized Phase II Trial of Cetuximab/ Bevacizumab/Irinotecan (CBI) versus Cetuximab/Bevacizumab (CB) in IrinotecanRefractory Colorectal Cancer. Abstr 3508, 41st Annual Meeting of American Society of Clinical Oncology, Orlando, Florida, May 13–17, 2005. Alexandria, VA: American Society of Clinical Oncology (ASCO), 1964. 54. Pomerantz RG, Grandis JR. The epidermal growth factor receptor signaling network in head and neck carcinogenesis and implications for targeted therapy. Semin Oncol 2004; 31:734–43. 55. Baselga J, Pfister D, Cooper MR, et al. Phase I studies of anti-epidermal growth factor receptor chimeric antibody C225 alone and in combination with cisplatin. J Clin Oncol 2000; 18:904–14.

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56. Shin DM, Donato NJ, Perez-Soler R, et al. Epidermal growth factor receptor-targeted therapy with C225 and cisplatin in patients with head and neck cancer. Clin Cancer Res 2001; 7:1204–13. 57. Humblet Y, Vega-Villegas E, Mesia R, et al. Phase I study of cetuximab in combination with cisplatin or carboplatin and 5-fluorouracil (5-FU) in patients (pts) with recurrent and/or metastatic squamous cell carcinoma of the head and neck (SCCHN). Abstr 5513, 40th Annual Meeting of American Society of Clinical Oncology, New Orleans, Louisiana, June 5–8, 2004. Alexandria, VA: American Society of Clinical Oncology (ASCO), 1964. 58. Burtness B, Goldwasser MA, Flood W, et al. Phase III randomized trial of cisplatin plus placebo compared with cisplatin plus cetuximab in metastatic/recurrent head and neck cancer: an Eastern Cooperative Oncology Group study. J Clin Oncol 2005; 23:8646–54. 59. Trigo J, Hitt R, Koralewski P, et al. Cetuximab monotherapy is active in patients (pts) with platinum-refractory recurrent/metastatic squamous cell carcinoma of the head and neck (SCCHN): results of a phase II study. Abstr 5502, 40th Annual Meeting of American Society of Clinical Oncology, New Orleans, Louisiana, June 5–8, 2004. Alexandria, VA: American Society of Clinical Oncology (ASCO), 1964. 60. Baselga J, Trigo JM, Bourhis J, et al. Phase II multicenter study of the antiepidermal growth factor receptor monoclonal antibody cetuximab in combination with platinumbased chemotherapy in patients with platinum-refractory metastatic and/or recurrent squamous cell carcinoma of the head and neck. J Clin Oncol 2005; 23:5568–77. 61. Herbst RS, Arquette M, Shin DM, et al. Phase II multicenter study of the epidermal growth factor receptor antibody cetuximab and cisplatin for recurrent and refractory squamous cell carcinoma of the head and neck. J Clin Oncol 2005; 23:5578–87. 62. Bonner JA, Harari PM, Giralt J, et al. Radiotherapy plus cetuximab for squamous-cell carcinoma of the head and neck. N Engl J Med 2006; 354:567–78. 63. Cohen EE, Rosen F, Stadler WM, et al. Phase II trial of ZD1839 in recurrent or metastatic squamous cell carcinoma of the head and neck. J Clin Oncol 2003; 21: 1980–7. 64. Wheeler RH, Jones D, Sharma P, et al. Clinical and molecular phase II study of gefitinib in patients (pts) with recurrent squamous cell cancer of the head and neck (H&N Ca). Abstr 5531, 41st Annual Meeting of American Society of Clinical Oncology, Orlando, Florida, May 13–17, 2005. Alexandria, VA: American Society of Clinical Oncology (ASCO), 1964. 65. Cohen EE, Kane MA, List MA, et al. Phase II trial of gefitinib 250 mg daily in patients with recurrent and/or metastatic squamous cell carcinoma of the head and neck. Clin Cancer Res 2005; 11:8418–24. 66. Soulieres D, Senzer NN, Vokes EE, et al. Multicenter phase II study of erlotinib, an oral epidermal growth factor receptor tyrosine kinase inhibitor, in patients with recurrent or metastatic squamous cell cancer of the head and neck. J Clin Oncol 2004; 22:77–85. 67. Belón J, Irigoyen A, Rodríguez I, et al. Preliminary results of a Phase II study to evaluate gefitinib combined with docetaxel and cisplatin in patients with recurrent and/or metastatic squamous-cell carcinoma of the head and neck. Abstr 5563, 41st Annual Meeting of American Society of Clinical Oncology, Orlando, Florida, May 13–17, 2005. Alexandria, VA: American Society of Clinical Oncology (ASCO), 1964. 68. Kim ES, Kies M, Sabichi A, et al. Phase II Study of Combination Cisplatin, Docetaxel and Erlotinib in Patients with Metastatic/Recurrent Head and Neck Squamous Cell Carcinoma (HNSCC). Abstr 5546, 41st Annual Meeting of American Society of Clinical Oncology, Orlando, Florida, May 13–17, 2005. Alexandria, VA: American Society of Clinical Oncology (ASCO), 1964. 69. Vokes EE, Cohen EE, Mauer AM, et al. A phase I study of erlotinib and bevacizumab for recurrent or metastatic squamous cell carcinoma of the head and neck (HNC). Abstr 5504, 41st Annual Meeting of American Society of Clinical Oncology, Orlando, Florida, May 13–17, 2005. Alexandria, VA: American Society of Clinical Oncology (ASCO), 1964.

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70. Moore J, Goldstein D, Hamm J, et al. Erlotinib plus gemcitabine compared to gemcitabine alone in patients with advanced pancreatic cancer. A phase III trial of the National Cancer Institute of Canada Clinical Trials Group [NCIC-CTG]. Abstr 1, 41st Annual Meeting of American Society of Clinical Oncology, Orlando, Florida, May 13–17, 2005. Alexandria, VA: American Society of Clinical Oncology (ASCO), 1964. 71. Doi T, Koizumi W, Siena S, et al. Efficacy, tolerability and pharmacokinetics of gefitinib (ZD1839) in pretreated patients with metastatic gastric cancer. Abstr 1036, 39th Annual Meeting of American Society of Clinical Oncology, Chicago, Illinois, May 31–June 3, 2003. Alexandria, VA: American Society of Clinical Oncology (ASCO), 1964. 72. Dragovich T, McCoy S, Urba SG, et al. Southwest oncology group. SWOG 0127: Phase II trial of Erlotinib in GEJ and Gastric Adenocarcinomas. Abstr 49, 2nd Annual Gastrointestinal Cancers Symposium, Miami, Florida, January 27–29, 2005. Alexandria, VA: American Society of Clinical Oncology (ASCO), 1964. 73. Janmaat ML, Gallegos-Ruiz MI, Rodriguez JA, et al. Predictive factors for outcome in a phase II study of gefitinib in second-line treatment of advanced esophageal cancer patients. J Clin Oncol 2006; 24:1612–9. 74. Rich JN, Reardon DA, Peery T, et al. Phase II trial of gefitinib in recurrent glioblastoma. J Clin Oncol 2004; 22:133–42. 75. Prados M, Chang S, Burton E, et al. Phase I study of OSI-774 alone or with temozolomide in patients with malignant glioma. Abstr 394, 39th Annual Meeting of American Society of Clinical Oncology, Chicago, Illinois, May 31–June 3, 2003. Alexandria, VA: American Society of Clinical Oncology (ASCO), 1964. 76. Schwartz G, Dutcher JP, Vogelzang NJ, et al. Phase 2 clinical trial evaluating the safety and effectiveness of ABX-EGF in renal cell cancer (RCC). Abstr 91, 38th Annual Meeting of American Society of Clinical Oncology, Orlando, Florida, May 18–21, 2002. Alexandria, VA: American Society of Clinical Oncology (ASCO), 1964. 77. Motzer RJ, Amato R, Todd M, et al. Phase II trial of antiepidermal growth factor receptor antibody C225 in patients with advanced renal cell carcinoma. Invest New Drugs 2003; 21:99–101. 78. Dawson NA, Guo C, Zak R, et al. A phase II trial of ZD1839 in stage IV and recurrent renal cell carcinoma. Abstr 1623, 39th Annual Meeting of American Society of Clinical Oncology, Chicago, Illinois, May 31–June 3, 2003. Alexandria, VA: American Society of Clinical Oncology (ASCO), 1964. 79. Baselga J, Albanell J, Ruiz A, et al. Phase II and tumor pharmacodynamic study of gefitinib in patients with advanced breast cancer. J Clin Oncol 2005; 23:5323–33. 80. von MG, Jonat W, Fasching P, et al. A multicentre phase II study on gefitinib in taxane- and anthracycline-pretreated metastatic breast cancer. Breast Cancer Res Treat 2005; 89:165–72. 81. Albain K, Elledge R, Gradishar WJ, et al. Open-label, phase II, multicenter trial of ZD 1839 (‘Iressa’) in patients with advanced breast cancer. Breast Cancer Res Treat 2002; 76 (suppl 1; abstr 20):S33. 82. Robertson JFR, Gutteridge E, Cheung KL, et al. Gefitinib (ZD1839) is active in acquired tamoxifen (TAM)-resistant oestrogen receptor (ER)-positive and ER-negative breast cancer: results from a phase II study. Abstr 23, 39th Annual Meeting of American Society of Clinical Oncology, Chicago, Illinois, May 31–June 3, 2003. Alexandria, VA: American Society of Clinical Oncology (ASCO), 1964. 83. Francis P, Green M, Gebski V, et al. Gefitinib has a low clinical benefit rate in advanced breast cancer patients. Breast Cancer Res Treat 2005; 94(suppl 1; abstr 4080): S193. 84. ECOG E1100: a phase II trial of trastuzumab and gefitinib in patients with metastatic breast cancer that overexpress HER2/neu (erbB-2). Clin Adv Hematol Oncol 2003; 1:237. 85. Mita M, de Bono J, Mita A, et al. A phase II and biologic correlative study investigating anastrozole (A) in combination with geftinib (G) in post menopausal patients with estrogen receptor positive (ER) metastatic breast carcinoma (MBC) who have

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87.

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89.

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91.

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95. 96. 97.

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previously failed hormonal therapy. Breast Cancer Res Treat 2005; 94(Suppl 1; abstr 1117): S80 Polychronis A, Sinnett HD, Hadjiminas D, et al. Preoperative gefitinib versus gefitinib and anastrozole in postmenopausal patients with oestrogen-receptor positive and epidermal-growth-factor-receptor-positive primary breast cancer: a double-blind placebo-controlled phase II randomised trial. Lancet Oncol 2005; 6:383–91. Ciardiello F, Troiani T, Caputo F, et al. Phase II trial of gefitinib combined with docetaxel as first-line therapy in patients with metastatic breast cancer. Abstr 3080, 41st Annual Meeting of American Society of Clinical Oncology, Orlando, Florida, May 13–17, 2005. Alexandria, VA: American Society of Clinical Oncology (ASCO), 1964. Fountzilas G, Pectasides D, Kalogera-Fountzila A, et al. Paclitaxel and carboplatin as first-line chemotherapy combined with gefitinib (IRESSA) in patients with advanced breast cancer: a phase I/II study conducted by the Hellenic Cooperative Oncology Group. Breast Cancer Res Treat 2005; 92:1–9. Graham DL, Hillman DW, Hobday TJ, et al. N0234: Phase II study of erlotinib (OSI774) plus gemcitabine as first-or second-line therapy for metastatic breast cancer (MBC). Abstr 644, 41st Annual Meeting of American Society of Clinical Oncology, Orlando, Florida, May 13–17, 2005. Alexandria, VA: American Society of Clinical Oncology (ASCO), 1964. Dickler M, Rugo H, Caravelli J, et al. Phase II trial of erlotinib (OSI-774), an epidermal growth factor receptor (EGFR)-tyrosine kinase inhibitor, and bevacizumab, a recombinant humanized monoclonal antibody to vascular endothelial growth factor (VEGF), in patients (pts) with metastatic breast cancer (MBC). Abstr 2001, 40th Annual Meeting of American Society of Clinical Oncology, New Orleans, Louisiana, June 5–8, 2004. Alexandria, VA: American Society of Clinical Oncology (ASCO), 1964. Beeram M, De Bono J, Patnaik A, et al. Phase I and pharmacokinetics (PK) of combined erbB1 and erbB2 blockade with OSI-774 (Erlotinib; E) and trastuzumab (T) in combination with weekly paclitaxel (P) in patients (pts) with advanced solid tumors. In: San Antonio Breast Cancer Symposium. Abstract No.2034. 2005. Gomez HL, Chavez MA, Doval DC, et al. A phase II, randomized trial using the small molecule tyrosine kinase inhibitor lapatinib as a first-line treatment in patients with FISH positive advanced or metastatic breast cancer. Abstr 3046, 41st Annual Meeting of American Society of Clinical Oncology, Orlando, Florida, May 13–17, 2005. Alexandria, VA: American Society of Clinical Oncology (ASCO), 1964. Blackwell KL, Kaplan EH, Franco SX, et al. A phase II, open-label, multicenter study of GW572016 in patients with trastuzumab-refractory metastatic breast cancer. Abstr 3006, 40th Annual Meeting of American Society of Clinical Oncology, New Orleans, Louisiana, June 5–8, 2004. Alexandria, VA: American Society of Clinical Oncology (ASCO), 1964. Geyer CE, Forster JK, Cameron D, et al. Scientific Special Session: Lapatinib in Trastuzumab Resistant Breast Cancer. In: 42nd Annual Meeting of American Society of Clinical Oncology, Atlanta, Georgia, June 2–6, 2006. Alexandria, VA: American Society of Clinical Oncology (ASCO), 1964. Miller VA, Kris MG, Shah N, et al. Bronchioloalveolar pathologic subtype and smoking history predict sensitivity to gefitinib in advanced non-small-cell lung cancer. J Clin Oncol 2004; 22:1103–9. Kim YH, Ishii G, Goto K, et al. Dominant papillary subtype is a significant predictor of the response to gefitinib in adenocarcinoma of the lung. Clin Cancer Res 2004; 10:7311–7. Saltz L, Kies M, Abbruzzese JL, et al. The presence and intensity of the cetuximabinduced acne-like rash predicts increased survival in studies across multiple malignancies. Abstr 817, 39th Annual Meeting of American Society of Clinical Oncology, Chicago, Illinois, May 31–June 3, 2003. Alexandria, VA: American Society of Clinical Oncology (ASCO), 1964.

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98. Janne PA, Gurubhagavatula S, Yeap BY, et al. Outcomes of patients with advanced non-small cell lung cancer treated with gefitinib (ZD1839, “Iressa”) on an expanded access study. Lung Cancer 2004; 44:221–30. 99. Mohamed MK, Ramalingam S, Lin Y, et al. Skin rash and good performance status predict improved survival with gefitinib in patients with advanced non-small cell lung cancer. Ann Oncol 2005; 16:780–5. 100. Lynch TJ, Bell DW, Sordella R, et al. Activating mutations in the epidermal growth factor receptor underlying responsiveness of non-small-cell lung cancer to gefitinib. N Engl J Med 2004; 350:2129–39. 101. Paez JG, Janne PA, Lee JC, et al. EGFR mutations in lung cancer: correlation with clinical response to gefitinib therapy. Science 2004; 304:1497–500. 102. Pao W, Miller V, Zakowski M, et al. EGF receptor gene mutations are common in lung cancers from “never smokers” and are associated with sensitivity of tumors to gefitinib and erlotinib. Proc Natl Acad Sci USA 2004; 101:13306–11. 103. Kobayashi S, Boggon TJ, Dayaram T, et al. EGFR mutation and resistance of nonsmall-cell lung cancer to gefitinib. N Engl J Med 2005; 352:786–92. 104. Pao W, Miller VA, Politi KA, et al. Acquired resistance of lung adenocarcinomas to gefitinib or erlotinib is associated with a second mutation in the EGFR kinase domain. PLoS Med 2005; 2:e73. 105. Pao W, Wang TY, Riely GJ, et al. KRAS mutations and primary resistance of lung adenocarcinomas to gefitinib or erlotinib. PLoS Med 2005; 2:e17. 106. Eberhard DA, Johnson BE, Amler LC, et al. Mutations in the epidermal growth factor receptor and in KRAS are predictive and prognostic indicators in patients with nonsmall-cell lung cancer treated with chemotherapy alone and in combination with erlotinib. J Clin Oncol 2005; 23:5900–9. 107. Shigematsu H, Lin L, Takahashi T, et al. Clinical and biological features associated with epidermal growth factor receptor gene mutations in lung cancers. J Natl Cancer Inst 2005; 97:339–46. 108. Cappuzzo F, Hirsch FR, Rossi E, et al. Epidermal growth factor receptor gene and protein and gefitinib sensitivity in non-small-cell lung cancer. J Natl Cancer Inst 2005; 97:643–55. 109. Bell DW, Lynch TJ, Haserlat SM, et al. Epidermal growth factor receptor mutations and gene amplification in non-small-cell lung cancer: molecular analysis of the IDEAL/INTACT gefitinib trials. J Clin Oncol 2005; 23:8081–92. 110. Kosaka T, Yatabe Y, Endoh H, et al. Mutations of the epidermal growth factor receptor gene in lung cancer: biological and clinical implications. Cancer Res 2004; 64:8919–23. 111. Hirsch FR, Varella-Garcia M, Bunn PA, et al. Epidermal growth factor receptor in non-small-cell lung carcinomas: correlation between gene copy number and protein expression and impact on prognosis. J Clin Oncol 2003; 21:3798–807. 112. Tsao MS, Sakurada A, Cutz JC, et al. Erlotinib in lung cancer—molecular and clinical predictors of outcome. N Engl J Med 2005; 353:133–44. 113. Hirsch FR, Varella-Garcia M, McCoy J, et al. Increased epidermal growth factor receptor gene copy number detected by fluorescence in situ hybridization associates with increased sensitivity to gefitinib in patients with bronchioloalveolar carcinoma subtypes: a Southwest Oncology Group Study. J Clin Oncol 2005; 23:6838–45. 114. Hirsch FR, Varella-Garcia M, Bunn PA, et al. Molecular analysis of EGFR gene copy number, EGFR expression and Akt activation status in advanced non-small-cell lung cancer (aNSCLC) treated with gefitinib or placebo (ISEL trial). AACR-NCI-EORTC; 124 (abstr A268). 2005. 115. Moasser MM, Basso A, Averbuch SD, et al. The tyrosine kinase inhibitor ZD1839 (“Iressa”) inhibits HER2-driven signaling and suppresses the growth of HER2-overexpressing tumor cells. Cancer Res 2001; 61:7184–8. 116. Cappuzzo F, Varella-Garcia M, Shigematsu H, et al. Increased HER2 gene copy number is associated with response to gefitinib therapy in epidermal growth factor receptor-positive non-small-cell lung cancer patients. J Clin Oncol 2005; 23:5007–18.

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117. Cappuzzo F, Magrini E, Ceresoli GL, et al. Akt phosphorylation and gefitinib efficacy in patients with advanced non-small-cell lung cancer. J Natl Cancer Inst 2004; 96:1133–41. 118. Lee JW, Soung YH, Kim SY, et al. Somatic mutations of EGFR gene in squamous cell carcinoma of the head and neck. Clin Cancer Res 2005; 11:2879–82. 119. Cohen EE, Lingen MW, Martin LE, et al. Response of some head and neck cancers to epidermal growth factor receptor tyrosine kinase inhibitors may be linked to mutation of ERBB2 rather than EGFR. Clin Cancer Res 2005; 11:8105–8. 120. Chung KY, Shia J, Kemeny NE, et al. Cetuximab shows activity in colorectal cancer patients with tumors that do not express the epidermal growth factor receptor by immunohistochemistry. J Clin Oncol 2005; 23:1803–10. 121. Burtness B, Goldwasser MA, Flood W, et al. Phase III randomized trial of cisplatin plus placebo compared with cisplatin plus cetuximab in metastatic/recurrent head and neck cancer: an Eastern Cooperative Oncology Group study. J Clin Oncol 2005; 23:8646–54. 122. Chung CH, Ely K, McGavran L, et al. Increased epidermal growth factor receptor gene copy number is associated with poor prognosis in head and neck squamous cell carcinomas. J Clin Oncol 2006; 24:4170–6. 123. Moroni M, Veronese S, Benvenuti S, et al. Gene copy number for epidermal growth factor receptor (EGFR) and clinical response to antiEGFR treatment in colorectal cancer: a cohort study. Lancet Oncol 2005; 6:279–86. 124. Liu W, Innocenti F, Chen P, et al. Interethnic difference in the allelic distribution of human epidermal growth factor receptor intron 1 polymorphism. Clin Cancer Res 2003; 9:1009–12. 125. Amador ML, Oppenheimer D, Perea S, et al. An epidermal growth factor receptor intron 1 polymorphism mediates response to epidermal growth factor receptor inhibitors. Cancer Res 2004; 64:9139–43.

3

HER2 Inhibition and Clinical Achievements Toru Mukohara Division of Hematology and Oncology, Research Center for Innovative Oncology, National Cancer Center Hospital East, Kashiwa, Japan

Pasi A. Jänne Lowe Center for Thoracic Oncology, Department of Medical Oncology, Dana Farber Cancer Institute, Boston, Massachusetts, U.S.A.

PATHOPHYSIOLOGY OF HER2 IN BREAST CANCER The ErbB family consists of HER1 (also called epidermal growth factor), HER2, HER3, and HER4. With the exception of for HER2, each family member has cognate identified ligands. Upon ligand binding to the extra-cellular domain, the receptor forms homo- or hetero-dimers with another family member, which is required for its kinase activation. Each dimer can trigger various intra-cellular signaling pathways, including PI3K/Akt, Ras/Raf/MEK/ERK, and STATs pathways, each of which plays an important role in cellular oncogenic properties, such as proliferation, survival, motility, and angiogenesis. It has been suggested that under conditions of overexpression, HER2 can be constitutively active and be able to transform NIH 3T3 cells in the absence of ligand (1). While, theoretically, HER2 could form four different types of dimers (HER2/HER1, HER2, HER3 or HER4), the HER2/HER3 heterodimer is thought to be the most mitogenic and transforming receptor complex (2–5). HER2 in Pathogenesis of Breast Cancer HER2 expression contributes to carcinogenesis of breast cancer and a series of experimental studies have demonstrated that HER2 transfection into mammary epithelial cells induces oncogenic transformation (6). Emphasizing the importance of HER2/HER3 hetero-dimers as suggested above, it was reported that HER2 requires HER3 to drive breast cancer cell proliferation (7). In conjunction with that, another study suggested that breast cancer cell lines that express both HER2 and HER3 appear to have a higher degree of phosphorylation of Akt (8). HER2 is overexpressed in 20–25% of breast cancers and shown to be associated with a poor disease-free survival and resistance to some chemotherapeutic drugs (9,10). Gene amplification is considered the main mechanism leading to HER2 protein overexpression. HER2 Measurement in Breast Cancer With the clinical development of anti-HER2 monoclonal antibodies such as trastuzumab (Herceptin
; Genentech, Inc., South San Francisco, California, U.S.A.), methods for evaluating level of HER2 have also evolved. Presently, two different strategies are used clinically. They are immunohistochemistry (IHC), which is used to determine HER2 protein expression, and fluorescence in situ hybridization (FISH), used to determine HER2 copy number. IHC is a semiquantitative method to detect HER2 protein levels on the cell surface in breast cancer specimens. 45

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IHC scoring is generally performed subjectively as 0, 1þ, 2þ, or 3þ, based on the percentage of malignant cells stained and the intensity of membrane staining in these malignant cells. Two types of IHC system to determine HER2 status are approved by the U.S. Food and Drug Administration (FDA): HercepTest
(Dako,  Carpinteria, California, U.S.A.) and PATHWAY (Ventana Medical Systems, Inc., Tucson, Arizona, U.S.A.). FISH is a DNA based assay that directly measures HER2 gene amplification. FISH testing results are semiquantitative, based on the average ratio of HER2 signals to CEP17 (a probe against the chromosome 17 centromeric sequences) signals in nonoverlapping interphase nuclei of the lesion. Three types of FISH testing that determine HER2 copy number are approved by the U.S. FDA: PathVysion
HER2 DNA-probe kit (Vysis, Inc., Downers Grove, Illinois, U.S.A.; where a
2.0 ratio HER2:CEP17 is considered amplified), INFORM
HER2/Neu probe, (Ventana Medical Systems, Inc; where a
5.0 gene copies of HER2 is considered amplified), and HER2 FISH pharmDx
(Dako, Carpinteria, California, U.S.A.; where a
2.0 ratio HER2/CEN-17 is considered amplified). MECHANISMS OF ACTION FOR HER2-TARGETED AGENTS At the present time, trastuzumab is the only HER2-targeted agent that is U.S. FDA approved for clinical use. Many other agents targeting HER2 are under preclinical or clinical development and are listed in Table 1. In this chapter, however, we discuss the mechanisms of action mainly for trastuzumab, which has been most extensively studied clinically. The mechanisms of action for trastuzumab can be roughly divided into two components: inhibition of intracellular signaling, and induction of an immune mediated antitumor response. HER2 is known to trigger multiple signaling pathways, and thus inhibition of HER2 should result in inactivation of those pathways. Although it is not fully understood how trastuzumab inhibits HER2 activity, some studies suggested that trastuzumab might promote internalization and degradation of HER2 (11,12). This effect results in downregulation of signaling pathways, such as the PI3K/AKT and ERK pathways, and subsequent effects on breast cancer cell growth. One important response is cell cycle arrest at the G1-S boundary, which is often accompanied by an increase in p27 level, a decrease in cyclin D1, and a decrease in CDK2 activity (13,14). Another important cellular response is the promotion of cell death, as reflected by enhancement of cytotoxic effect of chemotherapeutic agents (15). A series of in vitro studies demonstrated the synergistic effects of trastuzumab when combined with chemotherapeutic agents, thus forming the basis for the clinical studies combining trastuzumab with chemotherapy (15). The synergistic effect may be explained by inhibition of the PI3K/Akt signaling pathway, which normally promotes cell survival. Data from several in vivo experiments have indicated that trastutumab can mediate the induction of immune-responses, such as antibody-dependent cellular cytotoxicity (ADCC) and complement dependent cytotoxicity (16). ADCC is the immune-response, in which the natural killer (NK) cells expressing Fc gamma receptor recognize and bind to Fc domain of the IgG1 antibody (trastuzumab) and subsequently lyse (tumor) cells attached by the antibody. The most convincing evidence of the contribution of ADCC to trastuzumab-induced antitumor activity is in mice lacking the FC receptor (FcR–/–) where trastuzumab treatment results in a significantly lower tumor regression rate than when trastuzumab is administered in FC receptor expressing mice (17). Although it is more difficult to directly prove

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HER2 Inhibition and Clinical Achievements TABLE 1 Summary of HER2 Targeted Agents in Development Agent

Type of agent and target

Clinical phase of development

Pertuzumab

mAb; HER2

I

CI-1033

TKI; EGFR, HER2

II

Lapatinib

TKI; EGFR, HER2

III

2B1

Bispecific antibody against HER2 and Fc RIII Bispecific antibody against HER2 and Fc RI mAb–toxin; HER2 Recombinant vaccine

II

MDX-H210 Trastuzumab–DM1 HER2/neu recombinant therapeutic vaccine

II Preclinical I

Manufacturer Genentech (San Francisco, California, U.S.A.) Pfizer (New York, New York, U.S.A.) GlaxoSmithKline (Research Triangle Park, North Carolina, U.S.A.) Chiron Corp. (Emeryville, California, U.S.A.) Medarex, Inc. (Annandale, New Jersey, U.S.A.) Genentech Corixa Corp. (Seattle, Washington, U.S.A.) and GlaxoSmithKline

Abbreviations: EGFR, epidermal growth factor receptor; Fc RI, type I Fc receptors for IgG (Fc gamma RI); Fc RIII, type III Fc receptors for IgG (Fc gamma RIII); HER2, human epidermal growth factor receptor 2; IGF1R KI, insulin-like growth factor 1 receptor kinase inhibitor; mAb, monoclonal antibody; mTORi, mammalian target of rapamycin inhibitor; TKI, tyrosine kinase inhibitor.

the role of ADCC in breast cancer patients receiving treatment with trastuzumab, Gennari et al. (18) found an increase in infiltration by lymphoid cells in tumor samples collected following trastuzumab treatment compared to pre-treatment tumor specimens. MECHANISMS OF RESISTANCE TO HER2-TARGETED AGENTS Several potential mechanisms of resistance to trastuzumab have been identified. In preclinical models, mechanisms where interaction between trastuzumab and HER2 is interfered have been proposed. These include the production of a truncated form of HER2, leading to HER2 protein lacking the extracellular domain, and thus unable to be inhibited by trastuzumab (19), and the presence of mucin-4 (MUC4) glycoprotein, leading to masking of HER2 so as to prevent it from binding to trastuzumab (20). Alternative signaling through other receptor tyrosine kinase, in particular insulin-like growth factor 1 receptor (IGF1-R), is also suggested to cause resistance in preclinical models (21). Furthermore, Nagata et al. (22) recently reported that patients whose breast cancers lacked PTEN had significantly poorer responses to trastuzumab-based therapy than those with retained PTEN. These resistance mechanisms all provide a means to maintain persistent HER2 or downstream signaling in the presence of trastuzumab. Strategies aimed at overcoming these different mechanisms of resistance are currently being evaluated in ongoing preclinical studies. CLINICAL ACHIEVEMENT OF HER2-TARGETD AGENTS IN BREAST CANCER Trastuzumab in Metastatic Breast Cancer Clinical development of trastuzumab initially occurred in the treatment for metastatic breast cancers in mid-1990s. The first pivotal phase III clinical trial was

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completed by a multinational group, and the results were published in 2001 (23). In this trial, standard chemotherapy alone as a reference arm was compared with standard chemotherapy in combination with trastuzumab in the experimental arm. Patients were treated with doxorubicin or epirubicin and cyclophosphamide (AC or EC) with (n ¼ 143) or without trastuzumab (n ¼ 138), if the patient had not previously received adjuvant therapy with an anthracycline. Patients who had previously received adjuvant anthracycline were treated with paclitaxel alone (n ¼ 96) or paclitaxel with trastuzumab (n ¼ 92). The trastuzumab and chemotherapy arm was superior to chemotherapy alone in virtually all efficacy parameters assessed: time-to-disease progression (median, 7.4 vs. 4.6 mo; p < 0.001); rate of objective response (50% vs. 32%, p < 0.001); duration of response (median, 9.1 vs. 6.1 mo; p < 0.001); rate of death at 1 year (22% vs. 33%, p ¼ 0.008); survival (median survival, 25.1 vs. 20.3 mo; p ¼ 0.046). The survival advantage produced by addition of trastuzumab was projected as a 20% reduction in the risk of death. A notable adverse event was cardiac dysfunction. The addition of trastuzumab increased the incidence of cardiotoxicity regardless of chemotherapeutic agents (AC or EC vs. AC or EC/trastuzumab, 8% vs. 27%; paclitaxel paclitaxel/trastuzumab, 1% vs. 13%). This data raised the concerns about concomitant use of trastuzumab with an anthracycline with overlapping cardiotoxicity from both drugs. Subsequently, results of several phase II trials evaluating efficacy of trasutzumab and vinorelbine were reported. In a multicenter phase II study (n ¼ 44), the overall response rate was 68% [95% confidence interval (CI), 54–80%] when patients with HER2-positive (IHC 3þ or FISH positive) metastatic breast cancer were treated with trastuzumab in combination with vinorelbine as first-line chemotherapy (24). Median time-to-treatment failure was 5.6 months, and 38% of patients were progression free after one year (24). A separate phase-II trial of trastuzumab and vinorelbine as a first line chemotherapy for metastatic breast cancer (n ¼ 40) also produced a promising objective response rate: 78% (95% CI, 62–90%) (25). Median time-to-progression was 18 months (95% CI 9.2–34.5 months) (25). Although neither phase II trial was a comparative study, combined treatment appeared to be more active than either agent alone when compared to historic response rates: 16–40% for vinorelbine, and 15–30% for trastuzumab. Because of these favorable results of clinical trials, taxanes and vinorelbine are the agents that are often used in combination with trastuzumab for the treatment of metastatic breast cancers. Other trastuzumab-containing combination regimens are currently under investigation (26). Trastuzumab for Adjuvant Breast Cancer In 2005, two important publications describing the use of trastuzumab in adjuvant treatment for operable breast cancer were published in the same issue of the New England Journal of Medicine. The first publication reported the outcome of an international, multicenter, randomized trial designated as HERA (HERceptin Adjuvant) study. The HERA study compared one or two years of trastuzumab given every three weeks with observation in patients with HER2-positive and either node-negative or nodepositive breast cancer who had completed locoregional therapy and at least four cycles of neoadjuvant or adjuvant chemotherapy (27). In this initial report, results only of treatment with trastuzumab for one year compared to those of observation

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were published. In total, 5,081 patients were enrolled in this study, 1,694 women were randomly assigned to two years of treatment with trastuzumab, 1,694 women were assigned to one year of trastuzumab, and 1,693 women were assigned to observation. At the first planned interim analysis (median follow-up of one year), 347 events (recurrence of breast cancer, contralateral breast cancer, second nonbreast malignant disease, or death) were observed: 127 events in the trastuzumab group, and 220 in the observation group. The unadjusted hazard ratio for an event in the trastuzumab group, as compared with the observation group, was 0.54 (95% CI, 0.43–0.67; p < 0.0001 by the log-rank test, crossing the interim analysis boundary), representing an absolute benefit in terms of diseasefree survival at two years of 8.4%. Although overall survival in the two groups was not significantly different at the point of interim analysis (27), survival curves from each group may ultimately separate with further follow-up. The other study was a combined analysis of two similar phase III trials that were originally conducted independently. One of them was conducted by the National Surgical Adjuvant Breast and Bowel Project (NSABBP) designated as B-31. The B-31 trial compared AC followed by paclitaxel every three weeks (group 1) as a reference arm with the same regimen plus 52 weeks of trastuzumab beginning with the first dose of paclitaxel (group 2) (Fig. 1). The other trial was conducted by the North Central Cancer Treatment Group, designated as N9831. This trial compared AC followed by weekly paclitaxel (group A) as a reference arm with two experimental arms: AC followed by 52 weeks of trastuzumab after paclitaxel (group B); AC plus 52 weeks of trastuzumab initiated concomitantly with paclitaxel (group C). The two studies were amended to include a joint analysis comparing groups 1 and A (the control) with groups 2 and C (the trastuzumab group). At the point of the first interim analysis planned, 394 events (recurrent, second primary cancer, or death before recurrence) had been reported. Of these, 133 were in the trastuzumab group and 261 in the control group (hazard ratio, 0.48; p < 0.0001). This result crossed the early stopping boundary. The absolute difference in disease-free survival between the trastuzumab group and the control group was 12% at 3 years. Trastuzumab therapy was associated with a 33% reduction in the risk of death (p ¼ 0.015). The three-year cumulative incidence of class III or IV congestive heart failure or death from cardiac causes in the trastuzumab group was 4.1% in trial B-31 and 2.9% in trial N9831 (28). More recently, Joensuu et al. compared docetaxel with vinorelbine followed by (in both groups) three cycles of fluorouracil, epirubicin, and cyclophosphamide (FEC) for the adjuvant treatment of early breast cancer, in which they separately assigned women with tumors overexpressing HER2 to receive concomitant treatment with or without trastuzumab. Of 1,010 patients in the study, 232 women whose tumors contained a HER2 gene amplification were further assigned to receive or not to receive nine weekly trastuzumab treatments. The primary end point of the study was recurrence-free survival. In entire population, recurrencefree survival at three years was better with docetaxel than with vinorelbine (91% vs. 86%; hazard ratio for recurrence or death, 0.58; 95% CI, 0.40–0.85; p ¼ 0.005), but overall survival did not differ between groups (p ¼ 0.15). Within the subgroup of patients who had HER2-positive cancer, patients who received trastuzumab concomitantly with docetaxel or vinorelbine had better three-year recurrence-free survival than those who did not receive the antibody (89% vs. 78%; hazard ratio for recurrence or death, 0.42; 95% CI, 0.21–0.83; p ¼ 0.01). This data suggested that even a short period (nine weeks as opposed to one year in the prior studies) of

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NSABP B-31

AC NCCTG N9831 Paclitaxel Trastuzumab

FIGURE 1 Study designs for two adjuvant trastuzumab clinical trials (NSABP B-31 and NCCTG N9831). In both studies, chemotherapy [consisting of Adriamycin
(doxorabicin) Cyclophosphamide (AC)] followed by paclitaxel is administered either prior to or concurrently (paclitaxel only) with trastuzumab.

trastuzumab treatment is associated with difference in outcome of operable breast cancer who have an amplified HER2 gene (29). The issue of how long trastuzumab should be given in an adjuvant setting will be partly answered with future analysis of HERA study, comparing one year with two years of trastutuzumab treatment. Trastuzumab Use in Neoadjuvant Therapy Preoperative (neoadjuvant) chemotherapy is considered a standard treatment approach for certain subsets of operable breast cancers. The neoadjuvant administration of chemotherapy was shown to result in survival outcome similar to that of postoperative (adjuvant) chemotherapy (30). There are several potential advantages of neoadjuvant chemotherapy compared to adjuvant chemotherapy. The first is clinical downstaging of bulky tumors with treatment allowing less extensive surgery (31). A further advantage is that one is able to visibly evaluate the effect of chemotherapy. In addition, pathologic survey of the response to the chemotherapy on the surgically excised tumor provides strong prediction for recurrence. Patients with a pathologic complete response (pCR) have the most favorable outcomes as measured by disease-free survival and overall survival (32). Thus, applying the best conceivable chemotherapy regimen that could produce the highest pCR is thus a reasonable treatment strategy. Given that, the use of trastuzumab in combination with chemotherapy in neoadjuvant setting is currently being vigorously investigated. In the early trials of this treatment strategy, trastuzumab was mainly combined with taxanes, while anthracycline-based chemotherapies were moved out to adjuvant treatment with the potential concern about cardio-toxicity (33). However, recently reported results of a phase III clinical trial by a group from the M. D. Anderson Cancer Center may change the paradigm. In this trial, they compared paclitaxel followed by fluorouracil, epirubicine, and cyclophosphamide (CEF) as a reference arm with the same regimen

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combined with trastuzumab (34). The primary endpoint of this study was pCR. At the interim analysis after 34 patients had completed therapy, pCR rates were 25% and 66.7% for chemotherapy (n ¼ 16) and trastuzumab plus chemotherapy (n ¼ 18), respectively (p ¼ 0.02). Based on the result, the trial was early terminated. Because of the early termination, the concerns about cardiac toxicity when trastuzumab combined with anthracyclines have not been fully answered, even though no clinical congestive heart failure was observed in the patients participating in this trial (35,36). This issue should be elucidated by future clinical trials. The combination of trastuzumab and vinorelbine in the neo-adjuvant setting is also being investigated. Lapatinib for Trastuzumab Resistance Most if not all patients with HER2 overexpressing or amplified breast cancer ultimately develop clinical resistance to trastuzumab based therapy. The mechanism(s) of such acquired resistance are presently not clear. However, it is possible that in some patients the tumor remains dependent on HER2 for its growth and thus an alternative HER2 inhibitor may be clinically effective. Lapatinib (Tykerb
; GlaxoSmithKline, Research Triangle Park, North Carolina, U.S.A.) is a dual HER1/HER2 tyrosine kinase inhibitor (Table 1) (37). In pre-clinical models of trastuzumab resistance, lapatinib was still able to inhibit the growth of HER2 over expressing breast cancer cell lines that were selected for in vitro resistance to trastuzumab (38). To test the potential benefits of lapatinib in patients with acquired resistance to trastuzumab, Geyer and colleagues randomized 324 HER2 positive (3þ on IHC or 2þ on FISH) patients with recurrent/metastatic breast cancer to receive either capecitabine (n ¼ 161) alone or in combination with lapatinib (n ¼ 163) (39). All patients had previously received therapy with an anthracycline, taxane and trastuzumab. The primary end-point was disease progression. The median time-to-progression was superior in the lapatinib containing arm (8.4 vs. 4.4 months; p < 0.001). The overall response rate was also higher in the lapatinib containing arm (22% vs. 14%; p ¼ 0.09). Data on overall survival is not mature at this point. There were no significant differences in cardiac toxicity. Future studies will need to focus on studying the mechanisms of resistance to trastuzumab. It is possible that subsets of trastuzumab resistant patients are more or less likely to derive clinical benefit from lapatinib. In addition, since many other HER2 targeted therapies are in clinical development (Table 1), it will be important to determine whether these are effective in the same or different populations of patients who have developed trastuzumab resistance as lapatinib. REFERENCES 1. 2. 3. 4.

Lonardo F, Di Marco E, King CR, et al. The normal erbB-2 product is an atypical receptor-like tyrosine kinase with constitutive activity in the absence of ligand. New Biol 1990; 2:992–1003. Wallasch C, Weiss FU, Niederfellner G, Jallal B, Issing W, Ullrich A. Heregulindependent regulation of HER2/neu oncogenic signaling by heterodimerization with HER3. EMBO J 1995; 14:4267–75. Zhang K, Sun J, Liu N, et al. Transformation of NIH 3T3 cells by HER3 or HER4 receptors requires the presence of HER1 or HER2. J Biol Chem 1996; 271:3884–90. Pinkas-Kramarski R, Lenferink AE, Bacus SS, et al. The oncogenic ErbB-2/ErbB-3 heterodimer is a surrogate receptor of the epidermal growth factor and betacellulin. Oncogene 1998; 16:1249–58.

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Alimandi M, Romano A, Curia MC, et al. Cooperative signaling of ErbB3 and ErbB2 in neoplastic transformation and human mammary carcinomas. Oncogene 1995; 10: 1813–21. Pierce JH, Arnstein P, DiMarco E, et al. Oncogenic potential of erbB-2 in human mammary epithelial cells. Oncogene 1991; 6:1189–94. Holbro T, Beerli RR, Maurer F, Koziczak M, Barbas CF,III,, Hynes NE. The ErbB2/ ErbB3 heterodimer functions as an oncogenic unit: ErbB2 requires ErbB3 to drive breast tumor cell proliferation. Proc Natl Acad Sci USA 2003; 100:8933–8. Knuefermann C, Lu Y, Liu B, et al. HER2/PI-3K/Akt activation leads to a multidrug resistance in human breast adenocarcinoma cells. Oncogene 2003; 22:3205–12. Slamon DJ, Godolphin W, Jones LA, et al. Studies of the HER-2/neu proto-oncogene in human breast and ovarian cancer. Science 1989; 244:707–12. Slamon DJ, Clark GM, Wong SG, Levin WJ, Ullrich A, McGuire WL. Human breast cancer: correlation of relapse and survival with amplification of the HER-2/neu oncogene. Science 1987; 235:177–82. Menard S, Pupa SM, Campiglio M, Tagliabue E. Biologic and therapeutic role of HER2 in cancer. Oncogene 2003; 22:6570–78. Rubin I, Yarden Y. The basic biology of HER2. Ann Oncol 2001; 12(Suppl 1):S3–8. Le XF, Pruefer F, Bast RC, Jr. HER2-targeting antibodies modulate the cyclindependent kinase inhibitor p27Kip1 via multiple signaling pathways. Cell Cycle 2005; 4:87–95. Lane HA, Beuvink I, Motoyama AB, Daly JM, Neve RM, Hynes NE. ErbB2 potentiates breast tumor proliferation through modulation of p27(Kip1)-Cdk2 complex formation: receptor overexpression does not determine growth dependency. Mol Cell Biol 2000; 20:3210–23. Pegram MD, Konecny GE, O'Callaghan C, Beryt M, Pietras R, Slamon DJ. Rational combinations of trastuzumab with chemotherapeutic drugs used in the treatment of breast cancer. J Natl Cancer Inst 2004; 96:739–49. Hynes NE, Lane HA. ERBB receptors and cancer: the complexity of targeted inhibitors. Nat Rev Cancer 2005; 5:341–54. Clynes RA, Towers TL, Presta LG, and Ravetch JV. Inhibitory Fc receptors modulate in vivo cytoxicity against tumor targets. Nat Med 2000; 6:443–6. Gennari R, Menard S, Fagnoni F, et al. Pilot study of the mechanism of action of preoperative trastuzumab in patients with primary operable breast tumors overexpressing HER2. Clin Cancer Res 2004; 10:5650–5. Scott GK, Robles R, Park JW, et al. A truncated intracellular HER2/neu receptor produced by alternative RNA processing affects growth of human carcinoma cells. Mol Cell Biol 1993; 13:2247–57. Nagy P, Friedlander E, Tanner M, et al. Decreased accessibility and lack of activation of ErbB2 in JIMT-1, a herceptin-resistant, MUC4-expressing breast cancer cell line. Cancer Res 2005; 65:473–82. Lu Y, Zi X, Zhao Y, Mascarenhas D, and Pollak M. Insulin-like growth factor-I receptor signaling and resistance to trastuzumab (Herceptin). J Natl Cancer Inst 2001; 93: 1852–7. Nagata Y, Lan KH, Zhou X, et al. PTEN activation contributes to tumor inhibition by trastuzumab, and loss of PTEN predicts trastuzumab resistance in patients. Cancer Cell 2004; 6:117–27. Slamon DJ, Leyland-Jones B, Shak S, et al. Use of chemotherapy plus a monoclonal antibody against HER2 for metastatic breast cancer that overexpresses HER2. N Engl J Med 2001; 344:783–92. Burstein HJ, Harris LN, Marcom PK, et al. Trastuzumab and vinorelbine as first-line therapy for HER2-overexpressing metastatic breast cancer: multicenter phase II trial with clinical outcomes, analysis of serum tumor markers as predictive factors, and cardiac surveillance algorithm. J Clin Oncol 2003; 21:2889–95. Jahanzeb M, Mortimer JE, Yunus F, et al. Phase II trial of weekly vinorelbine and trastuzumab as first-line therapy in patients with HER2(þ) metastatic breast cancer. Oncologist 2002; 7:410–17.

6. 7. 8. 9. 10. 11. 12. 13. 14.

15. 16. 17. 18. 19. 20. 21. 22. 23. 24.

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26. 27. 28. 29. 30. 31. 32. 33. 34.

35. 36. 37.

38. 39.

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Jackisch C. HER-2-positive metastatic breast cancer: optimizing trastuzumab-based therapy. Oncologist 2006; 11(Suppl 1):34–41. Piccart-Gebhart MJ, Procter M, Leyland-Jones B, et al. Trastuzumab after adjuvant chemotherapy in HER2-positive breast cancer. N Engl J Med 2005; 353:1659–72. Romond EH, Perez EA, Bryant J, et al. Trastuzumab plus adjuvant chemotherapy for operable HER2-positive breast cancer. N Engl J Med 2005; 353:1673–84. Joensuu H, Kellokumpu-Lehtinen PL, Bono P, et al. Adjuvant docetaxel or vinorelbine with or without trastuzumab for breast cancer. N Engl J Med 2006; 354:809–20. Fisher B, Bryant J, Wolmark N, et al. Effect of preoperative chemotherapy on the outcome of women with operable breast cancer. J Clin Oncol 1998; 16:2672–85. Kaufmann M, von Minckwitz G, Smith R, et al. International expert panel on the use of primary (preoperative) systemic treatment of operable breast cancer: review and recommendations. J Clin Oncol 2003; 21:2600–8. Kuerer HM, Newman LA, Smith TL, et al. Clinical course of breast cancer patients with complete pathologic primary tumor and axillary lymph node response to doxorubicin-based neoadjuvant chemotherapy. J Clin Oncol 1999; 17:460–9. Montemurro F, Aglietta M. Incorporating trastuzumab into the neoadjuvant treatment of HER2-overexpressing breast cancer. Clin Breast Cancer 2005; 6:77–80. Buzdar AU, Ibrahim NK, Francis D, et al. Significantly higher pathologic complete remission rate after neoadjuvant therapy with trastuzumab, paclitaxel, and epirubicin chemotherapy: results of a randomized trial in human epidermal growth factor receptor 2-positive operable breast cancer. J Clin Oncol 2005; 23:3676–85. Ahluwalia MS, Daw HA. Neoadjuvant therapy with trastuzumab, paclitaxel and epirubicin for HER-2-positive operable breast cancer. J Clin Oncol 2005; 23:7759–60; author reply 7760–51. Puglisi F, de Azambuja E, de Castro G, Jr, and Demonty G. Shrinking the tumor, shrinking the patient sample size: the early disclosure dilemma. J Clin Oncol 2005; 23:6803–4; author reply 6804–5. Rusnak DW, Lackey K, Affleck K, et al. The effects of the novel, reversible epidermal growth factor receptor/ErbB-2 tyrosine kinase inhibitor, GW2016, on the growth of human normal and tumor-derived cell lines in vitro and in vivo. Mol Cancer Ther 2001; 1:85–94. Konecny GE, Pegram MD, Venkatesan N, et al. Activity of the dual kinase inhibitor lapatinib (GW572016) against HER-2-overexpressing and trastuzumab-treated breast cancer cells. Cancer Res 2006; 66:1630–9. Geyer CE, Forster J, Lindquist D, et al. Lapatinib plus capecitabine for HER2-positive advanced breast cancer. N Engl J Med 2006; 355:2733–43.

4

Pan-HER Inhibitors Gérard Milano Laboratoire d'Oncopharmacologie, du Centre Antoine Lacassagne, Nice, France

INTRODUCTION Despite its long conceptual history, anticancer therapy targeting the erbB family of receptor kinases became clinically possible in recent years only. The current array of HER family inhibitors includes agents targeting single HER members, such as HER1 (epidermal growth factor receptor, EGFR) or HER2 with monoclonal antibodies and tyrosine kinase inhibitors and also compound with dual activity able to interfere with both HER1 and HER2 such as lapatinib (1–3). The so-called pan-HER inhibitors belong to the family of tyrosine kinase inhibitors which are able to inhibit with a comparable activity more than two members of the HER family. One of the more advanced agents in the domain of pan-HER inhibitor is CI-1033 (carnetinib dihydrochloride). This is an irreversible ATP-binding sitedirected inhibitor of erbB1, erbB2 and erbB4 tyrosine kinases with IC50 values in the low nanomolar range (4). This chapter will cover general aspects of HER1–4 expression in tumors and will focus on the current clinical development of dual HER inhibitors and pan-HER inhibitors with their limits and perspectives. ErbB RECEPTORS AND THEIR LIGANDS IN CANCER Subclass I of the receptor tyrosine kinase superfamily includes the erbB or EGFRs with four members: erbB1/EGFR, erbB2, erbB3, and erbB4 (1,5). They share common characteristics with an extracellular domain including two cysteine-rich regions, a single transmembrane region and an intracytoplasmic tyrosine-kinasecontaining domain with tyrosine autophosphorylation sites. Of note, erbB3 is devoid of intrinsic kinase activity (6) whereas erbB2 has no direct ligand (7). Interreceptor interactions are produced by ligands and erbB2-containing heterodimers are mostly formed (8). Activated erbB receptors stimulate intracellular signaling pathways with the mitogen-activated protein kinase (MAPK) and the phosphatidylinositol 3-kinase (PI3K)-AKT pathways. However, different receptors modulate down-stream pathways due to the capacity of individual erbB members to bind specific effector proteins. For example, unlike erbB1, the kinase-defective erbB3 cannot bind the adaptor protein and ubiquitin ligase c-Cbl, the adaptor protein Grb2, the second-messenger-generating enzyme phospholipase Cg or the Rasspecific GTPase-activating protein. However, it can interact with adaptors Shc and Grb7 (9). Of note, although neither erbB2 nor erbB3 alone can be activated by interaction with a ligand, the heterodimer erbB2–erbB3 is a particularly transforming and mitogenic receptor complex (10,11). Overexpression of erbB members has been found in various tumors including breast, lung, head and neck, ovary, gastrointestinal tract, and brain (Table 1). In cancer, there are several mechanisms activating the erbB pathways. These mechanisms may include overproduction of ligands, overproduction of receptors 55

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TABLE 1 ErbB Receptor Expression in Human Solid Tumors Tumor type

ErbB family expression, overexpression, or mutation

Breast

erbB1, erbB2, erbB3, erbB4

Colorectal

erbB1, erbB2, erbB3, erbB4

Head and neck

erbB1, erbB2, erbB3, erbB4

Gastric

erbB1, erbB2, erbB3, erbB4

Glioma

erbB1, erbB2

Non–small cell lung cancer

erbB1, erbB2, erbB3, erbB4

Ovarian Pancreatic

erbB1, erbB2, erbB3 erbB1, erbB2, erbB3

Prostate

erbB1, erbB2, erbB3

Tumors expressing receptor (%) erbB1 erbB2 erbB3 erbB4 erbB1 erbB2 erbB3 erbB1 erbB2 erbB3 erbB4 erbB1 erbB2 erbB3 erbB1 erbB2 erbB1 erbB2 erbB1 erbB1 erbB3 erbB1 erbB2 erbB3

37 25 25–71 49–60 25–100 54–100 28–89 47 29 21 26 33–81 38 35 86–92 54 40–81 30–35 35–70 30–50 47 40–89 34 54

Source: From Refs. 13,14.

or constitutive activation of receptors. The overproduction of ligands can be due to secondary induction by a primary oncogene; the stroma of the tumor can be responsible for ligand production; the ligand can be also expressed but remains unprocessed or sequestered in an inactive form. Many cancers have been shown to overexpress different members of the erbB receptor family and erbB signaling may be receptor-specific. Also, during the course of tumor progression, aberrantly activated erbB receptors may contribute to the major molecular events frequently reported in prostate cancer (12). This background sustains the development of pan-HER inhibitors. DUAL-HER INHIBITORS Lapatinib Among several candidates, lapatinib (GlaxoSmithKline, Brentford, Middlesex, U.K.) is the dual-HER inhibitor which is currently the most advanced in terms of clinical development. The rationale for development of this dual EGFR/HER2 tyrosine kinase inhibitor is mainly sustained by a synergistic inhibition of cancer cell growth which has been demonstrated with simultaneous targeting of EGFR and HER2, resulting in more potent inhibition in cell growth compared with targeting either EGFR or HER alone (15). For this compound, the IC50 values against purified EGFR and HER2 are of 10.2 and 9.8 nM, respectively (16). Lapatinib was found to have a slower dissociation rate from EGFR than the EGFR-selective tyrosine kinase

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inhibitors erlotinib and gefitinib, resulting in a greater duration of effect at the target site than that seen with either erlotinib or gefitinib (17). Interestingly, it has been demonstrated that lapatinib plus trastuzumab mediated supra-additive growth inhibition in breast cancer cell lines (18). It was shown that lapatinib retained significant activity against cell lines that were exposed long-term to trastuzumab. This preclinical context represents a potential new therapeutic strategy for patients with resistance to trastuzumab. In phase I trials, lapatinib was investigated as a non-first-line therapy in a small number of patients with a variety of tumors. Complete or partial responses were noted in 2–16% of subjects (19). Although the small sample sizes and variety of tumors do not allow conclusions regarding efficacy to be drawn, these trials do demonstrate lapatinib's chemotherapeutic activity. An analysis of dose and concentration relationships with response indicated that the majority of responders were receiving 1200 mg of lapatinib per day, orally, and had a serum trough concentration of 0.3–0.6 mg/mL (20). Findings from this study indicated that patients with higher pretreatment expression of both HER2 and activated HER2 (p-erbB2) correlated with breast cancer patients achieving a partial response. In addition, inhibition of activated EGFR after 21 days of therapy correlated with a partial response. A phase I study involving only patients with metastatic breast cancer examined the effects of lapatinib in combination with trastuzumab (21). A complete or partial response was noted in 22% of subjects and stable disease in 37%. Evidence of lapatinib's activity obtained during these phase I clinical trials has prompted the initiation of several phase II and III clinical trials. Phase II clinical trials investigated the safety and efficacy of lapatinib as monotherapy for metastatic breast cancer refractory to trastuzumab, anthracyclines, taxanes, and capecitabine (22). Efficacy data on the first 81 patients with metastatic breast cancer refractory to trastuzumab combined from both studies were recently presented. Investigators reported that around 10% of patients had a complete or partial response. Additional clinical trials are in progress to assess lapatinib activity in patients with a variety of solid tumors. Of note in advanced breast cancer is the phase II trial of lapatinib in HERþ breast cancer patients with brain metastases (23). Lapatinib was well tolerated at the dose of 750 mg p.os bid and there was evidence of clinical effects. The most frequently reported, drug-related adverse effects over a daily dosage range of 500–1600 mg were diarrhea (42%), rash (31%), nausea (13%) and fatigue (10%) (19). The majority of these were either grade 1 or 2. In all, data from phase I and II trials indicate that lapatinib is safe and generally well-tolerated at daily doses up to 1800 mg, with grade 1 and 2 diarrhea and rash being the most commonly reported, drug-related adverse events. The relative positioning of lapatinib versus trastuzumab is a focal question in current clinical trials of lapatinib in breast cancer. AEE788 AEE788 (Novartis, Basel, Switzerland) is a 7H-pyrrolo [2,3-d] pyrimidine-class receptor tyrosine kinase inhibitor that inhibits EGFR and HER2 at nM range with respective IC50 values for tyrosine kinases at 2 nM and 6 nM (24). AEE788 have shown antiproliferative activity against a range of EGFR and HER2-overexpressing cell lines. A potent antitumor activity was reported in various animal models of cancer overexpressing EGFR and HER2 (24). Of note, the oral administration of AEE788 to tumor-bearing mice was followed by a high and persistent level of the

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drug in the tumor. Because of extended inhibition of AEE788 on KDR (CI50 at 77 nM) and Flt-1 (CI50 at 50 nM), the compound was able to inhibit VEGF-induced angiogenesis in a murine implant model (24). Combined AEE788 with mTOR inhibitor RAD001 given orally to athymic mice bearing established human malignant glioma tumor xenografts gave rise to supra-additive antitumor effects (25). Mice with orthotopic grafts of HT29 human colon cancer cells and treated with a combination of AEE788 and CPT-11 had significantly smaller tumors and complete inhibition of lymph node metastasis (26). AEE788 is currently undergoing phase I clinical trials. Pan-HER INHIBITORS Natural Inhibitors Yu et al. (27) have previously isolated a cDNA clone from the rat gastroduodenum showing more than 80% homology to the external domain of the rat EGFR. The authors referred to the product of the newly isolated cDNA as EGFR-related protein (ERRP). Interestingly, transfection of ERRP cDNA into the colon cancer cell line HCT116 resulted in a marked reduction in proliferation and colony formation as compared with the vector-transfected controls (27). Using a tetracycline-regulated promoter system, the authors reported, in another cancer colon cancer cell line, Caco-2, that the induction of ERRP expression was associated with a marked reduction in EGFR activation and proliferation (27). Recent data by Xu et al. (28) demonstrated that the growth of different colon and breast cancer cells expressing varying levels of EGFR, HER2 and/or HER4 was inhibited by recombinant ERRP in a dose-dependent manner. These results suggest that ERRP may be an effective pan-erbB inhibitor. Wang et al. (29) recently reported an experimental study in order to find an answer to the question of the precise molecular mechanism(s) by which ERRP exerts its antitumor activity. The authors found that ERRP caused marked inhibition of pancreatic cancer cell growth and suggested that ERRP-mediated inactivation of EGFR, Notch-1, NFKB and its downstream target genes contribute to the inhibition of cell growth and invasion (29). Further studies are needed in order to translate these promising observations into innovative therapeutic strategies applicable to the clinical setting. A more complete description of potential natural negative regulators of erbB family receptors has recently been provided by Sweeney and Carraway (30). Pan-HER Inhibitors CI-1033 CI-1033 (canertinib dihydrochloride; Pfizer, New York, New York, U.S.A.) is an orally biovailable 3 chloro, 4 fluoro, 4-anilinoquinazoline (Fig. 1). It is an irreversible inhibitor by cysteine alkylation of the tyrosine kinase domain of all erbB members (31,32). The covalent binding of CI-1033 results in prolonged suppression of erbB activity in comparison with currently available oral reversible tyrosine kinase inhibitors such as erlotinib or gefitinib. Interestingly, this property of irreversible inhibition by CI-1033 is determinant in its ability to induce ubiquitylation and degradation of both erbB1 and erbB2 (32). This ability is not shared by reversible tyrosine kinase inhibitors. Preclinical studies with CI-1033 indicate antiproliferative and antitumor effects using a variety of in vitro cell lines and in vivo human xenografts (33,34). Interestingly, enhanced chemosensitivity was shown with the cisplatin-CI-1033 combination (35). Also, synergistic cytotoxic interactions were observed for the combination of CI-1033 with various chemotherapeutic agents and

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FIGURE 1 Chemical structure of CI-1033 (Pfizer, New York, New York, U.S.A.).

with irradiation (35–38). Oral administration was found to be satisfactory in animal studies showing rapid absorption (33). In treated animals, the most common side effects were skin lesions and diarrhea (33). Two phase I studies on CI-1033 have been recently reported. Thirty-two patients with advanced stage nonhematologic malignancy were enrolled in a phase I multicenter trial with oral CI-1033 at a starting dose of 300 mg/day (39). Head and neck cancer patients constituted one third of the study population. Grade 3 dose-limiting toxicity was observed in three of six patients at the 560 mg dose level with diarrhea/nausea vomiting (one patient), rash (one patient) and anorexia (one patient). The maximum tolerated dose was declared at the 450 mg dose level at which one in six patients presented grade 3 dehydration associated with grade 2 stomatitis. Overall, the most frequently reported adverse events occurred in the gastrointestinal system and skin. The frequency of diarrhea, nausea, stomatitis and vomiting appeared to be dose-dependent whereas the incidence of rash and acne-like skin toxicity seemed to be relatively dose-independent. Gastrointestinal toxicity occurred within the first week of treatment (median 1–6 days). Also, cutaneous toxicity typically occurred within a week of therapy. Fifteen patients with measurable disease were evaluable for objective response. No confirmed partial or complete responses were observed although six patients fulfilled criteria for stable disease. Pharmacokinetic data were available for 22 out of 32 study patients. Marked inter-patient variability was observed in plasma concentrations and there was no apparent dose dependence through concentrations. Less than 1% of the administered dose was recovered intact in urine in the majority of samples. Given the preclinical antitumor activity, a dose escalation study of CI-1033 in combination with docetaxel was conducted in solid tumors (40). This was an open-label, non-comparative, dose-finding multicenter phase I study. Docetaxel was given on day 1 of each 21-day cycle as a single 1-hour infusion. Starting on day 2, CI-1033 was given as a single oral daily dose for 14 days of each 21-day cycle, with a 6-day drug-free period per cycle. Twenty-six patients with advanced solid tumors were treated on the study. The most common tumor histologies

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were renal cell, cervix uteri, pancreatic and NSCLC. Four patients had received a prior paclitaxel-containing regimen. The CI-1033 (50 mg/day) þ docetaxel (75 mg/m2) schedule was declared the recommended phase II dose for this combination. The most common grade 2–4 non-hematologic adverse events were asthenia (31%), diarrhea (27%), alopecia (23%) and stomatitis (29%). Nine patients (35%) presented grade 1 skin rash. Overall, the incidence of skin rash was 50% with no reported grade 3 or 4 rashes. The most frequent hematologic adverse events were leucopenia (27%) neutropenia (54%) and anemia (27%). A relatively high incidence of febrile neutropenia was noted (38%). Twenty-one patients were evaluable for tumor response. Two patients had confirmed objective responses (one cervix uteri carcinoma metastatic to lung and one metastatic NSCLC). Analysis of CI-1033 pharmacokinetic data suggested that a docexaxel effect on CI1033 disposition was possible as CI-1033 plasma concentrations were approximately 60% higher than expected. Analysis of docetaxel pharmacokinetic variables indicated no significant alteration as compared with historical data. A phase II, open-label clinical trial has evaluated CI-1033 in patients with ovarian cancer following failure with prior platinum-based therapy (41). Two oral doses of CI-1033 were compared, a 50-mg and a 200-mg oral dose given daily during 21 days of a 28-day cycle. On the basis of a randomly assigned dose, a total of 146 cycles were delivered to 53 patients treated in the 50-mg arm and 138 cycles were given to patients in the 200-mg arm. Clinically significant differences in toxicities between the two doses were reported with a higher incidence of adverse events in the 200-mg treatment group. The most commonly reported toxicities in the 200-mg treatment group were stomatitis, diarrhea, rash, nausea and vomiting. Twenty-six patients (49.1%) in the 200-mg treatment group required at least one dose reduction whereas no dose reduction was applied in the 50-mg treatment group. In the 49 patients analyzed for efficacy and treated with the highest dose there were no objective responses and the rate of disease stabilization was 34%. A median survival of nine months was comparable to that seen with cytotoxic agents in this heavily-pretreated patient population. Interestingly, the authors evaluated the level of expression of all four subtypes of erbB members from archived tumor samples. This led to the immunohistochemistry analysis of erbB1 to erbB4 expression in 56 tumor samples. Fifty per cent of the samples had simultaneous expression of at least three receptors with erbB3 being the most frequently expressed. The authors did not observe a relationship between tumor expression of any of the erbB subtypes and response to treatment, including disease stabilization. BMS-599626 BMS-599626 (Bristol-Myers Squibb, New York, New York, U.S.A.) (Fig. 2) was identified by optimizing a series of pyrrolotriazine analogues for their ability to inhibit both erbB1 and erbB2. This compound inhibits the enzymatic activity of recombinant kinases with IC50 values at 20 and 30 nmol/L for erbB1 and erbB2 respectively (42). Interestingly, BMS-59926 also inhibits erbB4 but with reduced efficacy (IC50 of 190 nmol/L). A phase I study on BMS-59926 has recently been reported (43). The drug was orally administered on a daily basis during 28 day cycles. The first dose level was 100 mg. There were 41 patients receiving a total number of 80 cycles. The main toxicities were diarrhea, anorexia, ischemia, acneiform dermatitis and skin rash. The dose-limiting toxicities were a prolonged QTc interval and hepatic toxicity (ALT, AST). The recommended dose for phase II

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FIGURE 2 Chemical structure of BMS-599626 (Bristol-Myers Squibb, New York, New York, U.S.A.).

trials was 600 mg/day. The best anti-tumoral response was stable disease reported for 12 patients. An associated biological study by the same authors showed a significant decrease in Ki-67 expression from paired skin samples obtained before and during treatment by the BMS compound. HKI-272 HKI-272 (Wyeth, Collegeville, Pennsylvania, U.S.A.) is a potent irreversible inhibitor of EGFR, erbB2 and erbB4 with IC50 values for kinase inhibition (in nM) at 92, 59 and 19 (44,45). Interestingly, it was recently shown that HKI-272 was able to dramatically inhibit the growth of the EGFR mutant variant III-transformed cells in vitro and tumor growth in vivo (46). Also, it was reported that mutations in the cysteine 530 residue in EGFR variant III can confer resistance to HKI-272 sensitivity in EGFR variant III-transformed cells (46). These data provide a potential mechanism for the development of in vivo resistance in the clinical setting. HKI272 could be particularly effective for treating cancers such as glioblastoma that harbor the EGFR variant III. Interestingly, experimental data suggest that HKI-272 may exhibit efficacy against EGFR-mutant non-small cell lung cancer cells, including tumors that carry the T790M mutation rendering them resistant to gefitinib and erlotinib (47). A recently reported phase I study of HKI-272 indicated a maximal tolerated dose at 320 mg/day, orally (48). HKI-272-related adverse events were (any grade) diarrhea (84%), nausea (55%), asthenia (45%), anorexia (31%), vomiting (29%), chills (12%) and rash (10%). Grade 3 toxicity was diarrhea and asthenia. HKI-272 had antitumor activity in HER-2-positive breast cancer. Phase II studies are under development in breast and lung cancer. LIMITS AND PROSPECTS Current clinical results with development of Pan-HER inhibitors have not confirmed the initial hopes which were raised by this category of drugs which seemed capable of killing several birds with one stone. These limitations are due to several factors. The first is the absence of patient selection based on tumor predictive factors able to orientate treatment. The second limitation lies in the presence of significant side-effects. This problem was particularly evident for the CI-1033 with significant gastro-intestinal and cutaneous toxicities. A ray of hope for the development of this category of drugs may be found in combinations with other targeted therapies such as antiangiogenic agents, thus offering optimal targeting not only of the tumor itself but also of endothelial cells which are known to carry HER receptors. Dual-HER inhibitors such as lapatinib are more promising,

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particularly in the management of advanced breast cancer, and will be soon able to be positioned towards to trastuzumab. REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9.

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Burris HA III, Hurwitz HI, Dees EC, et al. Phase I safety, pharmacokinetics, and clinical activity study of lapatinib (GW572016), a reversible dual inhibitor of epidermal growth factor receptor tyrosine kinases, in heavily pretreated patients with metastatic carcinomas. J Clin Oncol 2005; 23:5305–13. Storniolo A, Burris H, Pegram M, et al. A Phase I, open-label study of lapatinib (GW572016) plus trastuzumab: a clinically active regimen (abstract). Proc Am Soc Clin Oncol 2005; 23:559. Blackwell K, Burstein H, Pegram M, et al. Determining relevant biomarkers from tissue and serum that may predict response to single agent lapatinib in trastuzumab refractory metastatic breast cancer (abstract). Proc Am Soc Clin Oncol 2005; 23:3004. Lin NU, Carey A, Liu MC, et al. Phase II trial of lapatinib for brain metastases in patients with HER2þ breast cancer. Proc Am Soc Clin Oncol 2006; 35:503. Traxel P, Allegrini PR, Brandt R, et al. AEE788: a dual family epidermal growth factor receptor/ErbB2 and vascular endothelial growth factor receptor tyrosine kinase inhibitor with antitumor and antiangiogenic activity. Cancer Res 2004; 64:4931–41. Goudar RK, Shi Q, Hjelmeland MD, et al. Combination therapy of inhibitors of epidermal growth factor receptor/vascular endothelial growth factor receptor 2 (AEE788) and the mammalian target of rapamycin (RAD001) offers improved glioblastoma tumor growth inhibition. Mol Cancer Ther 005; 4:101–12. Yokoi K, Thaker PH, Yazici S, et al. Dual inhibition of epidermal growth factor receptor and vascular endothelial growth factor receptor phosphorylation by AEE788 reduces growth and metastasis of human colon carcinoma in an orthotopic nude mouse model. Cancer Res 2005; 65:3716–25. Yu Y, Rishi AK, Turner JR, et al. Cloning of a novel EGFR-related peptide: a putative negative regulator of EGFR. Am J Physiol Cell Physiol 2001; 280:C1083–9. Xu H, Yu Y, Marciniak D, et al. Epidermal growth factor receptor (EGFR)-related protein inhibits multiple members of the EGFR family in colon and breast cancer cells. Mol Cancer Ther 2005; 4:435–42. Wang Z, Sengupta R, Banerjee S, et al. Epidermal growth factor receptor-related protein inhibits cell growth and invasion in pancreatic cancer. Cancer Res 2006; 66:7653–60. Sweeney C, Carraway KL III. Negative regulation of ErbB family receptor tyrosine kinases. Br J Cancer 2004; 90:289–93. Fry DW. Site-directed irreversible inhibitors of the erbB family of receptor tyrosine kinases as novel chemotherapeutic agents for cancer. Anticancer Drug Des 2000; 15: 3–16. Citri A, Alroy I, Lavi S, et al. Drug-induced ubiquitylation and degradation of ErbB receptor tyrosine kinases: implications for cancer therapy. EMBO J 2002; 21:2407–17. Slichenmyer WJ, Elliott WL, Fry DW. CI-1033, a pan-erbB tyrosine kinase inhibitor. Semin Oncol 2001; 28(5 Suppl 16):80–85. Allen LF, Lenehan PF, Eiseman IA, et al. Potential benefits of the irreversible pan-erbB inhibitor, CI-1033, in the treatment of breast cancer. Semin Oncol 2002; 29(3 Suppl 11): 11–21. Gieseg MA, de Bock C, Ferguson LR, et al. Evidence for epidermal growth factor receptor-enhanced chemosensitivity in combinations of cisplatin and the new irreversible tyrosine kinase inhibitor CI-1033. Anticancer Drugs 2001; 12:683–90. Erlichman C, Boerner SA, Hallgren CG, et al. The HER tyrosine kinase inhibitor CI1033 enhances cytotoxicity of 7-ethyl-10-hydroxycamptothecin and topotecan by inhibiting breast cancer resistance protein-mediated drug efflux. Cancer Res 2001; 61:739–48. Nelson JM, Fry DW. Akt, MAPK (Erk1/2), and p38 act in concert to promote apoptosis in response to ErbB receptor family inhibition. J Biol Chem 2001; 276:14842–7. Rao GS, Murray S, Ethier SP. Radiosensitization of human breast cancer cells by a novel ErbB family receptor tyrosine kinase inhibitor. Int J Radiat Oncol Biol Phys 2000; 48:1519–28. Nemunaitis J, Eiseman I, Cunningham C, et al. Phase 1 clinical and pharmacokinetics evaluation of oral CI-1033 in patients with refractory cancer. Clin Cancer Res 2005; 11:3846–53.

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Garland LL, Hidalgo M, Mendelson DS, et al. A phase I clinical and pharmacokinetic study of oral CI-1033 in combination with docetaxel in patients with advanced solid tumors. Clin Cancer Res 2006; 12(14 Pt 1):4274–82. Campos S, Hamid O, Seiden MV, et al. Multicenter, randomized phase II trial of oral CI-1033 for previously treated advanced ovarian cancer. J Clin Oncol 2005; 23: 5597–604. Wong TW, Lee FY, Yu C, et al. Preclinical antitumor activity of BMS-599626, a panHER kinase inhibitor that inhibits HER1/HER2 homodimer and heterodimer signaling. Clin Cancer Res 006; 12(20 Pt 1):6186–93. Soria JC, Cortes J, Armand JP, et al. The PAN-HER inhibitor BMS-599626: biological effects, pharmacokinetic profile, and early clinical evaluation of a phase I trial. EJC 2005; 3:420 (abstract 145). Rabindran SK, Discafani CM, Rosfjord EC, et al. Antitumor activity of HKI-272, an orally active, irreversible inhibitor of the HER-2 tyrosine kinase. Cancer Res 2004; 64:3958–65. Tsou HR, Overbeek-Klumpers EG, Hallett WA, et al. Optimization of 6,7-disubstituted-4-(arylamino)quinoline-3-carbonitriles as orally active, irreversible inhibitors of human epidermal growth factor receptor-2 kinase activity. J Med Chem 2005; 48: 1107–31. Ji H, Zhao X, Yuza Y, et al. Epidermal growth factor receptor variant III mutations in lung tumorigenesis and sensitivity to tyrosine kinase inhibitors. PNAS 2006; 103: 7817–22. Kwak EL, Sordella R, Bell DW, et al. Irreversible inhibitors of the EGF receptor may circumvent acquired resistance to gefitinib. Proc Natl Acad Sci USA 2005; 102:7665–70. Wong KK, Fracasso PM, Bukowski RM, et al. HKI-272, an irreversible pan erbB receptor tyrosine kinase inhibitor: preliminary phase I results in patients with solid tumors. Proc of ASCO 2006; 24:125 (abstract 3018).

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Inhibiting the Phosphoinositide 3-Kinase/Akt/ Mammalian Target of Rapamycin Pathway Janet E. Dancey Investigational Drug Branch, Cancer Therapy Evaluation Program, Division of Cancer Treatment and Diagnosis, National Cancer Institute, Rockville, Maryland, U.S.A.

INTRODUCTION The reasons for the considerable interest in identifying and developing cancer agents that inhibit the phosphoinositide 3-kinase (PI3K)/Akt/mammalian target of rapamycin (mTOR) pathway are multiple. Activation of this pathway plays a pivotal role in essential cellular functions such as survival, proliferation, migration, and differentiation. Aberrant activation of the pathway contributes to tumorigenesis, tumor metastasis, and resistance to standard cancer therapy. Nonclinical studies suggest that specific inhibitors impede tumor cell growth or induce apoptosis, and combining inhibitors of the PI3K/Akt/mTOR pathway with standard cancer therapies can attenuate tumor resistance. Recent studies indicate that blocking PI3K-Akt activation underlies the therapeutic efficacy seen with a number of approved drugs, including imatinib (Gleevec
Bcr-Abl inhibitor; Novartis, Basel, Switzerland), trastuzumab (Herceptin
, Her2/neu antibody; Genentech, South San Francisco, California, U.S.A.), gefitinib (Iressa
EGFR inhibitor; AstraZeneca, London, U.K.), and erlotinib (Tarceva
EGFR inhibitor; OSI Pharmaceuticals, Melville, New York, U.S.A.). Small molecules designed to specifically target components of the pathway are now being developed as single agents and in combination with standard cancer therapy. The biology of the pathway and inhibitory agents under clinical development are described in this chapter. THE PI3K-Akt-mTOR PATHWAY IN NORMAL AND CANCER CELLS PI3-kinases fall into three classes based on their primary structure and substrate specificity (1). PI3K are lipid kinases that phosphorylates phosphatidylinositol at the 30 position of inositol and affect multiple cellular functions, many related to growth and differentiation. PI3Kalpha is the human homolog of the viral oncogene ASV16, which causes rapidly growing hemangiosarcomas in young chickens and induces oncogenic transformation in cultures of chicken embryo fibroblasts (2). In response to extracellular stimuli, PI3K phosphorylates the 30 -hydroxyl of phosphatidylinositol-4,5-bis-phosphate (PIP2), to generate phosphatidylinositol-3,4,5-triphosphate (PIP3) (3) (Fig. 1). The tumor suppressor phosphatase and tensin homologue deleted on chromosome 10 (PTEN) opposes the action of PI3K by dephosphorylating 30 -phosphoinositides. The product of p110alpha, PIP3, functions as an important second messenger in the cell. It recruits proteins that have a pleckstrin homology domain to the cell membrane and facilitates their interactions (4). Among these downstream targets of PI3K are the serine–threonine protein kinase Akt and the phosphoinositide-dependent kinase PDK1 (1,3). 65

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FIGURE 1 Phosphoinositide 3-kinase (PI3K). In response to extracellular stimuli through membrane receptors or Ras, PI3K phosphorylates the 30 -hydroxyl of phosphatidylinositol-4,5-bisphosphate (PIP2) to generate phosphatidylinositol-3,4,5-triphosphate (PIP3). The tumor suppressor phosphatase and tensin homologue deleted on chromosome 10 (PTEN) opposes the action of PI3K by dephosphorylating 30 -phosphoinositides. PIP3 recruits proteins that have a pleckstrin homology domain to the cell membrane and facilitates their interactions. Among these downstream targets of PI3K are Akt and the phosphoinositide-dependent kinase PDK1. PI3K has additional targets involved in cell survival, motility, and transformation.

Akt was originally identified as an oncoprotein encoded by the genome of a murine lymphoma virus (5,6). Three mammalian isoforms of the serine/threonine kinase Akt have been identified: Akt1, Akt2, and Akt3, which differ in tissue distribution and substrate specificity. Akt kinase binds to the products of PI3K, phosphatidylinositol 3,4-biphosphate and phosphatidylinositol 3,4,5-triphosphate, with its pleckstrin homology domain (Figs. 2 and 3). It then becomes activated by phosphorylation at threonine 308 and serine 473 through the action of the 3-phosphoinositide-dependent kinases PDK1 and PDK2 [reviewed in (7,8)]. The identity of PDK2 has not been positively determined; currently the leading candidate is the mTOR/Rictor complex (9). Activated Akt signals to numerous substrates that control key cellular processes of transcription, translation, cell cycle progression, and apoptosis. There are two broad categories of Akt targets: (i) survival and death factors and (ii) proteins controlling translation [reviewed in (10–15)]. Among the first group are the pro-apoptotic proteins, Bad and caspase 9, the growth-inhibitory proteins, glycogen synthase kinase-3 beta (GSK3), and the forkhead transcription factors, FKHR, FKHR-L1, and AFX, all of which are downregulated by Akt. Also in this category is the I-Kappa K kinase (IKK), a positive regulator of NF-kappaB, which is up-regulated by Akt (16). The second category consists of the kinase mTOR and its downstream targets. Akt activates mTOR through at least two mechanisms, direct phosphorylation of mTOR itself, and via phosphorylation and inactivation of tuberous sclerosis complex (TSC) [reviewed in (7,8)]. TSC is composed of two proteins, TSC1 and TSC2. TSC2 is a GTPaseactivating protein (GAP) toward the Ras-related small GTPase Rheb (Ras-homologenriched-in-brain) (17), which activates mTOR (Fig. 4). Conversely, the energysensing pathway [i.e., signaling is triggered by the presence of amino acids and adenosine triphosphate (ATP)] is linked to mTOR through serine threonine kinase 11 (STK11; also called LKB1), a tumor suppressor inactivated in PeutzJeghers syndrome. LKB1 kinase is an inhibitor of the mTOR pathway in response to

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FIGURE 2 Phosphoinositide-dependent kinase (PDK1). PDK1 phosphorylates Akt at T308, which contributes to activation of Akt. PDK1 also activates other protein kinases related to Akt, including isoforms of p70 ribosomal S6 kinase (S6K), serum- and glucocorticoid-responsive kinases (SGK), and p90 ribosomal S6 kinase (p90RSK), atypical and novel PKC isoforms, PRK1/2, and others. In addition, it functions as a nucleo-cytoplasmic shuttling protein. Activation of these substrates leads to an increase in glucose uptake, protein synthesis, and inhibition of pro-apoptotic proteins.

FIGURE 3 Akt kinases are activated by phosphorylation at threonine 308 and serine 473 through the action of and PDK2 (mTOR/Rictor complex). Akt targets include: (i) survival and death factors and (ii) proteins controlling translation. Among the first group are the pro-apoptotic proteins, Bad and caspase 9, the growth-inhibitory proteins, glycogen synthase kinase-3 beta (GSK3beta), and the forkhead transcription factors, FKHR, FKHR-L1, and AFX, all of which are down-regulated by Akt as well as I-Kappa K kinase (IKK), a positive regulator of NF-kappaB, which is up-regulated by Akt. The second category consists of the kinase mTOR and its downstream targets. Akt activates mTOR through at least two mechanisms, direct phosphorylation of mTOR itself, and via phosphorylation and inactivation of tuberous sclerosis complex (TSC). Activated Akt signals to numerous substrates that control key cellular processes of transcription, translation, cell cycle progression, and apoptosis.

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FIGURE 4 Mammalial target of rapamycin (mTOR). mTOR is phosphosphorylated via Akt and through inactivation of tuberous sclerosis complex (TSC). TSC2 is a GTPase-activating protein (GAP) toward the Ras-related small GTPase Rheb (Ras-homolog-enriched-in-brain), which activates mTOR. Conversely, the energy-sensing pathway is linked to mTOR through STK11 (serine threonine kinase 11; also called LKB1). LKB1 kinase is an inhibitor of the mTOR pathway in response to energy starvation through its activation of 50 AMP-activated protein kinase (AMPK). mTOR functions in complexes with other proteins. One complex is a formed regulatory-associated protein of mTOR (Raptor) and mammalian ortholog of LST8 (mLST8), another is with Rictor. Only the Raptor–mTOR complex is rapamycin-sensitive. Activated mTOR-Raptor phosphorylates eukaryotic initiation factor 4E (eIF-4E) binding protein-1 (BP1) and protein S6 kinase 1 (S6K1). Rictor– mTOR complex phosphorylates Akt at the Ser473 position, resulting in its full activation.

energy starvation through its activation of 50 AMP-activated protein kinase (AMPK) (18,19). The net result of these signaling interactions suggests a model in which growth factor signaling through PI3K-Akt is coordinated with nutrient availability signaling through LKB1-TSC1/2 to Rheb and mTOR (8). mTOR functions are dependent on its forming complexes with other proteins. One complex is formed with regulatory associated protein of mTOR (Raptor) (20) along with mammalian ortholog of LST8 (mLST8) (21); another is formed with Rictor. The functions of these complexes are distinct: Raptor–mTOR complex is rapamycin-sensitive, whereas the Rictor–mTOR complex is rapamycin-resistant (9,22,23). In addition, Rictor–mTOR complex phosphorylates Akt at the Ser473 position, resulting in its full activation. Activated mTOR, in complex with Raptor, leads to phosphorylation of two key proteins that regulate protein translation: eukaryotic initiation factor 4E (eIF-4E) binding protein-1 (4E-BP1) and protein p70S6 kinase 1 (S6K1) [reviewed in (7,8)]. mTOR is an essential component of the oncogenic signal emanating from PI3K or Akt. Phosphorylation of two downstream targets of mTOR, S6K and 4E-BP, is strictly correlated with PI3K and Akt-induced transformation (24). Both S6K and 4E-BP play important roles in the control of protein synthesis [reviewed in (7)]. S6K is a positive regulator activated by mTOR, and 4E-BP is a negative regulator of protein synthesis inactivated by mTOR. The phosphorylation status of S6K1 or 4E-BP1 is often used as a measure of mTOR activity in laboratory and clinical research studies.

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Although often depicted as linear, there is considerable crosstalk and feedback regulation within the PI3K/Akt/mTOR pathway. PDK1 can regulate translation independently of Akt by directly phosphorylating and activating a downstream effector of mTOR, S6K1 (Fig. 4). Negative feedback regulation of Akt can occur through S6K1, which can catalyze an inhibitory phosphorylation on insulin receptor substrate proteins, abrogating activation of PI3K. In addition, mTOR can phosphorylate Akt at its hydrophobic motif when bound to Rictor, potentially providing a level of positive feedback on the pathway [reviewed in (7,8)]. Such pathway complexity is required to regulate the many cellular processes, including proliferation, survival, carbohydrate metabolism, and motility, and likely contributes to its importance in the molecular mechanisms of diseases such as diabetes and chronic inflammation, as well as cancer [reviewed in (7,8)]. Numerous lines of data suggest that this pathway is critically important in mammalian growth and metabolic responses. Gene knock-out studies in mice have shown that Akt1 deficiency results in slightly diminished growth, and these mice exhibit increased frequencies of spontaneous and stress-induced apoptosis [reviewed in (15,25)]. Akt3-knock-out mice display a selective reduction in brain size resulting from fewer and smaller cells. In contrast, Akt2-deficient mice have a normal body size but are insulin resistant. However, the combined deletion of Akt1 and Akt2 genes results in perinatal lethality with multiple developmental defects, indicating a large degree of functional overlap between the different Akt isoforms. In the heart, Akt signaling regulates myocyte size, at least in part, through activation of mTOR-dependent pro-growth pathways and suppression of GSK3 and FOXO-dependent atrophy programs [reviewed in (15)]. These studies have documented functional differences between Akt isoforms that may result in differential toxicity for agents that are Akt isoform specific in their inhibition. Multiple components of this pathway are involved in oncogenesis (Table 1). Both activating mutations and amplification of oncogenes and loss of tumor suppressor genes occur within the pathway in human neoplasms with remarkable frequency (26). Growth factor receptor protein tyrosine kinases, integrin-dependent cell adhesion, and G-protein-coupled receptors either directly or indirectly through activation of Ras activate PI3K. The PI3K catalytic subunit p110alpha and Akt have been extensively studied as retroviral oncoproteins (27). The related isoforms p110beta, -delta, and -gamma induce oncogenic transformation as wild-type proteins. Activation mutations of growth factor receptors and PI3K, as well as amplification and or overexpression of PI3K and Akt, have been reported in different tumor histologies [reviewed in (28)]. Similarly, inappropriate activation of the TSC/Rheb/mTOR axis through the loss of tumor suppressor proteins has been linked to the pathobiology of a number of tumor predisposition syndromes, including tuberous sclerosis (TSC1/2), Peutz-Jeghers syndrome (LKB1), and Cowden’s syndrome (PTEN) (29). In laboratory models, the resultant aberrant pathway signaling through oncogene stimulatuion or tumor suppressor gene loss not only leads to a growth advantage during carcinogenesis but may also contribute to chemotherapy resistance (30). If such cancer cells are dependent on the growth and survival signaling effects of the PI3K-Akt pathway, it is possible that this dependency will result in cancer cell sensitivity to pathway inhibitors. In addition, persistent signaling through PI3K-Akt pathway has been described as a mechanism of resistance to cytotoxic agents (31), radiation (32), epidermal growth factor receptor inhibitors (33), and trastuzumab (34). The most frequently occurring mutants of p110alpha catalytic subunit are oncogenic in vitro

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TABLE 1 Oncogenes and Tumor Suppressor Genes in the PI3K-Akt-mTOR Pathway Target Protooncogenes PI3K

Mechanism Amplification Mutations in PI3K catalytic unit Mutation in p85alpha

Akt

Amplification Akt1, Akt2, Akt3

eIF4E S6K1 Cyclin D

Amplification Amplification Amplification Translocation

Tumor suppressors PTEN

Germline mutations in PTEN

Mutation/deletion/epigenetic silencing TSC1/2 complex

Germline mutations in TSC1 or TSC2 develop

LKB1

Germline mutations in LKB1 develop

Relevant human cancers Ovarian and cervical cancers Colorectal, gastric, breast, ovarian cancers, glioblastoma and hepatocellular carcinoma Colon and ovarian cancers Gastric adenocarcinoma Ovarian, breast, pancreatic Breast Breast, head and neck Breast cancers Breast cancers. mantle cell lymphoma

Hamartoma syndromes (Cowden disease, Bannayan-Riley-Ruvalcaba syndrome, Lhermitte-Duclos disease) and are at higher risk of developing cancer Brain, bladder, breast, prostate, endometrial lung, endometrial gastric, prostate, ovarian Tuberous sclerosis complex (TSC) characterized by the formation of hamartomas in many organs Peutz-Jeghers syndrome, characterized by hamartomas in the gastrointestinal tract

and in vivo, show gain of enzymatic function, activate Akt, and their oncogenic activity is sensitive to mTOR inhibitors (27,30,35). Thus, PI3K, PDK1, Akt, and mTOR are attractive targets for these drugs, as such agents might inhibit proliferation and reverse the repression of apoptosis and resistance to cytotoxic therapy in cancer cells and also effect proliferation, survival, and migration of endothelial cells. Because the PI3K/Akt/mTOR pathway plays a central role in many normal physiologic functions and is activated in many normal tissues, pathway inhibition may cause serious side effects and might lead to a limited therapeutic index. Certain complications, such as hyperglycemia and insulin resistance, may be manageable with short term exposures to the agents. The specificity of the inhibitor, not only for specific kinase but also for kinase isoforms, may also be clinically important to maximize therapeutic index. Obviously, any observed toxicities would have to be weighed against the benefits of receiving a potentially effective cancer therapy (36). AGENTS TARGETING PI3K-PDK1-Akt-mTOR PATHWAY PI3K Inhibitors Agents evaluated in nonclinical studies can broadly be divided into lipid analogues that impede membrane binding through PH domains and those that inhibit kinase activation by competing with ATP. Foremost amongst the agents used in nonclinical studies are wortmannin (37), a metabolite of the fungus

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Penicillium funiculosum, and LY294002, a morpholino derivative of the broadspectrum kinase inhibitor quercetin (38). Wortmannin is an irreversible inhibitor of all PI3K isoforms, whereas LY294002 is a competitive inhibitor of ATP. LY294002 effectively inhibits the growth of many types of tumor cells in vitro and in vivo, which is associated with inhibition of PI3K and downstream components of the pathway (39). Wortmannin is also effective in vitro and in vivo; however, it is not specific for PI3K, as some studies showed that its in vivo antitumor activity did not correlate with differences in PI3K activity or in vitro cytotoxicity (40,41). Combining either of the agents with chemotherapy can be effective, and lower concentrations may be required for therapeutic effect, suggesting that the combination of a PI3K inhibitor with conventional cancer therapy may provide therapeutic index for patients with cancers that have become resistant to standard therapies (39). Although these agents have been extensively used as pharmacological probes in laboratory models, their toxicity, lack of kinase selectivity, and poor pharmacology have limited their development as cancer therapeutics. Derivatives of LY294002 and wortmannin are being developed (42–44). The wortmannin derivative, PX-866, is more potent and less toxic than wortmannin, and inhibits the growth of human lung, ovarian, and colon cancer xenografts (42,43). Inhibition of Akt phosphorylation was only observed at doses that exceeded those that caused growth delay, suggesting the cytotoxicity of PX-866 may be independent of PI3K inhibition. Further optimization of wortmannin or LY294002 may yield inhibitors that are tolerable and effective; however, it is not clear whether derivatives of these rather promiscuous kinase inhibitors will be more specific and thus have an acceptable therapeutic index. The development of PI3K inhibitors that work through mechanisms other than inhibition of ATP binding, such as through inhibition of the association of the regulatory subunit p85 with growth factor receptors or with the catalytic subunit p110, might identify candidate inhibitors that possess better specificity, efficacy, and toxicity profiles (36).

PDK1 Inhibitors PDK1 is a serine/threonine protein kinase that phosphorylates several members of the conserved AGC kinase superfamily [comprising the prototype protein kinases A (PKA), G (PKG) and C (PKC)]. PDK1 phosphorylates Akt at T308, which contributes to activation of Akt. PDK1 also activates other protein kinases related to Akt, including isoforms of p70 ribosomal S6 kinase (S6K), serum- and glucocorticoid-responsive kinases (SGK), and p90 ribosomal S6 kinase (p90RSK), atypical and novel PKC isoforms, PRK1/2, and others [reviewed in (45)]. In addition, it functions as a nucleo-cytoplasmic shuttling protein [reviewed in (46)]. Activation of these substrates leads to an increase in glucose uptake, protein synthesis, and inhibition of pro-apoptotic proteins. Derivatives of staurosporine and celecoxib are being developed as PDK1 inhibitors. The 7-hydroxy-derivative of staurosporine, UCN-01 (Keryx, Biopharmaceuticals, Tucson, Arizona, U.S.A.), directly inhibits PDK1 with an IC50 value less than 33 nM in vitro and in vivo (47). Its inhibition of PDK1 directly correlates with inhibition of Akt and cell growth and induction of apoptosis (47). UCN-01 inhibits a number of serine-threonine kinases, including the Ca2þ- and phospholipiddependent protein kinase C isoforms (PKC) (48), cyclin dependent kinases (CDK) 2, 4, and 6 (49), and chk1 kinase (50). UCN-01 has synergistic effects on apoptosis

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when combined with cytotoxic chemotherapy in vitro and in vivo (51,52), but this synergism may be due to effects on Chk1 not PDK1. In phase I studies, pharmacokinetic data from the first patients administered UCN-01 revealed the agent binds tightly to human alpha-1 acid glycoprotein (hAGP), resulting in a long half-life of several weeks, small volume of distribution at steady state, and low systemic clearance (53–56). The recommended phase II dose of UCN-01 is 42.5 mg/m2/day for 3 days cycle 1, the same dose/day for 1.5 days for subsequent cycles, and 90 mg m2 over 3 hours cycle 1, with half of these doses subsequently given every 3–4 weeks (55,57) to prevent drug accumulation. Significant toxicities seen with this agent include hyperglycemia with metabolic acidosis, pulmonary dysfunction, nausea, vomiting, and hypotension. The mean total salivary concentration, which was evaluated as a surrogate measure of “free” drug, was 111 nmol/L of UCN-01, within the range predicted to inhibit PDK1 and Chk1 (55). The occurrence of hyperglycemia as a dose limiting toxicity suggests that modulation of insulin signaling pathway through inhibition of PDK1 may be occurring in patients. The ability of UCN-01 to inhibit many kinases makes it difficult to consider UCN-01 as a targeted pathway inhibitor, and pathway inhibition has not been directly demonstrated in tissues of patients enrolled on the clinical trials. Ongoing clinical trials are assessing the appropriate dose and antitumor activity of UCN-01 in combination with variety of cytotoxic agents (58,59). Celecoxib may mediate apoptosis through PDK1 inhibition; however, celecoxib is a weak PDK1 inhibitor (IC50, 48 mM), requiring at least 30 mM to exhibit discernable effects on the growth of tumor cells in vitro (60). The celecoxib derivatives OSU-03012 and OSU-03013 were designed to minimize cyclooxygenase-2 inhibition and maximize PDK1 antagonism (60). These analogues are more potent PDK-1 inhibitors than celecoxib and achieve total growth inhibition in multiple human tumor cell lines and overcome tumor cellular resistance to trastzumab and imatinib (60–62). However, the celecoxib derivatives also have other biological activities and induce apoptosis independently of PDK1 inhibition (63,64), suggesting that, like UCN-01, off-target effects contribute to their anticancer activity. In summary, all of the compounds in this class have targets outside the PI3K/Akt/mTOR pathway that may not be related to inhibition of PDK1 but nonetheless might contribute to associated antitumor effects. Determining the critical target(s) of these drugs will help guide their development as targeted therapeutics. Akt Inhibitors Inhibitors of Akt may be broadly categorized as those that are lipid-based and those that identified through high-throughput screening of small molecule libraries and rational design of peptides. The best-characterized inhibitor of Akt and the one in clinical development is the alkylphospholipid (APL) perifosine. Perifosine The first APL evaluated in cancer clinical trials was miltefosine (hexadecylphosphocholine), which had cytostatic and cytotoxic effects towards a variety of tumor cell lines in vitro and in vivo (65,66). Clinical studies with oral miltefosine were terminated due to absence of anti-tumor activity and significant gastrointestinal toxicity. At lower doses, miltefosine is an effective treatment for leishmaniasis (67), and topical formulations are used to treat cutaneous breast cancer metastases.

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Perifosine (D-21266; Kernyx) was chosen for further development because it was found to be more active and better tolerated than miltefosine in preclinical models (68,69). The exact mechanism of action of APLs remains unclear. Nonclinical studies indicate that perifosine and other APLs cause dose-dependent inhibition of Akt phosphorylation at concentrations that inhibit the growth of cancer cell lines (70–72). As myristoylated Akt, which bypasses the requirement for PH domainmediated membrane recruitment, abrogates perifosine-mediated decrease of Akt phosphorylation and cell growth inhibition, perifosine may decrease the plasma membrane localization of Akt. However, ALPs can have multiple effects in addition to inhibiting of Akt phosphorylation including inhibiting mitogenactivated protein kinase (MAPK) activation, activation of jun-kinase pathway [40] and release of intracellular calcium (73,74). These results suggest that perifosine is not a specific inhibitor of Akt. In phase I studies, the dose-limiting toxicities of chronic daily oral administration of perifosine were nausea, diarrhea, dehydration, and fatigue (75,76). Phase II single agent trials assessing perifosine in refractory cancers of the prostate, head and neck, sarcoma, and melanoma have not yielded promising results (77–80), suggesting that if the agent is to have a therapeutic role, it will be in combination with other active therapies and/or in selected patients with tumors having activation of the pathway. As no clinical study to date has assessed whether the agent modulates Akt in patient tissues, extrapolating results of these trials to anticipated outcomes other Akt inhibitors is uncertain. Inhibitors of the Mammalian Target of Rapamycin The macrocyclic lactone rapamycin, also known as sirolimus (Rapamune
; Wyeth, Madison, New Jersey, U.S.A.), is a product of the soil bacterium Streptomyces hygroscopicus, initially found on Easter Island (Rapa Nui). Sirolimus possesses fungicidal, immunosuppressive, and antiproliferative properties (81). Sirolimus initially received regulatory approval as an immunosuppressant for the prophylaxis of organ allograft rejection. More recently, sirolimus eluting arterial stents are approved to prevent coronary artery restenosis. Sirolimus and derivatives tirosel/ temsirolimus (Wyeth), everolimus (RAD001; Novartis), and AP23573 (Ariad Pharmaceuticals, Cambridge, Massachusetts, U.S.A.) also inhibit tumor cell proliferation and are under evaluation in cancer clinical trials. The antiproliferative effects of the various sirolimus derivatives have been evaluated in numerous in vitro and in vivo tumor models. In sensitive cells, these agents inhibit tumor and endothelial cell proliferation in picomolar to nanomolar concentrations and may add to the cytotoxicity of other chemotherapeutic agents and radiation. In selected models, sirolimus induces apoptosis (82,83). Selective apoptosis was seen in cells lacking functional p53 or p27Kip1 (83). In addition to its effects on tumor cells, sirolimus also potently inhibits angiogenesis and endothelial cell proliferation in vitro and in vivo (84–86). All the mTOR inhibitors in clinical development are well tolerated and have some evidence to suggest anti-tumor activity. The most common toxicities seen are mild to moderate skin reactions, stomatitis/mucositis, myelosuppression, and metabolic abnormalities, such hyperlipidemia and hyperglycemia. These adverse effects are reversible with interruption of dosing or, for hyperlipidemia and hyperglycemia, with specific treatment. To date, there has been no evidence of clinically significant

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immunosuppression with intermittent schedules, although reactivation of herpes zoster virus in patients treated with tirosel/temsirolimus has been reported. The recommended phase II doses of the agents appear to be within the range that can be expected to induce target inhibition. Effective target inhibition with the proposed phase II doses/schedules has been shown through pharmacodynamic assays assessing inhibition of either S6K1 or 4E-BP1 phosphorylation in surrogate tissue from patients treated with tirosel/temsirolimus, everolimus, or AP23573. More limited data from baseline and on treatment tumor tissue specimens supports that the agents do inhibit mTOR and downstream targets. However, optimal dose/ schedule for intratumoral target inhibition is not well defined due to limitations in numbers of specimens analyzed. For tirosel/temsirolimus and everolimus, these doses are below the agents’ MTDs; conversely, recommended phase 2 doses of AP23573 are MTDs. Whether more toxic doses will result in greater anti-tumor activity or whether there are differences in anti-tumor activity of the agents unrelated to their ability to induce toxicity will require further evaluation. Sirolimus may induce regression in astrocytomas associated with loss of TSC (87). In a study of 5 subjects with clinically definite TSC and either subependymal giant cell astrocytomas (n ¼ 4) or pilocytic astrocytoma (n ¼ 1) treated with oral rapamycin at standard immunosuppressive doses (serum levels 5–15 ng/mL), all lesions exhibited regression and, in one case, necrosis. Interruption of therapy resulted in regrowth of subependymal giant cell astrocytomas in one patient. Resumption of therapy resulted in further regression. These results suggest that oral sirolimus therapy can induce regression of astrocytomas associated with TSC and may offer an alternative to operative therapy of these lesions. They also provide the proof of concept that mutations in upstream tumor suppressor regulators may lead to enhanced signaling through mTOR that is amenable to inhibition and clinical benefit. Tirosel/temsirolimus is a soluble 42-[2,2-bis(hydroxymethyl)]-propionic ester of rapamycin. Results from four phase I studies evaluating increasing doses of tirosel/temsirolimus on different schedules and with differing formuations have been reported (88–92). The weekly IV schedule is the one that has been most extensively evaluated. Phase II studies of single agent tirosel/temsirolimus evaluating different doses of 25 mg, 75 mg, and/or 250 mg weekly IV have been undertaken in broad range of tumor histologies and results of these studies are summarized in Table 2. The most promising activity has been seen in mantle cell lymphoma (93) and endometrial carcinoma (94) with objective tumor response rates of 30–40%. Moderate activity has been reported in breast (95) and renal cell carcinoma (96). Minimal single agent activity has been seen in SCLC (97), melanoma (98), and GBM (99,100). In general, lower doses appear to be as active as higher doses, but with better tolerability. Recently, a 3-arm phase 3 trial of tirosel/temsirolimus, tirosel/temsirolimus with interferon, and interferon in poor prognosis patients with RCC has been reported (101). Of the 626 patients, overall survival of patients treated with tirosel/temsirolimus was significantly prolonged compared to those treated with interferon (median 10.9 vs. 7.3 mo, HR 0.73, p ¼ 0.0069). The combination of interferon and tirosel/temsirolimus did not confer greater benefit than interferon alone, possibly due to compromised dose delivery of the agent(s). Everolimus (RAD001, Novartis), or 42-O-(2-hydroxyethyl)rapamycin, has greater polarity than sirolimus and was developed in an attempt to improve the

25 78

626

RCC Phase III

RCC Sarcoma

65

GBM

86

SCLC 33

18

EC

43

35

MCL

GBM

109

MBC

MM

111

Patients (N)

RCC

Tumor

Dose/schedule

38 44

10 mg daily 12.5 mg per day · 5 days every 2 week

25 mg 15 mg/Interferon Interferon

250

250

25 250 250

33 5% decreasein PET uptake in 8 patients and symptomatic improvement in 13 patients

0

5

3

NR

31

9

25 25

ORR (%) 7 (26% MR)

25 75 250 75 250 250

Mg IV weekly

9 weeks 2% 2.3 months 8% PFS at 6 months Significantly longer survival in tirosel/temsirolimus arm (10.9 months) compared with interferon (7.3 months) NR NR

10 weeks

2.2 months

NR

5.5 months

6.5 months

12 weeks

5.8 months

TTP/PFS

Amato et al. (104) Chawla et al. (111)

Hudes et al. (101)

Galanis et al. (100)

Chang et al. (99)

Margolin et al. (98)

Pandya et al. (97)

Oza et al. (94)

Ansell et al. (116)

Witzig et al. (93)

Chan et al. (95)

Atkins et al. (96)

Reference

Abbreviations: EC, endometrial carcinoma; MBC, metastatic breast carcinoma; MCL, mantle cell lymphoma; MM, malignant melanoma; NR, not reported; ORR, objective response rate; PET, positron emission tomography; PFS, progression free survival; RCC, renal cell carcinoma; SCLC, small cell lung carcinoma.

Everolimus AP23573

Tirosel/temsirolimus

Agent

TABLE 2 Phase II and III Studies of mTOR Inhibitors

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pharmacokinetic characteristics of sirolimus, particularly to increase its oral bioavailability (102). Phase I studies of everolimus in cancer patients have only been published in abstracts; however, the agent has been extensively evaluated in organ transplant setting. Phase I study of everolimus administered orally one day a week in cancer patients was preliminarily reported (103). Among patients receiving doses of 5–30 mg once weekly, everolimus was well tolerated, with only mild-moderate anorexia, fatigue, rash, mucositis, headache, hyperlipidemia, and gastrointestinal toxicities. Phase II study in renal carcinoma reported promising activity, demonstrating a 33% partial response rate and time to tumor progression of greater than 3 months for 86% of patients (104). AP23573 (Ariad Pharmaceuticals) was identified among a series of semisynthesized phosphorus-containing C43-modified rapamycin analogs (105). To date, two phase I trials, evaluating single daily IV doses for 5 consecutive days every 14 days (106,107) and single weekly doses (108,109), have been completed. When administered as a 30 min IV weekly infusion, AP23573 dose-limiting toxicity was oral mucositis, which occurred at 100 mg dose. In the second study, AP23573 was administered as 30-min IV infusions daily · 5 days every 2 weeks for 4-week cycles (106,107). As with the weekly schedule, dose limiting toxicity was mucositis at 28 mg (140 mg total dose). The recommended phase II dose is 18.75 mg daily for 5 days. Early phase II clinical and pharmacodynamic data from a trial of AP23573 12.5 mg IV over 30 min daily for 5 days every 2 weeks in sarcoma patients were recently presented (110). In this study, patients with different sarcoma histologies were eligible. Among the first 82 patients, 78 were evaluable for clinical benefit response assessment defined as tumor response by standard criteria or stable disease >4 months. Three patients with osteosarcoma and one with malignant fibrous histiocytoma achieved confirmed partial responses (5%), 46 patients (59%) had stable disease and 29 patients (37%) progressed by the first assessment (111). AP23573 is being studied in patients with, hormone refractory prostate cancer, endometrial cancer, and lymphoproliferative malignancies. Initial studies of combinations of sirolimus and derivatives with standard therapies suggest enhanced toxicity at relatively modest doses may occur. Results from phase I studies evaluating tirosel/temsirolimus with interferon-alpha (112), and 5-fluorouracil (113) showed enhanced toxicities at relatively low doses. Everolimus combined with gemcitabine (114) and with gefitinib (115) demonstrated limiting toxicities at lower doses than the phase II single agent doses. Toxicities of both the standard agent and the mTOR inhibitor appear to occur more frequently. Across these studies, no apparent pharmacokinetic interactions were observed. Enhanced toxicity seen with combinations of mTOR inhibitors and standard cancer agents, may occur via overlapping toxicity of the agents or through alterations in expression of enzymes involved in drug metabolism. Modifications of dose and possibly schedule of administration of mTOR inhibitors may be required to optimize combination regimens. CONCLUSIONS In summary, agents in clinical development that specifically target the PI3K-AktmTOR pathway are currently limited to the rapamycins, which inhibit the downstream kinase mTOR. Multi-targeted inhibitors, such as UCN-01 and perifosine,

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are also under clinical investigation. Whether modulating downstream targets, such as mTOR rather than PI3K and Akt, will provide a better therapeutic index or whether specifically or broadly modulating multiple signaling pathways will have greater therapeutic effect remain unanswered questions. Initial clinical studies with mTOR inhibitors have shown the agents have anti-tumor activity and may confirm clinical benefit to patients with acceptable toxicity. Achieving a therapeutic index with inhibitors of the upstream components of the PI3K/Akt/mTOR pathway may be problematic. In vitro data show that pathway inhibitors are preferentially cytotoxic in tumor cells that exhibit increased activation of the pathway, suggesting that death of cancer cells without death of normal cells may be possible. To enhance the therapeutic index, identifying patients with tumors that selectively rely on activation of the PI3K/Akt/mTOR pathway for enrollment into clinical trials would be preferable; developing selective inhibitors to mutations in the kinases, such those described in the PI3K catalytic subunit, would be ideal. Inhibition of the pathway in tumor cells that depend upon it for survival might cause apoptosis, but would not kill normal cells that do not grow in these conditions and therefore do not have the same dependence. If a therapeutic index is not achievable when inhibitors are used as single agents, these agents might be valuable as radiation or chemotherapeutic sensitizers at lower doses, where toxicities might be less likely to develop. Given the frequent implication of the PI3K pathway in the pathophysiology of human malignancy, there is every reason to be optimistic that inhibition of the pathway will induce anti-tumor effects in cancer patients. A variety of agents designed to specifically target components of the pathway are in clinical testing and many more are in preclinical evaluation. The major clinical development challenges will be efficiently identifying the appropriate dose, schedule, and combination regimens for patients with susceptible malignancies and monitoring and managing toxicities to optimize therapeutic index. REFERENCES 1. Cantley LC. The phosphoinositide 3-kinase pathway. Science 2002; 296:1655–7. 2. Chang HW, Aoki M, Fruman D, et al. Transformation of chicken cells by the gene encoding the catalytic subunit of PI 3-kinase. Science 1997; 276:1848–50. 3. Vivanco I, Sawyers CL. The phosphatidylinositol 3-Kinase AKT pathway in human cancer. Nat Rev Cancer 2002; 2:489–501. 4. Corvera S, Czech MP. Direct targets of phosphoinositide 3-kinase products in membrane traffic and signal transduction. Trends Cell Biol 1998; 8:442–6. 5. Bellacosa A, Testa JR, Staal SP, Tsichlis PN. A retroviral oncogene, akt, encoding a serine-threonine kinase containing an SH2-like region. Science 1991; 254:274–7. 6. Staal SP. Molecular cloning of the akt oncogene and its human homologues AKT1 and AKT2: amplification of AKT1 in a primary human gastric adenocarcinoma. Proc Natl Acad Sci USA 1987; 84:5034–7. 7. Hay N, Sonenberg N. Upstream and downstream of mTOR. Genes Dev 2004; 18:1926–45. 8. Thomas GV. mTOR and cancer: reason for dancing at the crossroads? Curr Opin Genet Dev 2006; 16:78–84. 9. Sarbassov DD, Guertin DA, Ali SM, Sabatini DM. Phosphorylation and regulation of Akt/PKB by the rictor-mTOR complex. Science 2005; 307:1098–101. 10. Balaraman Y, Limaye AR, Levey AI, Srinivasan S. Glycogen synthase kinase 3beta and Alzheimer’s disease: pathophysiological and therapeutic significance. Cell Mol Life Sci 2006; 63:1226–35.

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Farnesyl Transferase Inhibitors in Cancer: Promise, but Limited Proof Laura Fanucchi Department of Hematology and Medical Oncology, Winship Cancer Institute, Emory University, Atlanta, Georgia, U.S.A.

Michael P. Fanucchi Department of Medical Oncology, St. Vincent's Comprehensive Cancer Center, New York Medical College, New York, New York, U.S.A.

Fadlo R. Khuri Department of Hematology and Medical Oncology, Winship Cancer Institute, Emory University, Atlanta, Georgia, U.S.A.

INTRODUCTION The transition from elegant regulation of cell growth, proliferation, and apoptosis to malignant transformation hinges on multiple complicated signaling pathways and cascades. There are several key proto-oncogenes that, when upregulated or mutated, become oncogenes and, through their gene products, greatly increase the malignant potential of their parent cell and progeny. There are several classes of oncogene protein products, including receptor tyrosine kinases like epidermal growth factor receptor (EGFR), cytoplasmic tyrosine kinases like Abl, cytoplasmic serine/threonine kinases like Raf kinase, transcription factors like Myc, and regulatory GTPases such as Ras. Advances in chemotherapeutic agents are increasingly aimed at these specific signaling pathways implicated in both carginogenesis and maintenance of malignant growth. This chapter focuses on Ras as a chemotherapeutic target. The biology of Ras and the role of Ras in human tumors is discussed, followed by the rationale for targeting Ras in chemotherapy. Finally, this chapter presents a summary of the preclinical and clinical development of farnesyltransferase inhibitors, one of the classes of Ras-targeting chemotherapeutics currently in active development and testing. BIOLOGY OF Ras The Ras proto-oncogene family encodes several membrane-bound monomeric G-proteins, which are approximately 21 kDa and have 85% sequence homology (1,2). The Ras gene is an important molecular switch in a complicated series of signaling pathways largely devoted to cellular differentiation and proliferation. Mutations in Ras have been identified in 30% of all human cancers (1). These mutations, particularly in codons 12, 13, and 61, lead to constitutively activated proteins in the Ras family (H, K, M, N, and R) (2). The most well-characterized mechanism of Ras activation occurs via a receptor tyrosine kinase, such as EGFR, 85

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which ultimately leads to conformational change in Ras and exchange of GTP for GDP. Ras has low GTPase activity, and GTPase-activating proteins (GAPs) are therefore required to stimulate hydrolysis of GTP. Mutated Ras proteins are constitutively activated via resistance to GAP-mediated hydrolysis (2). There are numerous Ras downstream effectors implicated in oncogenesis. The Raf serine/threonine kinase family initiates a mitogen-activated protein (MAP) kinase phosphorylation cascade, which leads to the activation of transcription factors for genes involved in cell growth and proliferation (1–3). MEKK is another serine/threonine kinase involved in cell survival and apoptosis (2)—it may also be involved in cell regulation (4). Phosphoinositide 30 -kinase (PI3-K) is an important oncogenic Ras effector through several downstream targets, of which the most well characterized are AKT and Rac/Rho. AKT is involved in apoptosis regulation (2,5) and the progression of cells from G1 to S via upregulation of cyclin D1 levels (1). Rac and Rho are integral to the regulation of the active cytoskeleton (1,6). These effectors are summarized in Figure 1. In addition to the above roles in cellular proliferation, cytoskeletal regulation, and apoptosis, activated Ras has been shown to be pivotal in tumor-induced angiogenesis (7). High levels of VEGF, a powerful growth factor that stimulates angiogenesis, have been found in tumors with Ras mutations. In particular, there is a correlation between expression of oncogenic K-Ras2 and high VEGF levels in pancreatic carcinoma and non–small cell lung carcinoma (7). Further adding to the role of activated Ras in angiogenesis is the observation that Ras acts to downregulate negative regulators of angiogenesis such as thrombospondin (7), which has direct effects on endothelial cell migration and survival (7,8).

Ras Activates Downstream Effector Pathways, but Do We Have Effective Inhibitors? Ras-GDP (inactive) Growth factors

Ras mutation Ras-GTP (active)

GAP (accelerator)

Ral-GDS PI-3 kinase

Raf-1

MEKK

Rac/Rho

AKT

MEK/MAPK

Cytoskeletal morphologic changes

Survival

Cell growth and proliferation

FIGURE 1 Ras signaling pathways modulate cytoskeletal morphology, cell survival, and cell death, as well as cell growth and proliferation in cancer cells. Source: From Ref. 3.

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Extensive post-translational modification is necessary for the hydrophilic Ras proteins to associate with the cellular membrane. Each of the Ras proteins has a CAAX motif on the carboxyl end (where C ¼ cysteine, A ¼ any aliphatic residue, and X ¼ any other residue), which is prenylated by either farnesyltransferase or geranylgeranyltransferase I or II (9). Prenylation forms a covalent thioether bond between the prenyl group and the thiol group of cysteine in the CAAX motif (10). In general, the final “X” determines which enzyme catalyzes the prenylation reaction, with farnesyltransferase preferring CAAX motifs that have X ¼ Met, Ser, Ala, or Gln, and geranylgeranyltransferase preferring Leu. Due to differences in enzyme kinetics, however, most Ras proteins will be farnesylated, except in the presence of farnesyltransferase inhibitors, in which case K-Ras and N-Ras will be geranylgeranylated (10). Then, the terminal AAX motif is cleaved via Rce1 or Afc1 proteases. Carboxymethylation follows, and lastly palmitoylation (with the exception of K-RasB) (2). The post-translational modification of Ras has been a focus of recent drug development, a topic that will be further explored in a later section. Ras in Human Tumors As stated earlier, high frequencies of Ras mutations have been identified in many human tumors, particularly lung, pancreatic, and colorectal malignancies. In light of this finding, there have been attempts to elucidate whether the presence of Ras mutations correlates with disease stage and prognosis. A meta-analysis of 43 publications published between 1990 and 2003 looking at the prognostic significance of K-Ras in lung cancers found that 20.9% of studies identified Ras mutations or P21 (the Ras protein) overexpression as negative prognostic factors for survival (11). Seventy-two percent of the studies found no association, and 2.3% found Ras to be associated with improved prognosis. For the 28 aggregable studies, Ras mutation or P21 expression was associated with decreased survival (overall hazard ratio 1.35; 95% confidence intervals 1.16–1.56) (11). It is important to note that though the overall analysis was statistically significant, in subgroup analysis Ras mutations had significant effect on survival only in adenocarcinoma (ADC), not squamous cell carcinoma (SCC). This may reflect the increased frequency of Ras mutations in ADC compared to SCC (23.12 vs. 7.09%) (11), or selection bias as ADC was assessed more frequently in the reviewed papers. Thirty-five percent of sporadic colorectal carcinomas contain K-Ras mutations. To further evaluate this relationship, the RASCAL I and II studies used data on 3439 patients with colorectal cancer from institutions around the world (12,13). Interestingly, these studies did not find an association between K-Ras mutations and tumor stage, but they did find an increased risk of relapse and death (12,13). In addition, the authors found that a specific point mutation in K-Ras, glycine to valine on codon 12, had a statistically significant effect on failure-free survival ( p ¼ 0.004, HR 1.3) and overall survival (p ¼ 0.008, HR 1.29) (12). K-Ras activation has been demonstrated in 75% to 90% of pancreatic cancers (1,14). Studies have shown a correlation between increasing incidence of K-Ras mutations with increasing grade of dysplasia in pancreatic intraductal neoplasias (14,15). To illustrate, one study using immunohistochemical staining found K-Ras expression in 0% of normal pancreatic duct tissue, 33% of hyperplastic ducts, 67% of dysplastic lesions, and 80% of the ADCs (14). In hematologic malignancies, the frequency of Ras mutations ranges from about 32% to 65% in chronic myelomonocytic leukemia (CMML), 25% to 44% in

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AML, and 6% to 18% in acute lymphoblastic leukemia (ALL) (16). The prognostic significance of Ras mutations in hematologic malignancies, however, is not clear, and may be related to Ras activation through unrelated mutations (16). Ras as a Therapeutic Target The rationale for Ras as target of cancer therapy is based largely on the functioning of the products of the Ras proto-oncogene. As described above, Ras proteins are involved in cell proliferation, regulation of the cell cycle and apoptosis, regulation of the active cytoskeleton, and control of angiogenesis—processes that are integral to oncogenesis. There is also support for the role of the Ras oncogene in tumorigenesis. Early research identified human Ras as homologous to the transforming Harvey and Kristen sarcoma viruses in bladder and lung carcinoma cell lines (17). Studies in mice have shown that oncogenic Ras induces oral tumor formation (18) and myeloproliferative states (19,20). Finally, there is evidence that inhibiting Ras activity in malignant cells decreases the survival and proliferation of these cells (2). For example, a dominant negative mutation of H-Ras, N116Y, inhibited growth of human esophageal (21) and pancreatic (22,23) tumor cells in mouse models. Therefore, considering that Ras mutations are so common in human cancers, and that the mutations are important in the development and persistence of malignant transformation, it is reasonable to target Ras in cancer therapy. FARNESYL TRANSFERASE INHIBITORS There are several compounds in preclinical and clinical trials that target various stages of the Ras signaling cascade. The most promising approaches have been: (a) inhibition of Ras expression via antisense oligodeoxynucleotides, (b) interfering with post-translational modification of Ras via farnesyltransferase inhibitors and geranylgeranyltransferase inhibitors, and (c) inhibition of Ras downstream effectors via MEK inhibitors, PI3-K inhibitors, and others (24). In the following sections, this chapter will focus on the rationale, biology, and preclinical and clinical development of farnesyltransferase inhibitors (FTIs). Currently, three FTIs are in clinical development: tipifarnib (R115777, ZarnestraTM; Johnson and Johnson Pharmaceutical Research and Development, LLC, Raritan, New Jersey, U.S.A.), lonafarnib (SCH66336, SarasarTM; Schering-Plough Research Institute, Lafayette, New Jersey, U.S.A.), and BMS214662 (Bristol-Myers Squibb, New York, New York, U.S.A.) (Table 1). Their development, and potential roles in solid and hematologic malignancies, are discussed later in this Chapter. Farnesylation is required for malignant transformation, invasion, and metastasis (9,10,25). Furthermore, though Ras undergoes extensive post-translational modification, farnesylation is the only obligatory step for Ras association with the plasma membrane (which is essential for Ras functioning) (26). Early research showed that oncogenic Ras cannot cause malignant transformation when farnesylation is inhibited by either mutation of the CAAX motif, or through inhibiting synthesis of farnesyl pyrophosphate (27). A peptidomimetic FTI revealed inhibition of v-Ras transformed cell lines, and no inhibition on v-Raf or v-mos transformed cells, thus demonstrating selective inhibition of Ras-dependent malignant transformation (26). Furthermore, this tetrapeptide and others were capable of inhibiting Ras-dependent transformation, but did not interfere with the growth of normal

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Farnesyl Transferase Inhibitors in Cancer TABLE 1 Chemical Structure and Pharmacology of the Three Farnesyl Transferase Inhibitors (FTIs) Currently in Clinical Testing FTI

Pharmacology

Tipifarnib (B)-6-[amino(4-chlorophenyl)(1-methyl-1H-imidazol-5-yl)methyl]-4-(3R115777 -chlorophenyl)-1-methyl-2(1H)-quinolinone TM Nonpeptidomimetic; orally active; competitively inhibits farnesylation of lamin B and Zarnestra K-RasB peptide substrates with IC50 values of 0.86 and 7.9 nM, respectively Lonafarnib [(þ)-4-[2-[4-(8-Chloro-3,10-dibromo-6,11-dihydro-5H-benzo[5,6]cyclohepta[1,2-b]SCH66336 pyridin-11(R)-yl)-1-piperidinyl]-2-oxo-ethyl]-1-piperidinecarboxamide SarasarTM Nonpeptidomimetic; tricyclic; orally active; inhibits farnesylation of H-Ras and K-Ras-4B in vitro with IC50 values of 1.9 and 5.2 nM, respectively BMS214662 [(R)-7-cyano-2,3,4,5-tetrahydro-1-(1H-imidazol-4-ylmethyl)-3-(phenylmethyl)-4-(2thienylsulfonyl)-1H-1,4-benzodi-azepine] Imidazole-containing tetrahydrobenzodiazepine; non-thiol; nonpeptide; CAAX-Comptetitive FTI

animal cell lines at effective concentrations (27). A pivotal study on peptidomimetic FTIs in low micromolar concentrations showed greater than 70% growth inhibition in 42 human tumor cell lines (28). Further research in transgenic mice with single (MMTV-v-Ha-Ras) (29) and multiple (30) oncogenes, which developed salivary and mammary gland carcinomas, revealed significant regression of existing tumors in response to FTIs, with minimal toxicity to normal tissues at effective dosages (29,30). Since these initial studies, there are currently several FTIs in preclinical and clinical trials, which are discussed in the following sections.

Mechanisms of Farnesyl Transferase Inhibitors The mechanisms behind the anticancer effects of FTIs are not completely understood. Several lines of evidence suggest that, though they were developed to target Ras oncoproteins, their efficacy as cancer therapeutics may not entirely be due to inhibition of Ras, but rather due to effects on downstream effectors of Ras or on other signaling cascades altogether. For example, in the presence of FTIs, K-Ras and N-Ras are geranylgeranylated and are able to associate with the plasma membrane (2). Also, there are many other proteins that are farnesylated, such as Rho-B, Rho-E, Rheb, and centromere proteins (CENP-E and CENP-F), which suggests that the action of FTIs extend to other signaling pathways besides Ras (2,31). The centromere proteins, CENP-E and CENP-F, are substrates for farnesyltransferase, but not geranylgeranyltransferase I. They are preferentially expressed during mitosis, and may be mediators of the G2 to M transition (32). The FTI SCH 66336 prevented the farnesylation of both centromere proteins in the human tumor cell line DLD-1, and altered the interaction between CENP-E and the microtubules in the lung carcinoma cell line A549 (32). These results, and others, (33) suggest that the action of FTIs may be attributed to cell-cycle effects, such as the accumulation of cells at the G2 to M transition. The antitumor activity of FTIs may be due in part to inhibition of P13K/Akt cell survival pathways, which are downstream effectors of Ras. In addition to their antiapoptotic activity, these proteins appear to be involved in several aspects of Ras-dependent oncogenesis (e.g., via cyclin D1 upregulation leading to cell-cycle progression) (34). Specifically, the FTI lonafarnib interferes with Akt activity in head and neck cells, (35) and with Akt expression in non–small cell lung cancer cells (31).

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Another protein implicated in the anticancer activity of FTIs is RhoB, one of the Rho proteins that is generally involved in regulation of the active cytoskeleton (1,31). RhoB is a target of farnesyltransferase or geranylgeranyltransferase I. When farnesylation is blocked by FTIs, the increase in geranylgeranylated RhoB appears to lead to apoptosis (31). It has also been demonstrated that SCH66336 induces phosphorylation and, therefore, inactivation of eukaryotic translation elongation factor-2, which leads to inhibition of protein synthesis (36). In addition, SCH66337 also inhibits the activation of NF-kB in the presence of tumor necrosis factor, phorbol 12-myristate 13-acetate, cigarette smoke, okadaic acid, and hydrogen peroxide, which are inflammatory and carcinogenic agents that normally activate NF-kB (37). FTIs may also have activity against other prenyl transferases, such as geranylgeranyl transferase I and II (GGT I and GGT II), which have similar active sites (31). Finally, via an unknown mechanism, FTIs appear to be involved in triggering the production of reactive oxygen species, leading to DNA damage (31). Further research into the various mechanisms underlying the anticancer activity of FTIs is warranted to improve their evidence-based use in the clinical setting. Preclinical Data in Solid Tumors Tipifarnib (R115777) R115777 (tipifarnib) is a selective nonpeptidomimetic FTI. It is a competitive inhibitor of the CAAX peptide binding site of farnesyltransferase with a Ki of 0.5 nM, and inhibits the farnesylation of lamin B1 and K-Ras (38). In vitro studies on intact cells showed that cell lines with N-Ras or H-Ras mutations were more sensitive to R115777 than those with K-Ras mutations (39). In pancreatic, colon, and melanoma in vivo mouse xenograft models, R115777 had significant antitumor effects with oral twicedaily dosing (39). Specifically, R115777 demonstrated an antiproliferative effect via apoptosis of host endothelial cells of the pancreatic tumor vasculature, an antiangiogenic effect in colon tumors, and a pro-apoptotic effect in melanoma tumors (39). A study on the in vitro and in vivo activity of R115777 in the MCF-7 estrogen receptor-positive, wild-type Ras, wild-type p53 model of breast cancer revealed significant growth inhibition both in cells and in nude mice (38). Furthermore, these experiments found that, when compared with xenograft data using cisplatin, R115777 appeared to have more of a cytostatic rather than a cytotoxic effect, suggesting that the drug may have greater utility in chemotherapeutic combinations (38). Several preclinical studies have been done investigating the role of R115777 as a cancer therapeutic agent in combination with other drugs. A study on the combination of aminobisphosphonates and R115777 in epidermoid cancer cells showed a strong synergistic effect on growth inhibition and apoptosis in KB and H1355 cells at concentrations feasible in vivo (42). Synergism between R115777 and docetaxel leading to growth inhibition has also been demonstrated in human epidermoid KB, colon HT-29, and breast HCC1937 cancer cell lines (43). Lonafarnib (SCH66336) SCH66336 (lonafarnib) is an orally bioavailable, nonpeptide, competitive, tricyclic farnesyltransferase inhibitor (44). In an HaRas transgenic mouse model, SCH66336 prophylaxis led to delayed tumor onset and reduced tumor burden, and treatment with SCH66336 caused significant tumor regression (44). In a soft agar cloning assay of 70 primary human tumor specimens, SCH66336 showed a concentrationdependent growth inhibition response in 50% (three of six) of breast tumors, 40%

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(6 of 15) of ovarian tumors, and 38% (5 of 13) of non–small cell lung tumor colony forming units (45). SCH66336 showed further preclinical activity in studies that combined it with other chemotherapeutic agents. In an in vitro study of SCH6636 and taxanes against NCI-H460 human lung tumor xenografts in nude mice and against mammary tumors in wap-Ras transgenic mice, the combination increased tumor growth inhibition in the lung cancer model and sensitized the mammary tumors to paclitaxel (46). SCH6636 was also found to act synergistically with cisplatin at clinically reasonable concentrations in A549 non-small cell lung cancer cells and T98G human glioblastoma cells, but not in MCF-7 breast, HCT116 colon, or BxPC-3 pancreatic ADC cells (47). BMS-214662 BMS-214662 is a tetrahydrobenzodiazepine, non-thiol, nonpeptide, highly selective FTI, with a mean IC50 and IC90 of 1.3 and 1.8 nM, respectively, and is more than 1000 times more selective for farnesyltransferase over geranylgeranyltransferase I (48). In preclinical testing, BMS-214662 was demonstrated to inhibit intracellular Ras processing in H-Ras-transformed Rat1 CVLS cells, and in HCT-116 human colon tumor cells (48). Furthermore, in contrast to other classes of FTIs, BMS214662 was shown to have potent apoptotic activity in vitro and in vivo (48,49). In vivo experiments in mice with HCT-116 human colon tumors treated with oral BMS-214662 resulted in the cure of eight out of eight mice at 600 mg/kg/administration, and intravenous administration at 400 mg/kg/injection yielded six out of seven mice cured (48). In mice harboring human lung carcinoma, Calu-1, oral administration of 800 mg/kg/administration cured four of eight mice; and in mice with EJ-1 bladder carcinoma, 600 mg/kg/administration of BMS-214662 cured eight of eight (48). Preclinical Data in Hematologic Malignancies Tipifarnib (R115777) In an in vitro study of the effects of R115777 on normal and leukemic hematopoisis, KG1a, U937, HL60, THP-1, K562, and UT-7 cell lines were used, along with blood or marrow aspirates from patients with acute myelogenous leukemia (AML) with high blast percentages (Table 2) (50). R115777 at concentrations of 10–50 nM inhibited growth of cell lines, AML blasts, and normal cells, but it did not affect AML blast cell adhesion or transmigration, nor did it induce significant apoptosis (50). An in vitro sensitivity study of samples from 52 pediatric cases of AML and 36 pediatric cases of ALL, found that AML cells were significantly more sensitive to tipifarnib in comparison to B-cell precursor ALL (although T-cell ALL was very sensitive), and that, within AML, the most sensitive subtype was French-American-British M5 (51). In addition, R115777 demonstrated in vitro activity at clinically achievable concentrations in myeloid progenitor cells from patients with myelofibrosis with myeloid metaplasia (52). Furthermore, R115777 induced significant dosedependent growth inhibition and apoptosis in multiple myeloma cell lines (53,54) and in fresh and cloned myeloma cells (55). The in vitro activity of R115777 in multiple myeloma cells appears to be independent of Ras mutation status (55,56). R115777 was found to act synergistically with paclitaxel and docetaxel, but not with doxorubicin, 5-fluorouracil, or cisplatin (and others), to inhibit multiple myeloma cell proliferation and to induce apoptosis—including cells resistant to

Phase I Adjei (75) Phase II Sharma (78) Phase I Ryan (82)

Advanced cancers Colorectal Advanced cancers

NSCLC

Colorectal

Phase II Adjei (69)

Phase III Rao (73)

Cancer types

Advanced cancers

Phase author

Phase I Crul (65)

Doses

Toxicity

Outcomes

6/43 SD; MS 7.7 months No difference in progression-free survival 1/7 PR No objective responses No objective responses

Acceptable profile Gastrointestinal fatigue Diarrhea Neutropenia

350 mg bid · 7 days for 3 wk 200 mg continuous 200 mg/m2 over 1 hr for 21 days

3 SD; 1 PR

300 mg bid · 21 days for 4 wk

Myelo-suppression, neuropathy

300 mg bid · 21 days for 4 wk

300 mg bid continuous

Abbreviations: CR, complete response; FTI, farnesyl transferase inhibitor; MS, median survival; NSCLC, non–small cell lung cancer; PR, partial response; SD, stable disease.

BMS-214662

Lonafarnib

Tipfarnib

FTI

TABLE 2 Summary of Clinical Trials of Farnesyl Transferase Inhibitors as Single Agents in Solid Tumors

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taxanes and R115777 (57). Finally, tipifarnib was found to act synergistically with bortezomib in multiple myeloma and AML cell lines, and the combination appeared to overcome cell adhesion-mediated drug resistance (58). Lonafarnib (SCH66336) In an in vivo mouse model of Bcr/Abl ALL, a Bcr/Abl leukemia that does not respond well to imatinib, SCH66336 reversed early signs of leukemia and significantly prolonged survival (59,60). Based on the rationale that multiple myeloma is IL-6 dependent, and that IL-6 is secreted via the Ras/Raf/MAPK pathway, a trial of lonafarnib in combination with the proteasome inhibitor bortezomib was undertaken in multiple myeloma cell lines (61). The two agents were found to act synergistically to induce apoptosis more rapidly and to downregulate phosphorylated AKT (61). BMS-214662 Most of the preclinical data on BMS-214662 are in nonhematologic malignancies (48); however, the drug also induced apoptosis in B-cell chronic lymphocytic leukemia (B-CLL) cells from 18 patients with B-CLL (62). Clinical Data in Solid Tumors Tipifarnib (R115777) Phase I studies of orally-administered R115777 in advanced cancers where no standard therapy was available recommended 500 mg orally bid for 5 consecutive days followed by 5 days of rest, (63) 300 mg orally bid for 28 days followed by 1–2 weeks of rest, (64) and 300 mg orally bid in continuous dosing (65). Doselimiting toxicities included grade 3 neuropathy and grade 2 fatigue at 1300 mg bid, (63) grade 4 neutropenia in one of six patients at 300 mg bid, (64) grade 4 myelosuppression in two of four patients at 400 mg bid, and grade 3 neuropathy in one of five patients at 500 mg bid (65). In the continuous dosing trial, three patients with pancreatic, colon, and cervical carcinomas had stable disease, and one with non–small cell lung cancer refractory to platinum therapy had a partial response lasting five months (65). Phase II trials of R115777 as monotherapy were disappointing, revealing minimal clinical activity in metastatic colorectal carcinoma (66), advanced transitional cell carcinoma (67), pancreatic cancer (68), stage IIIB and stage IV non–small cell lung cancer (69), and sensitive relapse small-cell lung cancer (70). In a study of 76 patients with advanced breast cancer comparing continuous and intermittent dosing, however, 10% and 15% of the continuous dosing group and 14% and 9% of the intermittent dosing group had partial responses and stable disease, respectively (71). The toxicity profile was significantly less in the intermittent dosing group (300 mg bid for 21 days followed by 7 rest days) (71). Several phase I trials of R115777 in combination with other agents have also been undertaken. A study of R115777 at 300 mg bid with gemcitabine at 1000 mg/ m2 and cisplatin at 75 mg/m2 in 27 patients with advanced solid tumors, revealed inhibition of protein farnesylation (as demonstrated by accumulation of prelamin A in buccal mucosa cells of patients), one complete response, and eight partial responses (71). The combination of R115777 at 300 mg bid for 14 days every 3 weeks and irinotecan at 350 mg/m2 demonstrated a clinically relevant response in one trial (72).

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A multicenter, double-blind, placebo-controlled phase III trial of R115777 in 368 patients with refractory advanced colorectal cancer did not reveal a statistically significant difference in progression-free survival between the R115777 and placebo groups, although the experimental drug was found to have an acceptable toxicity profile (73). Nor, in a phase III trial, was tipifarnib in combination with gemcitabine found to prolong overall survival in advanced pancreatic cancer compared to gemcitabine alone (74). Lonafarnib (SCH66336) A phase I trial of SCH66336 in 20 patients with metastatic or locally advanced solid tumors for which there was no established curative or life-prolonging therapy, recommended a dosing schedule of 350 mg bid for 7 days out of every 3 weeks (75). Dose-limiting toxicities were gastrointestinal (nausea, vomiting, diarrhea) and fatigue at 400 mg bid. The study also had one partial response in a patient with metastatic non-small cell lung cancer (75). Another study of SCH66336 in 20 patients with solid tumors found similar dose-limiting toxicities with continuous dosing, with the addition of grade 4 neutropenia, grade 3 neurocortical toxicity, and grade 3 fatigue at 300 mg bid. These authors recommended 200 mg bid as maximally tolerated dose for continuous dosing (76). In a phase I trial of lonafarnib in combination with paclitaxel in 24 patients with solid tumors, the maximally tolerated dose was determined to be lonafarnib 100 mg bid and paclitaxel 175 mg/ m2, with dose-limiting toxicities of grade 3 hyperbilirubinemia (lonafarnib 100 mg bid and paclitaxel 175 mg/m2), grade 4 diarrhea and grade 3 peripheral neuropathy (lonafarnib 125 mg bid and paclitaxel 175 mg/m2); and grade 4 neutropenia with fever and grade 4 diarrhea (lonafarnib 150 mg bid and paclitaxel 175 mg/m2) (77). The authors also reported that 6 of 15 previously treated patients had a durable partial response, including two previously treated with taxanes (77). A phase II study of SCH66336 given continuously at 200 mg to 21 patients with metastatic colorectal cancer refractory to 5-fluorouracil and irinotecan, revealed no objective responses, and grade 3 diarrhea in 42% of patients (78). A trial in 19 patients with advanced unresectable or metastatic transitional cell carcinoma at 200 mg lonafarnib given continuously, also revealed no responses, with 9 of the patients having symptomatic progression while on study (79). Phase II trials of lonafarnib in combination with other chemotherapeutic agents have been more promising. In one study of lonafarnib in combination with paclitaxel in 33 patients with taxane-refractory/resistant non–small cell lung cancer, 10% of the 29 evaluable patients had partial responses and 38% had stable disease (80). The treatment regimen of continuous lonafarnib 100 mg orally bid with paclitaxel 175 mg/m2 intravenously over three hours on Day 8 of each 21-day cycle was well tolerated with minimal toxicity (80). In another phase II study, the combination of SCH66336 and gemcitabine was evaluated as a potential second-line treatment in 33 patients with advanced urothelial tract cancer (81). Patients received SCH66336, 150 mg in the morning and 100 mg in the evening, and gemcitabine, 1000 mg/m2 on day 1, 8, and 15 per 28-day cycle, with minimal toxicity, and the authors reported an overall response rate of 32.3% (95% confidence intervals 17–51%), corresponding to 9 partial responses and one complete response (81). BMS-214662 A phase I trial of BMS-214662 in escalating doses from 36 to 225 mg/m2, as single one-hour infusions every 21 days, was completed in 54 patients with advanced

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solid tumors (predominantly pancreatic, lung, and colorectal cancers), and established 200 mg/m2 as the recommended dose for phase II studies (82). The most frequent adverse effects were noted as reversible grade 3 transaminitis, nausea, and vomiting (82). A phase I study of BMS-214662 as weekly one-hour infusions in 27 patients with solid tumors reported grade 3 and 4 neutropenia as the most common dose-limiting toxicity at doses ranging from 28 to 220 mg/m2 for four weeks (83). There were no objective clinical responses documented in either of these phase I trials. Another phase I trial compared weekly one-hour infusions with weekly 24-hr infusions and found that the 24-hr infusion resulted in decreased maximum farnesyltransferase inhibition in peripheral blood mononuclear cells, but increased duration of enzyme inhibition, and no objective therapeutic benefit over the one-hour schedule (84). BMS-214662 was also evaluated in the phase I setting in combination with paclitaxel and carboplatin (85). Thirty patients with solid tumors received BMS214662 on a one-hour weekly infusion schedule following paclitaxel and carboplatin on the first day of a 21-day cycle, with dose-limiting toxicities of neutropenia, thrombocytopenia, nausea, and vomiting, and a MTD of BMS-214662 at 160 mg/ m2, paclitaxel 225 mg/m2 and carboplatin (area under the curve ¼ 6 on day 1), every 21 days (85). The objective responses were one partial response in a patient with taxane-resistant esophageal cancer, and stable disease over more than four cycles in eight other patients (85). Clinical Data in Hematologic Malignancies Tipifarnib (R115777) A phase I trial of orally-administered R115777, at doses from 100 mg bid to 1200 mg bid for up to 21 days, in 35 adults with refractory and relapsed acute leukemia (AML, ALL, CML in blast crisis, or AML in poor prognostic subgroups) revealed dose-limiting toxicity of central neurotoxicity at 1200 mg bid, with additional toxicities of reversible nausea, renal insufficiency, polydipsia, and myelosuppression (Table 3) (86). In this trial, R115777 was demonstrated to accumulate in bone marrow in a dose-dependent fashion. Clinical responses occurred in 29% of patients, including two complete remissions, in a non-dose-dependent fashion at doses from 100 to 900 mg bid (86). A phase I trial of R115777 in escalating doses with a starting dose of 300 mg bid, in 21 patients with myelodysplastic syndrome, revealed a MTD of 400 mg bid (dose-limiting toxicities were grade 4 hematologic toxicity) and 6 objective responses (87). A trial of R115777 at 600 mg bid for four weeks every six weeks, in adults with Philadelphia chromosome-positive CML, myelofibrosis, or multiple myeloma, revealed that out of 22 patients with CML, 7 achieved a complete or partial response (the majority of these were in the chronic phase); of the 8 patients with myelofibrosis and of the 10 patients with multiple myeloma, there were no objective responses (88). The combination of tipifarnib with idarubicin and cytarabine was tested in 33 patients with newly diagnosed AML or high-risk myelodysplastic syndrome and resulted in 67% complete remission and 6% partial response (89). The treatment protocol was idarubicin 12 mg/ m2/d on days 1–3, cytarabine 1.5 g/m2 IV over 24 hr daily on days 1–4 and tipifarnib 300 mg BID for 21 days. Patients achieving CR received consolidation (5 courses) with idarubicin 8 mg/m2/d on days 1–2, cytarabine 0.75 g/m2/d on days 1–3, and tipifarnib 300 mg BID for 14 days every 4–6 weeks. Maintenance was with tipifarnib 300 mg BID for 21 days every 4–6 weeks for 6 months (89).

Diarrhea, nausea

200 mg bid 56–156 mg/m2 over 1 hr for 1 wk; 300 mg/m2 over 24 hr

AML, ALL, high risk MDS

Phþ CML

600 mg bid · 4 wk for 6 wk

MDS

Phase II Kurzrock (90)

Phase I Borthakur (91) Phase I Cortes (92)

Myelosuppression, neurotoxicity, rash (41% required dose reduction) Diarrhea, nausea

600 mg bid · 4 wk for 6 wk

Toxicity Central neurotoxicity, myelosuppression

Phþ CML, myelofibrosis, multiple myeloma

Doses 100–1200 mg bid

Phase I Cortes (88)

Cancer types

AML, ALL, CML in blast crisis

Phase author

Phase I Karp (86)

Outcomes

2/30 CRi; 1/30 CR for 14 weeks

2/13 clinical responses

8/11 PR at 900 mg; 2/8 PR, 1/8 CR at 600 mg; 4/5 PR at 300 mg 7/22 CR or PR for CML; no responses for other cancers 2/28 CR; 1/28 PR

Abbreviations: ALL, acute lymphoblastic leukemia; AML, acute myeloid leukemia; CML, chronic myeloid leukemia; CR, complete response; CRi, complete response with incomplete platelet recovery; FTI, farnesyl transferase inhibitor; MDS, myelodysplastic syndrome; MM, multiple myeloma; MS, median survival; Phþ, Philadelphia chromosome positive; PR, partial response; SD, stable disease.

BMS-214662

Lonafarnib

Tipfarnib

FTI

TABLE 3 Summary of Clinical Trials of Farnesyl Transferase Inhibitors as Single Agents in Hematologic Malignancies

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A phase II trial of R115777 in 28 patients with myelodysplastic syndrome treated at 600 mg bid in cycles of four weeks on, two weeks off, resulted in three responses (two had refractory anemia with excess blasts and one had refractory anemia with excess blasts in transformation) (90). The authors found that 600 mg bid was not well-tolerated (myelosuppression, neurotoxicity, rash), and 41% of the patients required dose-reduction (90). Lonafarnib (SCH66336) To date, there has only been one clinical trial of lonafarnib in hematologic malignancies. This phase I trial investigated lonafarnib, at 200 mg bid starting dose, in 13 patients with Philadelphia chromosome-positive CML in the chronic or accelerated phases, and who failed or were intolerant of imatinib therapy (91). Lonafarnib was generally well-tolerated, with diarrhea (84% of patients) and nausea being the most common adverse effects, and the authors noted two clinical responses (91). BMS-214662 A phase I trial of BMS-214662 in refractory or relapsed high-risk myelodysplastic syndromes and acute leukemias was undertaken to determine dose-limiting toxicity and MTD (92). Thirty patients in total were treated at doses of 56, 84, 118, and 156 mg/m2 in weekly one-hour infusions, and 300 mg/m2 given as a 24-hr continuous infusion. Nausea and diarrhea were the most common adverse effects, and dose-limiting toxicity ( grade 3 diarrhea and grade 2 nausea in one patient) was observed at 156 mg/m2. Seventeen percent of patients showed clinical responses, including two with complete remission with incomplete platelet recovery, one with hematologic improvement, and two with morphologic leukemia-free state (92). CONCLUSIONS AND FUTURE DIRECTIONS Despite their development as a Ras-targeting therapy, the farnesyltransferase inhibitors are now known not to inhibit Ras exclusively. Although clinical testing of the farnesyltransferase inhibitors has been largely disappointing in solid tumors, more recent data in hematologic malignancies are more promising. Tipifarnib has advanced farthest in clinical trials, and been most promising in treatment of AML. AML persists as a treatment challenge, with poor long-term survival, low remission rates, and high treatment-related mortality. An active area of research in AML focuses on identification of aberrant signaling pathways that may be targets of specific drug design. Farnesyltransferase inhibitors show potential in this area, especially given their relatively mild toxicity profile. A recent study on tipifarnib sensitivity in pediatric AML and ALL samples compared to normal bone marrow found large interindividual differences in the in vitro sensitivity of AML and ALL blasts to tipifarnib, with greater overall sensitivity in AML (93). The authors also reported a correlation between resistance to tipifarnib and resistance to cytarabine or 6-thioguanine. Additional studies are warranted to further explore potential biomarkers that are predictive of clinical response to tipifarnib in leukemias. Another area of active and future research is the use of farnesyltransferase inhibitors in combinations with other chemotherapeutic and targeted agents. Recent preclinical work on the combination of lonafarnib and bortezomib, a proteasome inhibitor, in mutiple myeloma was based on the hypotheses that the

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two agents would target different signaling pathways, and that lonafarnib would inhibit IL-6, which is a well-known multiple myeloma growth factor (61). The authors demonstrated significant and synergistic myeloma cell death at low, clinically acheiveable concentrations of the drugs. Furthermore, the order of administration of the two drugs was essential, with sequencing bortezomib before lonafarnib being most effective. The combination resulted in rapid caspase activation and downregulation of p-AKT expression, which correlated with rapid induction of cell death. It is possible that p-AKT expression could be used as a biomarker for response and dosing in further studies (61). Along with increasing understanding of the complicated and myriad signaling pathways involved in cell growth, proliferation, and apoptosis, and where these pathways may lead to carcinogenesis, will come better molecular agents to target aberrant signals.

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Lubet RA, Christov K, You M, et al. Effects of the farnesyl transferase inhibitor R115777 (Zarnestra) on mammary carcinogenesis: prevention, therapy, and role of HaRas mutations. Mol Cancer Ther 2006; 5:1073–8. Yao R, Wang Y, Lu Y, et al. Efficacy of the farnesyltransferase inhibitor R115777 in a rat mammary tumor model: role of Ha-ras mutations and use of microarray analysis in identifying potential targets. Carcinogenesis 2006; 27:1420–31. Caraglia M, D'Alessandro AM, Marra M, et al. The farnesyl transferase inhibitor R115777 (Zarnestra) synergistically enhances growth inhibition and apoptosis induced on epidermoid cancer cells by Zoledronic acid (Zometa) and Pamidronate. Oncogene 2004; 23:6900–13. Caraglia M, Giuberti G, Marra M, et al. Docetaxel induces p53-dependent apoptosis and synergizes with farnesyl transferase inhibitor r115777 in human epithelial cancer cells. Front Biosci 2005; 10:2566–75. Liu M, Bryant MS, Chen J, et al. Antitumor activity of SCH 66336, an orally bioavailable tricyclic inhibitor of farnesyl protein transferase, in human tumor xenograft models and wap-ras transgenic mice. Cancer Res 1998; 58:4947–56. Petit T, Izbicka E, Lawrence RA, et al. Activity of SCH 66336, a tricyclic farnesyltransferase inhibitor, against human tumor colony-forming units. Ann Oncol 1999; 10:449–53. Shi B, Yaremko B, Hajian G, et al. The farnesyl protein transferase inhibitor SCH66336 synergizes with taxanes in vitro and enhances their antitumor activity in vivo. Cancer Chemother Pharmacol 2000; 46:387–93. Adjei AA, Davis JN, Bruzek LM, et al. Synergy of the protein farnesyltransferase inhibitor SCH66336 and cisplatin in human cancer cell lines. Clin Cancer Res 2001; 7:1438–45. Rose WC, Lee FY, Fairchild CR, et al. Preclinical antitumor activity of BMS-214662, a highly apoptotic and novel farnesyltransferase inhibitor. Cancer Res 2001; 61:7507–17. Manne V, Lee FY, Bol DK, et al. Apoptotic and cytostatic farnesyltransferase inhibitors have distinct pharmacology and efficacy profiles in tumor models. Cancer Res 2004; 64:3974–80. Liesveld JL, Lancet JE, Rosell KE, et al. Effects of the farnesyl transferase inhibitor R115777 on normal and leukemic hematopoiesis. Leukemia 2003; 17–1806–12. Goemans BF, Zwaan CM, Harlow A, et al. In vitro profiling of the sensitivity of pediatric leukemia cells to tipifarnib: identification of T-cell ALL and FAB M5 AML as the most sensitive subsets. Blood 2005; 106:3532–7. Mesa RA, Tefferi A, Gray LA, et al. In vitro antiproliferative activity of the farnesyltransferase inhibitor R115777 in hematopoietic progenitors from patients with myelofibrosis with myeloid metaplasia. Leukemia 2003; 17:849–55. Le Gouill S, Pellat-Deceunynck C, Harousseau JL, et al. Farnesyl transferase inhibitor R115777 induces apoptosis of human myeloma cells. Leukemia 2002; 16:1664–7. Beaupre DM, McCafferty-Grad J, Bahlis NJ, et al. Farnesyl transferase inhibitors enhance death receptor signals and induce apoptosis in multiple myeloma cells. Leuk Lymphoma 2003; 44:2123–34. Ochiai N, Uchida R, Fuchida S, et al. Effect of farnesyl transferase inhibitor R115777 on the growth of fresh and cloned myeloma cells in vitro. Blood 2003; 102:3349–53. Beaupre DM, Cepero E, Obeng EA, et al. R115777 induces Ras-independent apoptosis of myeloma cells via multiple intrinsic pathways. Mol Cancer Ther 2004; 3:179–86. Zhu K, Gerbino E, Beaupre DM, et al. Farnesyltransferase inhibitor R115777 (Zarnestra, Tipifarnib) synergizes with paclitaxel to induce apoptosis and mitotic arrest and to inhibit tumor growth of multiple myeloma cells. Blood 2005; 105:4759–66. Yanamandra N, Colaco NM, Parquet NA, et al. Tipifarnib and bortezomib are synergistic and overcome cell adhesion-mediated drug resistance in multiple myeloma and acute myeloid leukemia. Clin Cancer Res 2006; 12:591–9. Reichert A, Heisterkamp N, Daley GQ, et al. Treatment of Bcr/Abl-positive acute lymphoblastic leukemia in P190 transgenic mice with the farnesyl transferase inhibitor SCH66336. Blood 2001; 97:1399–403.

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Mishra S, Zhang B, Groffen J, et al. A farnesyltransferase inhibitor increases survival of mice with very advanced stage acute lymphoblastic leukemia/lymphoma caused by P190 Bcr/Abl. Leukemia 2004; 18:23–8. David E, Sun SY, Waller EK, et al. The combination of the farnesyl transferase inhibitor lonafarnib and the proteasome inhibitor bortezomib induces synergistic apoptosis in human myeloma cells that is associated with down-regulation of p-AKT. Blood 2005; 106:4322–9. Marzo I, Perez-Galan P, Giraldo P, et al. Farnesyltransferase inhibitor BMS-214662 induces apoptosis in B-cell chronic lymphocytic leukemia cells. Leukemia 2004; 18:1599–604. Zujewski J, Horak ID, Bol CJ, et al. Phase I and pharmacokinetic study of farnesyl protein transferase inhibitor R115777 in advanced cancer. J Clin Oncol 2000; 8:927–41. Punt CJ, van Maanen L, Bol CJ, et al. Phase I and pharmacokinetic study of the orally administered farnesyl transferase inhibitor R115777 in patients with advanced solid tumors. Anticancer Drugs 2001; 12:193–7. Crul M, de Klerk GJ, Swart M, et al. Phase I clinical and pharmacologic study of chronic oral administration of the farnesyl protein transferase inhibitor R115777 in advanced cancer. J Clin Oncol 2002; 20:2726–35. Whitehead RP, McCoy S, Macdonald JS, et al. Phase II trial of R115777 (NSC #70818) in patients with advanced colorectal cancer: A Southwest Oncology Group study. Invest New Drugs 2006; 24(4):335–41. Rosenberg JE, von der Maase H, Seigne JD, et al. A phase II trial of R115777, an oral farnesyl transferase inhibitor, in patients with advanced urothelial tract transitional cell carcinoma. Cancer 2005; 103:2035–41. Macdonald JS, McCoy S, Whitehead RP, et al. A phase II study of farnesyl transferase inhibitor R115777 in pancreatic cancer: a Southwest oncology group (SWOG 9924) study. Invest New Drugs 2005; 23:485–7. Adjei AA, Mauer A, Bruzek L, et al. Phase II study of the farnesyl transferase inhibitor R115777 in patients with advanced non-small-cell lung cancer. J Clin Oncol 2003; 21:1760–6. Heymach JV, Johnson DH, Khuri FR, et al. Phase II study of the farnesyl transferase inhibitor R115777 in patients with sensitive relapse small-cell lung cancer. Ann Oncol 2004; 15:1187–93. Johnston SR, Hickish T, Ellis P, et al. Phase II study of the efficacy and tolerability of two dosing regimens of the farnesyl transferase inhibitor, R115777, in advanced breast cancer. J Clin Oncol 2003; 21:2492–9. Adjei AA, Croghan GA, Erlichman C, et al. A Phase I trial of the farnesyl protein transferase inhibitor R115777 in combination with gemcitabine and cisplatin in patients with advanced cancer. Clin Cancer Res 2003; 9:2520–6. Rao S, Cunningham D, de Gramont A, et al. Phase III double-blind placebo-controlled study of farnesyl transferase inhibitor R115777 in patients with refractory advanced colorectal cancer. J Clin Oncol 2004; 22:3950–7. Van Cutsem E, van de Velde H, Karasek P, et al. Phase III trial of gemcitabine plus tipifarnib compared with gemcitabine plus placebo in advanced pancreatic cancer. J Clin Oncol 2004; 22:1430–8. Adjei AA, Erlichman C, Davis JN, et al. A Phase I trial of the farnesyl transferase inhibitor SCH66336: evidence for biological and clinical activity. Cancer Res 2000; 60:1871–7. Eskens FA, Awada A, Cutler DL, et al. Phase I and pharmacokinetic study of the oral farnesyl transferase inhibitor SCH 66336 given twice daily to patients with advanced solid tumors. J Clin Oncol 2001; 19:1167–75. Khuri FR, Glisson BS, Kim ES, et al. Phase I study of the farnesyltransferase inhibitor lonafarnib with paclitaxel in solid tumors. Clin Cancer Res 2004; 10:2968–76. Sharma S, Kemeny N, Kelsen DP, et al. A phase II trial of farnesyl protein transferase inhibitor SCH 66336, given by twice-daily oral administration, in patients with metastatic colorectal cancer refractory to 5-fluorouracil and irinotecan. Ann Oncol 2002; 13:1067–71.

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Winquist E, Moore MJ, Chi KN, et al. A multinomial Phase II study of lonafarnib (SCH 66336) in patients with refractory urothelial cancer. Urol Oncol 2005; 23:143–9. Kim ES, Kies MS, Fossella FV, et al. Phase II study of the farnesyltransferase inhibitor lonafarnib with paclitaxel in patients with taxane-refractory/resistant nonsmall cell lung carcinoma. Cancer 2005; 104:561–9. Theodore C, Geoffrois L, Vermorken JB, et al. Multicentre EORTC study 16997: feasibility and phase II trial of farnesyl transferase inhibitor & gemcitabine combination in salvage treatment of advanced urothelial tract cancers. Eur J Cancer 2005; 41: 1150–7. Ryan DP, Eder JP, Jr, Puchlaski T, et al. Phase I clinical trial of the farnesyltransferase inhibitor BMS-214662 given as a 1-hour intravenous infusion in patients with advanced solid tumors. Clin Cancer Res 2004; 10:2222–30. Papadimitrakopoulou V, Agelaki S, Tran HT, et al. Phase I study of the farnesyltransferase inhibitor BMS-214662 given weekly in patients with solid tumors. Clin Cancer Res 2005; 11:4151–9. Eder JP, Jr, Ryan DP, Appleman L, et al. Phase I clinical trial of the farnesyltransferase inhibitor BMS-214662 administered as a weekly 24 h continuous intravenous infusion in patients with advanced solid tumors. Cancer Chemother Pharmacol 2006; 58:107–16. Dy GK, Bruzek LM, Croghan GA, et al. A phase I trial of the novel farnesyl protein transferase inhibitor, BMS-214662, in combination with paclitaxel and carboplatin in patients with advanced cancer. Clin Cancer Res 2005; 11:1877–83. Karp JE, Lancet JE, Kaufmann SH, et al. Clinical and biologic activity of the farnesyltransferase inhibitor R115777 in adults with refractory and relapsed acute leukemias: a phase 1 clinical-laboratory correlative trial. Blood 2001; 97:3361–9. Kurzrock R, Kantarjian HM, Cortes JE, et al. Farnesyltransferase inhibitor R115777 in myelodysplastic syndrome: clinical and biologic activities in the phase 1 setting. Blood 2003; 102:4527–34. Cortes J, Albitar M, Thomas D, et al. Efficacy of the farnesyl transferase inhibitor R115777 in chronic myeloid leukemia and other hematologic malignancies. Blood 2003; 101:1692–7. Ravandi-Kashani F, Kantarjian H, Garcia-Manero G, et al. Tipifarnib in combination with idarubicin and cytarabine in patients with newly diagnosed acute myeloid leukemia (AML) or high-risk myelodysplastic syndrome (MDS). J Clin Oncol, ASCO Proc 2006; 24:6557. Kurzrock R, Albitar M, Cortes JE, et al. Phase II study of R115777, a farnesyl transferase inhibitor, in myelodysplastic syndrome. J Clin Oncol 2004; 22:1287–92. Borthakur G, Kantarjian H, Daley G, et al. Pilot study of lonafarnib, a farnesyl transferase inhibitor, in patients with chronic myeloid leukemia in the chronic or accelerated phase that is resistant or refractory to imatinib therapy. Cancer 2006; 106:346–52. Cortes J, Faderl S, Estey E, et al. Phase I study of BMS-214662, a farnesyl transferase inhibitor in patients with acute leukemias and high-risk myelodysplastic syndromes. J Clin Oncol 2005; 23:2805–12. Gotlib J. Farnesyltransferase inhibitor therapy in acute myelogenous leukemia. Curr Hematol Rep 2005; 4:77–84.

7

Protein Kinase C Inhibitors in the Treatment of Non–Small Cell Lung Cancer Yun Oh Department of Thoracic/Head and Neck, Medical Oncology, M. D. Anderson Cancer Center, University of Texas, Houston, Texas, U.S.A.

Michael Lahn and Asavari Wagle Oncology Product Development, Eli Lilly and Company, Indianapolis, Indiana, U.S.A.

Roy Herbst Department of Thoracic/Head and Neck, Medical Oncology, M. D. Anderson Cancer Center, University of Texas, Houston, Texas, U.S.A.

INTRODUCTION Since its recognition as a critical signaling pathway in cancer, a variety of approaches have been developed to block protein kinase C (PKC) signaling in cancer cells. Here, we review the different PKC inhibitors currently in clinical investigation and their presumed role in the treatment of non–small cell lung cancer (NSCLC). Based on the knowledge of staurosporine, nonselective and selective PKC inhibitors have been developed. Among the nonselective PKC inhibitors, bryostatin 1 has more extensively been evaluated for its use in the treatment of NSCLC. Recently, the selective PKC inhibitor enzastaurin completed a phase II study to evaluate its activity as a second- and third-line treatment for NSCLC. Finally, we discuss the challenges for the future development of PKC inhibitors in NSCLC. PKC is a family of isoenzymes that are products of distinct genes and are activated in response to growth factors, hormones, and neurotransmitters (1,2). PKC isozymes have been classified into three groups: Group A, or classical (PKC-a, -bI, -bII, and -g), which requires a lipid cofactor (e.g., phosphatidyl-serine, PS), Ca2þ, and 1,2-diacylglycerol (DAG) for activation; Group B, or new (PKC-d, -e, -h, -, and -m), which are Ca2þ-independent; and Group C, or atypical (PKC-l and -z). All PKC isozymes are composed of one polypeptide chain divided into an Nterminal regulatory domain and a C-terminal catalytic domain; four regions (C1–C4) are conserved across all isozymes, and five regions (V1–V5) are variable between isozymes (2). The C1 regulatory region contains the pseudosubstrate domain, which is autoinhibitory and masks the catalytic domain. Activation is initiated by removal of the pseudosubstrate so that the catalytic domain is exposed. The Group A isozymes contain an additional Ca2þ activated regulatory binding site that is absent from the other groups. The C3 (ATP binding site) and C4 (substrate binding site) catalytic regions are found in all groups; phosphorylation is an essential step in enzyme activation (2). After its synthesis, PKC undergoes a series of ordered priming and phosphorylation events in which a conserved threonine at the activation loop is trans-phosphorylated by a phosphoinositide-dependent kinase (PDK-1), followed by the phosphorylation of a turn motif and a hydrophobic 103

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FXXFS/TF/Y motif (2). This priming process via phosphorylation results in a catalytically competent, but inactive, mature PKC, maintained in an autoinhibited conformation by the pseudosubstrate in the cytosol. The matured but inactive PKCs are believed to randomly diffuse in the cytosol. Upon stimulation of agonists, the activation of cell surface receptors (for example, growth factor receptors and G-protein-coupled receptors) results in the activation of phospholipase C at the plasma membrane, followed by a second phase of activation via phospholipases at the cytoplasm (3). The hydrolysis of phosphatidylinositol 4,5-bisphosphate by phospholipase C generates second messengers (DAG and calcium) and causes the translocation of cytosolic PKC to the plasma membrane. Pharmacologic stimulation of PKC has been shown to induce malignant transformation and proliferation, apoptosis, cell migration, and cell activation (4). The pharmacologic inhibition of PKCs is associated with decreased growth and survival of tumors, reduction of neoplastic properties, promotion of apoptosis, and sensitization of tumor cells to chemotherapeutic agents (4–13). Hence, a number of PKC inhibitors have been developed and their activity in the treatment of cancer has been pursued for the past 20 years. In the following sections, we review the experience of the various inhibitors currently explored in clinical investigation.

INHIBITORS OF PKCS A number of PKC inhibitors were identified in the mid-1980s (14–17), which were subsequently found to be nonspecific for particular PKC isoenzymes. Some of these inhibitors were evaluated in lung cancer cell lines (18) and have also been considered for the treatment of lung cancer. However, in the past decade progress in the basic understanding of PKC biology and the growing understanding of medicinal chemistry have resulted in the identification of selective PKC inhibitors, including inhibitors of novel pharmacological platform, such as antisense oligonucleotides (ASO) (19–21). When comparing the various inhibitors, it becomes apparent that they all possess multiple activities that are not limited to the kinase inhibition of the PKC isoenzymes, but also can have significant impact on cell cycle (22). Therefore, the definition of PKC inhibitors may not be precise, but for the sake of this review we will use the definition of PKC inhibitors for those molecules that were originally designed to inhibit PKC and its isoenzymes. We will first review the PKC inhibitors with a broad activity on multiple PKCs (i.e., nonspecific PKC inhibitors) and then review those PKC inhibitors with a specific PKC-inhibitory profile (i.e., specific PKC inhibitors) (Table 1).

Nonspecific PKC Inhibitors Staurosporine Staurosporine is an indole-carbazole originally isolated from the bacterium Streptomyces staurosporeus in 1977 (23). It was identified as an anticancer agent from an original screen of over 50 alkaloids in 1994 (24). Staurosporine is comprised of a sugar molecule and a heterocyclic indole-carbazole group. Staurosporine is a potent PKC inhibitor acting on the binding pocket of PKC with a broad spectrum of activity on many other protein kinases (14,25,26). Staurosporine is known to induce apoptosis through a mitochondria-mediated pathway (27), causing an oxidative stress through mitochondrially generated reactive oxygen species (ROS)

IV

Oral

Peptide

Acyclic bisindolylmaleimide

Phase III

N/A

Phase III (development stopped)

Phase I-II

Phase I-II

N/A Phase I-II

Clinical phase

DLBCL, glioblastoma NSCLC

N/A

NHL

NHL, CLL

AML

N/A Lymphoma, melanoma

Single agent activity

No MTD fatigue

N/A

MTD established thrombocytopenia, coagulopathy

MTD established myalgias

No MTD as single agent; diarrhea/ nausea in combination with chemotherapy

N/A MTD established Insulin resistance

Drug-related toxicities N/A

Pharmacodynamic markers

N/A

AKT, GSK-3b ribosomal Protein S6

None

Stimulation of various kinases

Decreased GSK3-b phosphorylation in tumor and PBMCs

N/A

None

None

None Cyclin-dependent kinase; broad serine-threonine kinases None Flt-3, PDGFR, c-KIT, tyrosine kinases, multidrug resistance, gene Pgp, ZNF-198 FGF receptor fusion protein

Various Protein Kinases

Other cancer-related target inhibition

Abbreviations: AML, acute myeloid leukemia; CLL, chronic lymphocytic leukemia; DLBCL, diffuse large B-cell; GSK, glucogen synthase kinase; IV, intravenous; MTD, maximum tolerated dose; NHL, non-Hodgkin's lymphoma; NSCLC, non–small cell lung cancer; PBMC, peripheral blood mononuclear cell; Pgp, P-glycoprotein; PKC, protein kinase C.

IV continuous infusion

IV

Oral

N/A IV

Administration

Phosphorothioate antisense oligonucleotide

C-20 (E,E)-octa2-dienoate ester group

Bryostatin

Specific PKC inhibitors Aprinocarsen (Affinitak
, LY900003, ISIS 3521) KAI-9803 (KAI-/CS-9803) Enzastaurin (LY317615)

Indolocarbazole

Indolocarbazole Indolocarbazole

Chemical class

Midostaurin (PKC-412, CGP 41252, 4- N Benzoyl-staurosporine)

Unspecific PKC inhibitors Staurosporine UCN-01 (7-hydroxystaurosporine)

PKC inhibitor

TABLE 1 Overview of PKC Inhibitors

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in a variety of cells (28,29). Based on its potent activity, staurosporine was investigated for anticancer activities (30) after it had been successfully synthesized in 1996 (31,32). Staurosporine has not been evaluated as a therapeutic agent, but many PKC inhibitors synthesized subsequently have been derived from staurosporine. By increasing its diverse structural and biological attributes, more selective therapeutic PKC inhibitors have been developed (33,34). UCN-01 7-Hydroxystaurosporine (UCN-01) occurs naturally in a soil bacterium and is a relatively nonspecific PKC inhibitor. Its anticancer activity appears predominantly related to its effect as a check-point-dependent kinase (CDK) inhibitor. It is no longer being developed by pharmaceutical companies, but its clinical activity continues to be investigated by the National Cancer institute (NCI) (35,36). UCN-01 inhibits serine-threonine kinases including the Ca2þ and phospholipid-dependent PKC. UCN-01 is a potent (<100 nmol/L) inhibitor of calciumdependent PKC-a, -b, and -g isoforms. Calcium independent PKC-d and -e were inhibited only at higher concentrations (>500 nmol/L), whereas PKC-z was not inhibited by UCN-01 (37,38). At even higher concentrations (>1 mmol/L), UCN-01 inhibits many other kinases. Subsequent studies revealed that it also potently inhibits the DNA damage response to regulatory kinases chk1, possibly chk2 (39–41) and phosphatidylinositide-dependent kinase 1 (PDK-1) (42). UCN-01 mediates distinct effects in vitro/in vivo: cell cycle arrest in G1, abrogation of G2 arrest by inhibiting chk1, induction of apoptosis and potentiation of cytotoxicity of S-phase-active chemotherapeutics (43). UCN-01 was also found to have potent activity in the National Cancer Institute's (NCI) 60 human cancer cell line panel, and has demonstrated antineoplastic activity in a number of preclinical animal models (44,45). Preclinical evidence that UCN-01 has activity in lung-cancer cells is based on a number of studies. One of the first established that UCN-01 can inhibit tumor cell proliferation and oncogene phosphorylation in A549 lung-cancer cell lines (46,47). However, under certain conditions lung-cancer cell lines seem to develop resistance to UCN-01, depending on status of cell cycle and phosphorylation of retinoblastoma (Rb) (44,48,49). On the basis of its novel antineoplastic properties, an initial phase I clinical trial was completed (35,50). Of 47 patients with refractory neoplasms, one patient (2%) with melanoma had a partial response and a second patient (2%) with anaplastic large cell lymphoma had stabilization of disease for >2.5 years. Although a safe dose and treatment schedule could be defined, dose-limiting toxicities included unexpected hyperglycemia, with frequent lesser degrees of hyperglycemia documented at lower doses. Insulin resistance in peripheral tissues has been implicated because circulating insulin and C-peptide levels increased (35), and the mechanism of insulin resistance appears to involve inhibition of AKT activation and subsequent GLUT4 translocation in response to insulin (51). A phase I trial of UCN-01 (70–90 mg/m2 on day 1) and the topoisomerase I inhibitor, topotecan (0.75–1.0 mg/m2 on days 1–5), was also conducted. This combination showed grade 3/4 hematologic toxicity in 16 of the 33 treated patients (48%). Of 22 evaluable patients, 12 had stable disease (54%) and one patient with ovarian cancer had a partial response (5%) (52). Another combination phase I trial of UCN-01 and perifosine has also been conducted in solid tumor patients (communication by University of Maryland, G.C.C., UCN-01 and Perifosine phase I clinical trial for solid tumors, 2006). A recent study completed a

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molecular profiling study to better understand the activity when UCN-01 is combined with cisplatin. Previous nonclinical studies suggested that this combination was particularly active (48,53). However, platinum-associated toxicities were increased in this study and the study was stopped prior to enrolling its targeted patient number. While these observations are consistent with the proposed effect of UCN-01 increasing cisplatin activity, the study also suggests that the therapeutic window of UCN-01 in conjunction with standard chemotherapy may be too narrow for its future development. Other single agent phase II clinical trials are ongoing for patients with lymphoma at the National Cancer Institute (communication by National Cancer Institute, UCN-01 phase II clinical trial for patients with relapsed or refractory lymphomas, 2006) and should reveal whether its single agent use has a place in the treatment of hematologic malignancies. PKC412 PKC412 (40 -N-benzoyl-staurosporine, CGP 41251, or midostaurin) is an oral multitargeted kinase inhibitor (54). It potently inhibits the Flt-3 receptor tyrosine kinase, mutated in approximately one-third of acute myeloid leukemia (AML) patients, as well as multiple other molecular targets thought to be important for the pathogenesis of AML (55,56). PKC412 has been recognized to also inhibit VEGFR-2, PDGFR, c-KIT, MDR, and the zinc-finger-198 (ZNF-198)-fibroblast growth factor receptor 1 fusion tyrosine kinase that is implicated in myeloproliferative disorders (57). In in vitro and in vivo studies, PKC412 inhibits multiple isoforms of the serine/threonine PKC and the Flt-3 kinase. In mice with the mutated form of Flt-3, PKC412 was shown to prolong survival (57). Furthermore, in preclinical studies PKC412 has demonstrated a broad antiproliferative activity against various tumor cell lines, including those that were resistant to several other chemotherapeutic agents. PKC412 suppresses growth of AML cells and neoplastic mast cells (55,57). Its activity appears to be determined by the presence of specific mutations in the ATP binding pocket of Flt-3 (58). PKC412 also appears to be effective in suppressing growth of gastrointestinal stromal tumors (GIST) that become resistant to imatinib, possibly by inhibiting PDGFR and Flt-3 kinase signaling together (56,59). While PKC412 has not been examined extensively in solid tumors, such as lung cancer, recent studies demonstrate that PKC412 can sensitize lung-cancer cell lines to apoptosis (60). This effect is reminiscent of the effect seen with UCN-01 and perhaps both PKC inhibitors share a similar molecular mechanism of sensitizing tumor cells to standard chemotherapy. PKC412 can be safely administered as a chronic oral therapy, and 150 mg/day was identified as an acceptable dose for phase II studies. Steady-state PKC412 plasma levels at the top three dose cohorts (150–300 mg) were 5–10 times the cellular 50% inhibitory concentration (IC50) for PKC412 of 0.2–0.7 mmol/L. The pharmacokinetics and lack of conventional toxicity indicate that pharmacodynamic measures may be additionally needed to optimize the drug dose and schedule (61). Currently, PKC412 is being investigated in phase I/II trials, including patients with mast cell leukemia (communication by Stanford University Cancer Center, PKC412, Midostaurin, phase II clinical trial for Aggressive Systemic Mastocytosis and Mast Cell Leukemia, 2006), AML and myelodysplastic syndrome (61). A phase I trial of PKC412 in conjunction with gemcitabine and cisplatin for front-line therapy of metastatic NSCLC has already demonstrated feasibility, with grade 3 diarrhea or nausea above the maximum tolerated dose (MTD) of 50 mg/day (62).

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Bryostatin Bryostatin (Bryostatin 1) was identified in a series of cyclic macrolides first isolated from the marine bryozoan Bugula neritina (Order Cheilostomata) in the late 1960s (63). This arborescent bryozoan is found in temperate and subtropical environments worldwide, but only B. neritina from California and the Gulf of Mexico is known to contain bryostatins 1, 2 and 3 that are characterized by the C-20 (E,E)octa-2-dienoate ester (16,64). Unfortunately, material is limited due to the fact that Bryostatin 1 needs to be extracted from bryozoans, which contain small amounts of the active forms (to extract one gram of bryostatin, roughly one ton of the raw bryozoans is needed), and the synthesis has proven difficult (65–67). Bryostatin 1 has been investigated as an anticancer agent in a number of preclinical models (68–70). In vitro studies have shown that bryostatin 1 is an effective anticancer agent that may work by modulation of the PKC receptor. In vitro, bryostatins have direct cancer cytotoxic properties as well as tumor-targeting T lymphocyte activation effects. Bryostatins can also act synergistically with other anticancer drugs to achieve potent antileukemic effect and inhibit growth of lung cancer, prostate and non-Hodgkin's lymphoma tumor cells (68,71–76). Unlike most chemotherapy drugs that are myelosuppressive, bryostatin directly enhances maturation of neutrophils by increasing granulocyte colony stimulating factor secretion from bone marrow stromal cells, but does not appear to affect significantly normal myeloproliferation in patients (77). Bryostatin has been evaluated in lung-cancer cell lines (78), and was found to have a biphasic effect characterized by a transient activation of PKCs followed by inhibition of PKC-d (79). Human clinical trials have suggested that bryostatins have a potential synergistic action with other chemotherapeutic agents (68,80–82). Phase I clinical trials have demonstrated a dose-limiting toxicity of myalgias developing at 48 hours (83–86). Responses have been observed in phase II clinical trials with bryostatin as a single agent. These studies included patients with relapsed lowgrade lymphoma and chronic lymphocytic leukemia (CLL) (87). In this study, Bryostatin 1 administration demonstrated one complete response (4%) and 2 partial responses (8%) out of 25 patients. Single agent activity of Bryostatin was also seen in patients with renal cell carcinoma (88), where it was administered as a weekly dose of 35–40 mg/m2. Partial responses were observed in 2 (6%) out of 32 patients and disease stabilization of longer than 6 months were documented for 6 patients (19%). By contrast, no single agent responses have been seen in other cancer types, including melanoma, colorectal, or ovarian cancer (83,89–91). Early phase I and II data suggested that tumor responses to cisplatin or paclitaxel were enhanced by adding bryostatin (80,92). One study examined the combination of bryostatin with cisplatin in patients with NSCLC (93). A larger phase II study investigated the activity of Bryostatin in combination with paclitaxel in patients with NSCLC (94). In this study bryostatin did not add clinical benefit to paclitaxel. Currently, Bryostatin 1 is in clinical evaluation for the treatment of CLL and NHL by the National Cancer Institute in the United States (communication by National Cancer Institute, Bryostatin 1 and Rituximab in Treating Patients With B-Cell Non-Hodgkin's Lymphoma or Chronic Lymphocytic Leukemia, 2006) and by the Cancer Research Campaign in Great Britain (83). In addition, studies are being conducted to determine whether bryostatin-stimulated T lymphocytes show an enhanced immunotherapeutic effect when combined with the treatment of interleukin 2 (70). Finally, the myelostimulatory effect of

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bryostatin seen in treated patients suggest that bryostatin administration could have additional application, such as a myeloprotective agent during chemotherapy or as a stimulant of hematopoiesis between courses of chemotherapy (77,86). Selective Inhibitors Aprinocarsen Aprinocarsen (Affinitak
; LY900003, ISIS 3521) belongs to a class of drugs called ASO (95). ASOs are designed to bind to a specific gene's messenger RNA (mRNA), resulting in the mRNA's degradation by the endogenous enzyme RNAse H and an overall reduction of protein synthesis at the mRNA level (96,97). Aprinocarsen is a phosphorothioate ASO, or a first-generation ASO, targeting human PKC-a. Aprinocarsen can reduce PKC-a mRNA and protein expression in various cancer cell lines (98–100). Sensitivity to aprinocarsen correlates with high PKC-a expression. In the glioblastoma cell line A172, which expresses high levels of PKCa, aprinocarsen exposure caused a marked increase of p53 and IGF-BP-3, which was associated with enhanced tumor cell growth arrest (101). In contrast, such an effect was not observed in the breast cancer cell line MCF-7, which does not express high PKC-a levels (5,101). Aprinocarsen has been given to patients in 21 clinical studies of various malignancies (102). Some encouraging results have been seen in single agent studies in patients with non-Hodgkin's lymphoma (103). Single agent phase II study of aprinocarsen as a continuous infusion over 21 days did not demonstrate objective responses in advanced ovarian carcinoma patients, although one (3%) of 36 patients showed reduction in serum CA125 levels and stable disease for 8 months (104). In combination with chemotherapy, however, aprinocarsen was hypothesized to have synergistic activity. In a phase I/II study for metastatic NSCLC patients, aprinocarsen was given 3 days prior to paclitaxel and carboplatin with a median survival of 15.9 months, almost twice as long as historical controls (105). A phase III study in advanced NSCLC comparing carboplatin and paclitaxel in combination with aprinocarsen versus chemotherapy alone, however, failed to show any difference in response or survival (106), prematurely terminating another phase III NSCLC study of cisplatin and gemcitabine with or without aprinocarsen, which also showed no benefit for the aprinocarsen treated patients (107). KAI-/CS-9803 (KAI-9803) KAI Pharmaceuticals is a specialized company that develops a number of isoenzyme-specific PKC inhibitors. In addition to early lead inhibitory candidates targeting PKC-b, -e, and -g, KAI Pharmaceuticals has advanced the PKC-d inhibitor KAI-/CS-9803 to clinical investigation. This agent was derived from combinatorial modification of the substrate consensus sequences of an individual PKC isozyme. This approach has led to the identification of potent and isozymeselective peptide inhibitors of PKC-a, PKC-d and PKC-z (108). Currently, KAI-/ CS-9803 is being pursued for reducing ischemia and reperfusion injury during the treatment of acute myocardial infarction (AMI). Unlike other agents that have been studied for reperfusion injury, KAI-/CS-9803 acts upstream of both the apoptotic and necrotic pathways by inhibiting PKC-d translocation into the mitochondria. KAI-/CS-9803 is not being evaluated in cancer patients, mainly because the drug platform of pure peptides such as KAI-/CS-9803 is unstable as a systemic therapy. However, similar specific peptide inhibitors of PKCs

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conceivably could be injected intratumorally or perfused into isolated organs as part of loco-regional treatment strategies. Enzastaurin Enzastaurin (LY317615) is an oral serine-threonine kinase inhibitor designed to suppress tumor growth through multiple mechanisms (109). Preclinical studies demonstrate inhibition of cell proliferation, increase in apoptosis, and inhibition of angiogenesis (109). Enzastaurin inhibits signaling through the PKC-b and PI3K/ AKT pathways—signaling pathways activated in a wide variety of cancers— which in turn inhibit the phosphorylation of ribosomal protein S6 and glycogen synthase kinase 3 beta (GSK-3b), an enzyme mediator of energy metabolism and neuronal cell development (109). For example, enzastaurin produced in vitro growth inhibition of SCLC and NSCLC cell lines accompanied by modulation of GSK-3b (110). Furthermore, enzastaurin was found to have synergistic growth inhibition when combined with pemetrexed (111). In addition, low mRNA expression of GSK3-b and high expression of the pro-angiogenic chemokine IL-8 have been shown to correlate with in vitro sensitivity of enzastaurin in freshly explanted human tumor cells. This implicates GSK3-b and IL8 as potential predictive markers for enzastaurin response (112). An ovarian cancer study corroborated the use of GSK3-b phosphorylation as a marker for enzastaurin activity in ovarian cancer models (113). This study demonstrated that taxane-resistant ovarian cancer cells may respond to enzastaurin treatment in low concentrations and suggests that enzastaurin should be investigated in women with taxane-resistant ovarian cancer. In the first phase I study of single-agent enzastaurin in cancer patient (study JCAD), 47 patients were enrolled (mean age, 58 years). Patients received at least one dose of enzastaurin, with a median of two cycles (range 1–17 cycles) (114). Prevalent malignancies in this study were lung cancer (n ¼ 10) and head and neck cancers (n ¼ 9). Although no MTD was identified up to 700 mg/day, 525 mg was chosen as the recommended dose, and 12 additional patients were accrued at that level. This level was found to be associated with a steady-state exposure of 1400 nm, a concentration associated with maximum PKC-b inhibition in cell lines. PKC phosphorylation inhibition was confirmed by flow cytometry in peripheral blood mononuclear cells (115). Exposure of enzastaurin and its metabolites increased proportionally with dose up to 240 mg, and appeared to plateau at 525 and 700 mg. Grade 1 chromaturia, fatigue, and other GI toxicities were the most common, while no clinically significant grade 3/4 toxicities occurred. Twenty-one patients (45%) achieved stable disease for 2–16 cycles, including 4 patients with NSCLC (19%). Hence, the recommended phase II dose was declared at 525 mg for the capsule formulation, which subsequently was replaced by a 500 mg tablet formulation. In advanced NSCLC patients who had progressed through one prior therapy, a phase II trial of single-agent enzastaurin 500 mg daily has demonstrated eighteen of fifty-three patients (34%) to have a best response of stable disease, while no patients had a partial or complete response (116). In this interim analysis of 53 patients, 10 (19%) received therapy for
6 cycles, 3 (6%) of whom received enzastaurin beyond 9 cycles of treatment, and one (2%) who received 11 cycles before progression. The most common toxicity was a varying degree of fatigue (n ¼ 21), noted within 1 week of starting treatment, but fatigue was not reported in patients with disease stabilization. Based on this interim analysis, 10.4% of the

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patients were progression-free at 6 months, and the final study analyses are being expected in 2007. Additional evaluations for the application of enzastaurin in NSCLC are ongoing, including studies to determine how to best combine enzastaurin with other agents active in NSCLC. Other clinical studies with enzastaurin are currently being conducted in NHL and glioblastoma. For advanced diffuse large B-cell lymphoma (DLBCL), patients who had progressed through 2 prior therapies were enrolled in a phase 2 multicenter clinical trial of enzastaurin, 500 mg once daily (117). Twenty-two percent of the 55 study participants were free of disease progression for 2 months. A quarter of those patients remained progression free, with continued responses from 1.5 years to more than 3 years in duration. Treatment was well tolerated, with seven reports of grade 3 toxicity including fatigue, thrombocytopenia, headache, motor neuropathy, and edema. Only one patient experienced grade 4 toxicity for hypomagnesemia. For recurrent glioblastoma multiforme and anaplastic glioma, a phase II study of enzastaurin included 92 patients who had progressive disease after more than one prior regimen of chemotherapy (118). Patients' treatment consisted of an oral fixed dose of 500 mg of enzastaurin, administered daily. Treatment was allowed to continue indefinitely depending upon the patient's response to the drug. Tumor shrinkage was evident in patients who received enzastaurin, with a corresponding response rate of 23% and a stabilization rate of 7%. Overall, enzastaurin was well tolerated in this patient population with the most common side effect being thrombocytopenia (16%). Based on these results in NHL and glioblastoma, two phase III trials of enzastaurin have been launched. The NHL phase III trial (PRELUDE—Preventing Relapse in Lymphoma Using Daily Enzastaurin) is a randomized, placebo controlled study of patients in their first remission from DLBCL (communication, Eli Lilly and Company). The study will compare the efficacy, safety and tolerability of enzastaurin, taken orally for up to three years, versus placebo. This study is planned to enroll 459 patients across 100 sites worldwide. The primary end point of this study will be overall disease-free survival. Additionally, Lilly will be assessing any biomarkers relevant to enzastaurin as a basis for correlating patient response to clinical trial outcomes. The glioblastoma phase III trial (STEERING—Study Evaluating Enzastaurin in Recurrent Glioblastoma) was a randomized, open label registration study, which was to enroll 397 patients to compare the efficacy, safety and tolerability of enzastaurin versus CCNU (lomustine). However, the study's planned interim analysis suggested that enzastaurin would not meet its primary end point of improved progression-free survival over CCNU, and the study has been discontinued (communication, Eli Lilly and Company). Other clinical trials combining enzastaurin with standard chemotherapy regimens are underway or have been completed. Preliminary phase I clinical trial experience combining enzastaurin with gemcitabine and cisplatin (119), pemetrexed (120), or capecitabine (121) have demonstrated no added toxicity when enzastaurin is combined with standard chemotherapies generally used in the treatment for NSCLC, breast, and colorectal cancer. Each study showed that enzastaurin was generally well tolerated across all dose levels and studies in combination with chemotherapy. Finally, there were no observations of significant alterations in pharmacokinetics, suggesting that enzastaurin will have no or limited drug–drug interactions with standard chemotherapies. Further data on

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enzastaurin's safety were reported from a review of data gathered from all early phase I and II studies comprising about 130 patients treated with enzastaurin. This review suggested that enzastaurin is generally well tolerated across all doses and for extended durations with few grade 3/4 toxicities (122). According to this analysis and given the severity of the disease and absence of controlled studies for comparisons, no event other than chromaturia or fecal discoloration (due to reddish-orange color of the active ingredient) and possibly fatigue appear to be definitively attributable to enzastaurin. CONSIDERATIONS FOR THE FUTURE DEVELOPMENT OF PKC INHIBITORS IN NSCLC Despite modest improvements in treatment-related toxicities, the efficacy of firstand second-line chemotherapy treatment for metastatic NSCLC has not changed appreciably during the last decade (123). Equally important is the fact that many patients will not benefit from the current chemotherapies (i.e., the response rate is generally between 20% and 30% at best), and other patients will not be eligible to receive chemotherapy due to their co-morbidities or other ineligibility reasons (performance status, etc.). Therefore, some of the most significant therapeutic advances for metastatic NSCLC during the last several years have focused on developing agents that are more selective in their antitumor effect with the hope of reducing toxicities. One approach was directed at targeting the EGFR and VEGFR pathways (124). As a result of this effort, the addition of bevacizumab, a monoclonal antibody against VEGF, to standard front-line chemotherapy with carboplatin and paclitaxel for NSCLC has been shown to improve response rates, time to progression, and overall survival compared to chemotherapy alone (125,126). Based on these results, the U.S. Food and Drug Administration has approved bevacizumab for use in combination with chemotherapy to treat metastatic NSCLC. In contrast to first-line therapy, standard second-line therapy for metastatic NSCLC has been limited to several single-agent chemotherapy drugs, all associated with a similar 10% to 15% rate of tumor response and overall survival (127,128). This situation changed with the arrival of molecular targeted agents; in particular, EGFR tyrosine kinase inhibitors, such as gefitinib and erlotinib (129,130). These agents have shown a marked benefit for appropriately selected patients with metastatic NSCLC. This recent experience of bevacizumab and the tyrosine kinase inhibitors suggests that inhibition of pathways associated with tumor proliferation or angiogensis can alter the clinical outcome of patients with NSCLC. It is understandable that many other VEGFR and EGFR pathway antagonists are currently being evaluated with great interest, either separately or in combination for the treatment of NSCLC (131–137). The success of the EGFR and VEGFR pathway antagonists in treatment of metastatic NSCLC has important implications for development of novel targeted therapies such as the PKC inhibitors. The development of EGFR and VEGFR pathway antagonists has been blessed by a clear appreciation of the biology targeted by these therapeutic agents. Bevacizumab, gefitinib, and erlotinib by design have exquisitely well-defined molecular targets that facilitate rigorous testing of their mechanisms of action and pharmacodynamics. Fortuitously, these drugs also have clinically obvious pharmacodynamic end points—hypertension for bevacizumab and skin toxicity for erlotinib and gefitinib. Even despite these clear molecular, biologic, and clinical endpoints of therapy, the results of clinical

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trials with these agents still have been marked by unexpected results and discoveries that have reinvented their therapeutic indications. It appears that PKC inhibitors act on similar and, in some instances, distinct pathways compared to those of the EGFR and VEGFR inhibitors. Hence, it is not unlikely that they will also provide clinical benefit in the treatment of lung cancer. PKC inhibitors can inhibit cell cycle (138), tumor cell proliferation, and neo-angiogensis, and can induce apoptosis (109). This multivalent activity of PKC inhibitors is based on a broader antitumor biology than the EGFR and VEGFR inhibitors. In contrast to the approved VEGFR and EGFR inhibitors, PKC inhibitors are only at the beginning of their discovery path, and their development path will likely be as challenging as the one for the VEGFR and EGFR inhibitors. Several aspects need to be considered for the successful clinical development of PKC inhibitors in NSCLC. Because the earliest PKC inhibitors do not exhibit an isozyme-specific inhibitory profile, it is difficult to apply well-defined pharmacodynamic endpoints to measure their activity in patients. For instance, the activity of UCN-01 was found to be dependent on the phosphorylation status of Rb (48). Similarly, PKC412 was found to be active in AML cells with a particular mutation (58). These two observations raise the question of whether patient selection would have improved the outcome of the first phase I studies. Perhaps, if patients had been appropriately selected, a different trial design with a focus on pharmacokinetic/ pharmacodynamic (PK/PD) assessment would have more appropriately estimated the activity of these PKC inhibitors. Instead, the original development had to rely on establishing the MTD dose level prior to moving into phase II development. In fact, this alternative approach was subsequently pursued for UCN-01, but by that time the interest level for this molecule had dropped and was superseded by other more attractive compounds in cancer therapy (53). Furthermore, the relevance of PKC expression and its isoenzymes or their effect on downstream proteins has not been studied sufficiently in human tumor tissue samples. In comparison to the EGF and VEGF pathway, little is known about the PKC expression and its activation pathways in humans. This lack of comprehensive assessment of PKC and its activation pathways in human lung cancer tissue may have contributed to the failure of specific PKC inhibitors in the clinical investigation, such as aprinocarsen. Assuming that PKC-a expression was correlated with clinical outcome as cell line studies suggested (100,139), inhibition of PKC-a expression with aprinocarsen should have resulted in clinical benefit. This assumption was not confirmed in larger phase III studies (106,107). Whether this was a result of insufficiently blocking PKC-a levels during administration of aprinocarsen (i.e., an unfavorable PK/PD relationship), or whether the expression of PKC-a was only relevant to the tumor growth within a small patient population enrolled on the larger phase III studies (i.e., leading to the underestimation of the required study sample size), cannot be determined today. But, these experiences underscore the need for designing clinical studies with PKC inhibitors differently than in the past, including the use of PK/PD modeling in early studies. They also suggest that integrating strategies to identify patients who may benefit from PKC inhibitor treatment will be valuable tools to determine the activity of PKC inhibitors in NSCLC patients. Some of these lessons have been applied to the development of the PKC-b inhibitor, enzastaurin. Enzastaurin was originally developed as an antiangiogenic agent, based on implication of PKC-b in angiogenesis and its preclinical activity (109). One of the striking differences of enzastaurin compared to the other PKC

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inhibitors investigated clinically is its favorable toxicity/tolerability profile. In the first study in cancer patients, no clinically relevant grade 3/4 toxicities were observed (114). Also, the subsequent safety review of about 130 patients enrolled in the early phase clinical trials indicated few grade 3/4 drug-related toxicities (2/134, or 2%) (122). This favorable tolerability profile has resulted in a more attractive development path for enzastaurin compared to the more toxic nonspecific PKC inhibitors. However, the lack of not having established an MTD has been challenged by many oncologists and casts some doubt as to whether the actual antitumor dose has been or can be achieved (140). Therefore, it was critical for the enzastaurin program to prove that its targeted exposure levels of 1400 nM was associated with significant inhibition of PKC phosphorylation (115). The favorable tolerability/toxicity profile and the evidence of a PK/PD relationship in patients has allowed the enzastaurin development to proceed in a different fashion than the previous PKC inhibitors. While the PK/PD relationship has been generally addressed for enzastaurin, the greatest challenge for the clinical development of enzastaurin remains in identifying the patient population likely to benefit from this treatment. This will require the discovery and validation of predictive biomarkers, which for most biologic agents have been unpredictable and elusive. The process of biomarker discovery and validation in treatment of metastatic NSCLC continues to be hindered by the inaccessibility of tumor tissue for analysis in the majority of the patients. Optimizing the success of molecular targeted therapies in NSCLC will require a concerted effort to overcome inherent hazards and reluctance associated with performing biopsies of lung cancer or metastatic lesions. Alternatively, one should consider the development of high sensitivity assays to detect tumor-related biomarkers in the circulating bloodstream, such as circulation tumor cell fragments or whole cells (141). Although predictive markers of response to enzastaurin have not yet been determined, mechanisms of anticancer activity have been determined in preclinical studies. Enzastaurin treatment in vitro suppresses the phosphorylation of GSK-3bSer9, ribosomal protein S6S240/244, and AKTThr308, and may thus directly inhibit cancer cell growth (109). In in vivo studies, enzastaurin treatment suppresses GSK-3b phosphorylation in both tumor tissue and in PBMCs, suggesting that GSK-3b phosphorylation may serve as a reliable pharmacodynamic marker for enzastaurin activity. Whether or not the presence or degree of GSK-3b or AKT phosphorylation prior to enzastaurin might predict response to treatment has not been reported, and will be explored in upcoming clinical studies. In summary, PKC inhibitors have not yet established themselves as a new anticancer drug treatment. The success of PKC inhibitor therapy for cancer will depend on demonstrating inhibition of specific PKC targets, discovering the critical biologic profile in tumors required for its anticancer activity, and creating tests to predict which patients would benefit from treatment. The first step towards this goal has been the evaluation of potent isozyme-specific PKC inhibitors. Future studies will need to incorporate tumor biopsies, blood, and/or surrogate tissues to evaluate potential predictive markers, such as GSK-3b AKT, ribosomal protein S6, and IL-8. PKC inhibitors will require much broader clinical and scientific investigations before they can be optimally utilized, but they offer the possibility of multi-valent anticancer effects, including induction of apoptosis, cell cycle inhibition, and interruption of angiogenesis, and have already demonstrated promising clinical efficacy.

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Signal Transduction Inhibitors: PDGFR and c-KIT Inhibitors Jean-Yves Blay and Jérome Fayette Department of Médecine, Centre Leon Berard, Laennec, Lyon, France, and Unité de Jour d'Oncologie Médicale Multidisciplinaire Hôpital Edouard Herriot, Place d'Arsonval, Lyon, France

Laurent Alberti, Severine Tabone-Eglinger, and Hiba El Sayadi Department of Médecine, Centre Leon Berard, Laennec, Lyon, France

Philippe Cassie and Armelle Dufresne Department of Médecine, Centre Leon Berard, Laennec, Lyon, France, and Unité de Jour d'Oncologie Médicale Multidisciplinaire Hôpital Edouard Herriot, Place d'Arsonval, Lyon, France

Dominique Ranchère and Isabelle Ray-Coquard Department of Médecine, Centre Leon Berard, Laennec, Lyon, France

INTRODUCTION The term }targeted molecular treatment} refers to treatment strategies directed against molecular targets considered to be involved in the process of neoplastic transformation, as the result of the identification of alterations characteristic of neoplastic cells, such as specific translocations, activating mutations, or gene amplifications. The identification of these alterations has brought considerable changes to the nosological classification of cancers. It has allowed the development and evaluation of a new class of drugs that aim to block, more or less specifically, the activity of these activating proteins. Molecular targeted therapies can be divided into different categories [(1) EJO]: 1. Therapies that target molecular defects directly contributing to the initiation of malignant transformation. 2. Therapies that target later molecular defects involved in tumor progression but not in the onset of malignant transformation. 3. Therapies that target molecular defects with no direct mediating effect on cell transformation. The KIT, PDGFR, and PDGF proteins were the among first described as mutated activated in specific tumor types in humans most likely as the initial causal event of the oncogenic transformation (2–5). Tyrosine kinase inhibitors targeting these mutated activated kinases, namely imatinib (Glivec
), and, more recently, sunitinib (Sutent
), AMN107, nilotinib (Tasigna
), AMG706, valatinib, or masatinib have hence been among the earliest characterized active agents in this family of compounds. In the present chapter, we review the molecular basis and clinical activity of tyrosine kinase inhibitors targeting (KIT) and PDGFR. 123

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KIT AND PDGFR KIT and Stem-Cell Factor The product of the KIT proto-oncogene, KIT protein, is a transmembrane receptor with tyrosine-kinase activity mediated by its physiological ligand, stem-cell factor (SCF). The KIT gene is located on the long arm of chromosome 4 (4q11–q12). KIT is a 145-KD type III transmembrane tyrosine kinase receptor whose extracellular portion binds a ligand known as SCF, also called Steel factor. The intracellular portion of KIT contains the actual enzymatic domain. KIT is similar in structure to other receptor tyrosine kinases (RTK) with oncogenic capabilities, including platelet-derived growth factor receptors (PDGFRs) A and B, CSF1R and FLT3. KIT is expressed by hematopoietic progenitor cells, mast cells, germ cells, melanocytic cells, and the interstitial cells of Cajal (ICC) (2,3). KIT activation normally occurs when two adjacent receptors are brought together binding to ligand dimers. This process, known as homodimerization, is accompanied by structural changes in the receptors, resulting in activation of the kinase domains. The activated kinases then crossphosphorylate tyrosine residues in the opposed homodimer partner. The phosphotyrosines also serve as binding sites for various substrates, many of which are phosphorylated by KIT or by each other. In many cases, these substrates are themselves kinases and serve as effectors of intracellular signal transduction. As for most growth factor RTKs, multiple physiological functions have been ascribed to signal transduction mediated through KIT. These include cell survival, proliferation, differentiation, adhesion, and apoptosis (programmed cell death) (2,3). In addition, KIT function is essential for normal hematopoiesis, melanogenesis, and for the development and function of mast cells in many tissues and differentiation and proliferation of ICCs in the gut. KIT expression has been documented in a wide variety of normal cells of the hematopoietic lineage, and of the neural crest in humans as well as in their malignant counterparts. The constitutional kinase activation of KIT has been involved in the pathophysiology of tumors derived from these cell types, including mastocytosis/mast cell leukemia, germ cell tumors, small-cell lung carcinoma (SCLC), acute myelogenous leukemia (AML), neuroblastoma, melanoma, ovarian carcinoma, and breast carcinoma (2,3). PDGFRs and PDGFs Similarly, the product of PDGFRA and PDGFRB proto-oncogenes, PDGFR-a and PDGFR-b, are transmembrane receptors with tyrosine-kinase activity located on chromosomes 4 (4q11-q13) and 5 (5q31-q32) respectively. PDGFR chains associate as homo or heterodimers upon the binding of their specific ligands. The family of PDGFs includes four different polypeptides: PDGF-A, PDGF-B, PDGF-C and PDGF-D linked with an amino acid disulfide bond forming homo- or heterodimers (4–7) whose genes are located on chromosomes 7, 22, 4 and 11, respectively (4,6). These factors exert their cellular effects through PDGFR-a and PDGFR-b protein tyrosine kinase receptors. PDGFR-a is activated by PDGF-AA, PDGF-AB, PDGF-BB and PDGF-CC, while PDGF-BB and PDGF-DD bind and activate PDGF-b. A heterodimeric PDGFR-a/b complex has also been identified which can be activated by PDGF-AB, PDGF-BB, and PDGF-CC. Ligand binding induces receptor dimerization, activation and autophosphorylation of the tyrosine kinase domains.

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Platelet-derived growth factors are produced by a wide variety of different cell types. Their production is regulated by a large number of cytokines and growth factors, and also by hypoxia. PDGF are mitogens for connective tissue, in particular during wound healing, and play an important role during embryonal development. Overexpression of PDGF through dysregulation of production, or constitutional activation of receptors has been found associated with different types of malignancies, including gastrointestinal stromal tumors (GIST), DFSP, hypereosinophilic syndromes, and subsets of chronic myelomonocytic leukemias. TYROSINE KINASE INHIBITORS OF KIT AND PDGFR Imatinib Imatinib mesylate, a derivative of 2-phenylamino pyrimidine is an orally administered, small molecule selective tyrosine kinase inhibitor. In vitro studies showed this drug to bind to and specifically inhibit the activity of a small number of related tyrosine kinases, in particular Bcr-Abl, the platelet-derived growth factor (PDGF) receptors, the wild-type and mutant c-KIT (SCF receptor), and recently MCSFR/ CSF1R (9–19). Imatinib is a competitive antagonist of ATP binding that blocks the ability of c-KIT to transfer phosphate groups from ATP to tyrosine residues on substrates proteins, which in turn interrupts c-KIT mediated signal transduction. At the molecular level, this is similar to the way imatinib binds with high affinity to the conserved ATP-binding site in the tyrosine kinase domain of Abl and Bcr-Abl. Sunitinib Sunitinib malate (SU11248; Sutent) is an oral multitargeted tyrosine kinase inhibitor with direct antitumor and antiangiogenic activities. Although both sunitinib and imatinib bind within the ATP-binding domain of both KIT and PDGFRs, they are members of different chemical classes and presumably have different binding characteristics and affinities. Sunitinib inhibits multiple receptors for signalling pathways fundamental to tumor growth and survival, including PDGFR-a and -b; KIT, RET, CSF-1R; and FLT3, but also VEGFR-1, -2 and -3 in marked contrast with imatinib or nilotinib (Fig. 1) (20–23). VEGFR kinases are essential for tumor-related angiogenesis, and this property is not shared by imatinib. Sunitinib, at the recommended dose of 50 mg once daily on a 4/2 schedule (4 weeks on treatment followed by 2 weeks off), has shown significant efficacy and acceptable tolerability in prior phase I/II and III trials of advanced GIST (24). Sunitinib was approved by the U.S. Food andDrug Administration (January 2006) and the EMEA (July 2006) for the treatment of GIST after disease progression on or intolerance to imatinib mesylate therapy, as well as for the treatment of advanced renal cell carcinoma (conditional approval for cytokine-refractory renal cell carcinoma in the European Union (EU). Nilotinib Nilotinib is a second-generation oral inhibitor of selected tyrosine kinases including KIT, PDGFR, and Bcr-Abl, designed to inhibit mutated activated tyrosine kinases resistant to imatinib (25–28). In vitro data in human GIST882 cells show reduction of KIT autophosphorilation similar to imatinib, but nilotinib inhibits cell proliferation in imatinib-sensitive and -resistant GIST cell lines (GIST882, GIST430, and GIST48) more potently than imatinib. This may be related to a preferential accumulation of nilotinib intracellular concentrations, which were found higher

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FIGURE 1 Typical aspect of a false progression to imatinib in a patient with advanced GIST, with hypodense lesions becoming more visible after two months of imatinib treatment (top, before treatment; bottom, after treatment). This would be a progression according to RECIST criteria.

than those of imatinib in GIST cell lines. Nilotinib is currently being tested in imatinib (and/or sunitinib) resistant CML and in GIST (26–28). AMG706 AMG706 is a novel, orally bio-available, small-molecule multikinase inhibitor (KIT, PDGFR, VEGFR1-3 and RET) which, like sunitinib, exerts both strong antiangiogenic (VEGFR) and direct antitumor activity on activated tyrosine kinasess on tumor cells (29,30). After a phase 1 clinical trial in patients with advanced

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solid tumor malignancies, this agent is being developed currently for the treatment of GIST failing imatinib, and RET dependent tumors. Valatinib PTK787/ZK222584 is a novel, oral inhibitor of the receptor tyrosine kinases essential for molecular pathogenesis of GIST (KIT, PDGFRs, VEGFR-1, and VEGFR-2) (31,32). Initially developed for the treatment of advanced colorectal cancer in combination with chemotherapy (33), this agent has also been tested for the treatment of advanced GIST (34). TUMORS WITH MUTATIONS OF KIT, PDGFR, PDGF GIST Gastro intestinal stromal tumors are the most frequent sarcoma of the gastrointestinal tract. It arises from precursors of the interstitial cells of Cajal, the pacemaker cell of the gastrointestinal tract (35–38). The incidence of gastrointestinal stromal tumors is 1.45/100,000 persons per year (35). GIST can occur at any site of the GI tract: the most frequent primary sites are gastric (50%) and small bowel (25%). Colorectal, esophageal and peritoneal GIST are less frequent. GIST can be diagnosed at any age, with a median of 60 in large series, and are generally revealed by an abdominal mass, GI bleeding, anemia, or incidentally. Immunohistochemical analysis show that these tumors are CD117þ (95%), CD34þ (70%), smooth muscle actinþ (40%), while PS100þ in 5% of cases and desminþ in 2% of cases (37–39). Eighty five percent of GIST exhibit mutations in the KIT and PDGFRA genes. A mutation in exon 11, 9, 13 or 17 of the KIT gene is observed in 66%, 13%, 1% and 0.6% of the tumors respectively; within the PDGFRA gene, mutations of exon 18 or 12 are observed in 6% and 1% of the cases (35,40–43). Intra-abdominal tumors suspected to be a GIST in which CD117 immuno-staining is negative, should be considered for molecular analysis for KIT or PDGFR alpha mutations in expert laboratories (37,38). GIST occuring during childhood have a lower incidence of KIT and PDGFRA gene mutations. For localized tumors, risk assessment profile is based on the size and mitotic index. Serosal breaching, primary site, and the nature of the mutations in the KIT and PDGFRA genes may also be prognostic factors (40). Treatment of Localized and Advanced GIST: Role of Imatinib The treatment of localized GIST is surgery. In this setting, adjuvant treatment with imatinib is experimental (37,38). For localized tumors, wedge resection of the stomach and segmental resection of the intestine are considered adequate treatments since GIST tend to grow out of the primary organ. Adjacent organs adherent to the mass should be resected en-bloc with the tumor, in order to avoid capsule rupture and intra-abdominal spillage. Though positive resection margins have not been definitely demonstrated to compromise survival, a re-excision should be considered in cases of intramural, intra-lesionally excised tumors, without infiltration of the serosal surface (37,38). Adjuvant imatinib is not a standard option and should only be given in randomized clinical trials. Neo-adjuvant imatinib should only be given in inoperable tumors, or in tumors when function sparing surgery is the goal (37,38). Conversely, for metastatic or relapsing GIST,

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imatinib is currently the standard first-line treatment while the role of surgery is not known (37,38). In 2001, the first phase I clinical studies evaluating imatinib in advanced or metastatic GIST were started, soon followed by phase II and III trials (14–18). Two parallel phase III studies have compared a 400 mg/day imatinib dose to an 800 mg dose: the US–Canada SO033 study, and the EORTC 62005 study, in 756 and 946 patients, respectively, with advanced GIST. The SO033 trial demonstrated no difference between the two arms in terms of response to treatment, progression-free survival and overall survival. Conversely, the 62005 EORTC study that included more patients with slightly longer follow-up, reported a significant progression-free survival gain in the 800 mg arm. At 24 months of follow up, progression-free survival was 55% in the group of patients receiving 800 mg imatinib, versus 40% in the group receiving 400 mg in the 62005 trial (18). No difference in overall survival was observed in either trial. Imatinib treatment yields 60% to 70% objective responses on conventional radiography (CT-scan/MRI), with disease stabilization in 15% to 20% of the patients and 10% to 15% primary resistant tumors. PET-scan may allow for an earlier detection of imatinib efficacy in this disease. Secondary resistance to the treatment (recurrence after initial response) is now being reported in 30% to 50% of the patients, a number of whom will react positively to secondary treatment with more active, broader spectrum tyrosine kinase inhibitors as described hereafter. The one-year survival rate of patients with advanced GIST, that was approximately 35% before imatinib, is currently close to 90%. Interestingly, median progression-free survival is close to 24 months while median overall survival extends beyond 60 months in the B2222 study, the longest follow up so far (M. von Mehren et al., personal communication). Treatment with imatinib is recommended to be started in all patients with advanced GIST regardless of the presence of residual disease. It is not demonstrated that complete surgical removal of the tumor is useful in this setting (37,38). The 400 mg/day dose is the currently recommended first-line treatment in the advanced phase. Imatinib interruption at 1 year is associated with a high risk of relapse, even for patients in complete remission (45). This was demonstrated in the prospectively randomized BFR14 phase III trial comparing treatment interruption versus continuation in GIST patients responding to imatinib (47). Although most (24/26 patients) responded to imatinib reintroduction, the drug should not be discontinued outside of a clinical trial. Therefore, imatinib is given continuously, generally for several years in the majority of advanced GIST patients. At ASCO 2007, the results of the follow-up of this trial testing imatinib interruption after 3 years will be presented. Molecular Alterations of KIT and PDGFR in GIST and Response to Imatinib Molecular biology of KIT mutations remains a crucial element for treatment prognosis and response to imatinib. Heinrich et al. (41,46) demonstrated that patients bearing mutations of exon 11 had higher response rate, progression-free survival, and overall survival than patients with mutations located in exon 9 or any other part of the molecule.

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The prognostic value of the nature of mutation of KIT for response to imatinib was later on confirmed on a larger dataset of more than 355 patients of the 62005 study, confirming the better outcome of patients with exon 11 KIT mutations in GIST cells, but also the differential impact of the dose of imatinib on the subset with exon 9 mutation: in this subgroup, median PFS was 6 months with 400 mg/day, versus 18 months in the 800 mg/day arm, with a trend towards improved overall survival (44). If confirmed at the ASCO 2007 conference within the MetaGIST metaanalysis project, which pools together 1640 patients of the US-CDN SO033 study and EORTC 62005 datasets, this observation may guide the upfront dose of imatinib in the future for GIST. Activating mutations of PDGFRA are found in 36% of GIST patients presenting no detectable mutation of KIT. The presence of PDGFRA exon 12, 14 and 18 mutations was observed in 1%, 0.3% and 6% of a series of more 1000 GIST (41–43). Exon 18 point mutation (mutation D842V) was associated with no response to imatinib. Sunitinib and the Management of Resistance in GIST The management of advanced GIST failing imatinib at the dose of 400 mg/day includes several options (37,38): First, it is critical to confirm the progression— distinguishing false progressions frequently observed in the first months of imatinib treatment (Fig. 1) to identify the type of progression, that is, localized or multifocal, and to discuss the clinical case in a multidisciplinary setting to design the appropriate treatment strategy. The first important issue in cases of progression is to check that the patient is indeed taking the medication appropriately. Indeed, the compliance with the treatment may be suboptimal in some patients (75% if the drug is taken by the patient in compliance studies performed in the U.S.A.). It is therefore essential to stress with the patient that the medication be taken appropriately. The issue of compliance will probably be a major concern associated with oral TKI for the long-term treatment of cancer patients, and of course this problem is not limited to KIT and PDGFR inhibitors. In cases of genuine progression at the dose of 400 mg/day, dose escalation up to 800 mg/day is the recommended approach outside of a clinical trial in most up-to-date clinical practice guidelines (47). In cases of focal progression amenable to surgical removal of a progressive metastatic lesion, the role of surgery has not been demonstrated, and actually is not supported by data published so far. However, when complete surgical removal of all lesions is performed by an experienced team, a median progressionfree survival close to 6–8 months is observed in most studies. Additional prospective evaluation of this approach is needed. Surgery is not recommended most often in cases of multifocal progression. When available, if dose escalation up to 800 mg/day fails or is not feasible for toxicity reasons, sunitinib at the dose of 50 mg/day for 4 weeks every 6 weeks is generally recommended. In a phase III trial, sunitinib, given at 50 mg/day for 4 weeks every 6 weeks, demonstrated a significant improvement in PFS over placebo, translating in a 50% reduction in the risk of death compared to placebo (24). Yet, the control arm in this trial was placebo and some investigators now consider that some patients with focal progression on imatinib still benefit from the drug. Imatinib withdrawal probably results in a flare up of sensitive clones in some if not most patients.

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Of note, preliminary results suggest that GIST with mutations of exon 9 or WT KIT may have a longer TTP than patients with exon 11 mutations (48), while in the latter group, the presence of secondary mutations on exon 13 or 14 is associated with a better outcome than the presence of secondary mutations on exon 17 or 18 (48). This points out possible adaptations of doses of imatinib or selection of secondary agents according to the nature of mutations. Interestingly, a phase II trial has recently been reported investigating a continuous daily dosage of 37.5 mg/day, based on the observation that some patients seem to experience disease progression during the 2-week break of the standard dosing schedule. Response and PFS was found to be similar to that of the standard 4/6 weeks schedule (49). Sunitinib is now the standard option of GIST patients failing or intolerant to imatinib. Therapeutic Agents Under Clinical Investigation AMG706 The results of a multi-center single arm phase II study of AMG706 for the treatment of advanced imatinib-resistant GIST were presented at the recent CTOS 2006 meeting. The primary endpoint was objective response per RECIST by independent review and secondary efficacy endpoints including week 8 FDG-PET response (>25% decrease in average SUVmax of target lesions) and Choi response (
10% decrease in uni-dimensional tumor size or
15% decrease in tumor density Hounsfield units) by contrast-enhanced CT (50). Patients received AMG706 125 mg/day orally until PD or toxicity. One hundred thirty-eight patients received at least 1 dose of AMG706. Response rate according to RECIST, PET or Choi criteria (50) were 3%, 31%, 38%, respectively. AMG706 therapy induced durable SD (
22 weeks) in 30 of the 120 evaluable patients (25%). Estimated median TTP for responders according to Choi criteria is 23 and 15 weeks for non-responders. Treatment-related adverse events that occurred in
20% of the patients were: diarrhea (49%), hypertension (47%), fatigue (30%), headache (25%) and nausea (20%). Thrombo-embolic events occurred in nine patients (7%). AMG706 demonstrated an encouraging clinical benefit rate (PR þ durable SD
22 weeks) of 28% in patients with advanced high-dose imatinib-resistant GIST. Nilotinib A Phase I study of AMN107 alone and in combination with imatinib has been performed in patients with imatinib-resistant gastrointestinal stromal tumors (GIST) as well as sunitinib resistance (for 67% of patients) (51). The results were presented at the recent ESMO 2006 conference (51). Cohorts of imatinib-resistant GIST patients with radiological progressive disease (PD) were treated with AMN107 alone (400 mg p.o. bid) or with escalating doses of AMN107 (200 mg/day, 400 mg/day, or 400 mg bid) in combination with imatinib (400 mg p.o. bid). Thirty-seven patients (15 women and 22 men), median age 50 years (range 24–83) received AMN107 alone (n ¼ 18) or in combination up to 400 mg bid with imatinib (n ¼ 19) for 9–193 days (median 114 days). Serious adverse events reported in 10 patients included nausea, vomiting, abdominal pain, peritonitis, rectal bleeding and anemia. The combination of AMN107 400 mg bid and imatinib 400 mg bid is associated with excessive skin toxicity. Four patients treated with AMN107 400 mg bid and imatinib 400 mg bid required dose reductions because of grade 3 skin rash. Thirtysix patients are evaluable for efficacy. Sixteen patients exhibited PD (44%),

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17 patients (47%) achieved stable disease (SD) lasting 2 to more than 6 months, and 2 patients had partial response (PR) after the first month of treatment. One of these two patients is receiving AMN107 alone and the other AMN107 plus imatinib. Preliminary efficacy data suggest there may be relevant activity of AMN107 alone and in combination with imatinib in imatinib-resistant metastatic GIST patients. Valatinib A small phase II trial of valatinib 1250 mg/day was presented at ASCO 2006 on 15 patients failing imatinib (34). Two (13%) patients achieved PR, eight (53%) had SD for 3 months or longer, and five progressed. The clinical benefit rate (PR þ SD) was 67% (95% CI, 38–86%). The duration of the 2 PR was 290 and 393þ days, and for the 8 SDs from 137 to 498þ days. The median time-to-progression was 8.9 months. This agent needs further investigation in a larger cohort of patients. Masatinib A phase I study of masatinib, an inhibitor of KIT and PDGFR in pretreated GIST and other tumor types, as well as a window phase II study in untreated GIST will be presented at ASCO 2007. Towards Multiple Lines of Therapy in Advanced GIST Tumor control and responses have now been reported with at least four different agents in patients with advanced GIST failing imatinib, including heavily pretreated patients in fourth or fifth line of TKI (Table 1). Long-term survival in patients failing imatinib is therefore not infrequent and it is interesting to note that, while the median PFS of first-line GIST patients failing imatinib is 24 months, median overall survival of the same series extends beyond 60 months in the B2222 trial (the trial with the longest follow-up). Since most patients will experience long-term survival following imatinib resistance, it has been recently stressed in consensus conferences and clinical practice guidelines that continuous inhibition of KIT should be provided even in patients progressing under imatinib 400 mg/day (37,38). TABLE 1 Response and Progression-Free Survival (PFS) in GIST

Imatinib first line 400 mg/day 800 mg/day Imatinib second line 800 mg/day Sunitinib second line 50 mg/day 4/6 wk Nilotinib 800 mg/day or Imatinib þ Nilotinib Valatinib 1250 mg/day AMG706 125 mg/day

n

Response (%)

Median PFS (month)

473 473

50 54

19 24

18

108

3

3

47

207

8

6

24

48

4

5

51

15

13

8.9

34

120

3

4

30

Abbreviation: GIST, gastrointestinal stromal tumors.

References

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Other Diseases Involving PDFR or PDGF Mutations GIST is the paradigm of a solid tumor treated by tyrosine kinase inhibitors specifically targeting the kinase causing neoplastic transformation. In 2007, a growing number of tumor models benefits from this type of therapy. Chronic Myelomonocytic Leukemia A subset of chronic myelomonocytic leukemia (CMML), characterized by a translocation involving PDGF receptor beta fused with the TEL gene, is a rare variant of CMML but has been demonstrated to be efficiently treated with imatinib in several reports (52–59). Darier–Ferrand Dermatofibrosarcoma Protuberans Darier–Ferrand dermatofibrosarcoma Protuberans (DFSP) is a rare connective tissue disorder of the skin associated with a translocation fusing the collagen 1a1 gene with the beta chain of PDGF (60–62). This molecular alteration results in an autocrine loop driving tumor cell proliferation. Although most often treated with surgery only, some patients may experience local or metastatic relapse (63–65), or may present initially with large unresectable primary lesions (66). In this rare condition, imatinib has been demonstrated to induce prolonged responses in more than 50% of the patients (63–66). Clinical trials are ongoing to establish the exact level of activity of this compound. Hypereosinophilic Syndromes In the last decade, the understanding of the molecular pathophysiology of eosinophilic disorders has considerably improved with the identification of recurrent molecular abnormalities. The majority of these genetic lesions result in constitutively activated fusion tyrosine kinases, resulting in an eosinophiliaassociated myeloid disorder. The recent discovery of the cryptic FIP1L1-PDGFRA gene fusion in karyotypically normal patients with systemic mast cell disease with eosinophilia or idiopathic HES, has redefined these diseases as clonal eosinophilias. In these cases, PDGFR inhibitors such as imatinib or nilotinib have been tested with demonstrated antitumor efficacy (67,68). Empirical Use of KIT and PDGFR Tyrosine Kinase Inhibitors Aggressive Fibromatosis Aggressive fibromatosis also known as desmoid tumors (AF/DT) are rare connective tissue tumors with a malignant loco-regional behavior. When local treatments have failed, cytotoxic agents and hormonal treatment have been reported to induce tumor control in some patients, but only few prospective phase II trials have been reported in the literature. Recently, anti-tumor activity of imatinib in AF/DT was reported in 3 phase II trials. In a first series of 15 patients, a response rate of 17% and a 1 year PFS of 37% was reported (69). In a recently reported phase II trial including 40 patients treated with one year imatinib at the dose of 400 mg/day, PFS at one year was found to be 71% with a median treatment duration of imatinib of 9 months (range 0.8–13.8) and a median follow-up of 13.8 months, 13 of the 40 patients had progressed, 2 of the 4 patients who have interrupted treatment after progression have reprogressed after 4 and 6 months of imatinib interruption. Imatinib induces prolonged disease stabilization in the majority of evaluable patients with AF/DT (70). The exact role

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of KIT and PDGFR modulation in imatinib activity is under investigation. Interestingly, mutations in exon 10 of the KIT gene were observed in patients responding to imatinib (71). However, these alterations are neither sufficient nor necessary for response to imatinib. Chordomas Chordoma is a rare (0.1/100,000/year) neoplastic disorder arising from notochordal remnants. Activated PDGF receptor PDGFRB and its ligand PDGFB has been reported in a series of chordoma patients (72). PDGFRB, and to a lesser extent PDGFRA and KIT, were found highly expressed and phosphorylated, suggesting a possible autocrine/paracrine loop. These results may account for the antitumor activity of imatinib reported by the same group in a prospective series of patients (73). Adenoid Cystic Carcinomas Adenoid cystic carcinoma are rare tumors with frequent overexpression of the KIT protein. PDGFR has also been reported expressed in these tumors, using immunohistochemical analysis of protein expression. Although occasional responses were observed in some patients (74–76), the majority of the reported observations found no evidence of tumor control. The molecular basis for response to imatinib in this disease is not known. Gliomas Autocrine PDGFR stimulation has been demonstrated to contribute to the development of brain tumors: gliomas express epithelial growth factor receptors and/or PDGFR, and overexpression of PDGFR-a and PDGF ligands has been documented in glioblastoma. Cell lines and xenograft models exhibit reduced growth when treated with PDGFR tyrosine kinase inhibitors (79,80). Interestingly responses to imatinib have been reported in patients with gliomas and glioblastoma, in particular when combined with hydroxyurea (81–83). Prostate Cancer Prostate cancer is a major cause of mortality in men. PDGFR has been reported to be overexpressed in the majority of prostate metastases to the bone as well as in primary prostate cancer, and has been suggested to contribute to tumor progression in preclinical models of prostate cancers (84,85). Interestingly, in these models, imatinib has been found to delay tumor progression, reduce tumor cell proliferation, and promote apoptosis while reducing significantly phosphorylated PDGF-R phosphorylation (86). CONCLUSION KIT and PDGFR expression using immunohistochemistry (IHC) on tumor cells are neither necessary nor sufficient criteria to predict the efficacy of a KIT tyrosine kinase inhibitor in a clinical setting. Negative phase II trials in KIT positive melanoma, NSCLC, Ewing tumors, sarcomas, and so on, have well demonstrated this point, despite anecdotal responses in some subtypes (87). This is even more critical for PDGFR for which the reproductibility of IHC detection has been questioned. The existence of activating mutations of these kinases may enable more efficient identification of sensitive tumor types (88). Screening for these and

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other mutations is likely to be the most efficient strategy to identify novel nosological entities susceptible to tyrosine kinase inhibition in the future. ACKNOWLEDGMENTS Supported by a grant from the 2003 Emergence Fund of the French Institut National du Cancer (INCa) through the Canceropole CLARA, an unrestricted grant of the Comité de l'Ain de la Ligue Contre Le Cancer, a Grant from the Comité du Rhône de la Ligue Contre Le Cancer, and the CONTICANET Network of Excellence of the 6th Framework Program of the European Commission. SUMMARY KIT, PDGFR, or PDGF are mutated and activated in several different tumor types, including hematological malignancies and solid tumors, most often arising from the connective tissue. The identification of the specific genetic alterations (translocation, deletion, point mutations, amplifications), which most often distinguishes specific nosological entities (GIST, DFSP, HES, CMML), has paved the way for testing inhibitors of these activated tyrosine kinases in clinical settings. As of 2007, GIST, DFSP, CMML, and HES have been clearly demonstrated to be highly sensitive to these targeted therapies in the clinical setting. The impact of these treatments in other diseases, such as gliomas, chordoma, aggressive fibromatosis, and prostate carcinomas, where a contribution of PDGFR and/or KIT is suspected, is currently under investigation. Future clinical trials testing KIT and PDGFR inhibitors will have to be based upon a strong biological rationale—the identification of a constitutional activation of one of the targets contributing to neoplastic transformation or progression—and to be combined with translational research programs designed to evaluate the response of the molecular target in vivo in the clinical setting. REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9.

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9

The Insulin-Like Growth Factor 1 Receptor: A Target for Cancer Treatment Yungan Tao, Jean Bourhis, and Eric Deutsch Department of Radiation Oncology, Institute Gustave Roussy, Villejuif, France

INTRODUCTION The success of kinase inhibitors, such as imatinib (Glivec
; Novartis, Basel, Switzerland), targeting tyrosine kinase (TK)–c-abl, and monoclonal antibodies, such as trastuzumab (Herceptin
; Genentech, South San Francisco, California, U.S.A.), interfering with the function of the human epidermal growth factor receptor-2 (HER-2)/Neu receptor, provides strong evidence for the disruption of signal transduction as an effective anticancer approach (1). Many other new agents are in the process of transfer into the clinic or are already approved [Bevacizumab (Avastin
; Genentech), Bortezomib (Velcade
; Millenium, Cambridge, Massachusetts, U.S.A.), Sorafenib (Nexavar
; Bayer, Leverkusen, North Rhine-Westfalia, Germany), Sunitinib (Sutent
; Pfizer, New York, U.S.A.)]. The insulin-like growth factor 1 receptor (IGF-1R) (2), which belongs to the tyrosin kinase(TK) receptor family, appears to be promising as a new target. IGF-1R is a transmembrane TK, consisting of 2a- and 2b-subunits. The extracellular a-subunits are required for ligand binding, while the transmembrane b-subunits contain the TK catalytic site and the ATP-binding site. Two ligands, IGF-1 and IGF-2, bind to IGF-1R (3). The local bioavailability of ligands is subject to complex physiological regulation and is probably abnormally high in many cancers. Ligands can be delivered from remote sites of production through the circulation or can be locally produced. IGF-binding proteins (IGFBPs) (3) and IGFBP proteases have key roles in regulating ligand bioavailability. IGFBPs prolong the half-life of IGFs, which have the potential to increase IGF-1R activation. On the other hand, these proteins have affinity for IGFs comparable to IGF-1R, and there is competition between IGFBPs and IGF-1R for available ligands in tissue microenvironment. This provides a basis for the inhibitory roles of IGFBPs on IGF-1 signaling. There is evidence that certain IGFBPs also have direct, IGF-independent, growth-regulatory actions. The IGF-2R binds IGF-2, but has no TK domain, and appears to act as a negative influence on proliferation by reducing the amount of IGF-2 available for binding to IGF-1R. Certain IGFBP proteases (often produced by neoplastic cells) that cleave IGFBPs can release free ligand and thereby increase IGF-1R activation. Following ligand binding to IGF-1R, its TK activity stimulates signaling through intracellular networks that regulate cell proliferation and cell survival. Pathways Activated by IGF-1R Binding of IGF-1 and IGF-2 to IGF-1R causes its auto-phosphorylation and leads to activation of multiple signaling pathways. There are four insulin receptor substrate (IRS) proteins in mammalian cells, but IRS-1 and IRS-2 are the most prominent in transmitting signals from either the IGF-1R or the insulin receptor (IR). At least two different major signal transduction pathways have been identified for IGF-1R. One activates Ras, Raf, and mitogen-activated protein kinase 141

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(MAPK)/extracellular signal-regulated kinase (ERK), the main mitogen–transduction pathway, and another pathway is responsible for antiapoptotic signal transduction, involving phosphatidylinositide-3-kinase (PI3K–AKT pathways (Fig. 1). Binding of extracellular ligands to IGF-1R, their cell-surface receptors, activates Ras and this initiates Raf activation (4). This leads to activation of the dual-specificity protein kinases MEK1 and MEK2 (MAPK and ERK kinase) and subsequently the MAPK/ERK proteins ERK1 and ERK2. Depending on the cellular context, this pathway mediates diverse biological functions such as cell growth, survival, and differentiation predominantly through the regulation of transcription, metabolism and cytoskeletal rearrangements. The PI3K–AKT pathway (5) is one of the most frequently altered pathways in all sporadic human tumors. The binding of a growth factor (IGF-1) to its TK receptor (IGF-1R) results in the recruitment and activation of the PI3K to the plasma membrane receptor, which in turn phosphorylates the phosphoinositides, increasing the local concentration of PIP3 and PIP2 at the plasma membrane. The PI3K activity is counteracted in the cell by PTEN, a lipid 3-phosphatase which is the second most common sporadically mutated tumor suppressor. This increase in lipid second messengers recruits and activates the PDK and AKT protein kinases at the plasma membrane where AKT is then fully activated by phosphorylation of ser-473 and thr-308. Through the phosphorylation of a diverse set of substrates, AKT regulates four intersecting biological processes: cell survival, cell-cycle progression, cell growth, and IGF-IR

IGF-IR

GRB2

p85 p110

GDP

SOS

GTP Ptdlns(4,5)P2

Ras Ptdlns(3,4,5)P3

GDP GTP

Raf

MEK

PDK 4EBP1

RSK

AKT TSC2

ERK

(A) Ras-Raf

Rheb

mTOR

eIF4E

eIF4B

S6K

S6

(B) PI(3)K-AKT

FIGURE 1 The insulin-like growth factor 1 receptor (IGF-1R) is a tyrosine kinase cell-surface receptor (RTK) that binds to either IGF-1 or IGF-2. Growth factors activate RTKs and activate two key signal-transduction components: (A) the GTPase Ras-Raf; and (B) the lipid kinase PI(3)K.

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cell metabolism. The AKT substrates that mediate some of these biological processes have been identified. AKT controls cell-cycle progression through several substrates. AKT can phosphorylate and inhibit Glycogen synthase kinase-3 (GSK-3) (6) which phosphorylates several cell-cycle regulators such as c-Myc, cyclin D1 and cyclin E and controls a number of critical cell-cycle events. AKT may also enhance the functions of some transcription factors by inactivating GSK-3. AKT controls cell survival through its inactivation of the proapoptotic protein BAD and its activation of the IkB kinase (IKK)–NFkB (nuclear factor-kB) pathway. In addition, the activated AKT protein moves to the cell nucleus where it phosphorylates the FOXO family of forkhead transcription factors, which is a set of highly conserved substrates of AKT, resulting in their removal from the nucleus into the cytoplasm and producing a change in the forkhead transcriptional activity. FOXOs are well established as being critical in the regulation of lifespan and metabolism downstream of AKT (4). PI3K and AKT are implicated in the activation of the mammalian TOR (mTOR) protein kinase by the phosphorylation of the TSC2 (4). The TSC2 tumor suppressor, tuberin, and its obligate binding partner, hamartin (TSC1), are mutated in a familial tumor syndrome called tuberous sclerosis complex (TSC). Of particular interest, recent studies have shown that AKT and mTOR can mediate activation of the hypoxia-inducible factor-1a (HIF-1a) transcription factor which increases expression of the glucose transporter GLUT1 and glycolytic enzymes, ultimately leading to increased glucose uptake. Stimulation of IGF-1R activates the PIK3–AKT–mTOR pathway causing an mTOR-dependent loss in IRS-1 expression leading to feedback downregulation of signaling through the pathway. The mTOR inhibition induces IRS-1 expression and abrogates feedback inhibition of the pathway, resulting in AKT activation in cancer cell lines and in patients treated with the mTOR inhibitors, such as rapamycin, tirosel/temsirolimus (Wyeth, Madison, New Jersey, U.S.A.) (7) or RAD001 (8). Rapamycin enhances basal AKT activity, AKT phosphorylation, and PI3K activity in multiple myeloma cells and prolongs activation of AKT induced by exogenous IGF-1. Rapamycin prevents serine phosphorylation of IRS-1, enhances IRS-1 association with IGF-1 receptors, and prevents IRS-1 degradation. Thus, mTOR inhibitors activate PI3-K/AKT in multiple myeloma cells (7); activation depends on basal IGF-R signaling; and enhanced IRS-1/IGF-1R interactions secondary to inhibited IRS-1 serine phosphorylation may play a role in activation of the cascade. This feedback inhibition could paradoxically reduce the antitumor effects of mTOR inhibitors by enhancing IGF-1 signaling. IGF-1R inhibition could prevent rapamycin-induced AKT activation and sensitize tumor cells to inhibition of mTOR. In contrast, IGF-1 antagonizes the antiproliferative effects of rapamycin in serum-free medium (8). It suggests that feedback downregulation of receptor TK signaling is a frequent event in tumor cells with constitutive mTOR activation. Reversal of this feedback loop by rapamycin may attenuate its therapeutic effects, whereas combination therapy of inhibition of IGF-1R that ablates mTOR function and prevents AKT activation may have improved antitumor activity. Evidence for IGF-1R Involvement in Cancer Many experimental IGF-1R-positive cancers could be stimulated by IGF-1 produced in tissues remote from the cancer or synthesized locally in an autocrine or paracrine manner. There is evidence from experimental systems and studies of

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clinical specimens that neoplastic progression, particularly in prostate cancer, might be associated with increased expression of IGF-1R. However, IGF-1R amplification does not seem to be frequently observed. IGF-1R seems to have a role in regulating proliferation and differentiation even if its expression levels are low; there is evidence that certain IGFBPs have IGF-independent growth-inhibitory or proapoptotic influences, and that neoplastic cells can develop resistance through these mechanisms. Tumor and IGF-1R Expression The IGF-1R has been implicated in promoting oncogenic transformation, growth, and survival of cancer cells. Several studies, both experimental and clinical, have demonstrated that the IGF-1R is overexpressed compared to normal tissues (9,10) (Table 1). Moreover, IGF-1R is ubiquitously expressed in tissues (11). Strong evidence has been provided that IGF-1R is not an absolute requirement for normal growth. IGF-1R has been shown to be crucial for anchorage independent growth (12), which is unique for malignant cells. This property of IGF-1R also implicates the function of this receptor in tumor progression since the degree of anchorage independency reflects the level of malignancy. This means that metastasis has acquired more anchorage independency and more IGF-1R dependency, compared to the primary tumor. There is a great deal of evidence based on in vitro and in vivo studies demonstrating the importance of IGF-1R signaling in mammalian cell transformation TABLE 1 IGF-1R Expression, IGF Level or Polymorphism in Tumors and Correlation with Cancer Risk, Prognosis Tumor type Prostate cancer

Breast cancer

Colorectal cancer

Lung cancer

Gastric cancer

Pancreatic cancer Bladder cancer Sarcoma Adrenal neoplasia Central nerve system

IGF-1R expression

IGF level or polymorphism

Expression in most prostate cancer cell lines, overexpression in PC-3 and DU-45 cells, etc. Higher in estrogen-dependent cell lines, presence of IGF-1R in biopsy specimens Presence on HCT 116 and CoLo-205, and human colon cancer specimens Expression common in SCLC and NSCLC

High circulating IGF-1 levels, 19-CA-repeat allele associated with worse survival, haplotypes Circulating levels of IGF and IGF-BP, 19-CA-repeat allele, A-202 C polymorphism in the IGF-BP 3 A high IGF-I/IGF-BP-3 ratio high risk cancer, CA 17 repeat allele

Overexpression in primary tumor correlated with increased lymph node metastasis Overexpression Expression Expression Overexpression in pheochromocytomas Gliomas meningiomas express receptor

IGF stimulate growth in SCLC and NSCLC cell lines, A-202C polymorphic variation of IGF-BP-3

Haplotypes for IGF-2R

Abbreviations: 1GF-1R, insulin-like growth factor 1 receptor; NSCLC, non–small cell lung cancer; SCLC, small cell lung cancer.

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and the development of tumors (13). Findings based on prostate cancer studies raised the possibility that tumor cell dependency on IGF-1R may be stage-specific. The multistep transformation of the prostate epithelium is initially IGF-1R dependent. IGF-1 has been shown to stimulate the proliferation of human prostate epithelial cells in culture and to be necessary for normal growth and development of the rat and mouse prostate. IGF-1R appears to be abundantly expressed in most prostate cancer cell lines (11). In cell lines PC-3 and DU-45, IGF-1R mRNA has been found to be overexpressed. The concentration of IGF-1R is higher in estrogen-dependent breast cell lines than in estrogen-independent cell lines. There is a positive correlation between the estrogen, progesterone, and PRL receptors and IGF-1R expression. IGF-1R expression, however, is ubiquitous or nearly ubiquitous, and its activation has been demonstrated to be a potent stimulus for growth (11). IGF-1R overexpression is observed in 43.8% of tumors in primary breast cancer patients, although IGF-1R overexpression has no correlation with prognosis or with other clinicopathologic parameters (14). IGF-1R is overexpressed in 62% of primary tumor and lymph node metastasis of gastric cancer when compared with adjacent tumor-free gastric mucosa. IGF-1R overexpression in primary tumor correlates with increased lymph node metastasis (15). IGF-1R is expressed on the human colon cancer cell lines (16), such as HCT116 and CoLo-205, and several human colon cancer specimens. A high IGF-1/IGFBP-3 ratio may increase the risk of colon cancer development. IGF receptor expression is common in lung cancer. Presence of IGF-1R mRNA has been found in all of the cell lines and most primary lung carcinomas (11). IGF-1 is a potent mitogen, stimulating growth 1.6- to 4.2-fold in a panel of small cell lung cancer (SCLC) cell lines and 1.1- to 2.7-fold in a panel of non–small cell lung cancer (NSCLC) cell lines such as NCI-H1299. Significant overexpression of the IGF-1R in human pheochromocytomas suggests IGF system involvement in the pathogenesis of adrenal neoplasia (17). Gastrointestinal neuroendocrine tumors (NET) frequently express IGFs and IGF-1R and apoptosis, and cell-cycle arrest could be induced by the IGF-1R-TK inhibitor, NVP-AEW541, in NET cells. The inhibition of the IGF/IGFR system appears to be a promising novel approach for future treatment strategies of NET disease (18). CIRCULATING LEVEL OF IGFs, POLYMORPHISMS, AND CANCER Circulating levels of IGF-1 are associated with the risk for developing prostate, breast, colorectal, and lung cancer (Table 1). IGF-2 appears to be overexpressed in most tumor cell lines. The presence of millions of genetic variations (polymorphisms) in the human genome may provide extensive biological variations that affect cancer physiologies, treatment outcome, and prognosis. Polymorphisms of genes encoding growth factors may be good candidates for a possible determinant of treatment outcome and prognosis. IGF-1 is a potent mitogen for the prostate cancer cell lines. Men with high levels of serum IGF-1 are at increased risk of developing clinically evident prostate cancer (19). Circulating levels of IGF-1 and IGFBP-3 may predict the risk of developing advanced-stage prostate cancer (20). Men in the highest quartile of IGF-1 level have a five-fold increased risk of advanced-stage prostate cancer than men in the lowest quartile. A known genetic cytosine–adenine (CA) repeat polymorphism in the promoter region of the human IGF-1 gene may be associated

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with circulating IGF-1 levels. The 19-CA-repeat allele is more frequent in prostate cancer patients than controls. Males homozygous for the 19-allele have a significantly increased risk of prostate cancer (21). The >19 repeats of IGF-1 is significantly associated with a worse cancer-specific survival and the presence of >19 repeats of IGF-1 is an independent risk factor for death along with clinical parameters. IGF-1 (CA) repeat may be a novel predictor in prostate cancer patients with bone metastasis (22). Haplotype analysis and two SNPs reveal significant associations with prostate cancer risk (23). Women with 19-CA-repeat allele homozygote and high IGF-1 levels have a much higher risk of breast cancer (24). The polymorphisms in the IGF-1 and IGFBP-3 genes are associated with an increased risk of breast cancer in familial cases carrying the variant alleles (25,26). Women in the highest quantile of circulating levels of IGF and IGFBP have more than twice the risk of developing breast cancer than those in the lowest, although this effect is only apparent at young ages (27). Colorectal cancer is modestly associated with having an IGF-1 genotype other than homozygous for 19 repeats and having the GG IGFBP-3 genotype. IGF-1 and IGFBP-3 genotype are significant effect modifiers of the relationship between risk factors (body mass index, postmenopausal hormone use and physical activity) and colorectal cancer (28). Elevated IGF-1 levels are associated with sporadic colorectal cancer (CRC) risk in hereditary nonpolyposis colorectal cancer (HNPCC). Patients carrying a shorter IGF1 CA-repeat length polymorphism (17 repeats) have higher CRC risk in HNPCC (29). A-202C polymorphic variation of IGFBP-3 gene constitutes a risk factor for NSCLC. The NSCLC risk correlated significantly with AA genotype (30). The dysregulation of IGF axis could now be considered as another important risk factor for NSCLC. IGF polymorphisms are also associated with osteogenic sarcoma (31). In addition to its role in proliferation of cancer cells, the IGF-1R protects cells from apoptosis caused by growth factor deprivation, anchorage independence, or cytotoxic drug treatment. Downregulation of IGF-1R function by antisense and dominant-negative techniques reduces the growth and tumorigenicity of several cancer cell lines in vivo and in vitro, including colon cancer, melanoma, lung cancer, ovarian cancer, glioblastoma, and neuroblastoma, and others. It is also shown that administration of a blocking antibody directed against this receptor slowed the in vivo proliferation of human breast cancer xenografts. IGF-1R is thus an attractive therapeutic target based on the hypothesis that inhibition of IGF-1R function would result in selective apoptosis and growth inhibition of tumor cells. The key role of IGF-1R signaling in cancer is underscored by the fact that IGF-1R deficient cells fail to be transformed by oncogenes such as Ras, EGFR SV40 and E6 (12). Interplay Between IGF-1R and Other Tyrosine Kinase Receptors IGF-1R signaling inteferes with numerous other growth factors or receptors such as epidermal growth factor receptor (EGFR), vascular endothelial growth factor (VEGF), and so on. Elevated VEGF levels are correlated with the lymph node metastasis, increased progression, and poor prognosis of cancer. VEGF expression is regulated by IGF-1. IGF-1 stimulates VEGF secretion and induces VEGF promoter activation. IGF-1 stimulates PI3K/Akt and Erk/MAPK pathways in

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SCC-9 cells, each contributing to Hif-1a expression and VEGF secretion (32). Increased Hif-1a expression leads to HIF-1 formation and the production of VEGF. IGF-1 regulates VEGF expression and secretion via HIF-1-dependent and independent pathways (33). Androgen ablation in LNCaP cells upregulates the transcriptional activity of VEGF-C and downregulates the IGF-1R pathway (34). Functional inhibition of IGF-1R signaling upregulates VEGF-C mRNA level. IGF-1R and EGFR are overexpressed in pancreatic cancer and their expression patterns through the cell have been shown to correlate with pancreatic cancer grade and prognosis. Membrane-dominant EGFR and cytoplasm-dominant IGF-1R are more frequent in lower-grade tumors and correlated with favorable prognosis in primary invasive ductal pancreatic carcinomas, whereas cytoplasmdominant EGFR and membrane-dominant IGF-1R are more frequent in highergrade tumors and correlated with poor prognosis (35). IGF-1R and Resistance to Targeted Therapies Signaling via IGF-1R has been associated with resistance to anti-EGFR and HER2-based therapies in the experimental system. Anti-EGFR targeting has been demonstrated to enhance apoptosis and reduce both cellular invasion and angiogenic potential in various tumor settings. However, primary resistance to anti-EGFR therapy has been observed as in preclinical studies as well as in clinical practice in tumors such as lung cancer and glioblastoma (36). Resistance to an EGFR TK inhibitor, AG1478, is associated with an up-regulation of IGF-1R levels in a glioblastoma model. Conversely, IGF-1R overexpression is found to correlate with decreased efficacy of EGF-R targeting in clinical trials, suggesting the importance of IGF-1R signaling in EGF-R inhibitors resistance. IGF-1R mediates resistance to anti-EGFR therapy in primary human glioblastoma cells through continued activation of PI3K-AKT signaling (37). Nevertheless, the recent identification of a novel mutation of the EGFR gene in lung tumors in the TK domain (T790M) of EGFR, rendering cells resistant to the EGFR TK inhibitor gefitinib, strongly suggests that tumors cells remain dependent on an active EGFR pathway for their proliferation (38). Interestingly, co-targeting IGF-1R with EGFR greatly enhances both spontaneous and radiation-induced apoptosis of a glioblastoma model. IGF-1R signaling through PI3K-AKT may represent a novel and potentially important mechanism of resistance to anti-EGFR therapy (39). The addition of an anti-IGF-1R strategy to EGFR targeting treatment may be more effective than a single-agent approach (40) and dual EGFR/ IGFR targeting compounds are currently in development. Tyrphostin AG1024 (an inhibitor of IGF-1R) is used with gefitinib for treatment of MDA468, MDA231, SK-BR-3, and MCF-7 breast cancer lines, which express similar levels of IGF-1R but varying levels of EGFR. Gefitinib and AG1024 when used in combination revealed an additive-to-synergistic effect on cell growth inhibition. Overexpression of IGF-1R in SK-BR-3 cells is sufficient to cause a marked enhancement in gefitinib resistance. IGF-1R signaling reduces the antiproliferative effects of gefitinib in several breast cancer cell lines. Similar findings of an involvement of IGF-1R in EGFR resistance mechanism were also found in pancreas and prostate cancer cell lines (41,42). Co-targeting HER2 and IGF-1R improved the efficacy of therapies directed against HER2/erbB2. In two cell lines, MCF7 and BT474 cells, IGF-1R antagonists enhance the effect of HER2 and ER antagonists. While these agents produce

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a small amount of apoptosis individually, their combination causes a dramatic degree of apoptosis, most striking for the IGF-1R/HER2 antagonist combination (43). Hence such combinations may be useful in tumors in which single drugs are inactive. In breast cancer cell models that overexpress HER2, an increased level of IGF-1R signaling appears to interfere with the action of trastuzumab. Thus, strategies that target IGF-1R signaling may prevent or delay development of resistance to trastuzumab (44,45). Trastuzumab inhibited the growth of MCF-7/ HER2-18 cells, which overexpress HER2 and express IGF-1R, only when IGF-1R signaling is minimized. In SKBR3 cells, which overexpress HER2 but express few IGF-1R, trastuzumab reduced proliferation by 42% regardless of IGF-1 concentration. When SKBR3 cells are genetically altered to overexpress IGF-1R and cultured with IGF-1, trastuzumab has no effect on proliferation. However, the addition of IGFBP-3, which decreased IGF-1R signaling, restored trastuzumab-induced growth inhibition. A strong synergistic interaction has been found in combining trastuzumab and reduction of IGF-1R signaling by expression of dominant-negative IGF1R in HER2—overexpressing MCF7her18 breast cancer cells—and this resulted in potentiation of growth inhibition in transfected cancer cells (46). Simultaneously co-targeting TK receptors may be therapeutically useful, and may provide a specific rationale for combining IGF-1R and HER2 targeting strategies in antineoplastic approaches. We recently found that Bcr-Abl expressing cells harboring imatinib (an inhibitor of the SCF-KIT loop) resistance due to Bcr-Abl gene amplification are sensitive to AG1024 (47,48); whether the effect is a direct consequence of IGF-1R or due to an “off target effect” of AG1024 remains to be determined. Several lines of evidences demonstrated that IGF-1R targeting inhibitors are effective against leukemia, multiple myeloma, and lymphoma models (49–52). IGF-1R blockade by ADW742, a small molecule specific for this receptor, alone and in combination with imatinib, on Ewing tumor cell lines has been studied (53). Addition of imatinib to ADW742 synergistically augmented these effects and is especially effective in inhibiting AKT/mTOR phosphorylation and reducing vascular endothelial growth factor expression in cell lines having high IGF-1R activation levels. Combination of ADW742 with imatinib induces a significant reduction of tumor cell growth, mainly by the increase in apoptosis with a pattern depending on IGF-1R activation levels.

MOLECULAR RESPONSE TO IONIZING RADIATION (CHECKPOINT OF CELL CYCLE, DNA DAMAGE SIGNALING) AND IGF-1R ATM plays a central role in cellular response after DNA damage triggering cellcycle arrest, apoptosis, and DNA repair mechanisms. ATM gene is mutated in the ataxia telangiectasia syndrome (AT), a syndrome characterized by an extreme radio-sensitivity. AT cells express low levels of IGF-1R and show decreased IGF1R promoter activity compared with wild-type cells. Complementation of AT cells with the ATM cDNA results in increased IGF-1R promoter activity and elevated IGF-1R levels, whereas expression in wild-type cells of a dominant-negative fragment of ATM specifically reduces IGF-1R expression. These results are consistent with a role for ATM in regulating IGF-1R expression at the level of transcription. When expression of IGF-1R cDNA is forced in AT cells via a heterogonous viral promoter, near normal radio-resistance is conferred on the cells. This

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suggests that IGF-1R may participate in DNA damage signaling especially after irradiation (54). Besides ATM, the interplay between IGF-1R and p53, Brca1, 14-3-3 d, all proteins involved in DNA damage signaling, also suggests the involvement of IGF-1R signaling in radiation response. Transcription of the IGF-1R gene is controlled by a number of tumor suppressors, including the DNA repair protein BRCA1 (55–57). BRCA1 represses the activity of co-transfected IGF-1R promoter reporter constructs in a number of cell lines (58). BRCA1 does not inhibit IGF-1R gene expression and promoter activity in the presence of a mutant p53 whereas expression of wild-type p53 does not prevent BRCA1 action (55). BRCA1 and p53 suppress IGF-1R promoter activity in an additive manner, suggesting that BRCA1 and p53 cooperate in the regulation of IGF-1R gene transcription. Loss-of-function mutation of p53 and/or BRCA1 in familial and/or sporadic breast cancer may result in aberrant regulation of IGF-1R gene expression. IGF-1R gene is a downstream target for p53 action. Wild-type p53 inhibits transcription of the IGF-1R gene, whereas a number of tumor-derived, mutant forms of p53 enhance IGF-1R gene expression (55). The role of p53 in regulating IGF-1R seems to be complex. The mechanism of action of p53 does not involve direct DNA binding to IGF-1R promoter sequences. P53 is capable of suppressing the activity of the IGF-1R promoter as well as lowering the endogenous levels of IGF-1R mRNA. Tumor-derived, mutant p53 significantly stimulated promoter activity. Upon inhibition of wild-type p53 in malignant melanoma cells overexpressing IGF-1R, the cells respond with a drastic IGF-1R downregulation and cell death. The oncoprotein MDM2 protein, which controls p53 degradation, seems to be involved in the control of IGF-1R expression. The MDM2 has recently also been found to associate with certain cell surface receptors and regulate their functions (59). When p53 is inhibited, MDM2 is redistributed and binds to the IGF-1R (60). MDM2 ubiquitinates and targets IGF-1R for degradation in a proteasome-dependent manner, eventually leading to cell death. Thus, a selective IGF-1 inhibition in cancer cells could be achieved by an increase of MDM2 levels by pharmacological modulations. Reciprocally, the IGF-1 system has been shown to influence the activity of MDM2 in a p38 MAPK-dependent manner. 14-3-3 d is originally identified as a p53-inducible gene responsive to DNAdamaging agents. 14-3-3 d induces G2 arrest by sequestering the mitotic initiation complex, cdc2-cyclinB1, within the cytoplasm after DNA damage and allows the repair of damaged DNA. It has been shown that 14-3-3 d is a positive mediator of IGF-1R-induced cell proliferation. Treatment with IGF-1 increased 14-3-3 d mRNA and protein levels (61); this effect occurs via the PI3-K/Akt pathway and is p53 independent. 14-3-3 d positively mediates IGF-1R-induced cell-cycle progression. The interplay between IGF-1R and Brca1, p53, ATM and 14-3-3 d, all proteins involved in DNA damage signaling and repair, suggests potential synergy between drugs targeting IGF-1R and agents targeting DNA. INHIBITION OF IGF-1R AND RADIO-SENSITIVITY Recent studies have identified IGF-1R as a protein capable of inducing radioresistance in breast cancer, while inhibition of IGF-1R function enhances tumor response to classical therapy (i.e., irradiation and chemotherapy) in breast and colon cancer models. IGF-1R is expressed on NSCLC cells and is involved in the radio-sensitivity in lung cancer cells lines (39). Blocking of IGF-1R function is

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effective in potentiating the effects of radiation in NSCLC cell lines. IGF-1R blockade by a recombinant adenovirus expressing truncated IGF-1R enhances chemotherapy and radiation responses and inhibits tumor growth in human gastric cancer xenografts. We have evaluated, in our laboratory, the effects of the tyrosin kinase IGF-1R inhibitor, AG1024 on radiation response and found a marked enhancement in radio-sensitivity and amplification of radiation-induced apoptosis in the human breast cancer cell line MCF-7 (47,48). INHIBITION OF IGF-1R A variety of approaches, including dominant-negative mutants, kinase defective mutants, antisense oligonucleotides, IGF binding proteins, soluble forms of the receptor, antagonistic and/or neutralizing antibodies or small molecule kinase inhibitors have been used to inhibit IGF-1R signaling. Reducing the levels of the ligands (IGF-1 and IGF-2) has given good results in mice which express only IGF-1 in adult life. However in adult humans, IGF-1 and IGF-2 are both expressed and both of them would have to be targeted. Antisense strategies are the first to be used successfully in vitro and in vivo. Antagonistic antibodies and TK inhibitors represent the most probable clinically viable options (62). Humanized monoclonal antibodies such as: EM164 (63,64) (AVE1642) (65), IMC-A12 (41) and CP-751, 871, h7C10 (Table 2) (66), have been successful in inducing apoptosis of cancer cells, and their usefulness is further supported by the observation that antibodies to the IGF-1R, like antisense strategies, downregulate the receptor. The feasibility of inhibiting IGF-1R function with a specific antibody is first demonstrated using a mouse monoclonal antibody (a-IR-3) directed against the a-subunit of IGF-1R. This antibody inhibits the binding of IGF-1 to its receptor, thereby preventing downstream signaling, tumor cell proliferation in vitro, and tumor growth in vivo. Numerous groups have recently described the identification and characterization of antagonistic and/or neutralizing humanized antibodies targeting the extracellular domain of IGF-1R. Although generated by applying TABLE 2

Specific IGF-1R Targeting Compounds

Compounds

Type of targeting

CP-751, 871 EM164 (AVE1642)

Antibody Antibody

IMC-A12

Antibody

h7C10

Antibody

INSM18 PPP

TK inhibitor TK inhibitor

NVP-ADW742, NVP-AEW541 BMS-536924, BMS-554417

TK inhibitor TK inhibitor

Abbreviation: TK, tyrosine kinase.

Company Pfizer, New York, New York, U.S.A. ImmunoGen, Cambridge, Massachusetts, U.S.A.; Sanofi-Aventis, Paris, France ImClone, New York, New York, U.S.A. Pierre Fabre, Boulogne, France; Merck, Whitehouse Station, New Jersey, U.S.A. Insmed, Richmond, Virginia, U.S.A. Karolinska Institute, Stockholm, Sweden Novartis Pharma, Basel, Switzerland Bristol-Myers Squibb, New York, New York, U.S.A.

Phase of development Phase I Preclinical

Phase I Preclinical

Phase I Preclinical Preclinical Preclinical

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different strategies, such potential biopharmaceuticals have been shown to bind specifically to IGF-1R, thereby preventing the activation of IGF-1R-mediated signaling (62). Parallel to the efforts directed at blocking the physical interaction between IGF-1R and its growth factors, drug discovery activities have also been aimed at modulating IGF-1R TK activity by targeting its intracellular kinase domain. The identification of specific low-molecular mass kinase inhibitors of IGF-1R kinase activity has proven to be a major challenge for medicinal chemistry. In theory, a specific inhibitor of IGF-1R TK activity would be the best solution. The problem is that this type of inhibitor will have to distinguish the TK domain of the IGF-1R from the one of the insulin receptor. The two domains are highly homologous, but there are small differences that could be exploited. These kinase inhibitors could be divided into two groups: ATP antagonists such as: NVP-ADW742 (49), NVPAEW541 (67) and BMS-536924 (68), BMS-554417 (69) and non-ATP antagonists such as: picropodophyllin (PPP), AG538 (70) and INSM18. PPP is a cyclolignan derivative developed at the Karolinska Institute and is a selective inhibitor of IGF-1R kinase activity. PPP potently inhibits IGF-1R autophosphorylation (IC50 of 0.04 mM) and is selective against a panel of other receptor TKs without interfering with insulin receptor activity (51,71–73). PPP did not compete with ATP but interfered with phosphorylation in the activation loop of the kinase domain. PPP reduces phosphorylated Akt and induces apoptosis and tumor regression in xenografted mice. IGF-1Rs of PPP treated cells undergo rapid downregulation. This downregulation may be important for the strong apoptotic effect of this compound. PPP treatment of IGF-1R overexpressing cells results in the preferential inhibition of the PI3K/PKB pathway. QUESTIONS REMAINING Recent successes in the development of small-molecule TK inhibitors, blocking antibodies against the IGF-1R, pose challenges to translational scientists seeking to design clinical trials. There are still many questions to be answered. Kinase inhibitors have potential advantages, including convenient oral administration. It is difficult to predict a priori to what extent these agents will be specific for IGF1R during long term in vivo use, where tissue concentrations might vary. As there are no examples of genetic alterations of this pathway in human tumors, it is unclear how tumors should be selected for treatment using this approach. Is activation of the receptor in a tumor likely to predict responsiveness? What effects may the inhibitors have on IGF-1R in normal tissues and even on the insulin receptor (74)? While several small molecules have a much lower affinity for the IGF-1R than the insulin receptor, the relative affinities in patients and on different tissues remain unknown. It is hoped that intermittent therapy with these or similar agents may have minimal effects, perhaps only on tissues that demonstrate a high level of cellular turnover, such as bone marrow and the gastrointestinal tract. These side effects may therefore be similar to those seen with chemotherapy and may be limited in extent and duration; clinical trials will be required to establish this. Regarding the insulin receptor, intermittent therapy may worsen insulin resistance and diabetes, which may be limited and easily treatable. Finally, because of its strong antiapoptotic activity, downregulation of the IGF-1R could be used in combination with other anticancer therapies that cause

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apoptosis of cancer cells. Blockade of the IGF-1R may be an important form of adjunct therapy for cancer patients. It may reduce side effects by lowering the doses of chemotherapeutic agents, perhaps making chemotherapy more effective. Whether the agent used is a humanized antibody, small peptide inhibitor, or small molecule, it is becoming clear that the IGF system plays a critical role in the development and treatment of cancer. Last, sequence of coadministration in the case of IGF-1R targeting drugs and chemotherapy seems to be critical. REFERENCES 1. 2. 3. 4. 5. 6. 7.

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10

Aurora Kinase Inhibitors Mitesh J. Borad, Steven L. Warner, and Daniel D. Von Hoff Clinical Translational Research Division, Translational Genomics Research Institute, Phoenix, Arizona, U.S.A.

DISCOVERY OF THE AURORA KINASES In 1993, the first homologue of the Aurora kinase family was reported to be isolated using a genetic screen to find mutations in yeast that confer an increasein-ploidy phenotype (Ipl1) (1). Shortly thereafter, subsequent reports further characterized the function of Ipl1 to be involved in the process of chromosome segregation (2,3). A Drosophila homologue was discovered in 1995, when it was shown that mutations in a gene named aurora led to mitotic arrest in which condensed chromosomes were attached to circular monopolar mitotic spindles (4). This name was chosen due to the localization of the aurora protein to the poles of the mitotic spindle, similar to the way an aurora borealis is observed at one of the poles of the earth. Furthermore, the name polo was already used to describe a gene of related function (5), and, to keep with a similar theme, aurora was named after the northern lights. In 1997, Sen and colleagues (6) showed that a putative serine/threonine kinase encoding gene that had been previously mapped to chromosome 20q13 (7) was amplified in human breast cancer cell lines, and they named this human homologue breast tumor amplified kinase (BTAK). In that same year, Kimura et al. further characterized this human homologue by reporting its cell cycle-dependent expression and spindle pole localization in HeLa cells (8), and Bischoff and colleagues showed its amplification in human colorectal cancers and its ability to transform rodent fibroblasts (9). These key early reports catapulted the aurora family of kinases to be closely studied as important mitotic kinases, contributors to tumorigenesis, and potential therapeutic targets. By 1998 it had become clear that there were at least three homologues of the aurora kinases that, despite having a high degree of amino acid sequence similarity, were quite distinct in function. However, due to the various groups making key discoveries, the nomenclature for this family of kinases became confusing. For the human homologues, it has been suggested that Aurora A, B, and C become the convention (10,11). BIOLOGICAL FUNCTIONS OF THE AURORA KINASES Aurora A Aurora A (also known as Aurora-2/BTAK/AIK1/ARK1/STK15) is a centrosome-associated kinase that functions to establish mitotic spindles by regulating centrosome duplication and separation. The loss of Aurora A function (or expression) leads to cell cycle arrest and monopolar mitotic spindles (12) and is therefore orthologous to the initial aurora discovered in Drosophila. Although it is well established that Aurora A regulates centrosome function and duplication, the mechanisms underlying this control are yet to be fully 157

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understood. It has been shown that similar to other kinases, Aurora A kinase activity is dependent upon the phosphorylation status of a key threonine residue (T288) located on the activation loop of the enzyme. This phosphate group is removed by Protein Phosphatase 1 or 2A (PP1/2A) rendering Aurora A kinase inactive. The kinase that adds the phosphate group to T288 went many years undiscovered until it was recently shown to be Aurora A kinase itself (13). In this manner, Aurora A is held inactive by its close association with PP1/2A and the switch to the active form requires a signaling pathway involving the small GTPase Ran and the microtubule-associated protein TPX2 (14). Prior to mitotic spindle assembly, Ran releases TPX2 from binding to importin a and b, which allows TPX2 to associate with Aurora A and target it to the microtubules of the mitotic spindle (15). In addition to controlling its localization, TPX2 also activates Aurora A by inducing a small conformational change that protects T288 from PP1/2A and thereby promotes the autophosphorylation of Aurora A (16). Once activated, Aurora A phosphorylates microtuble-associated proteins such as the kinesin Eg5 (17), the acidic coiled-coil protein, TACC (18), and TPX2 itself in order to assemble the mitotic spindle. Aurora B Aurora B (also known as Aurora-1/AIK2/ARK2/STK12) localizes to the centromere during the early stages of mitosis (prophase to anaphase), whereupon it relocates to the midbody throughout cytokinesis (19). It functions along with its binding partners and substrates, INCENP and survivin, to ensure proper kinetochore-microtubule attachments (20). Additionally, Aurora B is required for chromosome segregation and cytokinesis. Therefore, inhibition of Aurora B function results in an increase-in-ploidy phenotype making it orthologous to Ipl1 in yeast. In order for chromosomes to properly segregate during mitosis, microtubules from opposing spindle poles must attach to sister chromatids in a process called chromosome biorientation. An increase in mal-oriented chromosomes was noted in Aurora B-inhibited cells and another study showed Aurora B to be required for correcting non-bioriented chromosomes (21,22). The mechanism by which Aurora B contributed to biorientation was recently ascribed to its interaction with the microtubule-destabilizing mitotic centromere-associated kinesin (MCAK) (23). The emerging model for establishing biorientation is that the microtubule destabilizing activity of MCAK disconnects the stochastic attachment of microtubules to the kinetochore until biorientation is achieved, which is detected by Aurora B. Once established, Aurora B phosphorylates MCAK, which inhibits the catalytic activity of MCAK and the destabilization of microtubules at the kinetochore-microtubule interface halts, resulting in stable chromosome biorientation. In addition to its role in detecting proper kinetochore-microtububle attachments, Aurora B functions in the spindle checkpoint and is required for cytokinesis (24–26). The role of Aurora B in the spindle checkpoint is to recruit checkpoint proteins, such as BubR1, Mad2 and Cenp-E to the kinetochores (27). The inhibition of Aurora B function leads to a bypass of the spindle checkpoint and premature exit from mitosis without undergoing cytokinesis. This series of events leads to polyploidization and potentially to continued re-entry into the cell cycle resulting in multinucleated cells.

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Importantly, Aurora B phosphorylates Histone H3 at the Serine 10 position during mitosis. It was initially believed that Aurora B-mediated phospho-Histone H3 was required for chromosome condensation and mitosis; however, this has recently come under question (28,29). The biological significance of Histone H3 phosphorylation by Aurora B is not fully understood. Regardless, it has proven to be a very important pharmacodynamic endpoint in the preclinical and clinical development of Aurora kinase inhibitors. Aurora C Little is known about the function of Aurora C (also known as Aurora-3/AYK1/ AIK3/AIE1/STK13). The earliest reports show the expression of Aurora C to be primarily in meiotically active cells and to be important for processes such as spermatogenesis (30). Subsequently, Aurora C was shown to be expressed during mitosis when its localization was primarily to the centrosome (31). Most recently, reports suggest that Aurora C is a chromosome passenger protein that functions in a very similar, if not redundant, role to that of Aurora B (30). These seemingly conflicting reports in the literature show that the function of Aurora C needs further elucidation. THE AURORA KINASES IN CANCER Of the three human aurora kinases, Aurora A has been the family member most often and most closely associated with cancer; however, it is becoming evident that Aurora B likely contributes to tumorigenesis. An association between Aurora C and cancer has not been identified. Aurora A Aurora A is overexpressed in a variety of tumor types summarized in Table 1. Additionally, the amplification of the Aurora A gene has been associated with its overexpression in many tumor types, including breast, colorectal and liver cancers (6,9,32). The oncogenecity of Aurora A has been demonstrated in at least some tumorigenesis models, in which its overexpression led to the in vitro and in vivo transformation of rodent fibroblast cells and to the formation of multipolar mitotic spindles and genomic instability (33). Similarly, the overexpression of Aurora A in near diploid human breast epithelial cells revealed centrosome abnormalities, as well as induction of aneuploidy (33). The link between Aurora A overexpression and aneuploidy has led to the hypothesis that Aurora A overexpression and hyperactivity may be a major driving force in the acquisition of other genetic alterations required for tumorigenesis in some tumor types (34). In support of this, Aurora A overexpression has been shown to be an early event in rat mammary carcinogenesis (35) and present in high-grade PIN (prostatic intraepithelial neoplasia) lesions, indicating that this may be an early event that leads to the genetic instability seen in prostate carcinogenesis (36). Further implicating a role for Aurora A in tumorigenesis are discoveries showing its connection with genes and proteins typically associated with cancer. For example, BRCA1 activity is at least in part regulated by its binding to and phosphorylation by Aurora A (37). Furthermore, Aurora A plays a role in the regulation of c-Myc and telomerase expression levels (38) and p53 has been shown to be a substrate for Aurora A leading to the degradation and/or the inactivation

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TABLE 1 Reported Abnormalities of Aurora A and Aurora B in Human Tumors Cancer type Aurora A Breast

Specimen type Cell lines Primary

Colorectal

Cell lines Primary

Ovarian

Cell lines Primary

Prostate

Cell lines Xenografts Primary Cell lines Cell lines Cell lines Primary

Neuroblastoma Cervical Gastric

Bladder Pancreatic Laryngeal Non-Hodgkin’s lymphoma Endometrial Hepatocellular

Cell lines Primary Cell lines Primary Primary Primary Primary Primary

Esophageal Oral (tongue)

Primary Primary

Lung Head and neck Glioma

Cell lines Primary Primary Primary

Kidney Melanoma Medulloblastoma Mantle cell lymphoma

Cell lines Cell lines Primary Primary

Aurora B Colorectal Glioma Thyroid Lung Prostate Kidney Melanoma Breast

Primary Cell lines Primary Cell lines Primary Cell lines Primary Primary Cell lines Cell lines Cell lines

Findings

References

Amplification and overexpression Overexpression in 94% and amplification in 12% Amplification and overexpression Amplification and overexpression in >50% Amplification in 10–15%; Overexpression in 67% Overexpression in 50% Amplification and overexpression Overexpression Overexpression in 98% Amplification and overexpression Amplification and overexpression Amplfication in 29%; overexpression in 44% Amplfication in 5–13%; overxpression in 41–50% Overexpression Amplification and overexpression Overexpression Overexpression in 56–93% Overexpression in 68% Overexpression

(6) (33,75)

Amplification in 56% Amplification in 3%; overexpression in 61% Overexpression in 68% Amplification in 36%; overexpression in 100% Amplification and overexpression Overexpression in 69% Overexpression Amplification in 26–31%; overexpression in 60% Amplification and overexpression Amplification and overexpression Overexpression Amplification in 0%; overexpression

(93) (32)

Overexpression Overexpression Overexpression Overexpression Overexpression Overexpression Overexpression in 59% Overexpression Overexpression Overexpression Overexpression

(33) (9,76) (33,77) (78) (33) (79) (36) (33) (33) (80) (81)

(82) (83,84) (85) (12,86) (87–91) (92)

(94) (95) (9) (96) (97) (98) (9) (9) (99) (100)

(9,101) (102) (103) (104) (104) (9) (44) (105) (9) (9) (9)

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of p53 protein (39,40). Of further clinical significance, Aurora A overexpression induces resistance to tubulin-targeted agents such as paclitaxel by overriding the spindle assembly checkpoint in mitosis (41). Aurora B Although the biology of Aurora B in disease-free cells is arguably better understood than Aurora A, its role in cancer is not. Indeed Aurora B is overexpressed in a variety of tumor types (Table 1); however its amplification and ability to transform cells (oncogenicity) has not been noted. Therefore, some have speculated that its overexpression is possibly a characteristic of rapidly dividing cells with high mitotic indices, rather than an event that drives tumorigenesis. Contrary to this idea, although Aurora B overexpression alone did not transform rodent fibroblast cells, it did potentiate H-Ras(G12V)-induced transformation (42). In endometrial carcinomas the expression of Aurora B was significantly increased in high-grade tumors, and patients with Aurora B-positive carcinoma showed poor prognosis compared with those with Aurora B-negative tumors (43). Furthermore, Aurora B overexpression does correlate with the level of genomic instability within a tumor, suggesting Aurora B may contribute to the process of acquiring necessary genetic alterations required for tumorigenesis (44). THE AURORA KINASES AS THERAPEUTIC TARGETS Due to the strong association of Aurora A with cancer almost from its initial discovery, it was thought to be the more important therapeutic target. The validation of Aurora B as a potential drug target did not come about until it was discovered that small molecules intended to target either Aurora A or both aurora kinases produced a biological response entirely consistent with Aurora B inhibition alone. Aurora A The work validating Aurora A as a potential therapeutic target has been primarily carried out using gene silencing approaches such as siRNA and antisense oligonucleotides in pancreatic cancer cell lines. The first of these reports showed that Aurora A-specific inhibition by an antisense oligonucleotide resulted in cell cycle arrest in the G2/M phase and in the induction of apoptosis (12). The second study further showed that the specific knockdown of Aurora A in pancreatic cancer cells suppressed in vitro cell growth and in vivo tumorigenicity. Furthermore, a synergistic enhancement of the cytotoxicity of taxanes was shown when combined with siRNA-mediated knockdown of Aurora A (45). MLN8054 is a newly disclosed Aurora kinase inhibitor which is reported to show some selectivity towards Aurora A (46). In recent reports, drug-induced phenotypic changes consistent with Aurora A inhibition occurred at low concentrations; however phenotypic markers (such as phospho-Histone H3 levels) shifted toward a signature of an Aurora B-specific inhibitor when higher concentrations of the same drug were administered (47,48). With the available information it is unclear whether the therapeutic index between Aurora A and Aurora B inhibition is great enough to achieve selectivity in patients. Regardless of its ultimate target profile in patients, the emergence of a small molecule with preferential activity towards Aurora A is very exciting.

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Aurora B In contrast to Aurora A, target validation studies using gene silencing approaches targeting Aurora B are limited. However, several reports do characterize the anti-tumor effects of small molecule inhibitors. These small molecule inhibitors, with the exception of a few, were not intended to be Aurora B specific. In fact, in cell-free enzymatic assays most of them have comparable activity against all three Aurora kinases; however, when the compounds were evaluated in cell-based and animal models, the dual inhibitors produced all the phenotypic changes consistent with Aurora B inhibition alone. The reason for this was recently elucidated by showing that when Aurora B is inactivated, the requirement for Aurora A in mitosis is bypassed (49). Therefore, although a dual aurora kinase inhibitor does indeed inhibit the activities of both kinases, it functionally inhibits Aurora B only because the function of Aurora A is not required. Several aurora kinase inhibitors are in development as anticancer agents; four of them are described below. Hesperadin (Boehringer Ingelheim) was discovered from a cell-based screen to identify small molecules having effects on cell proliferation (21). Its target was identified as Aurora B as it was shown to decrease Histone H3 phosphorylation, induce chromosome segregation defects, and inhibit cytokinesis leading to polyploidization. Hesperadin has been a useful chemical tool to further understand the biology of Aurora B; however, it has not been further developed for potential clinical use. ZM447439 (AstraZeneca) is a dual aurora kinase inhibitor that has again been very instrumental in understanding Aurora B biology and validating Aurora B as a potential drug target (27,50). It also decreases phospho-Histone H3 levels, induces cellular polyploidization, inhibits tumor colony formation, and compromises the spindle checkpoint. It is an early analogue of AZD1152, which is an Aurora B-specific inhibitor currently in clinical trial. VX-680 (Vertex and Merck) potently inhibits all three aurora kinases. It inhibits the proliferation of several cancer cell lines resulting in the accumulation of cells with >4N DNA content (51). VX-680 inhibits Histone H3 phosphorylation and shows activity in mouse xenograft models (52). Importantly, VX-680 shows good activity in refractory acute myelogenous leukemia (AML) cells, which is likely due to its cross-inhibitory activity against FLT-3. Furthermore, VX-680 is currently under investigation as a BCR-ABL inhibitor in chronic myelogenous leukemia (CML) due to its potent activity against the imatinib-resistant T315I mutant of BCR-ABL (53). The fourth compound that has played an important role in the validation of Aurora B as a drug target is PHA-680632 (Nerviano). It has shown good activity on a wide range of cancer cell lines and significant tumor growth inhibition in in vivo tumor models (54). PHA-680632 induces a biological response consistent with Aurora B inhibition, such as decreased Histone H3 phosphorylation, endoreduplication and polyploidy. In cell-free evaluations, PHA-680632 is active against all three aurora kinases. CLINICAL DATA ON AURORA KINASE INHIBITORS Aurora kinase inhibitors for which data are available include MK-0457 (VX-680) and AZD1152. Other aurora kinase inhibitors that are actively being studied both preclinically and in clinical trials are listed in Table 2.

þ
þ þ þ þ

Merck/Vertex MK-0457 (VX-680) þ Millennium MLN8054 þ

þ þ

þ þ

AT9283 MP529

SNS-314 Resveratrol

Undesignated R763

CYC116 Hesperadin

Astex Supergen

Sunesis N/A

Avalon Rigel

Cyclacel Boehringer Ingelheim

Unknown


Unknown þ

Unknown Unknown

þ þ

þ


Unknown Unknown

Unknown

þ

Unknown

Rebamipide

N/A

Astra Zeneca ZM447439 Astra Zeneca AZD1152

Unknown

þ

þ

SU6668

Sugen/Pfizer

Unknown Unknown

Unknown Unknown

Unknown Unknown

Unknown Unknown

þ


Unknown þ

þ þ

þ þ

þ þ

PHA-680632 PHA-739358

Unknown

þ

þ

Oral Unknown

Unknown Oral/IV

Unknown Oral

Oral Oral

IV Oral

IV IV

Oral

Oral

Unknown IV

Unknown

Aurora Aurora Aurora A inhibition B inhibition C inhibition Formulation

JNJ-7706621

Compound

Johnson and Johnson Nerviano Nerviano

Source

TABLE 2 Aurora Kinase Inhibitors in Development Comments

Preclinical Preclinical

Preclinical

Preclinical

None None

None Natural product, also inhibits Polo-like kinase 1 None None

Also inhibits CDK1, CDK2 and CDK3 Preclinical None Phase II Schedule: days 1, 8, and 15 MTD: unknown DLTs: unknown No longer in Also inhibits several receptor development tyrosine kinases and TANK-binding kinase 1 Clinical use Inhibits Aurora B in Japan through survivin Preclinical None Phase I Schedule: days 1,8, and 15 MTD: 200 mg DLTs: neutropenia Phase II Also inhibits FLT3 Phase I At high doses also inhibits Aurora B Phase I None Preclinical None

Status Preclinical

References

www.cyclacel.com (21)

www.avalonrx.com www.rigel.com

(57,58) www.supergen.com (113) www.sunesis.com (114)

(51,55) (46–48,109–112)

(27,50,108) (56)

(107)

(60)

(54) (59)

(106)

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MK-0457 (VX-680; Merck/Vertex) MK-0457 is the Aurora kinase inhibitor that is furthest along in clinical development. It is currently in phase II trials in colorectal cancer and also in refractory acute myelogenous leukemia, poor risk myelodysplastic syndrome (MDS), B-cell acute lymphocytic leukemia (ALL) or chronic myelogenous leukemia (CML) in blast crisis (55). MK-0457 inhibits all three of the known Aurora kinases A, B and C. The inhibitory constants suggest that in terms of in vitro potency the inhibition is A > C > B. It also inhibits a multitude of other kinases to some degree including FLT-3, which is thought to be important in refractory acute myeloid leukemia and other hematological malignancies and as such may serve as an area of investigation for this compound independent of its Aurora kinase inhibition. Recently, results from a phase I study of this agent in advanced solid malignancies were presented (55). MK-0457 was administered as a continuous 24-hour intravenous for 5 days infusion every 28 days. An accelerated titration scheme was employed for dose escalation whereby 1–2 patients were treated per dose level until a grade 2 toxicity ensued at which time a more standard modified Fibonacci dose escalation scheme was followed. A total of 22 patients were treated in this first-in-man study. Characteristics of these patients were as follows: male:female (8:14), median age: 57 years, ECOG PS 0:1:2 (6:15:1), cancer types were colon (n = 6), lung (n = 3), pancreas (n = 4) and other (n = 5), and median number of prior therapies was three. The starting dose of MK-0457 was 0.5 mg/m2/hr. Virtually no toxicities were observed until a dose of 8 mg/m2/hr was achieved. One of seven patients had a neutropenic fever at 8 mg/m2/hr, one of six patients had a neutropenic fever at 10 mg/m2/hr, and two of four patients treated at 12 mg/m2/hr experienced grade 4 neutropenia. As such, the preclinically predicted toxicity of neutronpenia was the DLT for the drug. The dose of 10 mg/m2/hr was recommended as the dose most appropriate for future phase II investigations. Three patients had stable disease as their best response and two of these (patient with pancreatic cancer treated at the 2 mg/m2/hr dose and patient with non-small cell lung cancer treated at the 4 mg/m2/hr dose) completed 6 cycles of therapy. Pharmacokinetics were dose-related and showed an initial exponential decline in MK-0457 levels shortly after discontinuation of the infusion followed by a slower terminal phase. It was also noted that the levels of MK-0457 achieved during the infusion at all dose levels were higher than the IC50 (1 mM) during in vitro studies and above the effective doses in xenograft studies. The half-life of MK-0457 after completion of the infusion was about 15 hr. Biomarkers studied during this phase I study were the measurement of Histone H3 phosphorylation in skin (expected effect of this Aurora B substrate would be a decrease with administration of drug), Ki-67 measurement in skin (expected effect of this proliferation marker would be a decrease), and cyclin B1 measurement in skin (expected effect of this mitotic arrest marker would be an increase after administration of drug). Unfortunately, these markers did not help to demonstrate the proof-of-concept as they were found at very low levels at baseline and the changes in these markers were not substantial even at the higher dose levels of MK-0457 (8 and 12 mg/m2/hr) at which they were obtained.

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AZD1152 (AstraZeneca) AZD1152 is an aurora kinase inhibitor that is selective for Aurora B (IC50s: Aurora A ¼ 1369 nM, Aurora B ¼ 0.36 nM, Aurora C ¼ 17.0 nM). It did not exhibit any significant activity against any other kinases among a panel of >50 kinases studied. Data on results from the first phase I investigation in advanced solid malignancies was reported recently (56). The study was tiered into three parts: A, B and C. Part A involved weekly dosing on days 1, 8 and 15 of a 21-day cycle; Part B comprised bi-weekly dosing on days 1 and 15 of a 28 day cycle and; Part C was the expansion phase incorporating tumor biopsies. A total of 19 patients were described. The median age of the patients was 59 years (range 40–71 years). The male:female composition was 14:5. Tumor types were colorectal (n = 8), melanoma/schwannoma (n = 3), prostate cancer (n = 2), nasopharyngeal cancer (n = 1), mesothelioma (n = 1), esophageal cancer (n = 1), pancreatic cancer (n = 1), renal cancer (n = 1), and adenoid cystic cancer (n = 1). World Health Organization (WHO) performance statuses of the patients ranged from 0 to 2 (WHO PS 0, =1; WHO PS 1, n = 16, and WHO PS 2, n = 2). An initial accelerated titration scheme, using single patients per dose level, that reverted to the modified Fibonacci approach, drug-related toxicities examining was used for dose escalation. The starting dose for this agent was 100 mg. A flat dosing approach was employed for the purposes of this study. Dose escalation beyond the second dose level of 200 mg used the modified Fibonacci approach and the classic “3þ3” design. Doses studied ranged from 100 to 450 mg. AZD1152 was administered as a 2 hour-intravenous infusion in all cases. Grade 3/4 neutropenia was the DLT associated with AZD1152 in 3 of 6 patients at 450 mg and 2 of 6 patients at 300 mg. Neutropenia was both febrile and resulted in dose delays at both levels. The dose delays were 14 days and as such a schedule that involved dosing at two-week intervals could incorporate what would otherwise be dose-limiting neutropenia. Of note, no lymphopenia or thrombocytopenia ensued as a result of AZD1152 exposure, and as such from these preliminary data it appears that thrombocytopenia, which could also be dose-limiting, will not be encountered. AZD1152 is a pro-drug, which is converted to the active moiety AZD1152hQPA. Pharmacokinetics revealed rapid clearing of the parent drug and rapid detection of the active moiety, AZD1152-hQPA. The plasma exposure was above the IC50s noted in preclinical studies at all the doses tested. Variability in pharmacokinetic parameters was low between subjects. Stable disease greater than 12 weeks was the best response to therapy achieved in five patients. Of these, one with melanoma (at 450 mg that was dose reduced to 300 mg), one with nasopharyngeal carcinoma (at 450 mg that was also dose reduced to 300 mg), and one with adenoid cystic carcinoma (at 300 mg that had no dose reductions) enjoyed stable disease for 32þ, 25 and 35þ weeks, respectively. Part B of this study which employs dosing on days 1 and 15 (2 week intervals) on a 28-day schedule is currently underway. MLN8054 (Millennium) MLN8054 is an orally available, potent and selective inhibitor of Aurora A. A novel approach was utilized in determining activity of the agent in xenograft models which can be described as a growth modulation index whereby differences

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in growth rates of tumors between treated and untreated groups of animals were compared to find a “signal” that the drug may have activity. Although it is shown to be Aurora A selective, this is only the case at lower concentrations from in vitro studies. At higher concentrations the phenotypic changes in treated cells are more consistent with Aurora B inhibition. Given this preclinical information it will be interesting to see the pharmacodynamic data generated from the phase I study in advanced solid tumors that is currently underway (46–48). AT9283 (Astex) AT9283 is an orally available inhibitor of the Aurora A and Aurora B kinases. It has recently entered into phase I clinical trials in imatinib refractory chronic myelogenous leukemia. This molecule was developed using a structure-based approach towards drug discovery (57,58). PHA739358 (Nerviano) This compound is in a phase II clinical trial in patients with CML with T315I mutations. It is being administered on a 28-day schedule on days 1, 8 and 15 by way of a 6-hour intravenous infusion. Other than what is stated, very limited information is available publicly on this compound (54,59). SU6668 (Pfizer) SU6668 was developed as an oral multi-targeted kinase inhibitor. It is felt to inhibit VEGFR2, PDGFR-beta, c-KIT and FGFR-1. Additionally, recent work has shown that it is also a potent inhibitor in vitro of Aurora A and Aurora B kinases as well as the TANK-binding kinase 1 (60). As such, SU6668 is clearly not a selective Aurora kinase inhibitor. SU6668 has been the subject of four separate phase I studies reported thus far. Given that development of this compound had been discontinued in favor of the now approved agent sunitinib and that it has only been found to be an aurora kinase inhibitor in a single in vitro study, we have not described the clinical development of this compound in detail. ISSUES IN FUTURE CLINICAL DEVELOPMENT OF AURORA KINASE INHIBITORS Clinical Indications for Future Development of Aurora Kinase Inhibitors As noted previously MK-0457 (VX-680) is currently in phase II clinical investigations in patients with colorectal cancer. Extensive preclinical information supports the development of this compound in this tumor type. Many other tumor types have been found to have expression of aurora kinases and would be tumor types of interest from the perspective of clinical development (Table 1). Interestingly, preclinical studies have shown a relationship between estrogen levels and aurora kinase expression. From a drug development perspective, it may make sense to look for differences in response between men and women and between premenopausal and menopausal or post-menopausal women (61,62). Formulation Choice in Drug Development Thus far the two agents with the most extensive clinical experience, AZD1152 and MK-0457, have been developed as intravenous drugs. MLN8054 is being

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developed as on oral drug. Oral drugs will maintain an advantage over intravenous agents of equal efficacy only if they are dosed on a chronic schedule. Intermittent administration would likely favor parenteral agents because the variability in absorption would be removed from the variability and unpredictability equation with regards to the dose of the drug. Given that so far doselimiting neutropenia has been the major obstacle in drug development of this class of compounds, it appears that intermittent administration may be the way forward and that parenteral formulations could be the favored choice. Dose Escalation Schemes Accelerated titration employing one patient per dose level or initial dose level increments of 100% (63) followed by the traditional Fibonacci method has been the dose escalation scheme utilized in the development of both MK-0457 and AZD1152. Dose-limiting toxicities have been manageable with this approach and this will likely be the favored approach for the early development of other agents in this class. Novel Clinical Trial Designs Standard clinical trial approaches that employ investigation of the agent in a particular tumor type have historically had low success rates and have failed to uncover those populations of patients within a group as a whole who benefited but were not identified. A more rational approach that is increasing in popularity is treating only those patients who have either overexpression in the target felt to be affected by the drug, mutations in a gene that is felt to render responsiveness to the drug, or gene amplification of the target felt to be affected by the drug (as in the case of FISHþ HER2/neu breast cancer patients), etc. This approach can be concisely described as treating only targetþ patients after selection. In the case of aurora kinase inhibitor development targetþ could be: increased levels of expression of Aurora A, Aurora B, or Aurora C; amplification of Aurora A, Aurora B, or Aurora C or increased expression of aurora kinase substrates such as CENP-A, myosin regulatory light chain, protein phosphatase-1, TPX-2, INCENP, survivin, topoisomerase II alpha, vimentin, MBD-3, MgcRacGAP, desmin, Ajuba, and TACC (51), or mutations in aurora kinase. A major pitfall of this approach would be in tumor settings where Aurora (A, B or C) or an aurora-related target is abnormal but not a primary contributor to disease. In such a case, the target would not prove to be clinically meaningful because it is eclipsed by a more dominant or compensatory pathway which could potentially negate the effects of inhibiting the Aurora pathway. Regardless, a targetþ approach to select candidates for an aurora-based therapy is logical. Instead of a single arm phase II trial, it may make sense to utilize a randomized phase II whereby a “positive signal” may confer more confidence prior to pursuing a larger, more expensive trial (64). Such a trial design may employ two or three arms. In the case of the three arm design this may be: Arm 1: High Dose Aurora Kinase Inhibitor; Arm 2: Low Dose Aurora Kinase Inhibitor; and Arm 3: Standard Treatment or Placebo if appropriate. The determination of significant differences in a predetermined end-point such as time-to-tumor progression, relapse rate, response rate, or progression free survival can serve as the “signal” that would warrant further investigation of the agent in larger trials.

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If agents that are orally available and chronically dosed are advanced enough in clinical development a novel clinical strategy that can be employed is a randomized discontinuation design (65). Combination of Aurora Kinase Inhibitors with Other Agents Aurora kinase inhibitors, like many other cancer therapeutic agents, may find their optimal utility in combination with agents from other classes. In vitro studies have shown AZD1152 to be synergistic with irinotecan, gemcitabine, docetaxel, oxaliplatin, 5-FU and vinorelbine. Of interest in preclinical systems, it was found that administration of gemcitabine or irinotecan prior to the aurora kinase inhibitor inhibited its activity. This suggests that in the clinical setting with these combinations, perhaps the aurora kinase inhibitor should be administered prior to administration of either gemcitabine or irinotecan (66). In addition to traditional cytotoxics, it would be very worthwhile to explore the combination of agents that disrupt pathways that affect closely related or associated pathways. These include the survivin inhibitors, heat shock protein inhibitors (67–69), and KSP/Eg5 inhibitors (70,71). Even from a clinical perspective it makes more sense to use these combinations because the primary dose-limiting toxicity seen thus far with the aurora kinase inhibitors has been neutropenia, which along with the neutropenia of traditional cytotoxics would definitely preclude clinical development in an optimal fashion. Separate Trials in Heavily Pretreated Patients Given that neutropenia has been the major DLT in early clinical investigations, it would seem reasonable that separate phase II investigations ought to be conducted in patients heavily pretreated with cytotoxic chemotherapy versus minimally pretreated patients, as the MTD for the minimally pretreated patients may very well be higher. Development of Novel Pharmacodynamic Assays One of the most difficult aspects of drug development with new agents has been clinically validating the proof-of-concept regarding mechanisms of action and their correlation to clinically meaningful efficacy. Thus far, in patients receiving MK-0457, skin biopsies evaluating Ki-67, cyclin B1, and phosporylation of serine residue 10 in histone H3 have been used as pharmacodynamic tools. Unfortunately, these assays were not helpful in validating aurora kinase inhibition. It is possible that the tissue selected (skin in this case) may not have been appropriate in terms of reflecting events occurring in the tumor environment/microenvironment. Clearly, novel assays and appropriate tissue selection for observation for evidence of activity are required. Given that aurora kinases are expressed in significant proportions only during active cell division (i.e., M phase) it would be prudent to assess for inhibition of enzyme activity by an aurora kinase inhibitor in actively dividing cells. In addition to the skin, the oropharyngeal mucosa, hair follicles, gut, and bone marrow represent sites where collected samples would be replete in proliferating cells. As such, potential assays could include demonstration of polyploidy in buccal smear samples after administration of an Aurora B kinase inhibitor, hair follicle assay demonstrating cells having monopolar spindles after

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administration of an Aurora A kinase inhibitor, and stool samples containing cells shed from the bowel mucosa demonstrating loss of phosphorylation at the T288 autophosphorylation site after administration of an Aurora A kinase inhibitor (72–74). Analysis of cell populations obtained from tumor samples using FACS (Fluorescence Activated Cell Sorting) analysis to demonstrate a shift from predominantly normal 2N populations to 4N, 8N, 16N, etc. populations (i.e., cells that have not undergone cytokinesis) would also validate success of the compound under investigation from a proof-of-concept standpoint. Use of Imaging Traditional imaging modalities such as CT, MRI, or ultrasound would fail to provide conclusive and early evidence that aurora kinase inhibitors were exerting their desired effects. Given that aurora kinase inhibitors may have more cytostatic as opposed to cytotoxic effects, tumor shrinkage will likely be a rare event, and, as such, these traditional imaging modalities, despite being somewhat helpful from a clinical standpoint, will do nothing to build confidence in validation of the concept that disruption of the aurora kinase pathway is biologically meaningful. PET scans have overcome some of the shortfalls of the traditional imaging modalities in terms of providing a “metabolic” perspective of drug effects or disease status. Unfortunately, PET imaging may not be greatly valuable using the most widely used agent, FDG. This primarily stems from the mechanism of Aurora A inhibitors, which do not affect cellular proliferation but tend to affect disruption of cytokinesis. On the other hand FDG PET may be a valuable tool in evaluating Aurora B inhibitors. With these caveats, it is clear that development of novel imaging agents required to assess activity of this class of compounds is in dire need. CONCLUSION Aurora kinase inhibitors have enjoyed a rapid trajectory from the discovery of these enzymes to the first clinical studies of inhibitors of this novel target. The prompt development of these agents has been fueled by the interesting biology of the aurora family of kinases and by their implicated roles in tumor growth. Appropriate use of aurora kinase inhibitors as single agents, and in combination with other agents that affect complementary or related pathways, will be paramount in the successful integration of this class of drugs into the anti-cancer armamentarium. We remain optimistic that inhibitors targeting the aurora kinases will lead to increased disease-free survival of cancer patients. ACKNOWLEDGMENTS The preparation of this review was supported in part by NIH National Cancer Institute Grant CA95031 and the Drug Development Scholar Fund for the Translational Genomics Research Institute and for the Scottsdale Clinical Research Institute (MB). The authors also appreciate the assistance of Susan McCall with manuscript preparation.

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Apoptosis Modulators: p53 Targeting Sunil Chada, Dora Bocangel, and Kerstin Menander Departments of Clinical Research and Development, Introgen Therapeutics, Inc., Houston, Texas, U.S.A.

Jack A. Roth Thoracic and Cardiovascular Surgery, University of Texas, M. D. Anderson Cancer Center, Houston, Texas, U.S.A.

INTRODUCTION Cancer is a disease initiated, driven, and sustained by genomic instability; many pleiotropic and overlapping signaling pathways contribute to oncogenesis and pathologic progression. Despite accumulating multiple genetic, epigenetic, and chromosomal abnormalities, cancer cells can become dependent on a single or a few oncogenic pathways for both maintenance of the malignant phenotype and cell survival; this phenomenon—termed “oncogene addiction”—has been reported in both cultured cell lines and animal models (1). Thus, reversal of only one or a few of these abnormalities can trigger massive apoptosis resulting in inhibition of cancer cell growth. Considerable progress in the treatment of cancer in recent years stems from the development and clinical application of drugs targeted to specific molecular pathways. Examples of these pathway-specific drugs are Gleevec
(imatinib mesylate; Novartis Pharmaceutical Corp., East Hannover, New Jersey, U.S.A.) and Tarceva
(erlotinib; Genentech, San Francisco, California, U.S.A.), both of which act as selective tyrosine kinase inhibitors. However, clinical success with these pathway-specific agents has been idiosyncratic. Agents that affect not only one, but various, albeit similar, pathways are being developed and have demonstrated improved clinical results. Such is the case of Sorafenib (Nevaxar
; Wayne, New Jersey, U.S.A.), which was initially developed as a RAF-RAS kinase-targeted drug. Further studies showed that, in addition to targeting RAF kinase, Sorafenib also inhibits VEGF and PDGF receptor kinases, as well as KIT and FLT-3 kinases, culminating in tumor cell apoptosis and inhibition of angiogenesis. It appears that the combination of Sorafenib's actions on multiple tyrosine kinase pathways induces an enhanced therapeutic response by this drug. Additional drugs which target multiple kinases that have demonstrated clinical promise are sunitinib (Sutent
; Pfizer, Inc., New York, New York, U.S.A.), dasatinib (Sprycel
; BristolMyers Squibb, New York, New York, U.S.A.), and lapatinib (GlaxoSmithKline, Brentford, London, U.K.). In contrast, agents which have demonstrated specificity against one kinase target do not always show clinical activity; this concept was exemplified by the lack of robust clinical activity of Iressa
(gefitinib; Astrazeneca, Mississauga, Ontario, Canada), despite early enthusiasm. This ability to simultaneously target and interfere with multiple cancerpromoting pathways is a property shared by p53. The p53 gene is a critical tumor suppressor that plays a key role maintaining the integrity of cellular DNA. p53 regulates progression through the cell cycle and, in the presence of DNA damage, 177

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functions as a regulatory node to either facilitate DNA repair, or initiate apoptotic cell death when the damage is too extensive (2–5). The primary mode of action of p53 is transcription modulation; p53 activates or represses expression of hundreds of target genes involved in regulation of cell cycle arrest (p21WAF1/CIP1), apoptosis (bax, bcl-2), and/or DNA-repair processes (6–9). Additionally, p53 inhibits neovascularization by regulating expression of several key proteins in the process, including VEGF, BAI1, TSP1 (10,11), and bFGF-binding protein (12,13). A lack of functional p53 protein can, therefore, allow the accumulation of genomic instability, resulting in unregulated proliferation of damaged cells and tumor formation (14–17). Aberrant p53 pathways are present in virtually all cancer cells, either by mutation/deletion of the p53 gene, or by abnormal regulation of p53 gene expression, stability, or function in the absence of p53 gene mutations (Lane). Mutations have been detected in over 50% of human cancers tested, and up to 70% of non–small cell lung cancer (NSCLC) and squamous cell carcinoma of the head and neck (SCCHN) (18–22). Other alterations of this pathway include inactivation or sequestration of the wild-type p53 gene product (e.g., inactivation via overexpression of MDM2) (Fig. 1), inability to activate p53 protein (e.g., via post-translational modifications), and mutations of downstream p53 targets (e.g., inactive enzymes in the apoptotic cascade) (23,24). Importantly, the presence of altered protein function or mutation of the p53 gene has been associated with poor clinical outcomes in patients with several types of cancer (25–30), and the presence

FIGURE 1 p53 regulatory pathway and map of Advexin genome.

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of p53 mutations or disrupted p53 pathways correlates with resistance to chemotherapy and radiation. Advexin
(Introgen Therapeutics, Inc., Houston, Texas, U.S.A.) is an adenoviral vector, derived from adenovirus serotype 5 (Ad5), which mediates overexpression of the human wild-type tumor suppressor protein p53 under the control of the CMV promoter (Fig. 1) (31). The E1 region of the parental Ad5 DNA is deleted, thus preventing replication and expression of adenoviral genes. Numerous preclinical studies have demonstrated that transduction of cancer cells with a replication-incompetent adenoviral vector carrying the wild-type p53 gene (Ad-p53; Advexin), increases apoptosis and decreases proliferation of cancer cells with no apparent effect on normal cells (32). These studies have also shown that p53 sensitizes cancer cells to the effects of chemotherapy or radiation therapy and indicate that p53 may have utility both as monotherapy as well as a component of combination regimens. Significantly, increases in apoptosis and decreases in cancer cell proliferation have been demonstrated following administration of Advexin without observable effects on normal cells (33–35). Clinical studies have demonstrated that Advexin is safe and more easily tolerated than chemotherapy or radiation treatment. Initial clinical trials designed to assess the safety and tolerability of Advexin in patients with a variety of cancers had favorable outcomes, with safety profiles that are superior to those of chemotherapy and radiation (36–39). This review summarizes the extensive preclinical and clinical trials data gathered for Advexin in a variety of treatment settings as monotherapy and in combination with chemotherapy, radiation, and surgery. These studies indicate potential applications of Advexin for cancer prevention, initial cancer treatment, therapy of recurrent tumors and the treatment of disease refractory to standard therapies. Therefore, the use of wild type p53 to target genetic defects in tumor cells allows the application of this agent in a broad spectrum of tumors. PRECLINICAL STUDIES Targeting p53 In Vitro Advexin as Monotherapy As predicted from the known actions of p53 as a tumor suppressor, Advexin induces apoptosis and/or inhibits cell proliferation in cancer cell lines from numerous tumor types, while having comparatively little effect on normal cells. Apoptosis induction in tumor cell lines in response to Advexin monotherapy has been shown to be both dose- and time-dependent (40), and correlate with the dose- and time-dependent expression of p53 protein mediated by Advexin (up to 500-fold protein increase within 48 hours). Induction of apoptosis correlates with the changes in expression of various p53-responsive genes involved in apoptosis, angiogenesis, cell cycle progression, and DNA repair and replication [e.g., p21, MDM2 (41)]. Interestingly, the consequences of enhanced p53 expression last longer than the p53 protein levels, suggesting activation of a signaling cascade that leads to irreversible inhibition of proliferation, decreased viability, and/or apoptosis (42). As a general rule, the effects of Advexin on a cell line are not dependent on the p53 mutational status of that cell line. Very few cancer cell lines are resistant to Advexin; of over 100 tumor cell lines evaluated comprising 15 different tumor types, only two have been found to be completely resistant to Advexin (Table 1). Therefore the extremely high rate of response (i.e., cell death,

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TABLE 1 Cell Lines in which Advexin Inhibits Proliferation and/or Increases Apoptosis Indication Squamous cell carcinoma of the head and neck Non–small cell lung cancer

Breast Colorectal Prostate Cervical Osteosarcoma Esophageal Hepatocellular carcinoma Pancreatic Ovarian Glioma Endometrial Bladder Multiple myeloma

Normal cells a b

Cell lines Tu138, Tu167, Tu167, Tu177, Tu182, JSQ3, SQ-20B, FaDu, HN12, MDA 886, SCC97, HN30 H1299, H358, PC14, H322, H23, H226, H226Br, Calu-1, H596, H157, H460, A549 T47D, SK-BR-3, MDA-MB-231, MDA-MB-435, Calc-18, MCF-7, ZR-75-1 SW620, KM12L4, DLD-1, SW480, HT29, WiDr, HCT116, LoVo DU145, PC-3, LNCaP, C4-2 C33A, HT3, HeLa, C4-1, MS751, ME180, CaSki, SiHa Saos-2, U-20S, SA1 T.Tn, ECGI-10, TE8, YES6 HLE, HLF CFPAC-1, BxPC-3, PANC-1, Capan-1, AsPC-1, MIA PaCa-2, Capan2 SK-OV-3, OVCAR-3, OCC-1, OVCA432, OVCA420, OVCA429, OVCA433 U-251, U-251 MG, U-373 MG, A-172, U-87 MG, D54 MG, D54, EFC-2 SPEC-2 ECV304, KOTCC-1/P, KK47 8226, ARH-77, U266, ARP-1, MC-CAR,; HS-Sultan, IM9, MC-CAR,; HS-Sultan, IM9 MRC-9, CCD-16, HUVEC, MJ90, NHME, NHBE, CASMC, NHLF

Responsea 12/12

12/12

7/7 8/8 4/4 8/8 3/3 4/4 2/2 7/7 7/7 6/8b 1/1 3/3 7/7

0/8

Cell death/apoptosis: shown as number of lines affected/tested. Two glioma lines were resistant.

apoptosis) observed in tumor cells indicates that p53 can overcome apoptosis resistance that is a hallmark of tumor cells. Thus the apoptotic pathways downstream from p53 must be intact in the majority of tumor cells and can be activated to promote cell death. Furthermore, although more than 98% of tumor cells tested are sensitive to Advexin, no toxicity was observed in normal cells (Table 1). Advexin in Combination with Other Agents In addition to its effects as a single agent, there is a wealth of evidence that Advexin augments activity of radiation and other cytotoxic anticancer agents. The combinations demonstrate increased anticancer effects whereas Advexin does not cause an increase in toxicity mediated by conventional cytotoxic treatments (43). Advexin has been tested in vitro in combination with cisplatin, doxorubicin, etoposide, SN-38 (a metabolite of irinotecan), melphalan, carmustine (BCNU), fluorouracil, docetaxel, paclitaxel, radiation, and docetaxel plus radiation, in cell lines derived from SCCHN (44), NSCLC (43,45), glioma (46), breast cancer (47), colorectal cancer, prostate cancer, cervical cancer, epidermoid carcinoma and HCC (unpublished data). In the majority of these studies, Advexin increased tumor cell apoptosis in an additive or synergistic fashion when combined with standard

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Apoptosis Modulators: p53 Targeting TABLE 2 In Vitro and In Vivo Effects of Advexin Combined with Radiation or Cytotoxic Anticancer Agents Mechanism of action Forms DNA crosslinks Antimitotic, prevents microtubule disassembly Binds DNA and prevents nucleic acid synthesis Forms inter- and intra-strand Pt crosslinks Interferes with topoisomerase II Inhibits DNA synthesis Induces DNA single-strand breaks Induces DNA single and double-strand breaks

Drug/Agent Cisplatin Docetaxel Doxorubicin 5-Fluorouracil Etoposide Paclitaxel SN-38 Radiation Docetaxel and radiation

In vitro A, Aþ, S A, S A A, Aþ A, S A A, S A, Aþ, S S

In vivo Aþ Aþ, A Aþ Aþ NT NT NT Aþ Sa

a

Three-way combination more effective than two-way combination or single agents. Abbreviations: A, additive; Aþ, greater than additive; NT, not tested; Pt, platinum; S, synergistic.

anticancer modalities (Table 2). Importantly, inhibition of cytotoxic activity of conventional chemotherapy was not observed in any model evaluated. p53 Gene Therapy in Animal Models Advexin as a Single Agent Therapy Numerous studies have shown Advexin to be effective in animal tumor models, including SCCHN, NSCLC, breast, colorectal, prostate, cervical, ovarian, esophageal, bladder, glioma, hepatocellular carcinoma, and osteosarcoma. Early work using ex vivo models demonstrated that Advexin reduced the tumorigenicity of cells from several cancer types (e.g., Ref. 31). In later studies, intratumoral (IT) injection of Advexin into established SQ human tumor xenografts in nude mice resulted in a reduced growth rate or regression of tumors derived from a wide range of tumor types, including SCCHN, NSCLC, colorectal cancer and breast cancer. Advexin is effective against both p53 mutant and p53 wild-type xenograft tumors. As one would expect based on the broad spectrum of Advexin effects in vitro, Advexin appears to be effective in nearly all in vivo cancer models tested, with the possible exception of p53 wild-type gliomas (47,48). Some effects seen with in vivo models were dramatic, such as the complete inhibition of tumor growth after IT Advexin administration into human cervical cancer xenografts in nude mice reported by Hamada et al. (49). Most in vivo efficacy studies have been performed in SQ xenograft models, but Advexin also inhibits growth in disseminated xenograft cancer models (50) and in orthotopic and syngeneic models (31). As observed with in vitro studies, the antitumor effects of Advexin in animal models correlate with exogenous p53 expression, induction of p21 and mdm2 protein expression, and induction of apoptosis and/or decreased proliferation of cells within the tumor. Ohtani et al. (51) using a SQ NSCLC tumor model, demonstrated increased expression of p53, p21, MDM2, Noxa, and p53AIP1, and increased apoptosis, following a single IT injection of Advexin. Advexin in Combination with Other Agents Combination of Advexin with radiation or chemotherapy agents enhances the antitumor effects of Advexin in various animal models of cancer, and is summarized in Table 2. Enhanced efficacy has been demonstrated in SCCHN (unpublished data), NSCLC (43,52–54), breast cancer (55), colon cancer (56,57), prostate cancer

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3500

Tumor size (mm3)

3000

. .

2500

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2000

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.

1500

Control Control+5Gy dl312 dl312+5Gy Adp53 Adp53+5Gy

. .

1000 .

500 0 0

5

10

10

20 25

30 35

40

Day FIGURE 2 The combination of Advexin and radiation enhances antitumor effects in xenograft colorectal cancer tumors. Established SQ SW620 tumors were treated after reaching a size of 200 mm3 with three daily injections of PBS (control), dl312 (total dose of 2 · 1011 vp, 7.5 · 109 pfu), or Advexin (Adp53, total dose of 2 · 1011 vp, 7.5 · 109 pfu). Tumors were treated with 5 Gy on day 4. Tumors were measured every other day in two orthogonal dimensions, and tumor volume was calculated based on elliptical dimensions. n = 6 animals/group; points, mean values; bars, SE. Advexin combined with radiation resulted in the greatest degree of tumor growth inhibition. Source: Reproduced from Ref. 58.

(58,59), and glioma (47,60,61) animal models. As observed in vitro, the enhanced tumor growth inhibition observed with combination treatments correlates with increased apoptosis in the tumors (54,57,58). Representative data from one of these studies is presented in Figure 2. Nguyen et al. (57) treated established SQ colorectal cancer xenograft tumors in nude mice with radiation alone, or with Advexin with or without radiation. At the conclusion of the experiment, tumors treated with the combination were significantly smaller than those receiving Advexin or radiation alone. In a similar study using SQ NSCLC xenograft tumors in nude mice (43), the combination of docetaxel, radiation, and Advexin was shown to synergistically inhibit tumor growth. This Advexin-mediated increase in sensitivity to radiation or cytotoxic anticancer agents is not dependent on the p53 status of the tumor model tested. For example, Advexin increased sensitivity to radiation in p53-mutant tumor models of colorectal cancer (56), and NSCLC (43), prostate cancer (58) and NSCLC (53) with wild-type p53. These results are consistent with the in vitro data, which show sensitivity to Advexin-mediated growth inhibition in cell lines with wild-type or mutant p53 genes. This data also underscores the restoration of apoptotic competence provided by wt p53. Many (if not all) established tumor cell lines are considered to be resistant to apoptosis and understanding the molecular pathways responsible for apoptotic resistance is an area of intense research. Toxicity Preclinical Studies Effects of Advexin in Normal Cells Because both cancer and normal cells transduced with Advexin express supraphysiological levels of p53 in, there was an initial concern that treatment with

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Advexin might cause toxic effects in both. However, the toxic effects of Advexin appear to be selective for tumor cells. Various studies document that growth of normal cells is unaffected by Advexin transduction at levels that are cytotoxic to tumor cell lines: these include studies on normal human bronchial epithelial (NHBE) cells (35), normal human fibroblasts cultured from SCCHN tumors (33), normal human lung fibroblasts (41,54), normal human esophageal epithelial cells (60), and normal human hematopoietic stem cells and lymphocytes (62). Further investigation at the biochemical level by Sah et al. (41) found that Advexin had no effect on the DNA repair capacity of MRC-9 cells (normal human fibroblasts), nor on levels of two proteins involved in apoptosis (p21 and Bax). Toxicity studies in animal models have shown that Advexin can induce apoptosis in tumors while having little or no effect on normal cells and tissues in vivo. The reasons for these differential effects on normal and cancer cells are not completely clear, but some hypotheses proposed suggest that p53 may be more active at inducing the transcription of downstream genes in transformed cells than in normal cells (63). Additionally, normal cells may have biochemical differences that would make them less sensitive to the effects of supra-physiological levels of p53, such as a block to apoptosis downstream of p53 expression (54). Finally, p53 protein may be less stable and therefore less effective in normal than transformed cells (63). The selectivity index (i.e., ratio of IC50 for Advexin compared to control Ad-luciferase) varies between different tumor types but often exceeds 100 for tumor lines. In contrast, for normal cells, the selectivity index is 1, reflecting lack of toxicity to normal tissue by p53. Toxicity Studies of Advexin on Animal Models A series of preclinical acute toxicity studies have been conducted with Advexin, using seven routes of administration, including SQ, oral, intraperitoneal (IP), IV-portal vein, and IV. Three of the seven are particularly relevant to clinical studies in which Advexin is administered by IT injection to patients: SQ, the route in nontumor-bearing animals that most closely approximates IT administration; oral, since some of the vector may be swallowed when treating cancers such as head and neck, oral, or esophageal; and IV, because biodistribution analyses conducted during phase 1 and 2 clinical trials have shown some systemic distribution following IT administration. Many of these studies were conducted under GLP, with comprehensive analysis of clinical signs, gross pathology, hematology, clinical chemistry, and histopathologic evaluation. Relevant data from these studies, at doses similar to those used in the phase 3 clinical trials, is discussed below. Overall, Advexin as monotherapy was well-tolerated. SQ administration of Advexin induced minimal dissemination, with no toxicity detected even at the maximum dose tested, 3.7 · 1012 vp/kg (
100-fold higher on a per weight basis than the human phase 3 clinical trial dose of
3 · 1010 vp/kg). Oral administration, which also results in limited dissemination, gave a toxicity profile indistinguishable from the control; no toxicity was observed up to the maximum Advexin dose tested (8.3 · 1012 vp/kg). Locally, no toxic effects were observed at the injection site when Advexin was administered SQ. In an IT biodistribution study, Advexin caused some edema, hemorrhage, and a few inflammatory cells within the tumor, but no effects were observed in extra-tumoral tissue (unpublished data). Although lymphocyte infiltration occurred in other IT studies, no effects were observed on adjacent normal tissue (64).

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The safety of Advexin in combination with standard anticancer agents was also evaluated as part of preclinical studies evaluating efficacy of repeated IT administration with standard anticancer agents (docetaxel, cyclophosphamide, doxorubicin, cisplatin, fluorouracil, and radiation), in which mortality, histopathology, weight changes, and gross physical changes were measured. The combination of Advexin with standard anticancer agents administered at therapeutic doses did not increase mortality or histopathologic findings in liver, spleen, kidney, heart, or lung. Advexin does not appear to pose a risk of genotoxicity, mutagenicity, or carcinogenicity. No epidemiological evidence linking adenoviruses to human cancer has been reported and Ad5 DNA has not been found in human tumors. With respect to the potential for genotoxicity via integration of Advexin DNA, adenoviruses as a class are not integrating viruses, and a study specifically designed to evaluate integration of Advexin did not detect any integration events (unpublished results). In summary, preclinical studies show that the liver is the primary organ of toxicity of Advexin at high doses and with systemic routes of administration. Advexin is well-tolerated by routes of administration and at doses which approximate those used in clinical trials. Greater toxicity was generally seen with more systemic routes of administration, and the primary target organ for toxicity, the liver, was also the primary target organ for biodistribution. Advexin does not appear to cause DNA damage or otherwise pose a risk of genotoxicity. CLINICAL EXPERIENCE WITH ADVEXIN A total of 28 phase 1, 2, and 3 clinical studies have been conducted using Advexin; 23 have been finalized, completed, or closed, and five are ongoing. Of these, 16 are monotherapy studies and six have combined Advexin with chemotherapy or radiation. Patients in these studies had advanced cancers, most commonly lung cancer or SCCHN, although studies were also performed in patients with prostate cancer, breast cancer, colorectal cancer and other solid tumors. Advexin typically has been administered via intratumoral injection, although 17 patients in clinical studies have been treated intravenously. The majority of patients in these clinical trials have received multiple cycles of Advexin therapy (39), and the results of these have demonstrated the safety, tolerability, and utility of Advexin as monotherapy and in combination with chemotherapy, radiation, and surgery. These studies also identified a number of prognostic indicators that may be used to identify patients most likely to benefit from Advexin therapy. Below, we summarize the clinical experience of Advexin as a monotherapy (SCCHN), and in combination with chemotherapy (LABC) and radiotherapy (NSCLC). Advexin as Monotherapy Advexin has been evaluated as monotherapy for several types of cancer, including recurrent, unresectable, locally advanced SCCHN and radiation-resistant, locally advanced esophageal cancer (65,66). In this cohort of heavily pretreated patients, Advexin monotherapy was well tolerated with evidence of clinical activity. Patients with recurrent, unresectable, locally advanced SCCHN have a poor prognosis and recurrent disease is usually considered incurable. Median overall survival after first relapse in patients with recurrent SCCHN is dismally short

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regardless of the treatment: 6 months if treated with chemotherapy as monotherapy (67–71), and 6–9 months for patients treated with combination therapy with platinum- or taxane-based regimens (72–75). The rationale for use of a p53targeted therapy in treatment of SCCHN stems from loss of p53 function in approximately 70% of patients with SCCHN, which has been associated with tumorigenesis and resistance to radiation and chemotherapy (20–22). We conducted three phase II trials in this patient population; two studies used a higher dose of Advexin (5 · 1011 to 2.5 · 1012 viral particles (vp)/injection), and one used a lower dose (1–4 · 109 vp/injection). Patients had histologically confirmed SCCHN, with cytologically confirmed recurrence, excluding endolaryngeal recurrence, after first-line therapy administered with a curative intent (50 Gy radiotherapy and/or surgery with or without chemotherapy). All lesions in the head and neck region were accessible to intratumoral treatment; any inaccessible lesions had to be separately evaluable and unlikely to impair the patient's ability to complete the study. The total area of all measurable lesions had to be 30 cm2, and the sum of the longest diameter of each measurable lesion had to be 10 cm. In addition, patients had a Karnofsky performance status (KPS) 60%, a life expectancy >12 weeks, and a tumor tissue sample (primary or recurrent tumor) was made available for p53 genotyping. Results from one of the phase II trials (n ¼ 105) showed the overall tumor response rate to Advexin treatment in evaluable patients was 6%, while 20% showed evidence of durable tumor growth control lasting longer than 3 months. Dose response and survival analyses for patients with recurrent/refractory SCCHN treated in the high- and low-dose phase II trials revealed clinical benefit, as defined by durable tumor growth control. In patients who received at least one cycle of treatment, high dose for Advexin provided survival advantage, as compared to treatment with low dose Advexin. This suggests a dose–response effect induced by Advexin in this patient population. Multivariate analyses conducted on studies in SCCHN patients identified a long progression-free interval after initial therapy (12 months) as the major prognostic factor for all efficacy outcomes (65). The size of treated lesions (25 mm) was a favorable prognostic factor for both tumor response and tumor growth control, while prior irradiation of target lesions was a prognostic factor for the latter. Absence of ulcerated and/or necrotic lesions, and baseline tumor-pain identified tumors more suitable for intralesional Advexin treatment and were independent factors for response. Applying these selection criteria, subgroups of patients in these studies were defined; these groups exhibited overall response rates of 20–30% and tumor growth control rates of 50–60%, depending on the degree of selection. In contrast to previous findings regarding the negative impact of induction chemotherapy on subsequent treatments, our studies indicate that prior chemotherapy was a positive prognostic factor (65). Additionally, lesions treated in a prior radiation field had better tumor growth control than untreated lesions. These observations are consistent with p53-therapy induced apoptosis in the presence of DNA damage due to irradiation or chemotherapy, which result in cytotoxicity when p53 function is reactivated (76,77). Taken together, these results provide evidence that Advexin monotherapy can improve outcomes in heavily pretreated SCCHN patients and suggest that Advexin may provide recurrent SCCHN patients with an effective and well tolerated approach to controlling their disease.

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Advexin in Combination Therapy The key role of p53 as a regulator of cell cycle progression and apoptotic pathways following treatment with DNA damaging agents, in conjunction with the favorable safety profile and clinical activity of Advexin monotherapy supported its evaluation in combination with chemotherapy regimens. In this section we summarize the results of trials evaluating the use of Advexin in combination with chemotherapy for treatment of lung and breast cancer. Overall, the results are consistent with preclinical studies and report incorporation of Advexin into chemotherapy regimens with minimal additional toxicity and with improved efficacy. Advexin and Chemotherapy for Treatment of NSCLC Non–small cell lung cancer accounts for nearly 80% of all lung cancers, and one third of patients diagnosed with NSCLC present with locally advanced, unresectable tumors (78). Despite advances in chemotherapy and the recent approval of biologic therapies gefitinib and erlotinib HCl, the 5-year survival rate for all lung cancers is only 15% (American Cancer Society). Two-year survival for patients with advanced disease ranges from 20% (stage III) to 5% (stage IV), and treatments for patients with advanced disease frequently result in severe side effects that may significantly decrease quality of life (79–85). Cisplatin is the most active single agent in NSCLC, and the drug is a mainstay of combination chemotherapy for this disease. Although several other chemotherapy agents have shown evidence of activity in NSCLC, their use has not increased median survival and is associated with significant toxicity (86–91). Mutations in the p53 gene have been detected in approximately 70% of NSCLC samples tested (19,92,93), and preclinical studies demonstrated activity of Advexin in combination with chemotherapy (83). This provided the rationale for evaluation of the toxicity and antitumor activity of Advexin, delivered via computed tomography-guided percutaneous or bronchoscopic injection into NSCLC tumors obstructing the airway (78). The first Advexin study in lung cancer was conducted by Swisher et al. (94), who treated 28 NSCLC patients with intratumoral injections of 106–1011 pfu, and demonstrated wt-p53 transgene expression that was consistent with antitumor activity in a subset of patients. Below, we review two clinical studies evaluating Advexin in this population of patients. A two-arm phase I study was conducted to evaluate the feasibility, safety, humoral immune response and biologic activity of multiple IT injections of Advexin, and to characterize the pharmacokinetics in patients with advanced NSCLC. Fifteen patients, with life expectancy >12 weeks, histologically confirmed NSCLC resistant or refractory to standard therapies, with lesions accessible to repeated injection and measurable disease with p53 mutations, were enrolled (DNA mutation or protein overexpression). Patients in one arm (n ¼ 9) received escalating doses of Advexin monotherapy (1 · 106 to 1 · 1011 plaqueforming units), administered by fine-needle injection using a bronchoscope; the other arm (n ¼ 6) evaluated Advexin (escalating doses ranging from 1 · 109 to 1 · 1011 pfu), administered on day 4 of a 28-day schedule, in combination with intravenous (IV) cisplatin (80 mg/m2 over 2 hours) administered on day 1 (95). Patients received a total of up to 14 courses of study treatment (median ¼ 3, average ¼ 4.2) and were monitored for adverse events and clinical effects. Results of this study support the feasibility and safety of IT Advexin, alone or in combination with Cisplatin, in patients with advanced NSCLC. Of the

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15 patients enrolled, 13 were assessable for efficacy: one patient had a partial response, while 10 patients had stable disease (3 of these lasting >9 months), and two patients had progressive disease. Symptomatic improvement included reduction in dyspnea, cough, and hemoptysis, observed in 4 (26.7%) patients (95). There was no dose-limiting toxicity associated with the study treatment, and no patient was withdrawn from the study due to adverse effects. Of the adverse events (AE) reported, the most common was a transient, self-limited, fever. Patients usually recovered within 48 hours. Hematologic toxicity was limited (1 incidence of leucopenia, and 3 incidences of grade 2–3 anemia). Transient (lasting <10 days), mild increases in liver ALT and AST were observed in one patient treated with monotherapy. An open-label, dose escalating, phase I trial was conducted on patients with advanced NSCLC harboring p53 mutations, determined by DNA sequencing. Twenty-four patients were enrolled, and treated with IT injection of Advexin (1 ¼ 106 to 1 ¼ 1011 pfu) on day 4 and IV Cisplatin (80 mg/m2) on day 1. Apoptosis was measured using TUNEL assay. Vector dissemination and biodistribution was monitored. The study reports a best overall response of stable disease in 17 patients (74%), partial responses in two patients (9%), and progressive disease in four (17%). Consistent with p53 apoptotic functions, the mean apoptotic index in the tumor increased fourfold (p ¼ 0.011). As in study described above, the most common AE attributable to the study treatment was transient, self-limiting fever, reported in eight patients (33%). No changes in mean vital sign parameters, hematologic function, electrolytes, or renal or liver function were observed. There was no detectable dose-related effect on toxicity. The study concluded that IT injection of Advexin in combination with Cisplatin was well tolerated, and reported evidence of its clinical activity (96). Taken together, these clinical results support the use of direct bronchoscopic injection of Adp53 into endobronchial NSCLC in combination with platinum chemotherapy. Further, Advexin demonstrated relief of airway obstruction in heavily pretreated patients, thus supporting the use of Advexin in combination with chemotherapy or as monotherapy for localized lesions interfering with patient's quality of life. Neoadjuvant Advexin and Chemotherapy for Treatment of Locally Advanced Breast Cancer Breast cancer is the second-leading cause of cancer death in women, and it is estimated that 211,240 new cases of breast cancer will be diagnosed in 2005 in the United States and that 40,870 people will die of the disease (American Cancer Society). Locally advanced breast cancer (LABC) represents approximately 10–30% of all primary breast cancers diagnosed. Approximately 50% of patients with LABC express alterations of p53 in their tumors. Management of LABC has evolved, such that standard of care now incorporates neoadjuvant (preoperative or induction) chemotherapy as part of the multimodality approach (97–99). The taxanes and anthracyclines are considered to have the highest level of activity against breast cancer (BC), although as single agents they fail to produce a response in about half of BCs. Standard of care for this population of patients involves Induction Chemotherapy (IC), which is increasingly favored for management of LABC (disease stages IIA–IIIB) because: (1) it allows chemosensitivity testing; (2) it can downstage size of primary tumor and render it operable; (3) depending on the responses to primary systemic therapy, it may allow for

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breast-conservation surgery to be performed; and (4) allows for elimination of occult systemic metastases (100–104). Aberrations in p53 gene sequence or protein expression levels are frequently observed in primary breast tumors, particularly in LABC (105), and p53 dysfunction has been associated with poor prognoses, more aggressive tumors, early metastasis, chemoresistance, and decreased survival (105–107). Additionally, preclinical studies have shown that Advexin may increase chemosensitivity, especially to drugs that induce DNA damage (76,83,108,109). These observations provided the rationale for evaluating the efficacy of Advexin in combination with chemotherapeutic agents, in this case an anthracycline (doxorubicin) and a taxane (Docetaxel). Our group has conducted a prospective, open-label phase II study to assess the safety, efficacy and biological activity of the combination of doxorubicin and docetaxel with the intratumoral injection of Advexin in patients with newly diagnosed inoperable LABC (stage IIIB-IIIC) (110). Patients received four to six 3-week cycles of study treatment (IT injection of Advexin, at 2.5 · 1012 viral particles [vp] on day 1–2). Chemotherapy (doxorubicin 50 mg/m2 IV was followed by docetaxel 75 mg/m2 IV) was administered on day 1, after administration of Advexin. Patients that achieved clinical remission following study treatment were treated with surgery, followed by radiation therapy. Adjuvant hormonal therapy was given to those patients with hormone receptor-positive disease. One hundred percent of patients achieved clinical PR and underwent subsequent surgery; radiological assessment of response showed 79% median reduced primary tumor volume, and median reduced size of 67% for nodal disease. After 35 months of follow-up, 92% of the treated patients are alive and 83% have survived without evidence of disease recurrence. Overall clinical responses with a greater than 50 percent reduction in tumor size were seen following the combined therapy in all of the patients. The highly significant antitumor activity induced in the primary lesion, promising OS (82% at 3 years), and the 100% resectability rate, suggests that treatment with Advexin in conjunction with anthracycline-based IC dramatically reduces chemoresistance (98,110,111). The results of the therapy with the addition of ADVEXIN are better than what would be expected from neoadjuvant chemotherapy treatment alone. In a novel finding, activation of a local immune response at the site of the tumor was observed. Treated tumors were infiltrated with cells of the immune system that are known to participate in immune responses against tumors, which may be useful in controlling local disease as well as disease outside the breast. This study is the first to indicate safety and efficacy of a gene-based neoadjuvant therapy in breast cancer. The 100% PR rate observed in this study has not been observed in any other neo-adjuvant therapy trial for primary LABC (110,112,113). These data suggest that Advexin may be combined with neoadjuvant chemotherapy to further reduce tumor size and improve patient outcomes by facilitating complete surgical tumor removal. In addition, the results add to the very favorable safety profile observed in other Advexin clinical trials in patients with later stages of cancer and support clinical applications of Advexin in earlier phases of disease management. These data also suggest that Advexin can enhance the clinical benefit of chemotherapy without increasing this treatment's toxicity.

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Advexin in Combination with Radiation Therapy for Treatment of NSCLC A prospective, single arm, phase II study was conducted to evaluate the feasibility and mechanisms of apoptosis induced by IT bronchoscopic injection of Advexin (3 · 1011 to 1 · 1012 vp, administered on days 1, 18, and 32) in combination with radiation therapy (60 Gy, starting on day 4, administered over 6 weeks). The dose of Advexin was escalated in cohorts of 3 for the first 9 patients, while subsequent patients were treated with 3 · 1012 vp. A total of 19 patients with histologically proven, nonmetastatic, measurable, stage I-III NSCLC were enrolled; 9 of them had locoregional advanced NSCLC (stages IIIA–B). Patients were ineligible for chemoradiation or surgery because of significant comorbidities, age, or obstructed bronchi. The primary endpoint of the study was local control at 3 months after completion of radiation therapy, as assessed by CT scan (16 patients) and biopsy (3 patients). The study reports that 89% of the patients completed the study treatment. CT and bronchoscopic findings at the tumor site revealed complete response in 1 (5%), partial responses in 11 (58%), stable disease in 3 (16%), and progressive disease in 2 patients (11%); 2 patients were not evaluable due to progression or death. At the time the study was reported, 5 patients were alive 34–48 months after study initiation, and 11 had developed distant metastases. Median time to progression had not been reached for loco-regional disease and was 9.2 months for metastatic disease. The high number of pathologic negative biopsies (63%) and ORR are highly uncommon in this patient population (114,115). Importantly, combination of Advexin and radiation did not increase toxicity as compared with previously reported results for radiation alone (114,115), and no dose-limiting toxicities were observed in the study (116). These data suggest that Advexin can provide locoregional control of NSCLC for patients who are not candidates for surgery or chemoradiation, and have few treatment options. CONCLUSIONS AND FUTURE DIRECTIONS The results reviewed in this chapter show that transduction by Advexin induces high-level expression of biologically active wt-p53 protein. This p53 expression is time- and dose-dependent, and causes expression of other proteins known to be p53-responsive. Phenotypic consequences of p53 expression in cancer cells include inhibition of proliferation, decreased viability, and apoptosis, mechanisms of action which are consistent with known alterations in gene expression caused by Advexin. In many in vitro and in vivo preclinical models of cancer, Advexin is effective by various routes of administration and effective in nearly all types of cancer tested, regardless of p53 mutation status. Advexin directly affects the transduced cancer cells, but also has broader indirect effects on tumors, inhibiting angiogenesis, and stimulating an antitumor immune response. The combination of Advexin with standard anticancer modalities augments the effects of Advexin both in vitro and in vivo, and these effects are correlated with an increased incidence of tumor-specific apoptosis; little or no toxicity is observed in animal studies at doses equivalent to the highest doses used in human clinical trials. In clinical studies, Advexin has excellent safety and tolerability profiles both as a monotherapy and in combination with other agents. Of over 600 patients treated with Advexin in clinical studies, most side effects and AEs have been mild to moderate in severity, and transient in nature. No deaths have been attributed to Advexin treatment. Thus, the safety and clinical activity of Advexin have been

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demonstrated in different cancer indications and in combination with different chemotherapy regimens. Significantly, addition of Advexin to standard chemotherapy is associated with minimal increases in toxicity, supporting the broad potential application of Advexin in combination with a variety of chemotherapeutic agents and combination treatment strategies for both initial therapy and in the treatment of recurrent disease. REFERENCES 1. Weinstein IB. Addiction to oncogenes—the Achilles heal of cancer. Science 2002; 297:63–4. 2. Lane DP, Midgley CA, Hupp TR, et al. On the regulation of the p53 tumour suppressor, and its role in the cellular response to DNA damage. Philos Trans R Soc Lond B Biol Sci 1995; 47:83–7. 3. Levine AJ, Momand J, Finlay CA. The p53 tumour suppressor gene. Nature 1991; 351:453–6. 4. Haffner R, Oren M. Biochemical properties and biological effects of p53. Curr Opin Genet Dev 1995; 5:84–90. 5. Amundson SA, Myers TG, Fornace AJ. Roles for p53 in growth arrest and apoptosis: putting on the brakes after genotoxic stress. Oncogene 1998; 17:3287–99. 6. Bode AM and Dong Z. Post-translational modification of p53 in tumorigenesis. Nature Reviews/Cancer 2004; 4:793–805. 7. Nakamura Y. Isolation of p53-target genes and their functional analysis. Cancer Sci 2004; 95:7–11. 8. Vousden KH. p53: death star. Cell 2000; 03:691–4. 9. Zhang L, Yu J, Park BH, et al. Role of BAX in the apoptotic response to anticancer agents. Science 2000; 290:989–92. 10. Bouvet ML, Ellis M, Nishizaki M, et al. Adenovirus-mediated wild-type p53 gene transfer down-regulates vascular endothelial growth factor expression and inhibits angiogenesis in human colon cancer. Cancer Res 1998; 58:2288–92. 11. Nishizaki M, Fujiwara T, Tanida T, et al. Recombinant adenovirus expressing wildtype p53 is antiangiogenic: a proposed mechanism for bystander effect. Clin Cancer Res 1999; 5:1015–23. 12. Ueba T, Nosaka T, Takahashi JA, et al. Transcriptional regulation of basic fibroblast growth factor gene by p53 in human glioblastoma and hepatocellular carcinoma cells. Proc Natl Acad Sci USA 1994; 91:9009–13. 13. Sherif ZA, Nakai S, Pirollo KF, et al. Downmodulation of bFGF-binding protein expression following restoration of p53 function. Cancer Gene Ther 2001; 8:771–82. 14. Prives C. Signaling to p53: breaking the MDM2-p53 circuit. Cell 1998; 95:5–8. 15. Fisher DE. The p53 tumor suppressor: critical regulator of life & death in cancer. Apoptosis 2001; 6:7–15. 16. Ryan KM, Phillips AC, Vousden KH. Regulation and function of the p53 tumor suppressor protein. Curr Opin Cell Biol 2001; 13:332–7. 17. Bargonetti J, Manfredi JJ. Multiple roles of the tumor suppressor p53. Curr Opin Oncol 2002; 14:86–91. 18. Sager R: Tumor suppressor genes: the puzzle and the promise. Science 1989; 246:1406–12. 19. Chiba I, Takahashi T, Nau MM, et al. Mutations in the 53 gene are frequent in primary, resected non-small cell lung cancer. Oncogene 1990; 5(10):1603–10. 20. Boyle JO, Hakim J, Koch W, et al. The incidence of p53 mutations increases with progression of head and neck cancer. Cancer Res 1990; 53:4477–80. 21. Ahomadegbe JC, Barrois M, Fogel S, et al. High incidence of p53 alterations (mutation, deletion, overexpression) in head and neck primary tumors and metastases; absence of correlation with clinical outcome. Frequent protein overexpression in normal epithelium and in early non-invasive lesions. Oncogene 1995; 0:1217–27.

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Survivin Dario C. Altieri Department of Cancer Biology and the Cancer Center, University of Massachusetts Medical School, Worcester, Massachusetts, U.S.A.

INTRODUCTION: BIOLOGY OF SURVIVIN Except for rare malignancies that are driven by a single oncogenic lesion, most human tumors are extraordinarily heterogeneous. This involves a large spectrum of genetic mutations, extensive epigenetic changes, and activation of multiple signaling pathways, which differ not only between different tumor types, but also within seemingly histologically identical tumors. Such complexity has posed formidable challenges in generalizing the concept of targeted cancer therapy, as the identification, validation, and “druggability” of disease-causing “cancer genes” has in most cases remained elusive. Conversely, “crossroads” pathways that are differentially expressed in cancer and intersect multiple aspects of tumor cell maintenance may provide broader therapeutic prospects. One such pathway is centered on survivin, a cancer gene that contributes to tumor cell proliferation, resistance to programmed cell death (or apoptosis), and cellular adaptation to environmental stress. Targeted agents aimed at the “survivin network” have been developed and are being tested in cancer patients. This contribution will review the current state of survivin-based therapeutics, their rationale, and concrete prospects for clinical implementation. Survivin is a member of the inhibitor of apoptosis (IAP) gene family (1). These molecules contain 1–3 zinc finger folds and act as evolutionary conserved suppressors of caspases, the effector enzymes of apoptosis (1). Differently from other IAPs, survivin is a stable homodimer in solution, and is “essential,” in that deletion of the survivin gene in mice causes early embryonic lethality and immediate loss of tissue/organ viability. Two general features make survivin unique not only among IAP proteins, but also compared to other cell death regulators. First, survivin is intercalated in at least three pivotal signaling pathways required for tumor cell maintenance. These include the regulation of multiple aspects of mitosis (2), the inhibition of cell death by antagonizing mitochondria-dependent apoptosis (3), and the participation in the cellular stress response as one of the client proteins for heat shock protein-90, Hsp90 (4). Second, survivin is a bona fide “cancer gene,” overexpressed in histological and molecular analysis of nearly every human tumor, but undetectable or expressed at very low levels in most normal adult tissues (5). The differential expression of survivin in cancer is largely transcriptional, and may reflect a global deregulation of survivin gene expression in transformed cells. In particular, oncogenic transcription factors, including STAT-3 (6), E2F (7), T Cell Factor-4 (TCF-4) (8), or mutated Ras (9), have been shown to turn on the survivin promoter, whereas tumor suppressor genes that are frequently lost in cancer, including p53 (10) or the adenomatous polyposis coli (APC) protein (11), act as negative repressors of survivin gene transcription. The tumor-specific expression of survivin carries

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prognostic and predictive implications, and is consistently associated with high grade disease, abbreviated survival, resistance to therapy, and accelerated recurrences (2). Exploited for molecular diagnostics, survivin provides one of 16 genes predictive of recurrences in tamoxifene-treated, node-negative, breast cancer patients (12) and an easily accessible and sensitive urine biomarker for recurrences of bladder cancer (13). RATIONALE OF SURVIVIN-BASED THERAPEUTICS Because of the attributes listed above, survivin has been vigorously pursued as a cancer drug target. Compared to other apoptosis-based therapies (14), targeting survivin may provide critical conceptual advantages. First, interference with survivin affects multiple signaling pathways required for tumor cell maintenance. This may offer broader therapeutic advantages but also limit the emergence of drug resistance, which is commonly observed with targeted agents aimed at a single oncogenic pathway (15). Second, although not an enzyme or a cell surface receptor, survivin provides a flexible target, druggable with multiple independent approaches suitable for clinical development (see below). Third, survivin expression is regulated by developmental signaling pathways typically operative in stem cells, i.e., Wnt (8), and it is possible that survivin antagonists may affect cancer stem cells (16), a compartment largely untouched by cytotoxics or targeted agents (17). Finally, although survivin expression has been demonstrated in certain normal cells, including cytokine-stimulated hematopoietic progenitors and activated T cells, initial phase I studies of survivin antagonists revealed good tolerability and modest side effects, suggesting that such approaches may be safe for cancer patients. Because of these considerations, several scenarios can be envisioned for the use of survivin-based therapeutics as anticancer agents. First, targeting survivin is expected to dramatically hamper mitosis of tumor cells. During cell division, survivin is recruited to multiple aspects of the mitotic apparatus, and contributes to chromosomal segregation, assembly of a competent bipolar mitotic spindle, and completion of cytokinesis (2). Blocking survivin phosphorylation by Cdk1, or preventing its mitotic expression and/or localization is invariably associated with inhibition of cell-cycle progression and apoptosis, suggesting that survivin-based therapeutics may reproduce a similar phenotype (Fig. 1). This strategy may be particularly beneficial to enhance the anticancer activity of spindle poisons, taxanes in particular (18), whose efficacy is counteracted by elevated survivin levels in tumor cells (Fig. 1). Second, survivin functions as an apoptosis inhibitor downstream of a variety of signaling circuits, in particular growth factor stimulation and growth factor receptor signaling (2). Therefore, survivin antagonists would be expected to remove antiapoptotic mechanisms associated with stimulation through EGF or IGF receptor signaling, and to effectively enhance the activity of small molecule antagonists of these pathways currently in the clinic (Fig. 2). Third, survivin plays an important role in ancillary aspects of tumor formation and progression, particularly angiogenesis, the process of new blood vessel formation (19,20). Upregulation of survivin in response to stimulation by vascular endothelial cell growth factor (VEGF) or Angiopoietin-1 (Ang-1) has been shown to promote increased endothelial cell proliferation, vessel wall remodeling, and inhibition of endothelial cell apoptosis in the context of the tumor vasculature. Accordingly, survivin antagonists may successfully interfere with expansion and stabilization of tumor-associated blood vessels, thus serving a dual role as

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FIGURE 1 Effect of survivin targeting on mitosis. Survivin is recruited to various aspects of the mitotic apparatus during cell division, and is required for spindle assembly, chromosomal segregation, and proper completion of cytokinesis in a phosphorylation-dependent manner. Molecular antagonists of survivin have been shown to interfere with these processes and cause complete cell cycle arrest, spontaneous induction of apoptosis, and to enhance the anticancer activity of spindle poisons, especially taxanes.

anticancer agents on the transformed cell population, and inhibitors of angiogenesis on endothelial cells (Fig. 3). A brief overview of the various molecular strategies to target survivin for novel cancer therapeutics is described below. SURVIVIN-BASED THERAPEUTICS Antisense The first molecular antagonist of survivin was a phosphorothioate-modified antisense oligonucleotide characterized in 1999 (21). This reagent specifically

FIGURE 2 Survivin targeting eliminates antiapoptotic mechanisms downstream of growth factor receptor signaling. Growth factor receptor signaling (i.e., EGF, IGF receptor) increases survivin expression, thus contributing to apoptosis inhibition. Survivin-based therapeutics are expected to block these downstream survival mechanisms, promote spontaneous apoptosis, and enhance the activity of cytotoxics or targeted agents that affect growth factor receptor signaling.

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FIGURE 3 Survivin regulation of angiogenesis. As a downstream transcriptional target of VEGF and VEGF receptor-2 (VEGF-R2), survivin has been implicated in endothelial cell proliferation, remodeling, and protection from apoptosis during tumor-associated angiogenesis. Molecular antagonists of survivin are expected to suppress these responses and function as angiogenesis inhibitors, in addition to targeting the transformed cell population as anticancer agents.

suppressed survivin mRNA and protein expression without affecting other apoptosis regulators, and produced strong anticancer activity with inhibition of tumor cell proliferation, spontaneous apoptosis, synergy with cytotoxics or ionizing radiation, and inhibition of tumor growth in xenograft models (22). Supported by a favorable safety profile, the original survivin antisense oligonucleotide has now completed a phase I clinical trial in patients with advanced cancers, and a phase II study has been announced by Eli-Lilly & Co (Indianapolis, Indiana, U.S.A.). Similar antisense reagents have been developed by other drug companies, and are currently at various stages of advanced preclinical development or safety evaluation in the clinic. A parallel strategy to suppress survivin levels in tumor cells involved small interfering RNA (siRNA) (23,24), or hammerhead ribozymes (25). These reagents produced a phenotype similar to that of antisense, with induction of apoptosis and inhibition of tumor growth in xenograft models (23,25,26). However, given the challenges associated with delivery of such antagonists in humans, it is unclear whether the clinical development of siRNA or hammerhead ribozyme sequences to survivin is being pursued for human testing. Cancer Vaccine / Immunotherapy Because of its differential expression in cancer as compared to normal tissues, it has long been hypothesized that cancer patient immune systems may recognize survivin as a nonself protein and mount an immune response to it (27). This principle has been amply validated in the clinic, and sera from cancer patients have been shown to contain antibodies (28), as well as cytolytic T cells against survivin (29). This recognition has been mapped in detail (30,31), and dendritic cells pulsed with survivin peptides, or expressing survivin, have been used as a strategy to generate cytolytic T cells with MHC-restricted anticancer activity in vitro (32,33) and in preclinical models (34). When used as an oral DNA vaccine, the survivin-directed immune response affected both tumor cells and tumorassociated angiogenesis, eradicating pulmonary metastases without toxicity in preclinical studies (35). Survivin-directed immunotherapy has been quickly moved to the clinic, and several phase I trials with administration of survivin peptides or survivin-directed autologous CTL generated ex vivo, have been recently

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completed (36–38). Survivin-based vaccination was found to be safe, devoid of significant side-effects, and frequently associated with antigen-specific immunologic responses (36,37). Some of these protocols have now been moved to larger phase II trials to test their potential anticancer efficacy. Gene Therapy Several gene therapy approaches to target survivin have recently passed proof-ofprinciple in preclinical studies. One approach included plasmid or adenoviral delivery of survivin “dominant negative” mutants, in particular a Thr34!Ala variant of survivin that abolishes a phosphorylation site for the main mitotic kinase p34cdc2 (18). This mutant is unstable in vivo (39), and its dimerization with endogenous survivin may result in accelerated degradation of the complex, causing acute loss of survivin levels. In turn, this results in inhibition of cell proliferation, induction of apoptosis, suppression of tumor growth, and enhancement of cytotoxics or immunotherapy in preclinical models (40–43). A second approach involved the use of the survivin promoter to drive expression of a “payload” cytotoxic gene in tumor cells. This “suicidal” strategy (44) relies on the fact that the survivin gene has virtually no transcriptional activity in normal tissues, including the liver (45), as opposed to a 200-fold increased expression in tumor cells, in vivo (46). When coupled to a pro-apoptotic protein, administration of the suicidal construct as a DNA–liposome formulation resulted in complete tumor eradication in xenograft models (46), thus offering good prospects for further clinical development. Small Molecules Small molecules have recently become available to disable various cancer signaling pathways. Such approach is also being aggressively pursued to target survivin, and two classes of such antagonists have recently entered clinical testing. The first class comprises small molecules that were screened and selected for direct inhibition of survivin expression. Examples of these antagonists include YM155 and tetra-O-methyl nordihydroguaiaretic acid (M(4)N), which likely function as transcriptional inhibitors of survivin gene expression. YM155 was selected in a large screening for suppression of survivin promoter activity, and abolished survivin mRNA and protein expression, without affecting other apoptosis regulators. M(4)N is also likely a transcriptional inhibitor of the survivin promoter, potentially interfering with Sp1-dependent transcription (47), a main activator of the survivin gene. Both YM155 and M(4)N exhibit strong anticancer activity in preclinical models, with induction of apoptosis and complete tumor regression as single agents in xenograft models. YM155 has recently completed phase I evaluation in 41 heavily pretreated cancer patients. This study demonstrated encouraging clinical responses, in some cases leading to no evidence of residual disease. In addition, YM155 was well tolerated, and the relatively modest side effects observed were potentially unrelated to survivin inhibition. Multiple phase II studies to evaluate YM155 alone, or in combination with various cytotoxic agents, are currently underway. M(4)N is at an earlier stage of development, having recently completed preclinical evaluation (47). A phase I study to evaluate safety and optimal dosing of M(4)N has been recently announced. A second class of survivin antagonists includes small molecules that indirectly affect survivin levels. Representative examples of these agents are inhibitors

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of cyclin-dependent kinases, for example flavopiridol (48), antagonists of STAT3 (49), TCF (50), Hsp90 (51), and ErbB2 (lapatinib) (52). These compounds reduce survivin levels by different mechanisms. Flavopiridol may prevent a critical phosphorylation event of survivin on Thr34, thus destabilizing the protein via accelerated proteasomal destruction (18). Hsp90 antagonists, like the ansamycin antibiotic 17-AAG may also cause accelerated proteasomal destruction of survivin, but this is likely mediated by protein misfolding following disruption of an Hsp90–survivin complex. It is unclear how lapatinib inhibition of ErbB2, a member of the ErbB tyrosine kinase overexpressed in approximately 30% of breast cancer cases, results in reduction of survivin levels. However, this may involve inhibition of PI3 kinase/ Akt signaling, a general survival pathway activated by ErbB2 stimulation consistently associated with increased survivin expression in various tumors. Finally, inhibitors of STAT3 or TCF may act as transcriptional repressors of the survivin promoter, as both molecules have been shown to directly activate survivin gene expression. Regardless of the mechanism of action, the lowering of survivin levels below a critical threshold may further sensitize tumor cells to apoptosis and contribute to the anticancer activity of these small molecule antagonists. In addition, modulation of survivin expression under these conditions can provide an easily accessible biomarker for target validation in human trials. Shepherdin The specific disruption of the survivin–Hsp90 complex (4) has been pursued as an additional anticancer strategy. Both Hsp90 and survivin are upregulated in cancer, contribute to tumor cell maintenance, and provide proven, druggable targets, with 17-AAG as the leading small molecule Hsp90 antagonist in the clinic (51). A recent screening for antagonists of the survivin–Hsp90 complex identified Shepherdin, a cell-permeable peptidomimetic derived from the survivin sequence Lys79–Leu87 (53). Shepherdin physically inhibited the survivin–Hsp90 interaction, but also acted as a global antagonist of the chaperone function of Hsp90. This resulted in loss of mitochondrial integrity, degradation of multiple Hsp90 client proteins, and activation of caspase-dependent and -independent cell death, independently of the proliferative condition of tumor cells, overexpression of survival proteins, or p53 status (53). In preclinical studies, Shepherdin ablated tumor growth as single agent in multiple xenograft models, with no toxicity for normal tissues, including purified CD34þ progenitor cells (53,54). The much stronger anticancer activity of Shepherdin compared to 17-AAG may reflect a unique engagement of the Hsp90 ATPase pocket (53), which may affect chaperone functions of Hsp90 that are not adequately inhibited by 17-AAG. The clinical development of Shepherdin is currently underway through the National Cancer Institute (NCI) RAID (Rapid Access to Intervention Development) program. FUTURE PROSPECTS Despite its relatively recent discovery in 1997, survivin has provided unique opportunities for basic and clinical oncology. In a relatively short period of time, multiple strategies targeting the survivin network have quickly passed proofof-principle in preclinical settings, and many have entered clinical testing in humans. An intriguing common denominator emerging from these earlier studies is the relative safety of survivin-based therapeutics; an encouraging note,

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especially if one considers that survivin is an essential developmental gene, and has potentially important functions, at least in some normal adult tissues. This may reflect a critical differential dependence or “addiction” of tumor cells to survivin-dependent functions as compared to normal tissues, a concept that has been proposed for other signaling networks contributing to tumor cell maintenance. The diversified class of survivin antagonists being tested in the clinic provides a new paradigm for “targeted” cancer therapy, as these agents specifically affect a cancer signaling pathway, but are also broad enough to be applicable to disparate tumor types, regardless of their extraordinary functional and genetic heterogeneity. ACKNOWLEDGMENTS This work was supported by NIH grants CA78810, CA90917, and HL54131. REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18.

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TRAIL Modulators C. H. Mom, I. A. Sloots, S. de Jong, J. A. Gietema, and E. G. E. de Vries Department of Medical Oncology, University Medical Center Groningen, Groningen, The Netherlands

S. Sleijfer Department of Medical Oncology, Erasmus University Medical Center, Rotterdam, The Netherlands

INTRODUCTION Specific induction of apoptosis is an interesting therapeutic strategy for destroying tumor cells. Tumor necrosis factor (TNF)-related, apoptosis-inducing ligand (TRAIL or Apo2L) is an attractive candidate in this respect, as it induces apoptosis in a wide range of tumor cells and xenografts, without causing toxicity to normal cells (1). Other recombinant members of the TNF family—that is, Fasligand (FasL) and TNF—also induce apoptosis of cancer cells in preclinical models, but have failed to attain broad application in cancer treatment due to their side effects. Administration of FasL in preclinical studies induced severe liver toxicity, hampering its introduction in the clinic (2). TNF is only administered by isolated limb perfusion to achieve limb salvage in soft tissue sarcoma or melanoma, because systemic use of this agent, if not employed at very low doses, induces a sepsis-like syndrome (3,4). Recombinant human (rh) TRAIL and agonistic monoclonal antibodies that target the TRAIL pathway appear to have a more favorable safety profile and are currently in early clinical development. In this chapter we describe the TRAIL pathway, the factors involved in modulating this route, the physiologic role of TRAIL, and the therapeutic possibilities of targeting this pathway in oncology. TRAIL SIGNALING PATHWAY TRAIL is a member of the TNF-superfamily and was discovered in 1995 by the human genome project (1). TRAIL can, like the other TNF-members, immediately induce apoptotic cell death in the target cell after binding to one of its death receptors. Apoptosis can be induced in a cell via two distinct pathways: the intrinsic and the extrinsic pathways. TRAIL activates the extrinsic pathway at the cellular membrane, where it can bind to five different receptors. Death receptor 4 (DR4) and 5 (DR5) are transmembrane receptors that act as signal transducers containing intracellular death domains. After binding of the ligand, the receptor has the ability to recruit Fas-associated death domain (FADD) to its death domain (Fig. 1). Also, procaspases-8 or -10 are recruited, which, together with FADD, form the death-inducing signaling complex (DISC). The interaction between these molecules can be negatively regulated by cellular FLICE-like inhibitory protein (cFLIP), that competes with procaspase-8, thereby blocking the signal. After DISC 207

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FIGURE 1 TRAIL pathway. After binding of TRAIL or an agonistic antibody to the death receptors (DR4 or DR5), the extrinsic pathway of apoptosis is activated. The Fas-associated death domain (FADD) assembles the death-inducing signaling complex (DISC), leading to cleavage of procaspase-8. Activated caspase-8 subsequently cleaves the downstream effector caspases-3, -6, and -7, leading to apoptosis. DNA damage caused by chemo- or radiotherapy triggers the intrinsic pathway. Upon activation, proapoptotic members of the Bcl-2 family, BAK and BAX, translocate to the mitochondria, causing subsequent release of cytochrome c and other mitochondrial factors into the cytosol. Cytochrome c binds to the adaptor protein Apaf-1 and procaspase-9 in the presence of dATP. This complex, the apoptosome, cleaves caspase-9, followed by activation of the effector caspases. Crosstalk between the intrinsic and extrinsic pathway exists through Bid. After cleavage, this protein translocates to the nucleus to induce cytochrome c release. The activation of this pathway can be inhibited on various levels. Cellular FLICE-like inhibitory protein (cFLIP) is a regulatory protein, which can block the formation of the DISC by competing with procaspase-8. Bcl-2 and Bcl-XL prevent the release of cytochrome c from the mitochondria, thereby inhibiting the signal. Furthermore, inhibitor of apoptosis proteins (IAPs) can attentuate the signaling cascade at the level of the active caspases-9 and -3.

formation, active caspase-8 is released into the cytosol, leading to cleavage of executioner caspases-3, -6, and -7, and ultimately cell death (1,5). The other three receptors that TRAIL can attach to are thought to be decoy receptors. Two of them, decoy receptor 1 and 2 (DcR1 and DcR2), lack a functional intracellular death domain and are therefore unable to transmit an apoptotic signal. The third TRAIL binding decoy receptor is osteoprotegerin (OPG), a soluble receptor molecule (6). The other pathway leading to apoptosis is the intrinsic pathway. Standard anticancer treatments, such as radiation and chemotherapy, induce apoptosis through DNA damage, which subsequently triggers the intrinsic pathway.

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Activation of this pathway is dependent on p53 and results in translocation of BAX and BAK, proapoptotic members of the Bcl-2 family, to the mitochondria. Upon mitochondrial activation, cytochrome c and second mitochondria-derived activator of copase/direct inhibitor of apoptosis-binding protein (IAP) with low pI (SMAC/DIABLO) are released into the cytosol. Cytochrome c binds to APAF-1 together with procaspase-9, leading to the formation of an apoptosome, which then activates caspase-9. This caspase activates the effector caspases-3, -6, and -7, resulting in apoptosis. Inhibition of intracellular signaling via the intrinsic pathway occurs at various levels. Bcl-2 and Bcl-XL are antiapoptotic members of the Bcl-2 family and prevent the release of cytochrome c from the mitochondria (7). The IAPs block caspase activation further downstream. SMAC/DIABLO promotes apoptosis by binding to these IAPs, thus enhancing caspase activation (1). In addition to triggering the extrinsic pathway, TRAIL can induce apoptosis via the mitochondria, as crosstalk exists between the two pathways. Apoptosis can be amplified through cleavage of the Bcl-2 superfamily member BID by caspase-8 or -10. Upon activation, it interacts with BAX and BAK, thereby leading to mitochondrial cytochrome c release (5). The interaction of BID with BAX and BAK occurs downstream of p53. Therefore, TRAIL-mediated activation of the intrinsic pathway is, in contrast to activation through DNA damaging agents, independent of p53. The frequent occurrence of p53 mutations in human tumors provided an additional reason for exploring activators of the TRAIL pathway. PHYSIOLOGICAL ROLE OF TRAIL TRAIL is a naturally occurring ligand in the human body. Its biological function is not completely elucidated yet, but TRAIL seems to play a role in immune surveillance against oncogenically transformed and virally infected cells, as well as in autoimmunity (1,6). The involvement of TRAIL in surveillance against tumors is underscored by the observation that blocking of TRAIL with neutralizing antibodies increases the incidence and metastasis of mutagen-induced tumors in mice (8–11). TRAIL is expressed on the surface of immune cells, including natural killer (NK) cells, monocytes, macrophages T-cells, and dendritic cells (1,5,12). Activated NK cells and cytotoxic T-lymphocytes (CTL) use several mechanisms to destroy tumor cells. One of these mechanisms is to produce death ligands, such as TRAIL or FasL, leading to programmed cell death of target cells. Macrophages can become activated by tumor cells to produce TRAIL. Additionally, these macrophages can release substances inducing an increased DR4 and DR5 expression in tumor cells (7,13). In vitro studies have shown that TRAIL is implicated in immunosurveillance of virus-infected cells. In response to interferon (IFN)-g, infected cells that are initially resistant to TRAIL can become sensitive to apoptotic cell death. Sedger et al. showed that fibroblasts infected with human cytomegalovirus and treated with IFN upregulate both TRAIL and its DRs. IFN potentiates the apoptotic response in infected cells by upregulating TRAIL expression in uninfected neighboring cells and simultaneously downregulating their death receptors (6,12,14). Besides apoptosis-induction, TRAIL also has various antiapoptotic functions in normal cells, such as stimulating lymphocyte proliferation. The stimulating effect on proliferation has also been observed in certain tumor cell lines (15). In cell lines resistant to TRAIL-induced apoptosis, death receptor stimulation can activate an alternate,

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antiapoptotic pathway. This pathway can be triggered after interaction of FADD and receptor-interacting protein (RIP) and causes transcription factor NFkB to be translocated to the nucleus (16). Activation of NFkB by TRAIL has been shown to antagonize apoptosis-induction and inhibition of NFkB activation enables apoptosisinduction in otherwise TRAIL resistant cells (17,18). TRAIL-mediated tumor cell survival and proliferation may be caused by three different mechanisms: prevention of apoptotic death of otherwise dying cells, induction of proliferation in otherwise resting cells thereby being mitogenic, or by shortening the cycling time in proliferating cells (18). In cancer therapy, this proliferative effect might represent a potential risk for patients with tumors resistant to TRAIL-induced apoptosis. ROLE OF TRAIL RECEPTORS As previously mentioned, TRAIL can bind to several receptors. DR4 and DR5 are responsible for TRAIL-induced apoptosis, while DcR1 and DcR2 act as decoy receptors. In addition to being an apoptotic signal transducer, a study by Wang et al. (19) suggests that DR5 has other functions as well. The authors generated stable human colon cancer cell lines in which the function of DR5 was ablated using inducible RNA interference, and injected these cells in mice to create tumor xenografts. Loss of function of DR5 promoted colon tumor xenograft growth and resulted in a decreased sensitivity to chemotherapy-induced apoptosis. Therefore, DR5 may be an important determinant for tumorigenesis and chemosensitivity (19). Osteoprotegerin (OPG), the soluble TRAIL receptor, is a key regulator of bone metabolism by inhibiting osteoclast differentiation and function in vitro and in vivo. It can regulate bone resorption by osteoclasts through binding with RANKL, thereby blocking its interaction with RANK. Interaction between the TNF-family members RANKL and RANK stimulates differentiation, activation, and survival of RANK-expressing osteoclasts (20). In theory, TRAIL can increase osteoclast formation in vitro by binding to OPG and preventing its interaction with RANKL. Additionally, OPG plays a role in immunity; it is, for example, reported to be essential for normal B-cell development and function. Furthermore, several reports have described the production of OPG by different tumor cell types, including breast cancer, prostate cancer, and osteosarcoma (21–23). No OPG was produced in normal tissue or benign tumors. This suggests that OPG may act as a survival factor in tumors. This survival effect may be the result of OPG binding to TRAIL and thus protecting tumor cells from TRAIL- induced apoptosis. However, this hypothesis could be disputed because OPG has a low affinity for TRAIL at physiological temperatures (21). An alternate explanation may lie in the ability of OPG to promote survival of endothelial cells (24). Tumor cells and endothelial cells are able to produce OPG and have been shown to express high levels of this receptor in malignant tumors. OPG may support tumor growth by aiding in the development and maintenance of blood vessels in the tumor. The tumor-promoting effect of OPG has recently become highly relevant, as recombinant OPG and denosumab, a fully human monoclonal antibody to RANKL, have been administered clinically. Two patients with juvenile Paget's disease, a rare genetic bone disease caused by a deficiency in OPG and characterized by a highly increased bone turnover, were treated with rhOPG (25). This resulted in a marked reduction of bone resorption. However, the development of rhOPG is stopped. Currently, denosumab, which mimics the function of OPG, is being evaluated. Denosumab has been tested for the treatment of postmenopausal women

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with low bone mineral density (26,27). A total of 412 patients were randomized to receive denosumab in various doses, oral alendronate or placebo, for 12 months. Treatment with denosumab resulted in increased bone mineral density and a decrease in bone turnover. However, of the 314 patients in the denosumab group, five developed a malignant tumor during the one year treatment period as opposed to none in the other two groups (28). This was not a statistically significant difference, which could be due to the relatively small number of patients that received no denosumab (n ¼ 92). Thus, although promising for the future treatment of patients with osteoporosis or other conditions associated with bone loss, caution should be exerted with respect to the possible tumor-promoting effects of these forms of therapy. THE TRAIL PATHWAY IN PREMALIGNANT LESIONS In an immunohistochemical study investigating DR-expression in normal, premalignant and malignant colon tissue, the expression levels varied between these stages of carcinogenesis. DR4 and DR5 showed stronger staining in malignant than in normal cells (29). Furthermore, in vitro rhTRAIL-sensitivity increases during the conversion of an adenoma cell line to a malignant phenotype (30). These data suggest a potential role of rhTRAIL in the premalignant setting. Recently, Jalving et al. (31) have shown that rhTRAIL can induce apoptosis in adenoma cell lines and in short term explant cultures of adenomas with high grade dysplasia. These data could imply a new approach to the treatment of premalignant lesions, as normal colonocytes are relatively resistant to rhTRAIL-induced apoptosis. A potential application could be the downsizing of endoscopically irresectable adenomas with high-grade dysplasia which may allow endoscopic removal, or prevent resection of parts of the colon in the case of hereditary colon cancer syndromes (31). TRAIL and DR expression have also been investigated in cervical carcinogenesis. DR4 and DR5 were upregulated during the cervical cancer sequence, while TRAIL seemed to be downregulated, suggesting a possible functional role of these ligands and receptors during cervical carcinogenesis. The high expression of DR4 and DR5 renders these receptors as potential targets for therapy (32). THERAPEUTIC IMPLICATIONS Targeting of the TRAIL Signaling Pathway: rhTRAIL and Agonistic Antibodies RhTRAIL has antitumor activity against a wide variety of tumor cell lines, including cell lines derived from colon, lung and breast cancer (33). Substantial cytotoxic effects have also been demonstrated in mouse xenograft models with rhTRAIL as single agent, without causing harm to normal cells (33,34). Conventional anticancer chemotherapeutics stimulate the intrinsic apoptosis pathway via activation of p53, but many cancer cells have mutations in p53 causing resistance to chemotherapyinduced cell death. This resistance may be overcome by concomitant activation of the extrinsic pathway by rhTRAIL or agonistic antibodies. In cell lines of several human tumor types, combinations of rhTRAIL and chemo- or radiotherapy show synergistic antitumor effects (1). The safety of rhTRAIL in vivo has been evaluated not only in mice, but also in nonhuman primates, because of the homology in sequence identity of TRAIL and the TRAIL receptors in primates. An 84–99% extra-cellular protein sequence

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identity is shared between humans and cynomolgus monkeys and a 97–99% extracellular sequence identity exists between humans and chimpanzees. The in vivo studies showed a favorable safety profile, since no toxicity was observed in cynomolgus monkeys and chimpanzees after rhTRAIL administration. As FasL and earlier recombinant variants of TRAIL caused severe toxicity in hepatocytes, which precluded their use in the clinical setting, possible hepatotoxic effects were closely monitored (35). The early versions of rhTRAIL contained an exogenous sequence tag, for example polyhistidine or a leucine zipper (LZ), and were not optimized for zinc content. Because the zinc atom is crucial for stability and biologic activity, the non-zinc optimized rhTRAIL tended to form aggregates. This may have led to induction of apoptosis in normal cells, including hepatocytes, due to high-order multimerization of death receptors. The nontagged, zinc-optimized version of TRAIL is currently used. This rhTRAIL caused no liver toxicity in cynomolgus monkeys and chimpanzees. Furthermore, in the preclinical studies no antibodies against rhTRAIL have been detected (33–35). Pharmacokinetic evaluation showed a serum half-life of 21–31 minutes in nonhuman primates. RhTRAIL clearance is highly correlated to glomerular filtration rate in various species, suggesting that rhTRAIL is primarily eliminated by the kidneys (34). A phase I study with rhTRAIL (Genentech, San Fransisco, California, U.S.A.) is ongoing (Table 1) (36). So far, 58 patients with advanced or metastatic solid malignancies or non-Hodgkin's lymphoma have been treated with rhTRAIL once daily for five consecutive days, every three weeks. Patients received doses varying from 0.5 to 15 mg/kg. Due to concerns about liver toxicity, two staggered cohorts were formed: patients without liver metastases were treated before patients with liver metastases. In both cohorts, no drug-related, dose-limiting toxicities were observed, and rhTRAIL appeared to be well tolerated. One partial response was seen at 8 mg/kg in a patient with chondrosarcoma. Pharmacokinetic analyses revealed a serum half-life of approximately 36 minutes (37). No anti-rhTRAIL antibodies were detected. Enrolment into this study is continuing at 20 and 30 mg/kg, doses that are much higher than estimated to be effective in humans based on pharmacokinetic animal studies and mouse xenograft models (34). So far, no immunological adverse events have been reported following administration of rhTRAIL. However, since the physiological role of TRAIL in immune responses is not yet fully understood, close monitoring of immunological side effects in patients receiving rhTRAIL remains of importance.

TABLE 1 Agents Targeting the TRAIL Death Receptors Agent rhTRAIL HGS-ETR1 HGS-ETR2 HGS-TR2J LBY135 CS-1008 AMG 655 rhTRAIL variants

Target

(Pre)clinical development

DR4 and DR5 DR4 DR5 DR5 DR5 DR5 DR5 DR4 or DR5

Phase I Phase IIa Phase I Phase I Phase I Phase I Phase I Preclinical

a Also combinatory studies with gemcitabine/cisplatin and carboplatin/paclitaxel (phase Ib studies) and a phase II study with bortezomib.

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Activation of the apoptotic pathway through DR4 or DR5 can also be achieved by using agonistic antibodies. These antibodies bind selectively and with high affinity to their cognate receptors. In addition to the receptor specificity, another potential advantage of the use of antibodies is their longer half-life compared to rhTRAIL. Currently, six agonistic monoclonal antibodies are in clinical development. HGS-ETR1 (mapatumumab; Human Genome Sciences, Rockville, Maryland, U.S.A.) is a fully human agonistic antibody to DR4, whereas HGS-ETR2 (lexatumumab; Human Genome Sciences), HGS-TR2J (Human Genome Sciences), LBY135 (Novartis, Basel, Switzerland), CS-1008 (Sankyo, Tokyo, Japan), and AMG 655 (Amgen, Thousand Oaks, California, U.S.A.) are agonistic antibodies to DR5. HGS-TR2J appears to be a more potent DR5 antibody than HGS-ETR2, in view of its greater efficacy in inducing tumor regression in xenograft models (38). Whereas HGS-ETR2 needs cross-linking reagents for exertion of its apoptotic activity, HGS-TR2J induces apoptosis independently of cross-linking reagents, and is therefore capable of directly activating the apoptotic pathway. What the clinical implications of these differences are, especially with regard to efficacy and toxicity, is still unknown. The DR4 antibody HGS-ETR1 inhibits growth and induces apoptosis in a broad range of human tumor cell lines and in well established xenograft models in mice. Two phase I studies with HGS-ETR1 have been conducted in patients with advanced solid malignancies and non-Hodgkin's lymphoma (39,40). Patients received doses of HGS-ETR1 varying from 0.01 to 20 mg/kg every 28 days. The maximum tolerated dose has not been reached and preliminary data show that HGS-ETR1 was well tolerated. Stable disease was the best response observed in these groups of heavily pretreated patients. The plasma mean terminal elimination half-life was approximately 17 days. HGS-ETR1 was also well tolerated in three phase II studies. In these studies patients with colorectal cancer, non–small cell lung cancer and non-Hodgkin's lymphoma have been treated with single agent HGS-ETR1. Forty patients with relapsed or refractory non-Hodgkin's lymphoma were enrolled in one of two treatment groups, receiving either 3 mg/kg every 21 days (n ¼ 8) or 10 mg/kg every 21 days (n ¼ 32) (41). In three patients with follicular lymphoma (8%) tumor responses were observed (one complete and two partial responses). Stable disease was seen in 12 patients (30%). Another phase II study was conducted in 32 heavily pre-treated patients with relapsed or refractory non–small cell lung cancer, who received 10 mg/kg of HGS-ETR1 every 21 days (42). Stable disease was observed in nine patients (29%) with a median duration of 2.3 months. The third phase II study involved 38 patients with relapsed or refractory colorectal cancer (43). HGS-ETR1 was administered at a dose of 20 mg/kg every 14 days during cycles 1 and 2, and at 10 mg/kg every 14 days in cycles 3–6. Stable disease occurred in 12 patients (32%). When HGS-ETR1 is given in combination with chemotherapy, increased cytotoxic effects are seen in human tumor cells and mouse xenograft models. In two phase Ib studies combinations of HGS-ETR1 with cisplatin and gemcitabine, and with carboplatin and paclitaxel were well tolerated (44,45). The maximum tolerated dose has not been reached. No signs of pharmacokinetic interaction between the drugs were seen. The combination study with gemcitabine and cisplatin is still ongoing. The fully human DR5 antibody HGS-ETR2 shows growth inhibitory effects as single agent in various human tumor xenografts derived from glioma, non– small cell lung cancer, colorectal cancer and breast cancer. Two phase I studies have been conducted with HGS-ETR2 in patients with advanced solid tumors. In the first study, HGS-ETR2 was well tolerated at dose levels of 0.1–10.0 mg/kg

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every 14 days (46). Stable disease was observed in 10 out of 31 patients (32.3%). In the second study, 37 patients received HGS-ETR2 in doses up to 20 mg/kg every 21 days (47). Dose-limiting toxicity was observed in four patients in the 20 mg/kg cohort, consisting of liver enzyme abnormalities (n ¼ 2), elevated serum amylase levels (n ¼ 2), and acute renal failure (n ¼ 1). Therefore, 10 mg/kg was identified as the maximum tolerated dose. The best response was stable disease in 11 patients (29.7%). Preclinical studies with HGS-TR2J shows regression or growth inhibition in human cancer cell lines and xenograft models, both as single agent and in combination with several chemotherapeutics. There are no data of phase I studies available yet. The chimeric DR5 antibody LBY135 is currently evaluated in a phase I clinical trial. Also, a phase I study involving another, humanized antibody against DR5 (CS-1008) has been initiated (48). The fully human monoclonal DR5 antibody AMG 655 is in phase I clinical development as well (49). Potential of Combination Therapies In preclinical models combinations of rhTRAIL and agonistic antibodies with conventional chemotherapy or radiotherapy result in an enhancement of cytotoxicity. In addition, many new compounds targeting molecular pathways that are important in carcinogenesis are in development and potentially interesting for combined treatment. Part of these new agents are directed at proteins of the TRAIL signaling pathway, while others affect prosurvival routes or interfere with basic cellular processes. Sensitivity for rhTRAIL can be enhanced by targeting antiapoptotic proteins that are involved in the TRAIL pathway, such as Bcl-2, Bcl-XL, and the IAPs. Antisense therapy and small molecule inhibitors of these proteins are at different stages of development (50–52). Oblimersen, an antisense phosphorothioate oligonucleotide that decreases expression of Bcl-2, is currently evaluated in various clinical studies. An antisense oligonucleotide targeting XIAP, the predominant inhibitor of caspases-3, -7, and -9, is under investigation in a phase I clinical trial. Small molecule inhibitors directed at Bcl-2, Bcl-XL, and XIAP are advancing towards the clinic as well. Survivin is a member of the IAP family and, apart from its function as caspase inhibitor, has a role in cell cycle control. Various strategies that target this protein have been developed. Mimics of SMAC bind to IAPs and counteract the IAP induced inhibition of caspase activation (53,54). These SMAC agonists are being evaluated in preclinical studies. Also belonging to the group of promising anticancer therapeutics are the histone deacetylase (HDAC) inhibitors. They induce differentiation, growth arrest and/or apoptosis in tumor cells by activating transcription of target genes. Apoptosis-induction by HDAC inhibitors results from activation of the death receptor pathway, and occurs independently of p53 (55). The combination of HDAC inhibitors and rhTRAIL causes a synergistic apoptotic effect in cancer cell lines (56). HDAC inhibitors enhance the apoptosis-inducing potential of rhTRAIL in tumor cells by upregulation of DR4, DR5, and proapoptotic proteins of the TRAIL pathway, e.g., pro-caspase-8, BAX and BAK, and downregulation of antiapoptotic Bcl-2 family members and IAPs (55,56). Phase I and II studies with HDAC inhibitors are ongoing. Other antitumor drugs that enhance apoptosis in tumor cells when combined with rhTRAIL are proteasome inhibitors (57). The proteasome is responsible

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for the intracellular degradation of proteins. Interference with protein degradation by proteasome inhibitors affects many processes in the cell, including those involved in cell cycle progression and mitosis, resulting in reduced tumor growth and apoptosis. Tumor cells are more sensitive to the damaging effects of proteasome inhibition than normal cells. Proteasome inhibitors, such as bortezomib, have the ability to inhibit the activation of NFkB, decrease levels of the antiapoptotic protein cFLIP, and induce cell surface expression of DR4 and DR5. This may account for the sensitization of tumor cells to rhTRAIL (57). Bortezomib is registered for the treatment of refractory multiple myeloma. Recently, a randomized phase II study has been initiated to compare bortezomib in combination with HGS-ETR1 to bortezomib alone for the treatment of patients with relapsed or refractory multiple myeloma. Heat shock proteins (HSP) are cellular chaperone proteins that are frequently overexpressed in malignant cells. They are essential for the proper folding, assembly and intracellular transportation of proteins, and for the proteolytic turnover of many of the key regulators of cell growth and survival. Of the heat shock proteins, HSP90 is the most abundant and protects cancer cells from apoptosis through among others stabilisation of Akt and NFkB. Inhibition of HSP90 results in cell cycle arrest and apoptosis. Preclinical data show a synergistic apoptotic response when HSP90 inhibitors are combined with rhTRAIL or the antibodies HGS-ETR1 and -ETR2 (58–60). The HSP90 inhibitor 17-allylamino, 17-demethoxygeldanamycin (17-AAG) has been tested in phase I clinical trials (61,62). No responses were seen, but some patients had a prolonged period of stable disease. Phase II studies exploring 17-AAG and other HSP90 inhibitors are in progress. The synthetic triterpenoids 2-cyano-3,12-dioxoolean-1,9-dien-28-oic acid (CDDO) and its analogues are synthesized from the naturally occurring triterpene oleanic acid. These compounds inhibit tumor growth and induce apoptosis in tumor cells lines. They have also been shown to sensitize cancer cells to rhTRAIL-induced apoptosis. Furthermore, the combination of rhTRAIL and a CDDO derivative increased suppression of tumor growth in a mouse xenograft model (63,64). Sensitization to rhTRAIL was associated with downregulation of cFLIP and upregulation of DR4 and DR5. CDDO is tested as single agent in phase I studies (65). Peroxisome proliferator-activated receptor g (PPARg) agonists, such as troglitazone, are used as oral antidiabetics, but are also known for their antineoplastic effects in preclinical models (66). Troglitazone acts as a selective ligand for PPARg, a ligand dependent transcription factor that binds to the DNA to activate target genes. PPARg agonists sensitize tumor cells to apoptosis-induction by rhTRAIL by downregulation of cFLIP and survivin, and by upregulation of DR5 (67). Clinical application of this combination may however be hampered by the possible occurrence of liver toxicity, a rare side effect of troglitazone. It is not clear whether other PPARy agonists like pioglitazone induce the same toxicity. The combination of rhTRAIL with therapeutics targeting pro-survival routes can also be interesting. The PI3K/Akt signaling pathway is commonly activated in cancer cells, and is involved in the regulation of various processes, including proliferation, growth and survival. Inhibition of this route through RNA interference directed at PI3K and Akt results in increased sensitivity to rhTRAILinduced apoptosis in resistant colon cancer cells (68). Furthermore, enhanced apoptosis was observed in glioma cell lines with the combination of an Akt inhibitor and rhTRAIL (69). Inhibitors of PI3K and Akt are in late preclinical and early clinical development respectively. Mammalian target of rapamycin (mTOR)

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is a kinase that acts downstream of Akt, and has a key regulatory role in the pathway. Inhibition of mTOR by rapamycin or its analogues results in cell cycle arrest in dividing cells by preventing progression from G1 to S phase. Responses have been observed with a rapamycin analogue in several solid tumors in phase I and II studies (70). In the preclinical setting, enhanced sensitivity to rhTRAILinduced apoptosis has been observed with combined treatment of rhTRAIL and rapamycin (71). Pro-survival pathways are also activated by epidermal growth factor receptor (EGFR) and human epidermal growth factor receptor 2 (HER2), members of the epidermal growth factor receptor family. Upon activation these receptors play a role in proliferation, angiogenesis, invasion and survival of cancer cells. Trastuzumab, an antagonistic antibody directed towards HER2, is used for the treatment of HER2 overexpressing breast cancer. In HER2 overexpressing cancer cell lines treatment with the combination of trastuzumab and rhTRAIL enhances apoptosis (72). Trastuzumab treatment results in a downregulation of HER2, a reduction in Akt activity, and increased sensitivity for rhTRAIL. Enhancement of rhTRAILinduced apoptosis is also observed with EGFR tyrosine kinase inhibitors (73,74). FUTURE PERSPECTIVES Recent evidence suggests that apoptotic signaling may be preferably mediated by either DR4 or DR5 depending on tumor type. In chronic lymphocytic leukemia, the TRAIL pathway is only activated through DR4, whereas in some other tumor types that express both death receptors, DR5 signaling is most important (75,76). Thus, the apoptosis-inducing potential of rhTRAIL and the agonistic monoclonal antibodies against the TRAIL receptors may vary between different tumor types. Analogous to the agonistic antibodies, mutants of rhTRAIL have been developed that exclusively target either DR4 or DR5 in the preclinical setting (75–77). Which approach eventually will be preferred in the clinic for targeting the TRAIL receptors is yet unclear and depends on a variety of factors. RhTRAIL binds to all TRAIL receptors. As a consequence, rhTRAIL has the possibility to signal through both death receptors, which may enhance its therapeutic potential. However, the broad binding capacity of rhTRAIL could also lead to reduced apoptosis-induction, because rhTRAIL may attach to the functionally inactive decoy receptors. In addition, as the precise role of TRAIL in the human body remains unclear, administration of rhTRAIL may have unforeseen immunological effects, even though these have not been reported so far. The selectivity of the agonistic DR4/ DR5 antibodies could therefore be a therapeutic advantage. Another potential advantage of the agonistic antibodies is their longer half-life, although prolonged exposure to these antibodies could theoretically also lead to downregulation of the death receptors on the cell surface. RhTRAIL, as a result of its short existence in the human body following administration, may not exert this effect on death receptors. To enhance tumor-selective binding of rhTRAIL, fusion proteins have been developed that consist of rhTRAIL linked to a tumor-specific antibody fragment (78,79). This antibody fragment binds to a target antigen on the cell surface of a tumor cell, enabling apoptosis-induction by rhTRAIL in an autocrine or paracrine manner. In the same manner rhTRAIL has been linked to an EGFR-blocking antibody fragment. Exposure to this fusion protein results in potent apoptosisinduction in EGFR-positive tumor cell lines (80).

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As mentioned earlier, there are data supporting a proliferative role for rhTRAIL in tumor cells that are resistant to TRAIL-induced apoptosis (15,18). This might pose a risk for the use of rhTRAIL or monoclonal antibodies in patients harbouring tumor cells resistant to apoptosis-induction by TRAIL. Further research has to reveal what the implications for the clinic are and whether certain patients should be excluded from treatment with agents that activate DR4 and/or DR5. Several clinical studies with rhTRAIL and agonistic antibodies to the TRAIL receptors are currently ongoing. Although there are hints suggesting antitumor activity of TRAIL pathway activators as single agent in patients with specific cancer types, the evidence so far is not robust enough to foresee a future for these compounds as therapeutics in a single agent setting. It seems that more is needed for tumor cells to die. Therefore, adding other anticancer drugs will probably be necessary, not only to adequately switch on the apoptotic machinery, but also to avoid drug resistance. Combination therapy can aim at simultaneously activating both the extrinsic and intrinsic apoptosis pathways, for example by the addition of chemotherapy to rhTRAIL or agonistic DR4/DR5 antibodies, but can also be directed at removing the intracellular barriers to apoptosis. To elucidate the optimal treatment strategy, translation of knowledge about basic processes involved in apoptosis into rational therapeutic approaches is essential. In order to survive in a hostile environment with limited supply of oxygen and many other kinds of cell stress, tumor cells are highly dependent on prosurvival and antiapoptotic signals. Upregulation of various antiapoptotic proteins is the tumor cell's defence mechanism against death, which may be attacked by therapeutic intervention. Several strategies have been developed that reduce the levels of antiapoptotic proteins, resulting in sensitization of tumor cells to rhTRAIL-induced apoptosis. By using several agents or agents that target antiapoptotic proteins at multiple levels in the TRAIL pathway, elimination of obstacles on the route to cell death may even be enhanced. Proteasome- and HDAC-inhibitors are examples of agents that interfere with the TRAIL signaling pathway at various levels. Besides upregulating TRAIL receptors at the cell surface, they inhibit antiapoptotic proteins downstream. One can image that, if at multiple checkpoints along the pathway proapoptotic signals prevail, the balance may more easily tip towards apoptosis when the route is activated. In other words, if rhTRAIL, or a DR4/DR5 antibody, triggers the TRAIL route, and there are few roadblocks on the way preventing intracellular signaling, apoptosis of the tumor cell may more easily be achieved. But whether treatment combinations with agents like HDAC inhibitors will actually have a greater synergistic effect than combinations with agents directed at one specific antiapoptotic protein, remains to be established. Trials evaluating combination regimens will reveal what strategy or strategies will ultimately be most successful in killing cancer cells while limiting damage to normal cells. In the future, determination of tumor characteristics may also aid in clinical decision making, resulting in true patient tailored therapy with agents targeting the TRAIL pathway. REFERENCES 1. 2.

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Schultze K, Bock B, Eckert A, et al. Troglitazone sensitizes tumor cells to TRAILinduced apoptosis via down-regulation of FLIP and Survivin. Apoptosis 2006; 11:1503–12. Rychahou PG, Murillo CA, Evers BM. Targeted RNA interference of PI3K pathway components sensitizes colon cancer cells to TNF-related apoptosis-inducing ligand (TRAIL). Surgery 2005; 138:391–7. Puduvalli VK, Sampath D, Bruner JM, et al. TRAIL-induced apoptosis in gliomas is enhanced by Akt-inhibition and is independent of JNK activation. Apoptosis 2005; 10:233–43. Janus A, Robak T, Smolewski P. The mammalian target of the rapamycin (mTOR) kinase pathway: its role in tumourigenesis and targeted antitumour therapy. Cell Mol Biol Lett 2005; 10:479–98. Panner A, James CD, Berger MS, et al. mTOR controls FLIPs translation and TRAIL sensitivity in glioblastoma multiforme cells. Mol Cell Biol 2005; 25:8809–23. Cuello M, Ettenberg SA, Clark AS, et al. Down-regulation of the erbB-2 receptor by trastuzumab (Herceptin) enhances tumor necrosis factor-related apoptosis-inducing ligand-mediated apoptosis in breast and ovarian cancer cell lines that overexpress erbB-2. Cancer Res 2001; 61:4892–900. Park SY and Seol DW. Regulation of Akt by EGF-R inhibitors, a possible mechanism of EGF-R inhibitor-enhanced TRAIL-induced apoptosis. Biochem Biophys Res Commun 2002; 295:515–18. Teraishi F, Kagawa S, Watanabe T, et al. ZD1839 (Gefitinib, `Iressa'), an epidermal growth factor receptor-tyrosine kinase inhibitor, enhances the anti-cancer effects of TRAIL in human esophageal squamous cell carcinoma. FEBS Lett 2005; 579:4069–75. Kelley RF, Totpal K, Lindstrom SH, et al. Receptor-selective mutants of apoptosisinducing ligand 2/tumor necrosis factor-related apoptosis-inducing ligand reveal a greater contribution of death receptor (DR) 5 than DR4 to apoptosis signaling. J Biol Chem 2005; 280:2205–12. MacFarlane M. TRAIL-induced signalling and apoptosis. Toxicol Lett 2003; 139:89–97. Van der Sloot AM, Tur V, Szegezdi E, et al. Designed tumor necrosis factor-related apoptosis-inducing ligand variants initiating apoptosis exclusively via the DR5 receptor. Proc Natl Acad Sci USA 2006; 103:8634–9. Bremer E, Kuijlen J, Samplonius D, et al. Target cell-restricted and -enhanced apoptosis induction by a scFv:sTRAIL fusion protein with specificity for the pancarcinomaassociated antigen EGP2. Int J Cancer 2004; 109:281–90. Bremer E, Samplonius DF, Peipp M, et al. Target cell-restricted apoptosis induction of acute leukemic T cells by a recombinant tumor necrosis factor-related apoptosisinducing ligand fusion protein with specificity for human CD7. Cancer Res 2005; 65:3380–8. Bremer E, Samplonius DF, van Genne L, et al. Simultaneous inhibition of epidermal growth factor receptor (EGFR) signaling and enhanced activation of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) receptor-mediated apoptosis induction by an scFv:sTRAIL fusion protein with specificity for human EGFR. J Biol Chem 2005; 280:10025–33.

14

VEGF Targeting Lee M. Ellis Departments of Surgical Oncology and Cancer Biology, M. D. Anderson Cancer Center, University of Texas, Houston, Texas, U.S.A.

INTRODUCTION The process of angiogenesis, the development of a neovascular blood supply, is critical for tumor growth and metastasis formation. Numerous proangiogenic and antiangiogenic molecules orchestrate the process of vessel formation. Vascular endothelial growth factor (VEGF) has emerged as a central player in these pathways, and thus serves as a primary target for neoplastic therapy and, in fact, any disease where angiogenesis is involved in the pathophysiology of that disease (see below) (1–3). This chapter focuses on VEGF biology, the role of targeting the VEGF ligand as a component of anticancer therapy, and potential mechanisms of action of anti-VEGF therapy. THE VEGF LIGAND FAMILY The VEGF family comprises six secreted glycoproteins, VEGF-A, VEGF-B, VEGFC, VEGF-D, VEGF-E, and placenta growth factor (PlGF) (Fig. 1). The best characterized of the VEGF family members, VEGF-A [commonly referred to as VEGF, also known as vascular permeability factor (VPF)], has been demonstrated to be temporally and spatially associated with key events in angiogenesis. VEGF gene knockout (heterozygous and homozygous) models are lethal due to cardiovascular abnormalities (4). VEGF-A is important to a number of nonmalignant processes as well, including wound healing, ovulation, menstruation, maintenance of blood pressure, and pregnancy, and has been linked to several pathological conditions associated with increased angiogenesis, including arthritis, psoriasis, macular degeneration, and diabetic retinopathy (5). The VEGF-A gene undergoes alternative splicing to yield mature proteins of 121, 145, 165, 183, 189, and 206 amino acids (6). VEGF165 is the predominant isoform and is commonly overexpressed in a variety of human solid tumors. Recent studies suggest that expression patterns of certain VEGF isoforms are tissue-specific, implying that these isoforms have specific roles in development, and probably in tumor angiogenesis as well (6). THE VEGF RECEPTORS VEGF mediates its angiogenic effects via several different receptors. VEGFR1 (Flt-1) and VEGFR2 (KDR and the murine homologue Flk-1) are as tyrosine kinase receptors (TKRs) and were originally discovered on endothelial cells (ECs) (7). Following discovery of VEGFR-1/-2, an additional TKR, VEGFR3; this receptor is primarily associated with lymphangiogenesis (8). The various members of the

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PlGF VEGF-B

VEGF-A

VEGF-C,D

Isoforms 121 165 189 205

Cell membrane

Functions

VEGF-R1 (Flt-1) Migration Invasion

VEGF-R2 VEGF-R3 (Flt-4) (KDR/Flk-1) Proliferation Lymphangiogenesis Survival Permeability

FIGURE 1 VEGF family of ligands and receptors. Abbreviations: Flk, fetal liver kinase; Flt, fmslike tyrosine kinase; KDR, kinase-insert-domain-containing receptor; PIGF, placenta growth factor; VEGF, vascular endothelial growth factor.

VEGF family have differing binding affinities for each of these receptors, which have helped in elucidating the functions of each of these receptors (Fig. 1). VEGFR1 was initially thought to play a relatively minor role in VEGFmediated signal transduction, but recent studies have suggested that VEGFR1 is critical in developmental angiogenesis, as well as other processes including monocyte migration, recruitment of EC progenitors, and inducing growth factors from liver sinusoidal ECs (6). A naturally occurring soluble form (sVEGFR1) also exists, and has been shown to mediate inhibition of VEGF-induced EC proliferation (9). sVEGFR1 may function to modulate endogenous VEGF or PlGF activity, as it has been shown to be present in the serum and amniotic fluid of pregnant women (10) and in cycling human endometrium (11). In fact, high levels of sVEGFR are associated with preeclampsia (12). VEGFR1 is also expressed on specific tumor cells, and this topic will be addressed below. VEGFR2 (kinase-insert-domain-containing receptor, KDR, and the murine homologue, fetal liver kinase-1, Flk-1) mediates the majority of the downstream effects of VEGF in angiogenesis, including microvascular permeability, EC proliferation, and survival (13,14). VEGFR3 is expressed throughout the embryonic vasculature and later is limited to lymphatic ECs (15). In adults, VEGFR3 expression has been correlated with transient lymphangiogenesis in wound healing in patients (8). NRP-1 AND NRP-2 Neuropilin-1 (NRP-1) was initially identified as a receptor for semaphorins/ collapsins, a system that mediates axonal and neuronal development (16). NRP-1

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and -2 have been shown to serve as a co-receptors for VEGF, suggesting a potential role in angiogenesis (17). NRP-1 and -2 differ greatly from the tyrosine kinase VEGFRs in that it does not have an intracellular signaling domain (18). Its activities are, at least in part, mediated via its function as a co-receptor for VEGFR-1 and -2. NRP enhances the binding affinity of ligands to the receptors and affects downstream intracellular signaling (17). VEGF binding to NRP leads to increased endothelial mitogenesis and chemotaxis (17,19). Recent evidence suggests that NRP-1 may actually function on its own, causing increased EC migration independent of the VEGF TKRs (20). While VEGF is an EC activator, semaphorins—the other family of NRP ligands—are inhibitors of endothelial function and have been shown to inhibit EC migration and collapse the EC cytoskeleton by forming signaling complexes with another known semaphorin receptor, plexin, which can subsequently lead to downstream signaling (21). FUNCTIONS OF VEGF VEGF promotes angiogenesis through several mechanisms, including EC permeability, enhanced survival, migration, invasion, and proliferation of vascular ECs all important events in sprouting angiogenesis (3,5). VEGF was initially discovered as VPF in a guinea pig model of malignant ascites. It is one of the most potent vascular permeabilizing agents known, 50,000 times that of histamine, and can lead to pathological conditions, such as malignant ascites and malignant pleural effusions (1). VEGF exerts a number of different effects on the vascular endothelium and ECs including changes in EC morphology, cytoskeleton alterations, and stimulation of EC migration and growth. In ECs, VEGF increases expression of procoagulant tissue factor, urokinase plasminogen activator receptor, tissue-type plasminogen activator, specific matrix metalloproteases (MMPs), nitric oxide synthase, and integrins (1,22). Many of the latter proteins are involved in EC migration and invasion. VEGF has now been shown to promote EC survival by inhibiting apoptosis pathways, upregulating antiapoptotic proteins, such as bcl-2, and surviving and activating pathways, such as the PI3K/Akt pathway, a classic cell survival network (22,23). VEGF is a potent EC mitogen, probably mediated primarily via the activation of VEGFR-2 and downstream pathways, such as Erk-1/2, JNK/SAPK, and protein kinase C (PKC) (22). VEGF also serves as a chemokine for bone marrow derived EC precursors cells, which may contribute cellular components to the growing vascular bed (24). AGENTS TARGETING THE VEGF LIGAND Agents Targeting VEGF Although there are a few agents that bind to VEGF and prevent activation of its cognate receptors, the vast majority of anti-VEGF approaches involve the use of tyrosine kinase inhibitors. The use of tyrosine kinase to inhibit VEGF receptor signaling will be covered in another chapter in this book. Several agents, including antibodies and soluble receptor constructs, have been developed to inhibit the activity of soluble VEGF-A. Bevacizumab

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(recombinant humanized monoclonal antibody to VEGF, Avastin
; Genentech, Inc., South San Francisco, California, U.S.A.) is the only drug that has been extensively studied in clinical trials and is now approved, in combination with chemotherapy, by the U.S. Food and Drug Administration and the European Union for metastatic colorectal cancer (mCRC). This humanized monoclonal antibody was developed in the early 1990s. Preclinical studies showed that it markedly inhibited the growth of human tumors, xenographs, and metastasis (25,26). This agent was rapidly advanced into phase I clinical trials where doses as low as 0.3 mg/kg neutralized all free circulating VEGF in patients (27). In phase I trial, toxicities associated with bevacizumab included headaches and hypertension. The phase I trial demonstrated that the half-life of bevacizumab ranged from 11 to 50 days, with a mean half-life of approximately 20 days. Following the phase I clinical trial, investigators began a broad program evaluating the use of bevacizumab as a single agent or in combination with standard chemotherapy in a series of randomized phase II clinical trials. These individual trials will be addressed in more detail under specific disease types below. VEGF-Trap VEGF-Trap has undergone a number of modifications in order to obtain optimal pharmacokinetics and acceptable toxicity. The mature VEGF-Trap currently utilized in clinical trials consists of portions of the extracellular domain of VEGFR-1 and VEGFR-2 fused to the FC region IgG1 (28). This agent has shown significant activity in preclinical models and is currently being evaluated in multiple clinical trials. HuMV833 HuMV833 is a humanized monoclonal IgG4 antibody. The original antibody was raised utilizing recombinant human VEGF-121 as the immunogen to elicit an immune response in Balb/c mice. This agent has also been evaluated in early stage clinical trials (29,30). 2C3 2C3 is a monoclonal antibody that blocks the interaction of VEGF with VEGRF-2, but not VEGFR-1 (31). The rationale for the development of this selective antiVEGF antibody is that it would not inhibit the activity of VEGFR-1 on immune effector cells. There are no published reports on the early phase clinical utility of this agent. Inhibiting VEGFR-2, but not VEGFR-1, is a key difference in the antitumor activity of 2C3. VEGF receptor 2 has been shown to be the main receptor that cancer cells use to grow new vessels, whereas VEGFR-1 is utilized for normal cellular function of macrophages and monocytes. An inhibitor of VEGF that selectively blocks the function of VEGF receptor 2 should not interfere with macrophage infiltration into tumors. PIVOTAL TRIALS EVALUATING ANTI-VEGF-A THERAPIES The following text focuses on phase III clinical trials with anti-VEGF therapy, as it is not feasible to summarize all clinical trials in earlier stages of development for

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multiple disease types. Phase II results are referred to in the context of the subsequent development of the phase III trials. Bevacizumab in Combination with Chemotherapy for mCRC A phase II randomized trial was conducted in patients with mCRC where they were randomized to one of three arms. The control arm was the standard of care at the time of conceptualization of the trial: 5-fluorouracil (5FU) and leucovorin (LCV). The other two arms of the trial consisted of 5FU/LCV plus bevacizumab at two different doses (5 mg/kg every two weeks and 10 mg/kg every two weeks). At the time of this trial, the concept that lower dose may achieve better efficacy than a higher dose was postulated for antiangiogenic agents. Interestingly, the addition of bevacizumab to chemotherapy improved response rates and time to progression (Table 1) (32). However, the greatest improvement appeared to be in those patients who received the lower dose of bevacizumab (5 mg/kg). This dose was chosen for the pivotal phase III trial that is discussed below. However, in retrospect, it appears that there were more females in the arm of the study that received the 5 mg/kg dose of bevacizumab. This could have bias the results, and perhaps could explain why the lower-dose arm appeared to achieve better results than the higher-dose arm. Following these interesting results from the phase II trial, a phase III (AV 2107) trial was initiated where patients received IFL (Irinotecan, Fluorouracil, Leucovorin) chemotherapy plus placebo, IFL plus bevacizumab (5 mg/kg every two weeks), or 5FU/LCV plus bevacizumab (same dose) (33). The third arm was included because, at the initiation of the study, there was no data on the safety data of IFL plus bevacizumab. Once it was determined that IFL plus bevacizumab was relatively safe to administer, accrual to the 5FU/LCV plus bevacizumab arm was discontinued. An interesting part of the study design was that patients who received bevacizumab during first-line therapy could continue to receive bevacizumab at the discretion of the physician and the patient in subsequent lines of therapy. Patients in the IFL plus placebo arm, however, were not allowed to cross over to receive bevacizumab therapy following progression. This pivotal phase III trial demonstrated that the addition of bevacizumab to IFL significantly improved overall survival, progression-free survival (PFS), and response rate (Table 1) (Fig. 2). The results of this pivotal phase III trial led to the eventual approval of bevacizumab in combination with intravenous 5FU-based chemotherapy in the front line setting. The ECOG 3200 trial evaluated the addition of bevacizumab to FOLFOX [Folinic Acid (leucovorin), 5-FU, Oxaliplatin] chemotherapy in patients who had progressed on 5FU and/or based on irinotecan chemotherapy (34). In this trial, it is important to note that the bevacizumab dose utilized was 10 mg/kg every two weeks. A third arm in this trial utilized bevacizumab (10 mg/kg every two weeks) as a single agent. In a interim analysis, it was determined that single agent bevacizumab in the second line setting was not as efficacious as the other two arms of the study and accrual to this arm was discontinued, albeit at a relatively late date. The findings from ECOG 3200 demonstrated that the addition of bevacizumab to FOLFOX in the second line setting increased PFS, overall survival, and response rates. The magnitude of the increase in PFS and OS was less than that of the IFL
bevacizumab trial as would be expected in a refractory patient population (Table 1). The results from ECOG 3200 led to the approval

Refractory Front line Front line Front line Front line Refractory Front line

Disease type

Metastatic breast cancer Metastatic breast cancer Stage IIIb/IV NSCLC mCRC mCRC mCRC Pancreatic cancer

Capecitabine Paclitaxel Carboplatin/Paciltaxel IFL CAPOX or FOLFOX FOLFOX Gemcitabine

Chemotherapy regimen AVF2119 ECOG2100 ECOG4599 AVF2107 NO 16966 ECOG3200 CALGB80303

Trial designation 462 715 878 923 1400 829 602

Number of patients randomized to CTX or CTX þ Bev 0.7 5.3b 1.7b 4.2b 1.4b 3.0b 0.2

Improvement in PFS (months)a

10b 22b 20b 10b 0 13b 1

Improvement in response rate (%)a

b

Numbers subject to change as data matures in particular trials. P < 0.05. Abbreviations: Bev, bevacizumab; CTX, chemotherapy; FOLFOX, fluorouracil, leucovorin, oxaliplatin; IFL, irinotecan, fluorouracil, leucovorin; mCRC, metastatic colorectal cancer; NSCLC, non–small cell lung cancer; PFS, progression-free survival.

a

Front line or refractory

TABLE 1 Results of Phase III Trials of Chemotherapy with or Without Bevacizumab for Advanced Stage Disease

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Overall survival (%)

100 15.6

80

20.3

60 40

IFL+bevacizumab

20

IFL+placebo

0 0

10

20 Months

No.at risk IFL+bevacizumab IFL+placebo

402 411

362 363

320 292

30

178 139

73 51

40

20 12

1 0

0 0

FIGURE 2 Overall survival of patients with metastatic colorectal cancer treated with chemotherapy with or without bevacizumab. Source: From Ref. 33.

of bevacizumab in combination with chemotherapy in the second line setting and further substantiated the value of the addition of bevacizumab to oxaliplatin containing regimens. Concomitant to the conduct of the pivotal AV2107 trial, a randomized phase II trial was conducted for those patients who are not ideal candidates for irinotecan-based therapy (35). These patients were randomized to 5FU/LCV bevacizumab (5 mg/kg). The results from this study demonstrated an increase in PFS when bevacizumab was added to 5FU and leucovorin, but there was no statistically significant increase in overall survival. This is likely due to the fact that the number of patients on this trial was significantly less than that on the IFL
bevacizumab trial. Due to the fact that a small number of patients may have biased the outcome, a combined analysis of data from multiple phase II or phase III trials was conducted comparing those patients who received 5FU and leucovorin plus bevacizumab to chemotherapy arms alone (32). This combined analysis demonstrated an improvement in overall survival, PFS, and response rates, again demonstrating the benefit of adding bevacizumab to standard chemotherapeutic regimens. Recently, results were reported on the NO10966 trial at the 2006 European Society for Medical Oncology (ESMO) annual meeting. This trial was initially designed to show noninferiority of capecitabine (Xeloda; Hoffman-La Roche, Ltd., Basel, Switzerland) þ oxaliplatin (CAPOX/XELOX) compared to FOLFOX. After bevacizumab was shown to benefit patients when added to chemotherapy, the trial was modified to a 2 · 2 design, where the treatment arms were subdivided and patients were randomized to received bevacizumab or placebo. In a pooled analysis from the two chemotherapy regimens, the addition of bevacizumab improved PFS relative to placebo (HR 0.83, CI 0.72–0.95, p ¼ 0.0023). However, when the FOLFOX þ bevacizumab arm was compared to FOLFOX þ placebo, the difference in PFS was not significant (p ¼ 0.1871). The reason for the relative lack of efficacy compared to ECOG 3200 and AVF 2107 is not clear, but may be due to decreased duration of use of bevacizumab until progression. In addition, patients

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on the FOLFOX/bevacizumab arm had a shorted disease-free interval from adjuvant therapy, suggesting a more aggressive biology in this group of patients. Further maturation of the data and events, including overall survival may provide useful information (36). Bevacizumab and Chemotherapy for Non–Small Cell Lung Cancer VEGF has been found to be associated with vessel count and poor prognosis in patients with non–small cell lung cancer (NSCLC). Similar to studies in other tumor types, a randomized phase II trial was conducted in patients with previously untreated stage IIIB/4 NSCLC patients. Ninety-nine patients were randomized to receive paclitaxel and carboplatin (PC), PC þ “low dose bevacizumab” (7.5 mg/kg every three weeks), or PC þ “high dose bevacizumab” (15 mg/kg every three weeks). Patients who progressed on the chemotherapy alone arm could then receive high dose of bevacizumab as a single agent. The endpoints for this trial included time-to-progression, overall response rate, and overall survival (37). The results of this study demonstrated that the addition of high dose bevacizumab to chemotherapy improved time-to-progression (p ¼ 0.02) with a slight but not statistically significant improvement in response rate and median survival. For the most part, the addition of bevacizumab to chemotherapy was well tolerated. However, there were six life threatening bleeding events, and four patients died secondary to bleeding (6/66). In reviewing these patients with hemorrhagic events, all of them had centrally located tumors close to major blood vessels, and five of the patients developed tumor capitation or necrosis during therapy. Four of the patients had tumors of squamous cell histology, which is a major risk factor for this adverse event. This toxicity was of significant concern and played a role in the development of subsequent trials. Investigators from the Eastern Cooperative Oncology Group (ECOG) then initiated a phase III clinical trial, randomizing patients to PC or PC þ bevacizumab (15 mg/kg every three weeks). The primary endpoint of the E4599 study was overall survival, with secondary endpoints being PFS, overall response rate, and tolerability. Of note, due to the bleeding events noted in the phase II clinical trial, patients with squamous cell histology, central nervous system metastasis, and active cardiovascular disease were excluded from this study. Inclusion criteria included previously untreated patients with stage IIIB/4 NSCLC with an ECOG performance status of 0–1. Eight hundred and seventy-eight patients were randomized. Stratification between the groups was excellent. The results of the study were reported at the 2005 meeting of the American Society of Clinical Oncology (ASCO), where investigators demonstrated a significant improvement in overall survival (10.3 months in the PC arm vs. 12.3 months in the PC þ bevacizumab arm, p ¼ 0.003). In addition, PFS was likewise improved from 4.5 to 6.2 months (p < 0.001). Interestingly, response rates were improved from 15% in the PC arm to 35% in the PC þ bevacizumab arm (p < 0.001). Overall, the addition of bevacizumab to PC was well tolerated, with a slight increase in neutropenia and thrombocytopenia. However, despite excluding patients with the squamous histology, there was an increase equal to grade three hemorrhage (0.7% in the PC arm vs. 4.4% in the PC þ bevacizumab arm, p < 0.001). In addition, as expected, there was increase in hypertension, which was, for the most part, easily managed.

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Current trials are incorporating other targeted therapies in addition to bevacizumab for patients with advanced NSCLC. In addition, several trials are addressing the addition of bevacizumab to chemotherapy in the neoadjuvant setting for respectable tumors. Furthermore, bevacizumab is being added to chemotherapy in the adjuvant setting after resection of NSCLC. Bevacizumab in Metastatic Breast Carcinoma Similar to other disease types, VEGF expression has been correlated with poor prognosis and microvessel density in tumors from patient’s breast carcinoma. A phase II trial with single agent bevacizumab demonstrated a modest response rate (7–9%). On the basis of these results, as well as results from other trials, it appeared that bevacizumab may be best utilized when added to chemotherapy. Therefore, a phase III trial was initiated with metastatic breast carcinoma. Patients who had previously received anthracycline and taxane therapy were eligible for randomization. Patients who relapsed within 12 months of completing anthracycline and taxane containing adjuvant therapy were also eligible for the trial. These heavily pretreated patients were randomized to receive capecitabine or capecitabine þ bevacizumab (15 mg/kg every three weeks). Four-hundred and sixty-two patients were randomized, with a primary endpoint being PFS, and secondary endpoints being overall response rate and duration of response. Unfortunately, there was no significant improvement in PFS with the addition of bevacizumab to capecitabine in this group of heavily pretreated patients. However, it was interesting to note that there was a statistically significant 10% improvement in response rate with the addition of bevacizumab to capecitabine. This is relatively consistent across all tumor types, where the addition of bevacizumab appears to improve response rates when added to chemotherapy. The bevacizumab plus capecitabine regimen was relatively well tolerated and, as expected, there was an increase in the percent of patients who experienced hypertension when receiving bevacizumab. Due to the relative success of the addition of bevacizumab to chemotherapy and other tumor types, another phase III trial was initiated in the front line setting, where patients were randomized to receive paclitaxel
bevacizumab (ECOG 2100 trial). In this trial paclitaxel was administered weekly and bevacizumab was administered every two weeks at a dose of 10 mg/kg. The primary endpoint of this trial was PFS. Again, patients were well stratified among the two treatment regimens. In this trial in the front line setting, the addition of bevacizumab to paclitaxel led to a significant improvement in PFS (p < 0.0001) and, at a relative early analysis, also led to an improvement in overall survival. Again, the addition of bevacizumab to chemotherapy improved response rates and, in those patients with measurable disease, the improvement in response rates was >20%. The addition of bevacizumab to weekly paclitaxel was well tolerated, and again there was an increase in grade 3 hypertension, which was easily manageable. There was a significant increase of neuropathy in those patients who received paclitaxel þ bevacizumab, and although it is difficult to determine the reason for this increase in neuropathy, it may be due to the fact that these patients received paclitaxel therapy for a longer duration. As with lung cancer, the addition of bevacizumab to chemotherapy is now being studied in the adjuvant as well as the neoadjuvant. Furthermore, the other targeted therapies are being added to

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bevacizumab in the phase II setting in order to determine if phase III trials are warranted. POTENTIAL MECHANISMS OF ACTION OF BEVACIZUMAB The inhibition of VEGF signaling by bevacizumab may affect tumor growth and progression through several mechanisms, including (1) inhibiting the growth of new vessels, (2) regression of newly formed vasculature, (3) altering vascular function and tumor blood flow (normalization of the vasculature to transiently improve the delivery of and increase the efficacy of cytotoxic agents), and (4) direct effects on tumor cells (39–42). Effects on Vessel Numbers Using animal models of tumor growth, investigators have studied the mechanisms by which VEGF-targeted therapy affects the tumor vasculature. The vessel beds of most normal adult tissues are quiescent. In contrast, the immature vessels in tumors (defined as having a decrease in pericyte coverage) are more susceptible to VEGF-targeted therapy. In a mouse glioma xenograft model, disrupting the expression of VEGF in genetically-engineered mice pruned immature vessels (defined as vessels that lack pericytes) and spared pericyte-associated vessels (43). This observation supports the proposed role of VEGF as a maintainer of endothelial survival in newly formed vessels, with the subsequent appearance of pericyte coverage marking the transition to a less labile, non–VEGF-dependent state (44). Pericytes are believed to support the survival of vascular ECs through several mechanisms, including the production of VEGF (45) and angiopoietin-1, and through N-cadherin-mediated cell–cell interaction (13,46). These observations have prompted the idea that the regression of more-mature tumor vessels may require the additional targeting of pericytes through the blockade of platelet-derived growth factor or other factors that mediate pericyte function (47–49). The simultaneous inhibition of VEGF and platelet-derived growth factor receptor signaling in murine tumor xenografts leads to the dissociation of pericytes from vessels, with subsequent greater EC death and regression of pericyte-covered vessels than with the inhibition of VEGF signaling alone (48–50). It is difficult to obtain serial biopsies of tumors in clinical trials, so there are no large clinical trials that have studied the effects of single-agent bevacizumab on vessel counts before and after therapy. In a phase 1 trial, the investigators were able to obtain pre- and post-bevacizumab therapy biopsies in six patients with locally advanced rectal cancer who were treated with chemoradiotherapy plus bevacizumab. In this small study a single treatment with bevacizumab reduced tumor microvascular density by 29–59% (p < 0.05), tumor blood perfusion by 40–44% (p < 0.05), and tumor blood volume by 16–39% (p < 0.05), as assessed by functional computed tomography 12 days after treatment (51). Studies with other anti-VEGF agents have not shown such striking results; hence, it is necessary that such findings be reproduced in larger studies, to confirm that treatment with single agent bevacizumab can lead to the regression of tumor vessels. There is indirect evidence that anti-VEGF therapy is indeed cytostatic, as was first hypothesized. Clinical measures were recently evaluated in a study in patients with mCRC who were treated with bevacizumab but did not have tumor responses by the Response Evaluation Criteria in Solid Tumors (RECIST).

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These patients did, however, have a greater clinical benefit than patients who were treated with chemotherapy alone (52). The hazard ratio for death was 0.60 [95% confidence interval (CI), 0.40–0.90] in patients with a response (complete or partial) and was 0.76 (95% CI, 0.60–0.96) in patients without a response (stable disease, progressive disease, or nonassessable). Similarly, the hazard ratio for death or disease progression was 0.53 (95% CI, 0.38–0.74) in responders and 0.63 (95% CI, 0.49–0.80) in nonresponders. This suggests a cytostatic effect of bevacizumab, which indicates that it inhibits the growth of new blood vessels. Furthermore, the increase in PFS with higher response rates are greater in patients treated with chemotherapy plus bevacizumab than those treated with chemotherapy alone, supporting the cytostatic effects of bevacizumab (53). Effects on Vessel Function Structural irregularities are common in tumor vasculature. Owing to insufficient vascular supply, tumors lack adequate oxygen, which results in greater stabilization and expression of hypoxia-inducible factor 1. Signaling by this factor induces the expression of a large number of genes that are involved in tumor progression and metastasis (54,55), the most important of which is VEGF (56–58). Overexpression of VEGF causes vascular hyperpermeability. As a result, the tumors have high interstitial fluid pressure, sluggish blood flow, and uneven perfusion, with regions of hypoxia and acidosis (59). The abnormal hemodynamic environment of tumors may render them less sensitive to cancer therapies through several mechanisms (59,60). Uneven perfusion impedes the delivery of cytotoxic agents throughout the tumor. The loss of the vessel-to-exterior fluid pressure gradient and colloid osmotic pressure gradient inhibits the delivery of high-molecularweight agents, such as monoclonal antibodies. Some animal studies support the idea that the normalization of tumor vasculature—specifically, vasoconstriction to transiently improve flow dynamics and pruning of immature, functionally abnormal vessel sprouts—may improve the delivery of cytotoxic agents, with subsequent increases in the efficacy of chemotherapy and radiotherapy (61). Treatment with VEGF-targeted agents in murine xenograft models improves the intratumoral delivery of therapeutic agents. One week after the injection of a monoclonal antibody to VEGF (i.e., the murine precursor of bevacizumab), mice with colon adenocarcinoma xenografts had greater tumor uptake of the cytotoxic agent irinotecan (62). In another study, in mice with mammary carcinoma xenografts, a single dose of DC101, an antibody to VEGFR-2, increased tumor penetration by fluorescence-labeled bovine serum albumin protein (63). Subsequent studies in preclinical models by this group showed a window of opportunity for the normalization of blood vessels that occurs shortly after the anti-VEGF therapy has been delivered (3–5 days). After that, it is likely that the tumor vasculature undergoes further vasoconstriction, which I believe is due to the inhibition of EC-derived nitric oxide, a potent vasodilator. In fact, it has been hypothesized that this blockade of the production of nitric oxide, may in part explain the hypertension that is seen in patients who are treated with anti-VEGF therapy. It is clear that anti-VEGF therapy changes vascular function in addition to inhibiting classic angiogenesis. Activation of VEGFR-2 by VEGF leads to downstream signaling that induces the expression of vasodilators. Specifically, VEGF leads to the induction of nitric oxide and prostacyclin. These mediators lead to a

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relaxation of pericytes and vascular smooth muscle cells that regulate vessel tone. Thus, blocking VEGF signaling limits the secretion of these vasodilators, leading to a “relative vasoconstriction” associated with higher levels of VEGF that are released from tumors. This relative vasoconstriction is consistent with the findings in clinical studies that have used computed tomography scanning or magnetic resonance imaging to show changes in tumor blood flow, blood volume, transit time, and permeability. These imaging modalities show that anti-VEGF therapy causes a rapid decrease in tumor blood flow, blood volume, and permeability that occurs before any changes that would be expected in vessel density. As stated above, it is possible that the hypertension that is associated with anti-VEGF therapy may be due to this vasconstrictive effect. In fact, one clinical study has suggested that early hypertension may predict a response to anti-VEGF therapy (64). Obviously, more research is necessary to better understand the effect of antiVEGF therapy on the tumor vascular bed and on vessel tone in normal tissues. Effects on Tumor Cells In addition to its effects on tumor vasculature, anti-VEGF therapy may have direct inhibitory effects on tumor cells. For example, VEGF provides a survival signal for breast carcinoma cells in vitro (39). Treatment of breast cancer cell lines with SEMA3F, an endogenous protein that competitively inhibits the binding of VEGF to NRP-1 and NRP-2, decreased these cells’ capacity for chemotactic migration (65,66). A recent trial of a bevacizumab-containing regimen in patients with inflammatory breast cancer found that VEGFR-2 was present and activated on tumor cells and that treatment with bevacizumab blocked the activation of tumor cell VEGFR-2 (Fig. 3) (67). Studies in colon cancer and pancreatic cancer cell lines have found that VEGFR-1 is present on all cell lines that were studied. The activation of VEGFR-1 led to an increase in tumor cell invasion and migration, which was blocked by treatment with antibodies to VEGFR-1 (40,41). More recent work has shown that the activation of VEGFR-1 on tumor cells leads to an alteration in cell phenotype from an epithelial phenotype, in which cells are thought to be immobile, to a mesenchymal phenotype, in which cells shifts their molecular machinery to increase cell migration and invasion (i.e., epithelial–mesenchymal transition) (42). Adverse Effects of Anti-VEGF Therapy For the most part, the addition of bevacizumab to chemotherapy is well tolerated. However, there does appear to be some class effects of inhibiting the VEGF pathway as well as tumor-specific effects. Hypertension A very consistent yet easily manageable toxicity of anti-VEGF therapy is hypertension. Almost all patients will experience some increase in both diastolic and systolic blood pressures. However, it is estimated that 10–20% of patients will experience grade 3 or 4 hypertension (grade 4 is rare) requiring adjustment of current medications or the addition of agents to existing regimens. There are no clear cut recommendations for treating hypertension induced by anti-VEGF therapy. However, it is this author’s belief that diuretics should not be used, as many of the chemotherapeutic agents used along with bevacizumab therapy may have the potential for nephrotoxicity or are associated with GI toxicity, leading to

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FIGURE 3 Effect of bevacizumab monotherapy on vascular endothelial growth factor receptor (VEGFR-2) activation on breast carcinoma cells. VEGFR-2 was present and activated on breast carcinoma cells from biopsies from patients with locally advanced or inflammatory breast cancer (left panels). After single agent bevacizumab therapy, VEGFR-2 phosphorylation was decreased on breast cancer cells (two phosphorylation sites were investigated). Source: From Ref. 67.

diarrhea and potentially dehydration. At our institution most patients are treated with calcium channel blockers or ace inhibitors. Proteinuria In the phase I and phase II clinical trials, bevacizumab therapy appeared to be associated with proteinuria. The nephrotic syndrome was exceedingly rare but oncologist should be aware of the potential for the development of this syndrome. For the most part, investigators and oncologists utilizing bevacizumab perform a dipstick on the urine to be sure that there is not excessive protein in patients who are receiving bevacizumab. For patients with a positive dipstick for proteinuria, a 24-hour urinary protein excretion should be done in order to determine the amount of protein being lost in the urine. If this protein loss exceeds 2.5 g in 24 hours, then bevacizumab should be discontinued (Bevacizumab product insert). Disease Specific Toxicities One of the interesting findings from the trials in patients with mCRC was the finding that there was a slight increase in the number of patients who suffered from a bowel perforation. In the initial trial comparing IFL
bevacizumab, there were six bowel preparations in the bevacizumab containing arm versus only one in the chemotherapy alone arm. This finding of bowel perforation while receiving bevacizumab has been incredibly consistent among clinical trials; currently, the rate appears to occur in 1.5–1.7% of patients receiving bevacizumab for mCRC.

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Recently, the BRITE Study, a registry of patients who receive chemotherapy plus bevacizumab for mCRC has shed some light on the timing and perhaps etiology of this event. The BRITE registry has accumulated data on more than [1,900] patients who have received chemotherapy plus bevacizumab for mCRC in the United States. This registry is also demonstrated a bowel perforation rate of 1.7%, with the majority of perforations occurring within the first three to six months of therapy. Although the numbers are relatively small, it does appear that patients who have an intact primary tumor and had recent sigmoidoscopy or colonoscopy may have a slight (but not statistically significant) increased risk of bowel perforation. There is also a similar registry collecting data from patients receiving chemotherapy plus bevacizumab for mCRC representing other countries throughout the world (BEAT trial). Perhaps with greater numbers of patients on these two registries we will be able to determine potential indicators of those patients more likely to suffer from a bowel perforation. As stated earlier, patients with NSCLCs with predominant squamous cell histology may also be at risk for bleeding. In the phase II and phase III trials, many of these patients actually had a good response to therapy, though one should follow directions in the package insert after the anticipated approval of bevacizumab for patients with NSCLC. CONCLUSIONS In the past, most investigators would have thought that targeting a ligand would not be as efficacious as targeting a receptor. However, at this stage in clinical development of anti-VEGF/anti-VEGF-receptor therapy, it is clear that significant benefit can be achieved by targeting the ligand VEGF and preventing its binding to its receptors. In most situations, anti-VEGF therapy improved the effects of chemotherapy, but the exact mechanism of action of how this occurs has yet to be determined. Many questions remain with respect to the best use of anti-VEGF therapy in malignant diseases. Such questions/issues include continuation of therapy in subsequent lines of therapy, the efficacy and potential long-term toxicity when used in the adjuvant setting, and, most importantly in the mind of this author, the identification of predictive markers for efficacy and toxicity. Collectively, we still have a great deal to learn about the biology of the VEGF ligand/receptor system in both pathology and physiology, and it is imperative that clinicians and basic scientists keep an open line of communication in order to take knowledge from the laboratory to the clinic, and to try to address issues raised by clinical observation in the research laboratory. ACKNOWLEDGMENTS This work was supported, in part, by NIH Grant CA112390 and the Lockton Fund for Pancreatic Cancer Research. REFERENCES 1.

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15

Targeting Angiogenesis with Oral Agents Benjamin Besse Department of Médecine, Paris University XI, Institut Gustave Roussy, Villejuif, France

Jean-Pierre Armand Department of Medicine, Institut Gustave Roussy, Villejuif, France, and Department of Medicine, Institut Claudius Regaud, Toulouse, France

INTRODUCTION The advent of molecular targeted therapies through a better understanding of the cancer cell molecular circuitry has revolutionized treatment approaches in oncology. Targeting neovascularization offers tremendous potentialities (1). There is a finely regulated equilibrium between numerous natural antiangiogenic and proangiogenic factors. The tumor angiogenic phenotype is characterized by high microvessel density in the tumor with elevated vascular endothelial growth factor (VEGF) levels and is usually correlated with a worse prognosis. During angiogenesis, endothelial cells are stimulated by various growth factors that bind to membranous receptors, essentially tyrosine kinase receptors (TKRs). The TKRs directly involved in angiogenesis include receptors for VEGF, FGF, PDGF, angiopoïetin-1 (Ang-1) Ang-2, hepatocyte growth factor (HGF), Eph, and receptors belonging to the epithelial growth factor family. These receptors are expressed by endothelial cells or pericytes but not by tumor cells that secrete ligands. VEGF is the most potent inducer of angiogenesis. VEGF-A, commonly referred to as VEGF, can be induced by hypoxia and hypoxia-inducible factor 1 (HIF-1), inactivation of the von Hippel-Lindau (vHL) tumor suppressor gene, and a number of cytokines and growth factors, including platelet-derived growth factor (PDGF), tumor necrosis factor a (TNF-a), and transforming growth factor b (TGF-b). VEGF binds to VEGFR-1 (Flt-1) and VEGFR-2 (KDR), which are the major mediators of the mitogenesis-, angiogenesis-, and permeability-enhancing effects of VEGF. A large number of oral antiangiogenic agents, particularly TKR inhibitors, are currently under clinical development, with marketing approvals already granted for some of them (sunitinib and sorafenib). This review focuses on the most promising and advanced among them, with special emphasis on the VEGFR pathway (Tables 1 and 2). ANTIANGIOGENIC TYROSINE KINASE INHIBITORS IN CLINICAL TRIALS Sorafenib (BAY 43-9006) Sorafenib (Nexavar
; Bayer, West Haven, Connecticut, U.S.A) is a potent inhibitor of RAF-1, a key enzyme in the RAS/RAF/MEK/ERK signaling pathway, and an inhibitor of VEGFR-2 and PDGFR-b involved in angiogenesis (2). Based on phase I data, sorafenib was further investigated at a dose of 400 mg qd (3). Sorafenib activity was first studied in renal cell carcinoma (RCC), given its activity demonstrated in phase I trials. In a phase II study, 202 RCC patients were treated with 241

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TABLE 1 Oral Antiangiogenic Agents Targeting TKRs Agent PTK787/ZK222584 (Vatalanib) SU 11248 (Sunitinib) ZD6474 BAY 43-9006 (Sorafenib) AG 013736 AZD2171 CP-547,632

Targeted TKRs

Phase

VEGFR-1, VEGFR-2, PDGFR VEGFR, PDGFR, Flt-3, c-KIT VEGFR-2, VEGFR-3, EGFR VEGFR-2, PDGFR (and RAFa) VEGFR, PDGFR, c-KIT VEGFR-2 VEGFR-2, EGFR and PDGFR

III FDA approved III ongoing FDA approved II II II

a Not a TKR. Abbreviations: EGFR, epidermal growth factor receptor; FDA, U.S. Food and Drug Administration; MMP, matrix metalloproteinase; PDGFR, platelet-derived growth factor receptor; TKRs, tyrosine kinase receptors; VEGF, vascular endothelial growth factor; VEGFR, vascular endothelial growth factor receptor.

sorafenib and randomization took place at 12 weeks in patients with a stable disease (SD, 32%) while patients with tumor shrinkage >25% (36%) continued to receive sorafenib until disease progression (PD) (4). In the placebo arm, 18% of the patients were free of progression for 24 weeks compared to 50% in the sorafenib arm (p ¼ 0.008). Similarly, progression-free survival (PFS) was worse in the placebo group as compared to the sorafenib group (6 vs. 23 weeks, p ¼ 0.0001). The TARGET phase III trial confirmed the efficacy of sorafenib in 903 patients with RCC who failed prior treatment (5). PFS was 5.5 months in the sorafenib group and 2.8 months in the placebo group (hazard ratio for disease progression in the sorafenib group = 0.44; 95% confidence interval (CI) ¼ 0.35–0.55; p < 0.01), although an objective response was observed in only 10% of the patients in the sorafenib group (2% in the placebo group). In this setting (second-line treatment in RCC), objective response rates are usually just above 5%. The primary end point, the evaluation of overall survival (OS), was complex because crossover to an alternative treatment was subsequently allowed. Immediately before crossover was allowed, 220 deaths (41% of the protocol defined 540 deaths) had occurred: 97 of 451 patients (22%) in the sorafenib group and 123 of 452 patients (27%) in the placebo group died. At a median follow-up of 6.6 months, the median actuarial overall survival was 14.7 months in the placebo group but had not yet been reached in the sorafenib group (hazard ratio, 0.72; 95% CI ¼ 0.54–0.94; p ¼ 0.02). Grade 3/4 toxicities included a hand–foot skin reaction (6%), diarrhea (2%), fatigue (5%), and hypertension (4%). Grade 3/4 lymphopenia, hypophosphatemia, and elevated lipase levels were reported in 12–13% of the patients. Treatment was discontinued in 10% and 8% of the cases in the sorafenib group and the placebo group, respectively, because of skin and gastrointestinal toxicities. Doppler ultrasonography was performed in a subset of 30 patients (6). Changes in vasculature were positively correlated with PFS and OS, as early as three weeks. Sorafenib was approved for first- and second-line treatment of patients with RCC in December 2005 by the FDA. Its value as an adjuvant treatment is currently being investigated in the SORCE trial. Given that activating BRAF mutations are present in up to 80% of human melanomas, sorafenib has been investigated in this disease. In a phase II study in metastatic melanoma, little or no effect was detected when sorafenib was given alone, even in subgroup of patients with activating BRAF mutation (7). Phase II and III trials are ongoing to evaluate sorafenib in combination with tirosel/temsirolimus (Wyeth, Madison, New Jersey, U.S.A.) or carboplatin-paclitaxel. In a phase II study of sorafenib in untreated hepatocellular carcinoma, 46 out of the 137 patients (33%) exhibited a stable disease at week 16

FDA approved

Sunitinib

GIST

Phase II after imatinib Phase II

Phase III vs. imatinib

Phase II after imatinib and sunitinib

Colorectal

Phase II with capecitabine Phase II/III with FOLFOX Phase II after FOLFOX

Phase I/II with irinotecan and cetuximab Phase II with cetuximab Phase I/II with irinotecan and cetuximab

Breast

Phase II

Phase III with paclitaxel vs. paclitaxel þ bevacizumab Phase III with docetaxel Phase III with capecitabine Phase I/II with exemestane Phase II with trastuzumab Phase II after taxanes Phase II with letrozole

Phase I/II with anastrozole

NSCLC

Phase III with docetaxel (second line) Phase III vs. erlotinib Phase III with pemetrexed (second line) Phase II with paclitaxel-carboplatin

Phase II/II with paclitaxel-carboplatin

Phase II with erlotinib

Phase III with paclitaxel-carboplatin Phase II second line

Melanoma

Phase II

Phase I/II with temozolomide

Phase III with paclitaxel-carboplatin Phase I/II with tirosel/temsirolimus

Abbreviations: FDA, U.S. Food and Drug Administration; GIST, gastrointestinal stromal tumor; NSCLC, non–small cell lung cancer; RCC, renal cell carcinoma.

ZD6474

AZD2171

Vatalanib

FDA approved

Sorafenib

RCC

TABLE 2 Current Status of Oral Antiangiogenic Agents Targeting Tyrosine Kinase Receptors Regarding Cancer Type

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whereas 8% had a partial or minor response (8). With a median survival of 9.2 months, and a manageable toxicity, those results compared favorably to anthracyclin-based regimens. Combination trials with cytotoxic agents are ongoing. In stage III or IV non–small cell lung cancer, sorafenib was given alone to 52 pretreated patients (9). Although no partial response was reported, tumor shrinkage or cavitations were observed in 29% of the cases. One patient died of hemoptysis. Median PFS was 2.7 and 5.3 months in patients with SD. The more frequent grade 3/4 events were hand–foot skin reaction (10%) and hypertension (4%). Those encouraging results led to an ongoing phase III trial evaluating sorafenib as a maintenance treatment after a paclitaxel carboplatin combination in the first line setting. Phase I–III trials are also ongoing in head and neck, pancreatic and prostate cancer, as well as in sarcoma. Sunitinib (SU11248) Sunitinib (Sutent
; Pfizer, New York, New York, U.S.A.) inhibits VEGFR1, PDGFR, and c-KIT, a receptor of the pluripotent cell growth factor (SCF) implicated in malignant blood diseases. At higher concentrations, it inhibits FGFR1, another angiogenesis TKR. Sunitinib was synergistic with radiotherapy in murine models attaining tumor responses and sustained tumor control (10). A phase I study recommended a 50 mg/day dose given orally for four weeks every six weeks, and reported adverse effects such as asthenia, sore mouth, oedema and thrombocytopenia (11). In 38 patients, 6 objective responses were observed in three renal cell carcinomas, one neuroendocrine tumor, one stromal tumor and one unknown primary adenocarcinoma patient. In two phase II studies, a total of 169 patients with RCC received sunitinib after failure of a cytokine (12,13). In a pooled analysis, it resulted in 71 (42%) PR and 40 (24%) SD for at least three months (13). The median PFS for all 168 patients was 8.2 months (95% CI 7.8– 10.4). The most common toxicities experienced by patients were fatigue, diarrhea, neutropenia, elevation of lipase, and anemia. On the basis of these results, a phase III trial comparing sunitinib to interferon alpha in untreated RCC patients was conducted (14). Patients with brain metastases, uncontrolled hypertension, or clinically significant cardiovascular events during the preceding 12 months were excluded. Sunitinib was administered orally at a dose of 50 mg once daily, every four out six weeks, as in the phase II studies. Interferon a (INF-a) was given as a subcutaneous injection three times per week at 3 MU per dose during the first week, 6 MU per dose during the second week, and 9 MU per dose thereafter. The median PFS in the sunitinib group was longer than in the group INF-a (11 vs. 5 months, HR = 0.42, 95% CI 0.32–0.54; p < 0.001). The response rate was also higher in the sunitinib group compared with the INF-a group (31% vs. 6%, p < 0.001). Grade 3/4 toxicities were significantly higher among patients in the INF-a group (12% vs. 7%, p < 0.05); in particular, fatigue (12% vs. 7%) and lymphopenia (22 vs. 12%), whereas patients in the sunitinib group had higher rates of diarrhea (5% vs. no cases), hypertension (8% vs. 1%), and hand–foot skin reaction (5% vs. no cases). An alternative schedule (37.5 mg/day continuously) has been evaluated in a phase II trial: incidence of grade 3/4 adverse events might be lower and this regimen is still active (15). Note that in 66 RCC patients treated with sunitinib 50 mg/day (4 weeks out of 6), thyroid function tests were abnormal in 85% of them, consistent with hypothyroidism (16). Among patients with abnormal thyroid function tests, signs and symptoms possibly related to hypothyroidism were found in 84%

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of them, including signs usually linked to sunitinib as fatigue. Thyroid test abnormalities were detected relatively early in the treatment course (median = at cycle 2). The number of patients was too small to correlate thyroid dysfunction and outcome. Thyroid hormone replacement therapy improved symptoms in a subset of patients. Routine evaluation of thyroid function in patients treated by sunitinib may be therefore of value. Sunitinib has also achieved clinical activity in gastrointestinal stromal tumors (GIST), after failure on imatinib. Ninety-seven patients (92 evaluable) enrolled in a phase II study received sunitinib: 8 (8%) PR and 68 SD (70%, 36 of them for more than six months) were observed (17). The efficacy (response rate, OS) of sunitinib was higher in patients with wild-type KIT or exon 9 mutation compare to those with exon 11 mutation (18). Secondary KIT mutations (in exons 13, 14, 17, or 18) were found in 62% of GISTs with a primary KIT exon 11 mutation, in 16% with a primary KIT exon 9 mutation, but not in any of the GISTs lacking a primary KIT/PDGFRA mutation. Secondary kinase mutations of KIT exon 13 and 14 conferred in vivo and in vitro sensitivity to sunitinib compared to KIT exon 17 and 18 mutations. Given that sunitinib had activity against KIT mutants but not against PDGF-R mutants, it has been hypothesized that the activity of sunitinib against GIST could be distinct from its angiogenic effects (19,20). In a randomized trial, sunitinib was compared to a placebo in 312 patients with GIST after failure of imatinib (2:1) (21). Time to tumor progression (primary endpoint) was better for the sunitinib arm (6.3 vs. 1.5 months, p < 0.00001). Patients were switched from the placebo arm to the sunitinib arm in cases of progression, which could explain why OS did not differ between the two arms. PR and SD were similar to that obtained in the previous phase II study, 7% and 58%, respectively, in the sunitinib arm. The most common treatment–related adverse event was fatigue, occurring in 34% of the patients (any grade) in the sunitinib group, and in 22% of the cases in the placebo group. Sunitinib induced grade 3 (G3) fatigue in 5% of the patients, G3 hypertension in 3%, G3/4 neutropenia in 10%, and G3/4 thrombocytopenia in 5% of them. Results from preclinical models suggest that DCE-MRI could predict sunitinib activity, as shown with PTK/ZK (22,23). TEP-FDG could also be a sensitive surrogate marker of sunitinib efficacy in GIST patients. In 75 patients with imatinib-resistant GIST or intolerance to imatinib, the mean SUVmax (maximum standardized uptake value in up to five lesions) significantly decreased after 7 days of sunitinib (50 mg/day) and rebounded after 7–14 days of therapy (p < 0.001 in both cases) (24). Sunitinib efficacy was also evaluated in 64 patients with metastatic breast cancer pretreated with taxanes and anthracyclines (25). Hematologic toxicities were higher in this population: G3 neutropenia was seen in 39% of the patients and thrombocytopenia in 15% of them. In 51 evaluable patients, sunitinib exhibited significant activity: 7 (14%) had a PR and 1 (2%) a SD > 6 months. In non–small cell lung cancer (NSCLC), sunitinib was evaluated in 110 patients, previously treated with 1 (43%), 2 (44%) or 3 (13%) chemotherapy regimens (26). A first cohort of 63 patients was treated at 50 mg/day for four weeks followed by two weeks rest. Given the notable G3/4 toxicities (asthenia in 27%, myalgia in 18%, nausea 10%), a subsequent cohort was treated at 37.5 mg/day continuously. It resulted in a reduction of G3/4 asthenia, myalgia, and nausea (3%, 3%, and 0%, respectively), an increase of G3/4 neutropenia (11 vs. 5% in the first cohort), and a stable incidence of grade 3 hypertension (5%). Lethal hemorrhages were reported in three patients (two pulmonary and one cerebral hemorrhages). In the first cohort, 11% PR and 44% SD have been

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observed (27% of the patients not evaluated). Sunitinib activity is currently being evaluated in consolidation after first-line chemotherapy in stage IIIB-IV NSCLC. Vatalanib (PTK787/ZK 222584) Vatalanib (or PTK/ZK) inhibits the tyrosine kinase activity of VEGFR1, VEGFR2, and the platelet-derived growth factor receptor (PDGF-R) and is administered orally (27). Preclinical studies demonstrated activity through the inhibition of tumor vasculature, when it was administered alone or in combination with chemotherapy or radiotherapy. Phase I studies have evidenced good tolerability and efficacy in various tumors (28). The most commonly reported adverse events were nausea (59%), fatigue (41%), vomiting (35%), dizziness (29%), and headache (24%). Different solid tumor types have been explored with this agent: colon, brain, renal, and lung carcinomas. In a phase I/II study, patients with advanced colorectal cancer were treated every 14 days with oxaliplatin, 5-FU and folinic acid (known as FOLFOX 4) in combination with PTK/ZK (29). No increase was observed in oxaliplatin/5-FU toxicity and PTK/ZK was well tolerated at doses of 1500 mg/day. Similar results were obtained when PTK/ZK was combined with irinotecan-5-FU-folinic acid (30). PTK/ZK (1250 mg/day) combined with FOLFOX 4 was subsequently compared to a placebo in the CONFIRM-1 phase III trial, reported at the 2005 ASCO meeting (31). A total of 1168 with untreated metastatic colorectal cancer were randomized in that trial. Neutropenia, thrombocytopenia and neuropathy did not differ between the two groups. There were more cases of grade 3/4 hypertension (21% vs. 6%), venous thrombosis (7% vs. 4%) and pulmonary embolism (6% vs. 1%) in the PTK/ZK arm but similar grade 3/4 bleeding and arterial thrombosis. According to investigator-based assessment, there was a statistically significant increase in PFS in PTK/ZK-treated patients. However, a central review failed to document any significant difference. The CONFIRM 2 study investigated the same combination as second-line therapy in irinotecan pretreated patients (32). As in the CONFIRM 1 study, bone marrow toxicity was equivalent in the two arms whereas there were more grade 3/4 adverse events in the PTK/ZK arm: hypertension (22% vs. 5%), dizziness (9% vs. 1%) and venous thrombosis (6% vs. 1%). OS and time-to-progression (TTP) did not significantly differ between the two arms (12.1 vs. 11.8 months and 5.6 vs. 4.1 months, respectively). In a meta-analysis of the CONFIRM 1 and 2 studies, PFS was improved in the PTK arm in patients with elevated LDH (33). Two phase I/II trials have investigated PTK/ZK alone or in combination with either temozolomide or lomustine in patients with recurrent glioblastoma multiforme (34,35). As a single agent, 2 out of 55 patients (4%) experienced a PR and 31 (56%) an SD (median duration: 12.1 weeks). Sixty patients were treated with combined therapy that yielded 4 PR and 27 SD with a median TTP ranging from 12.1 to 16.1 weeks. Another study in 45 patients (37 evaluable) demonstrated the efficacy of PTK/ZK in metastatic renal cancer (36). Seven patients (19%) achieved a measurable response (1 partial and 6 minor) with a median TTP of 5.5 months (95% CI = 3.7–7.9 months), 17 (46%) had SD, and 5 (14%) PD. Rapid disease progression (within 3 months) occurred in only 28% of the patients (95% CI = 12.3–43.6%) treated with at least 1000 mg/day compared with an expected rate of 49.7% (95%

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CI = 43.5–55.9%) based on cytokine therapy in a similar patient population. Oneyear OS was 63.7% (95% CI = 41.9–85.5%). In another study, 55 NSCLC patients pretreated with a single line of platin-based chemotherapy received 1250 mg of PTK/ZK once a day and a further 55 patients received the same dose b.i.d. (37). Most frequent adverse events were nausea, vomiting, and dizziness. Best response by RECIST criteria for 55 evaluated patients (52 qd and 3 b.i.d.) include 2 PR (4%), 30 (55%) SD at week 4, hereof 18 (33%) for at least 12 weeks, and 23 (41%) PD. PTK/ZK efficacy has also been demonstrated in GIST resistant to imatinib (38). Studies have attempted to identify surrogate markers of response to PTK/ ZK. Dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI) was performed in patients treated with increasing doses of PTK/ZK (23). The bidirectional transfer constant (Ki), reflecting tumor permeability and vascularity, was negatively correlated with the oral PTK/ZK dose and plasma levels at day 2 (p = 0.01 and p = 0.0001, respectively). SU5416 and SU6668 SU5416, the first specific synthetic inhibitor of VEGF-TKR activity, inhibits the growth of human tumors xenografted into mice (mostly slowly growing tumors). However, despite encouraging preliminary results in pilot studies with a good safety profile, SU5416 proved disappointing in several phase II studies in different tumors and its clinical development was stopped. In particular, a randomized, multicenter, international prospective phase III study was conducted in 737 untreated metastatic colorectal cancer patients. Patients received a 5FU/leucovorin combination (Roswell–Park regimen) alone or concomitantly to SU5416. The toxicity was significantly worse in the SU5416 arm (diarrhea, cardiopulmonary arrest and vomiting) and there was no improvement on response rate, TTP and OS (39). SU6668 has a wide spectrum of activity and was first developed to inhibit tyrosine kinase activity of PDGFR, FGFR1 and VEGFR2. Regression of human tumor xenografts was obtained in mice with a complete histological response following rapid apoptosis in tumor microvessels. In two phase I studies, it was not known whether the plasma concentration of SU6668 at the MTD was sufficient to inhibit VEGFR and thus, induce tumor response (40). Interestingly, a recent study indicated that SU6668 has greater affinity for other tyrosine kinases, including other potential cancer drug targets such as TBK1 (also known as NAK or T2K) and two aurora kinases (41). SU6668 activity against angiogenesis could be minor, and the drug development could be reoriented based on these new preclinical data. Vandetanib (ZD6474) ZD6474 is an inhibitor of VEGFR-2, VEGFR-3 and HER1 (EGFR), albeit to a lesser extent. It has a certain degree of activity against other TKRs (PDGFR > VEGFR1 > Tie-2 > FGFR1). A phase I study demonstrated a safe clinical profile but no tumor response (42). The most common adverse events were diarrhea, rash and nausea. Asymptomatic QTc prolongation was noted in 7/77 patients (9%). In 46 previously treated patients with metastatic breast cancer, ZD6474 was neither effective at the 100 mg/day nor at the 300 mg/day dose (23 patients in each arm) (43). The most frequent grade 3/4 toxicity was diarrhea, concerning only 3 out of 22 patients at the 300 mg/day dose. A clinical trial with docetaxel and ZD6474 has been conducted in patients with advanced NSCLC who had progressed after

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first-line platinum-based chemotherapy (44). In this phase II trial, 127 patients were randomized to three arms: docetaxel plus ZD6474 100 mg/day, ZD6474 300 mg/day or a placebo. Common adverse events included diarrhea, rash and asymptomatic QTc prolongation. Results suggested efficacy with a PFS of 19 weeks versus 17 weeks versus 12 weeks, respectively. A confirmatory phase III trial is ongoing. In a Japanese NSCLC population, ZD6474 was given as a single agent at three different doses (100, 200 or 300 mg/day) after one or two platinum based chemotherapy regimens (45). A response rate of 13% was seen irrespectively of the dose level, but the median duration of treatment was longer in the 300 mg/ day arm. Grade 3/4 toxicities were more frequent in the 300 mg/day arm (67% vs. 39% and 29% in the 200 and 100mg/day arms). QTc-related events were reported in 72% of the patients at the level of 300 mg/day, 61% of the patients at 200 mg/day and 29% of the patients at 100 mg/day. ZD6474 (300 mg/day) has been compared to gefitinib (250 mg/day) in 168 Caucasian NSCLC patients after failure of 1–2 line platinum-based chemotherapy. In this randomized phase II trial, median PFS was significantly longer in the ZD6474 arm (11 vs. 8.1 weeks, p = 0.025). In 37 progressive patients in the gefitinib arm, ZD6474 achieved a disease control > 8 weeks in 16 patients, whereas only 7 out of 29 ZD6474 patients that switched to gefitinib achieved a disease control > 8 weeks (46). Axitinib (AG 013736) Axitinib inhibits VEGFR, PDGFR, and c-KIT TKIs. With continuous oral dosing, the main toxicities were similar to those that occurred with oral VEGR TKIs: hypertension, fatigue, diarrhea, stomatitis, nausea and vomiting (47). Hypertension was the most frequent toxicity reported in the first published phase I study, occurring in 22 patients (61%) and was mainly moderate (grade 1/2 in 18 patients). Impressive antitumor activity was demonstrated in 52 patients with metastatic RCC who failed prior cytokine-based therapy (48). Axitinib (5 mg b.i.d.) induced a PR in 46% of the patients and SD in a further 40%. Grade 3/4 adverse events were diarrhea in 8%, hypertension in 15% and fatigue in 8% of the patients. Evaluation of tumor vascularization by CT perfusion or DCE-MRI was performed in a subset of patients (49,50). The accuracy of the information is limited given the sample size, but it may be a valuable alternative for determining tumor activity. In 32 patients refractory or not suitable candidates for iodine (131I), axitinib induced a partial response in 23% of the patients and a stabilization in 47% of them (51). Grade 3 adverse events were fatigue (9%), hypertension (6%), and diarrhea, nausea, and proteinuria in 3% of the cases. Evaluation of axitinib efficacy in combination with gemcitabine is ongoing in pancreatic carcinomas (52). CP-547,632 CP-547,632 is an orally administered inhibitor of the tyrosine kinase activity of VEGFR2. A phase I study demonstrated good tolerability and 6 SD for more than 8 weeks and 1 for more than 6 months in 22 evaluable patients (53). The toxicity profile when combined with the paclitaxel/carboplatin doublet was reported to be safe in NSCLC patients. In a randomized phase II in the first-line setting (chemotherapy with or without CP-547,632), CP-547,632 conferred no apparent improvement in objective response rate when added to carboplatin and paclitaxel (54).

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AZD2171 AZD2171 is a highly potent and orally active inhibitor of VEGFR-2 tyrosine kinase activity that inhibited the growth of various established human tumor xenografts dose-dependently (55). Consistent with inhibition of pathologic angiogenesis in vivo, tumor perfusion, evaluated with [18F]fluoromethane PET, decreased within 24 hours of starting AZD2171 (56). As a once-daily oral therapy for the treatment of cancer, toxicity reported in a phase I trial consisted mainly of grade 3/4 hypertension (21% of the patients), as well as diarrhea, hoarseness and headache (57). Objective responses were reported in 2 out of 83 patients while 50% of them were stabilized. When combined with the EGFR inhibitor gefinitib (250 mg/day), dose-dependent hypertension was also reported (58). This doublet seemed, however, globally safe and may be further investigated in phase II trials. In combination with the paclitaxel–carboplatin combination, AZD2171 can be administered at the full dose of 45 mg/dose in NSCLC patients (59). AZD2171 has also been successfully combined to FOLFOX 4, pemetrexed, irinotecan and docetaxel in another phase I trial (60). FUTURE DEVELOPMENT Combination of Antiangiogenic Agents Antibodies and tyrosine kinase inhibitors that target the VEGF signaling pathway could be associated on the basis that the mechanism, spectrum of activity, and spectrum of toxicity of each agent is partially different. The significant antitumor activity of sunitinib in patients who had progressed on bevacizumab highlights different therapeutic spectrums and strengthens the rational to combine the two agents (61). This concept has been labeled }vertical blockade,} since the same pathway is targeted at two or more different levels by two or more different agents. In a phase I study, sorafenib was combined with bevacizumab (62). A significant increase in expected single-agent toxicity with mostly sorafenib toxicities was observed when adding bevacizumab. The maximal tolerated dose was sorafenib 200 mg b.i.d. and bevacizumab 5 mg/kg every two weeks. Grade 2–4 hypertension and hand–foot skin reaction were frequent and dose limiting (17/38 and 11/38, respectively), grade 3 proteinuria and thrombocytopenia were also dose limiting. A clinical benefit was seen in 34 out of 37 patients (92%) but PR was restricted to patients with ovarian cancer (4/14, 29%) leading to a subsequent phase II trial in this tumor type. Combination with Chemotherapy Bevacizumab activity has been demonstrated in phase III trials in combination with chemotherapy in colorectal cancer, NSCLC, and breast cancer (63–65). It has also demonstrated activity given as a single agent in renal cell carcinoma in a randomized phase II trial (66). However, large phase III trials combining valatinib with chemotherapy have failed to demonstrate an OS benefit, whereas different agents given alone reached this goal (13,33,67). It is striking to note that no trial with EGFR tyrosine kinase inhibitors has been positive when given concurrently with platinum-based chemotherapy, even if EGFR tyrosine kinase inhibitors trials were positive when given as a single agent (68–70). This raises the possibility of a class characteristic and favors the sequential regimen compared to the concomitant regimen when combining platinum-based chemotherapy and kinase inhibitors.

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Optimize the Use of Oral Antiangiogenic Agents Many issues are left unresolved at this time: What is the optimal duration of oral antiangiogenic agents? What is the role of oral antiangiogenic agent in the adjuvant setting and thus their long-term secondary effects? Are usual response criteria adequate to evaluate oral antiangiogenic efficacy? What are the biological or radiological predictive factors of efficacy? One of the key points is the definition of predictive factors to select the patients who will benefit from antiangiogenic treatments, given the toxicity profile of those agents. Most studies have focused on plasma markers, such as VEGF, soluble receptors (sVEGFR), and IL-8. Initial level of sVEGFR-2 and VEGFR were not predictive of a sensitivity to sorafenib (9). VEGF level increases at initiation of the treatment, but its predictive value remains debated (71,72). Circulating levels of endothelial cells (CEC) have not been related to sunitinib efficacy (73). BRAF mutations, frequent in melanoma, have not been correlated to sorafenib activity (7). Few tissue markers have been investigated even if activation of downstream markers may be of interest (8). The area of predictive markers of antiangiogenic therapy efficacy is yet to be fully explored. CONCLUSION During the past several years, rapid progress has been achieved in the understanding of angiogenesis, including signaling pathways and their regulation. This has enabled the development of numerous potentially interesting agents, many of which are oral agents. Angiogenesis-targeting is now a clinical reality accessible to more and more patients, due to formal approval of agents such as bevacizumab, sorafenib, and sutent. Further advances are awaited in the field. In particular, resistance to antiangiogenic treatments is now an established clinical reality. The molecular basis of this resistance needs to be better understood. Mutations of the p53 protein (observed in 50% of human cancers), which lower tumor cell oxygen requirements and thus their dependence on neovascularization, could be debated. The induction of bcl-2, a gene involved in resistance to apoptosis, has also been observed. Cross activation between the different signaling pathways must also be further elucidated. Preclinical and clinical studies show that VEGFR and EGFR inhibitors can be combined to enhance their efficacy (74,75). One of the present challenges is also to determine whether it is best to simply combine these new antiangiogenic agents or to combine them with conventional cytotoxics so that patient survival can be increased significantly. Ongoing clinical trials are applying these concepts with the prospect of using these antiangiogenic therapies in clinical practice. ACKNOWLEDGMENT The authors thank Lorna Saint Ange for editing. REFERENCES 1. 2.

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as second-line monotherapy in patients (pts) with stage IIIB or stage IV non-small-cell lung cancer (NSCLC). Ann Oncol 2006; 17:ix218–19. Joensuu H, De Braud F, Coco P, et al. A phase II, open-label study of PTK787/ ZK222584 in the treatment of metastatic gastrointestinal stromal tumors (GISTs) resistant to imatinib mesylate. J Clin Oncol (Meeting Abstracts) 2006; 24(18_suppl):9531. Longo R, Sarmiento R, Fanelli M, Capaccetti B, Gattuso D, Gasparini G. Antiangiogenic therapy: rationale, challenges and clinical studies. Angiogenesis 2002; 5:237–56. Kuenen BC, Giaccone G, Ruijter R, et al. Dose-finding study of the multitargeted tyrosine kinase inhibitor SU6668 in patients with advanced malignancies. Clin Cancer Res 2005; 11:6240–6. Godl K, Gruss OJ, Eickhoff J, et al. Proteomic Characterization of the angiogenesis inhibitor SU6668 reveals multiple impacts on cellular kinase signaling. Cancer Res 2005; 65:6919–26. Holden SN, Eckhardt SG, Basser R, et al. Clinical evaluation of ZD6474, an orally active inhibitor of VEGF and EGF receptor signaling, in patients with solid, malignant tumors. Ann Oncol 2005; 16:1391–7. Miller KD, Trigo JM, Wheeler C, et al. A multicenter phase II trial of ZD6474, a vascular endothelial growth factor receptor-2 and epidermal growth factor receptor tyrosine kinase inhibitor, in patients with previously treated metastatic breast cancer. Clin Cancer Res 2005; 11:3369–76. Heymach JV, Johnson BE, Prager D, et al. A phase II trial of ZD6474 plus docetaxel in patients with previously treated NSCLC: follow-up results. J Clin Oncol (Meeting Abstracts) 2006; 24(18_suppl):7016. Nakagawa K, Kiura K, Shinkai T, et al. A randomized double-blind phase IIa dosefinding study of ZD6474 in Japanese patients with NSCLC. J Clin Oncol (Meeting Abstracts) 2006; 24(18_suppl):7067. Natale RB, Bodkin D, Govindan R, et al. ZD6474 versus gefitinib in patients with advanced NSCLC: Final results from a two-part, double-blind, randomized phase II trial. J Clin Oncol (Meeting Abstracts) 2006; 24(18_suppl):7000. Rugo HS, Herbst RS, Liu G, et al. Phase I trial of the oral antiangiogenesis agent AG-013736 in patients with advanced solid tumors: pharmacokinetic and clinical results. J Clin Oncol 2005; 23:5474–83. Rini B, Rixe O, Bukowski R, et al. AG-013736, a multi-target tyrosine kinase receptor inhibitor, demonstrates anti-tumor activity in a phase 2 study of cytokine-refractory, metastatic renal cell cancer (RCC). J Clin Oncol (Meeting Abstracts) 2005; 23(16_suppl):4509. Rixe O, Meric JB, Bloch J, et al. Surrogate markers of activity of AG-013736, a multitarget tyrosine kinase receptor inhibitor, in metastatic renal cell cancer (RCC). J Clin Oncol (Meeting Abstracts) 2005; 23(16_suppl):3003. Liu G, Rugo HS, Wilding G, et al. Dynamic contrast-enhanced magnetic resonance imaging as a pharmacodynamic measure of response after acute dosing of AG-013736, an oral angiogenesis inhibitor, in patients with advanced solid tumors: results from a phase I study. J Clin Oncol 2005; 23:5464–73. Kim S, Rosen LS, Cohen EE, et al. A Phase II study of axitinib (AG-013736), a potent inhibitor of VEGFRs, in patients with advanced thyroid cancer. J Clin Oncol (Meeting Abstracts) 2006; 24(18_suppl):5529. Spano J, Moore M, Kim S, et al. A phase I study of axitinib (AG-013736), a potent inhibitor of VEGFRs, in combination with gemcitabine (GEM) in patients (pts) with advanced pancreatic cancer. J Clin Oncol (Meeting Abstracts) 2006; 24(18_suppl):13092. Tolcher A, O'Leary JJ, deBono JS, et al. A phase I and biologic correlative study of an oral vascular endothelial growth factor-2 (VEGFR-2) tyrosine kinase inhibitor, CP—547,632, in patients with advanced solid tumors. J Clin Oncol (Meeting Abstracts) 2003; 21. Cohen RB, Langer CJ, Simon GR, et al. A phase I/randomized phase II, noncomparative, multicenter, open label trial of CP-547,632 in combination with paclitaxel

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60.

61. 62. 63. 64. 65.

66. 67. 68. 69.

70. 71.

72.

Besse and Armand

and carboplatin or paclitaxel and carboplatin alone as first-line treatment for advanced non-small cell lung cancer (NSCLC). Cancer Chemother Pharmacol 2006; 60:81–9. Wedge SR, Kendrew J, Hennequin LF, et al. AZD2171: a highly potent, orally bioavailable, vascular endothelial growth factor receptor-2 tyrosine kinase inhibitor for the treatment of cancer. Cancer Res 2005; 65:4389–400. Miller KD, Miller M, Mehrotra S, et al. A physiologic imaging pilot study of breast cancer treated with AZD2171. Clin Cancer Res 2006; 12:281–8. Drevs J, Medinger M, Mross K, et al. Phase I clinical evaluation of AZD2171, a highly potent VEGF receptor tyrosine kinase inhibitor, in patients with advanced tumors. J Clin Oncol (Meeting Abstracts) 2005; 23(16_suppl):3002. van Cruijsen H, Voest EE, van Herpen CML, et al. Phase I clinical evaluation of AZD2171 in combination with gefitinib, in patients with advanced tumors. J Clin Oncol (Meeting Abstracts) 2005; 23(16_suppl):3030. Laurie SA, Arnold A, Gauthier I, et al. Final results of a phase I study of daily oral AZD2171, an inhibitor of vascular endothelial growth factor receptors (VEGFR), in combination with carboplatin (C) þ paclitaxel (T) in patients with advanced non-small cell lung cancer (NSCLC): A study of the National Cancer Institute of Canada Clinical Trials Group (NCIC CTG). J Clin Oncol (Meeting Abstracts) 2006; 24(18_suppl):3054. Lorusso PM, Heath E, Valdivieso M, et al. Phase I evaluation of AZD2171, a highly potent and selective inhibitor of VEGFR signaling, in combination with selected chemotherapy regimens in patients with advanced solid tumors. J Clin Oncol (Meeting Abstracts) 2006; 24(18_suppl):3034. Rini BI, George DJ, Michaelson MD, et al. Efficacy and safety of sunitinib malate (SU11248) in bevacizumab-refractory metastatic renal cell carcinoma (mRCC). J Clin Oncol (Meeting Abstracts) 2006; 24(18_suppl):4522. Azad NS, Posadas EM, Kwitkowski VE, et al. Increased efficacy and toxicity with combination anti-VEGF therapy using sorafenib and bevacizumab. J Clin Oncol (Meeting Abstracts) 2006; 24(18_suppl):3004. Sandler A, Gray R, Perry MC, et al. Paclitaxel-carboplatin alone or with bevacizumab for non-small-cell lung cancer. N Engl J Med 2006; 355:2542–50. Hurwitz H, Fehrenbacher L, Novotny W, et al. Bevacizumab plus irinotecan, fluorouracil, and leucovorin for metastatic colorectal cancer. N Engl J Med 2004; 350:2335–42. Miller KD, Wang M, Gralow J, Dickler M. A randomized phase III trial of paclitaxel versus paclitaxel plus bevacizumab as first-line therapy for locally recurrent or metastatic breast cancer: a trial coordinated by the Eastern Cooperative Oncology Group (E2100). Proc San Antonio Breast Cancer Symp 2005:3a. Yang JC, Haworth L, Sherry RM, et al. A randomized trial of Bevacizumab, an antivascular endothelial growth factor antibody, for metastatic renal cancer. N Engl J Med 2003; 349:427–34. Escudier B, Szczylik C, Eisen T, et al. Randomized phase III trial of the Raf kinase and VEGFR inhibitor sorafenib (BAY 43-9006) in patients with advanced renal cell carcinoma (RCC). J Clin Oncol (Meeting Abstracts) 2005; 23(16_suppl):LBA4510. Herbst RS, Prager D, Hermann R, et al. TRIBUTE: a phase III trial of erlotinib hydrochloride (OSI-774) combined with carboplatin and paclitaxel chemotherapy in advanced non-small-cell lung cancer. J Clin Oncol 2005; 23:5892–99. Gatzemeier U, Pluzanska A, Szczesna A, et al. Results of a phase III trial of erlotinib (OSI-774) combined with cisplatin and gemcitabine (GC) chemotherapy in advanced non-small cell lung cancer (NSCLC). J Clin Oncol (Meeting Abstracts) 2004; 22(14_suppl):7010. Shepherd FA, Rodrigues Pereira J, Ciuleanu T, et al. Erlotinib in previously treated non-small-cell lung cancer. N Engl J Med 2005; 353:123–32. DePrimo SE, Friece C, Huang X, et al. Effect of treatment with sunitinib malate, a multitargeted tyrosine kinase inhibitor, on circulating plasma levels of VEGF, soluble VEGF receptors 2 and 3, and soluble KIT in patients with metastatic breast cancer. J Clin Oncol (Meeting Abstracts) 2006; 24(18_suppl):578. Drevs J, Zirrgiebel U, Schmidt-Gersbach CIM, et al. Soluble markers for the assessment of biological activity with PTK787/ZK 222584 (PTK/ZK), a vascular endothelial

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74. 75.

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16

Endothelial Cell Propagation Targeting Gordon C. Tucker Cancer Drug Discovery Department, Institut de Recherches Servier, Croissy-sur-Seine, France

INTRODUCTION Endothelial cell (EC) propagation requires a fine adhesive balance during tumor progression. On the one hand, cell-to-cell contacts must be maintained to prevent loss of blood content—at least during the initial phases of tumor angiogenesis, whereas in more advanced stages, immature tumor vessels can be leaky. On the other hand, interactions with the extracellular matrix allow the expansion of microvessels towards and into solid tumor masses. The strength of the adhesive contacts will dictate whether ECs can migrate or not. Too strong an adhesion, whether intercellular or to the matrix, will prevent cell movement. The same goes for total loss of EC–matrix contact, with the additional consequence that this configuration will also trigger anoikis (homelessness in Greek), i.e., a process of active cell death induced to prevent the propagation of most cohesive normal cells into the body (1,2). Therefore, during neovascularization, ECs need appropriate receptors to interact with the extracellular matrix for migration and survival. They also need to shift from a resting state to a motile phenotype. The intracellular cytoskeleton must respond appropriately. Integrators between the outside and inside of the EC membrane would ideally perform this task. Such integrators do exist and constitute the main receptors for the extracellular matrix: the integrins (3). To gain access to tumor nodules, ECs also need the opening of pathways for cell migration. This event is triggered by the production of specific enzymes altering the extracellular matrix and released by ECs or tumor cells; for instance, matrix metalloproteinases, or MMPs (4) and heparanase (5)—clinical development of inhibitors of these enzymes as antiangiogenic and antimetastatic compounds is ongoing (see chapter 7 for an update on MMP inhibitors and comments in Miao et al. (6) for the PI-88 heparanase inhibitor currently in phase II clinical trials in patients with melanoma, liver, prostate, or lung cancers). This happens in the early stage of tumor angiogenesis when the underlying basement membrane is ruptured to allow microvascular cell escape, and also during the propagation stage itself. The ECs are then confronted with a new environment when compared to their quiescent state in the vessel (connective tissue and tumor stroma extracellular matrices vs. basement membranes). In addition to these events initially taking place near small tumor nodules (Fig. 1), the abnormal nature of solid tumors—often described as wounds that do not heal—can also exacerbate the remodeling of the matrix. In some cases, the matrix is altered by tumor-secreted proteases to the point that massive fragments can escape and reach the circulation; thus, bathing the body and potential metastases with new molecular entities. Some of these primary tumor-produced proteins have been shown to alter metastatic growth by blocking angiogenesis 257

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FIGURE 1 Angiogenesis: a balance between pro- and antiangiogenic factors. In response to the tumor-induced production of angiogenic factors, endothelial cells (ECs) converge as microvascular structures towards primary tumors or metastases. During this step, ECs adhere to and migrate on the extracellular matrix components via specific cell surface receptors. In addition, attachment is crucial for the survival of ECs (loss of matrix engagement triggers the process of active cell death). Primary tumors also secrete degrading enzymes remodeling the local matrix (basement membranes and interstitial matrix) or released into the circulation. As a result, some endogenous proteins in the matrix (like collagens) or in the circulation (like plasminogen) are fragmented. Some of these fragments are endowed with antiangiogenic properties that can act on metastases. Antiangiogenic proteins are also naturally present in the interstitial matrix, like thrombospondin. Black arrows point to the sites of intervention for the EC propagation inhibitors discussed in the text. At the matrix level (location 1), the cell surface adhesion receptors called integrins can be antagonized by antibodies (like Vitaxin
, Abegrin, CNTO 95, or Volociximab), peptides (like Cilengitide or ATN-161), or peptidomimetics, or modulated at the expression level (as with E7820) in order to prevent EC migration and survival. Another possibility to hinder EC propagation is to administer exogenous peptides with antiangiogenic potential (location 2) to shift the balance towards tumor growth arrest: recombinant forms of the protein fragments mentioned above, like plasminogen-derived angiostatin or collagen XVIII-derived endostatin (and its variant Endostar), or small peptides derived from natural angiogenic inhibitors (like the thrombospondin-derived ABT510 compound).

(7,8). If not produced locally near the tumor, they can be generated in the circulation through the action of tumor-derived proteases released into the vascular compartment (Fig. 1). The first example of such angiogenesis inhibitors is angiostatin, a fragment of circulating plasminogen (9). Again, among other mechanisms, endothelial receptors for the extracellular matrix, like integrins, were shown to participate in the antiangiogenic action of some of these compounds. Integrins are the main focus of the present chapter. The description of the integrin target strategy is followed by a short incursion into the field of large,

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tumor-derived protein fragments endowed with antiangiogenic potential, like angiostatin and endostatin (9–11). Finally, because it also reached clinical trial as an anti-EC propagation compound, an artificial peptidic fragment derived from the endogenous angiogenesis inhibitor thrombospondin (8,12) is described. In all cases, the emphasis is put on clinical achievements. INTEGRINS AS ENDOTHELIAL CELL PROPAGATION TARGETS The Integrin Family Integrins are cell-surface heterodimers of the so-called a and b subunits whose association dictates the nature of the ligands recognized (3). The protein sequences and structures of the a or b subunit types are similar. Only a subset of ab arrangements have been described, amounting to 24 identified members of the integrin family (Fig. 2). Their cellular expression is more or less specific and nonexclusive. For instance, platelets will selectively express the aIIbb3 integrin (responsible for platelet aggregation through recognition of fibrinogen dimers bridging platelets together); leukocytes express a subset of integrins formed

αE

α2β1 = VLA-2 ↔

β7 α4 α1

↔

α5β1 = VLA-5, FNR

α2

FN, endostatin

α3 α5

β1

α6

αL

β4

αD

α7 α8

β6

αIIb

αM αX

↔

β5

β8 β3

FN, VN, Fg OPN, vWF, TSP, tumstatin

Activated

αvβ3 = VNR ↔

α10

αv

β2

Upregulated & activated

αvβ5

α9 α11

Activated

Coll, LN

FN, VN, Fg OPN, vWF, TSP, endostatin

De novo expressed & activated

FIGURE 2 The integrin family. The lines between a and b subunits denote possible heterodimers associations. Solid lines indicate integrins that can be expressed by endothelial cells (ECs). Some have been shown to participate in EC cell propagation during pathological angiogenesis. Additional information on ligands, other names, and regulation during angiogenesis is provided for the integrins targeted in clinical trials, i.e., a2b1, a5b1, avb3, and avb5. Abbreviations: Coll, collagens; Fg, fibrinogen; FN, fibronectin; FNR, fibronectin receptor; LN, laminins; OPN, osteopontin; TSP, thrombospondin; VLA, very late antigen; VN, vitronectin; VNR, vitronectin receptor; vWF, von Willebrand factor.

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Tucker

around the b2 subunit, thus enabling them to interact with other cell types; but other integrins, like the fibronectin receptor a5b1, are more promiscuous. Considered initially as a mechanical link between the cytoskeleton and the extracellular matrix or other cell types, integrins were subsequently shown to act as classical receptors involved in signal transduction (13). The number of known cytoplasmic partners increased progressively, and complex arrangements of proteins are now depicted for, on the one hand, actin coupling in relation to cell adhesion, spreading, and movement and, on the other hand, for signaling. Functions assumed by the integrins range from the interaction with the environment (cell adhesion and motility) to other cell behaviors such as proliferation, survival or cell death, translating in tissue events linked to tumor progression as diverse as angiogenesis, tumor invasion, and metastasis (14–16). The activity of integrins is further complicated by the existence of distinct conformations, active and inactive (17). For instance, resting platelets express inactive aIIbb3 integrins that can be rapidly activated to engage in aggregation. Activation apparently results from a conformational change from a bent or folded state to an extended configuration of the integrin subunits, an event facilitated by the existence of a hinge between extracellular domains of both integrin subunits (18). Accompanying changes in the proximity of the short cytoplasmic tail domains modulate the intracellular binding of protein partners involved in cytoskeleton coupling and signaling. These changes are brought about by insideout (e.g., growth factor signaling) and outside-in (integrin ligation) signaling (19). In an active state, integrins bind their extracellular ligands with higher affinity. Relevance and Role of Integrins in Tumor Angiogenesis The role of integrins is not limited to angiogenesis during tumor progression but a subset of integrins have been associated with ECs during neovascularization (20,21). In vitro and in situ descriptive work showed constitutive integrin expression on quiescent microvessels (low levels of a1b1, a3b1, a5b1, a6b1, a6b4, and barely detectable levels of a2b1, avb3, avb5). Integrins can be either downregulated (a1b1, a6b4), upregulated (a5b1, a6b1), or de novo expressed (avb3, a4 integrins) on ECs during angiogenesis (16,22,23). The expression of some integrin ligands can also be modulated during tumor progression. For instance, the ligand fibronectin is upregulated along with its receptor, the a5b1 integrin (24). The situation is more complicated for other integrins that recognize many ligands, such as the avb3, and avb5 integrins. In addition, integrin activation can occur through the action of angiogenic factors; for instance, VEGF activates the a2b1, a5b1, avb3, and avb5 integrins (25). This pattern of expression and its modulation during angiogenesis must be considered as a general overview not reflecting the possible disparities from one tissue to another. Moreover, the repertoire of endothelial integrins expressed or activated depends on the pathophysiological context, as well as the progression of the pathology. Thus, at least in mouse models, the avb3 integrin can be de novo expressed during tumor angiogenesis, but not liver regeneration or healing (26), and its pattern of expression varies as a function of time during angiogenesis associated with retinopathy (27). Similar differences may occur during human tumor progression. Changes in pattern of integrin expression do not necessarily mean that integrins participate in angiogenesis. Knock-out experiments demonstrated causation in the process of angiogenesis for some integrins during embryonic development

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(a4, a5, a9, av, and b8) or tumor angiogenesis (for a1) and not mere epigenetic association (21). On the other hand, although the use of specific integrin antagonists confirmed avb3 or avb5 as targets to inhibit during pathological angiogenesis [reviewed in (21)], this conclusion was challenged by experiments conducted with b3 and b3/b5 knock-out animals: tumor angiogenesis was enhanced (28,29). This reveals that the mechanism thought to explain the role of the avb3 and avb5 integrins during angiogenesis is insufficiently understood. Several hypotheses have been advanced to explain the discrepancies between antagonist and knock-out experiments [reviewed in (22,23,30–32)]: compensation for the loss of integrins during development by other angiogenic pathways exacerbated during pathological angiogenesis or integrin-mediated death (33), a process whereby unligated integrins recruit caspase-8 to induce cell death. Whether unligated avb3 integrins exist in vivo is not known, but, if this happens, eradication of the integrin will suppress this pro-apoptotic pathway and enhance angiogenesis. Interestingly, the concept of vascular integrins as survival receptors has evolved to the point of considering them as dependence receptors (34). By acting as biosensors of the local extracellular matrix, some integrins, such as a5b1 and avb3, can transmit life and death signals. It is again a fine balance between the type of ligands encountered; some bound to the matrix scaffold—thus favoring survival—and others soluble after matrix degradation— soluble ligands can promote active cell death directly, independently of anoikis. The latter property may not necessarily be shared by all integrin ligands, especially small antagonists (35). The precise details of av integrin function in cell behavior are thus still not entirely known. However, the antiangiogenic success of preclinical experiments performed with various integrin inhibitors (antibodies, peptides, small heterocyclic peptidomimetics) directed against a2b1, a5b1, avb3, and avb5 convinced clinicians to evaluate integrin antagonists as antiangiogenic drugs. Incidentally, the term antagonist may be a misnomer if these integrins can play some part in angiogenesis as negative regulators—the compounds could act as agonists of negative regulatory functions rather than as antagonists of integrin positive functions like survival. A summary of the rationale for targeting some of the integrins is provided in Table 1. Of all the integrin antagonists tested in animal models, only antibodies and peptides made it to the clinic (Table 2). The host of heterocyclic inhibitors modeled as peptidic mimetics to target the avb3 integrin and described in the literature [examples in (36,37)] prudently stayed away from clinical trials, at least in oncology—one specific anti-avb3 integrin compound (SB-273005; GlaxoSmithKline, King of Prussia, Pennsylvania, U.S.A.) did enter a phase I trial for osteoporosis however, but its development was halted for undisclosed reasons. This may be envisioned as a cautious behavior in the light of the paucity of clinical favorable responses obtained with the first antagonists evaluated. Developing an antiangiogenic compound is a slow process as exemplified by the duration between the first clinical trial of bevacizumab (the Avastin anti-VEGF antibody) in April 1997 and its drug approval in 2004. Combination therapy was crucial to demonstrate the benefit of this compound. This might prove to be crucial for integrin antagonists too, as exemplified by preclinical data (38–40). Finally, when interpreting clinical—or preclinical—data, it must be kept in mind that integrins can be expressed by the tumor cell themselves—thus facilitating their motile or invasive capacities, as during melanoma vertical growth or metastasis—and also by some stromal cells. An example of the importance of the (Text continues on page 266)

Some endothelia, epithelia, and leukocytes, osteoclasts, fibroblasts, platelets, smooth muscle cells

Some endothelia, epithelia, and leukocytes, osteoclasts, platelets, smooth muscle cells

avb5

Normal human tissue distribution (adapted from Refs. 32,33)

avb3

Type

Activated by some angiogenic factors

de novo expressed and activated by some angiogenic factors

Modulation of expression on tumor endothelial cells (16,20–23)

Expressed in some carcinomas and associated with poor prognosis in some tumor types

Expressed in melanoma and some carcinomas and associated with poor prognosis in some tumor types

Expression in human tumor (adapted from Ref. 133)

TABLE 1 Rationale for Integrin Targeting during Endothelial Cell Progression

av subunit null: embryonic (E10) or perinatal lethal, placental or cerebral vascular defects; b3 subunit null: viable and fertile, hemorrhage, enhanced tumor growth av subunit null: see above; b5 subunit null: viable and fertile without obvious defects, enhanced tumor growth in the b3 null context

Mouse knockout studies (adapted from Refs. 3,135)

Antiangiogenic and antitumor activity demonstrated with numerous antibody, peptide, and non peptide antagonists

Antiangiogenic and antitumor activity demonstrated with numerous antibody, peptide, and non peptide antagonists

Effects of antagonists in preclinical models (reviewed in Ref. 21)

262 Tucker

Epithelium, endothelium, fibroblasts, cells of hematopoietic origin, platelets

a2 subunit

Activated by some angiogenic factors but lost in some tumors

Upregulated along with its ligand fibronectin (24) and activated by some angiogenic factors

Overexpressed—but possibly downregulated—in some tumors depending on the progression (e.g., elevated during melanoma invasion and in RCC with worsening clinical grade) Reduced levels in carcinomas (e.g., during tumor progression in the mammary gland) Antiangiogenic and antitumor activity demonstrated with antibody and peptide antagonists

Antiangiogenic activity demonstrated with an antibody antagonist (134) and the E7820 expression modulator (61)

a5 subunit null: embryonic lethal (E10-11) with vascular defects (dilated and disrupted endothelial tubes); b1 subunit null: embryonic lethal (E5.5) (note that b1 binds many a subunits, Fig. 2) a2 subunit null: viable with few immediate obvious developmental defects; no report of effect on tumor growth

Note: De novo expression or activation of the avb3 and avb5 integrins during tumor angiogenesis has been demonstrated in some but not all human cancers. In some cases, expression of av integrins correlates with a poor prognosis. The somewhat surprising results obtained with tumor grafts in b3 and b3/b5-null animals (enhanced tumor growth) blurred the previously straightforward accepted mechanism of action of these integrins during angiogenesis (see text for comments). Nevertheless, preclinical experiments with various antagonists validated the concept of targeting the avb3 and avb5 integrins to reduce tumor angiogenesis, at least in animal models. The same goes for the other two integrins targeted in the clinic, a5b1 and a2b1. However, a5b1 expression is not systematically enhanced in human tumors—it can be down-regulated in some instances. Contrary to avb3 and avb5 integrins, knock-out studies for a5b1 point to a positive regulatory role of this integrin in angiogenesis, but the embryonic lethality of the knock-out animals did not allow to study the effect of the integrin loss during tumor progression. The a2 subunit is generally expressed at low levels on tumor endothelial cells and downregulated in tumors. However, angiogenic factors like vascular endothelial growth factor (VEGF) can activate it, thus enabling some tumors to use it during angiogenesis. Since studies in a2-null animals failed to reveal abnormalities in vessels, and no report has been published on tumor growth in these animals, the most compelling evidence for its participation in tumor angiogenesis comes from the use of inhibitors of the a2b1 integrin (blocking antibodies and the expression modulator E7820) in animal models.

Widely distributed

a5b1

Endothelial Cell Propagation Targeting

263

Humanized version of the LM609 murine monoclonal antibody

Humanized version of the LM609 murine monoclonal antibody

Cyclic RGD peptide

Human monoclonal antibody

Humanized monoclonal antibody

Peptide Ac-PHSCN-NH2

Abegrin (MEDI-522, etaracizumab)

Cilengitide EMD 121974

CNTO 95

Volociximab Eos-200-4 M-200

ATN–161

Type

Vitaxin (MEDI-523)




Name(s)

Phase I started November 2002—phase II for the treatment of advanced melanoma and androgenindependent prostate cancer Phase I started August 1998—phase II for the treatment of solid tumours and acute myeloid leukaemia Phase I started December 2003—phase I/II in combination with dacarbazine in patients with stage IV melanoma started in April 2005 Phase I started May 2003— various phase II for the treatment of solid tumours Phase I started January 2003—phase II in advanced renal cell cancer, I/II in malignant melanoma

avb3 integrin

a5b1 & avb3 integrins

a5b1 integrin

av integrins

avb3 and avb5 integrins

Phase I started April 1997— phase II for the treatment of solid tumours

Clinical status

avb3 integrin

Target

TABLE 2 Clinical Trial Status of Inhibitors of Endothelial Cell Propagation Described in the Text

Scripps Research Institute; Applied Molecular Evolution; MedImmune, Inc. MedImmune, Inc.

Company

Well tolerated up to 10 mg/ kg for 2 wk, higher dose explored Safe

Good tolerance in phase I

Attenuon

Protein Design Labs

Centocor; Medarex

Safe and potential benefit for Merck KGaA; EMD glioma treatment (Orphan Pharmaceuticals; Drug designation) National Cancer Institute

Safe and potential benefit for metastatic melanoma

Safe but anecdotal response

Outcome

264 Tucker

Recombinant protein

Recombinant protein

Recombinant modified endostatin

Adenoviral construct

Peptide fragment from TSP

Angiostatin

Endostatin

Endostar

Endostatin gene therapy

ABT-510

CD36

Idem

Idem

a5b1, other receptors

Integrins(?), angiomotin

a2 integrin subunit

Phase I started—phase II in progress

Phase I started in May 2005

Phase I started January 2004—phase I/II planned in colorectal cancer Phase I started in 2000— phase II/development hindered by production issues Phase I started in 1999– phase II in 2002 in melanoma and neuroendocrine tumors; clinical studies stopped in February 2005 because of production issues Phase I started in 2001— phase III in lung cancer EntreMed

Safe, but no objective response found

No additional toxicity in combination chemotherapy, Orphan Drug designation for soft tissue sarcoma

Delayed disease progression, approved by the Chinese FDA in non– small-cell lung cancer in combination with chemotherapy Well tolerated

Sun Yat-sen University; Doublle Bioproduct Abbott Laboratories

Yantai MedGenn

EntreMed Safe and long-term stabilization in some patients, slow tumor shrinkage, but considered a “no response” according to traditional nomenclature

Eisai Medical Research, Inc.

Safe up to 100 mg/day (MTD ¼ 200 mg/day)

Note: No objective responses were observed so far with integrin antagonists or modulators in phase I and II clinical trials. With respect to the clinical development of antiangiogenic protein fragments, clinicals trials in the USA for angiostatin and endostatin were halted because of industrial production issues. However, an optimized version of endostatin, Endostar, has been evaluated in China, as well as an adenoviral construct for endostatin gene therapy (see text for details). Abbreviations: FDA, Food and Drug Administration; TSP, thrombospondin.

Aromatic sulfonamide derivative

E7820

Endothelial Cell Propagation Targeting

265

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avb3 integrin during local tissue growth is its role during osteoclast recruitment elicited by tumor cells and subsequent bone destruction in skeletal metastases (41). Incidentally, in this context, experiments with b3 knockout animals and avb3 integrin antagonists are reconciled (42), contrary to the angiogenic context as mentioned earlier. Also, since antiangiogenic compounds can affect the tumor response to ionizing radiation (43), integrin antagonists have been evaluated in preclinical models in combination with radioimmunotherapy (44) or external beam radiotherapy (45–47). Such combinations were shown to increase the efficiency of radiotherapy. Clinical Achievements with Integrin Antagonists Compounds in the Clinic The most advanced integrin antagonists in clinical trials are anti-avb3 compounds (Table 2). One is a monoclonal antibody, MEDI-522 or Abegrin, an affinityoptimized version of the former Vitaxin
antibody (MEDI-523) (48). Both antibodies are humanized antibodies derived from the LM609 murine antibody. MEDI-523 is claimed to be more stable and has a 7.2-fold greater affinity to avb3 than MEDI-523 (49,50). The second is a small cyclic pentapeptide comprising the three amino acid–long sequence Arg-Gly-Asp (RGD) present in the ligands of its targets, the integrins avb3 and avb5. It is called EMD 121974 or Cilengitide, cyclo(RGDf-N(Me)V) in amino acid letter coding (51). The earliest phase I trial for an integrin antagonist dates back to 1997 and implied the anti-avb3 antibody Vitaxin. A year after, the cyclic peptide Cilengitide entered human clinical trial. Both compounds are still in phase II. The latest antiavb3 compound entering clinical trial for cancer is another antibody, the human monoclonal antibody CNTO 95, that targets the av integrin subunit (52), thus enlarging the potential activity to other av integrins besides avb3 and avb5. ATN161 or Ac-PHSCN-NH2 in amino acid letter coding, a peptide derived from the fibronectin sequence (53–59) also entered clinical trial in 2003. ATN-161 targets the fibronectin receptor a5b1, but it is also claimed to inhibit other integrins, like avb3 and, contrary to the previous antibodies and peptide, to block integrin-dependent signalling and not integrin-dependent adhesion. Later on in 2003–2004, compounds inhibiting the integrin a5b1 or the a2 subunit were introduced into the clinic: the humanized Volociximab monoclonal antibody (60) and the aromatic sulfonamide derivative E7820 (61,62), respectively. An overview of the clinical achievements with each of these compounds follows. VITAXIN (MEDI-523) AND ABEGRIN (MEDI-522) The first published phase I of the anti-avb3 Vitaxin antibody (MEDI-523) involved 17 patients with advanced (stage IV), incurable malignancies refractory to standard therapy who were treated with 6 weekly infusion doses ranging from 0.1 to 4 mg/kg (63). Among the 14 evaluable patients, 7 disease stabilizations and 1 partial response are reported. Limited drug supply at the time did not permit to go higher than 4 mg/kg/wk. The pharmacokinetics were not different from those of other humanized monoclonal antibodies, with a dose-dependent half-life ranging from 14 (0.1 mg/kg) to 138 hours (4 mg/kg). The optimum dose or schedule could not be determined, but Vitaxin treatment appeared safe. The most frequent

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adverse effect reported were infusion-related fever, chills, nausea, and flushing. For three patients receiving continued treatment beyond the 6 weekly doses, no significant adverse effects were noted. Interestingly, with the exception of one patient, and in fact for only one of his biopsies, no increase in bleeding or woundhealing inhibition was observed after biopsies, suggesting that antiangiogenic treatment with Vitaxin does not increase the risk of bleeding. The partial response concerned a patient with a leiomyosarcoma metastatic to the liver and lasted till 93 weeks of treatment. At that time, the measurable lesions remained stable but the disease progressed outside of the liver and treatment was discontinued. A subsequent pilot study (64) conducted in 15 patients with advanced leiomyosarcomas failed to show objective responses or any significant stabilization after administration of Vitaxin (0.25 mg/kg/wk intravenously) for a median of six months and no concurrent therapy. Another pilot trial (65) in nine patients with metastatic cancer for which there was no standard therapy also failed to elicit objective responses after 1–4 administrations of Vitaxin (10, 50, or 200 mg every three weeks). The three patients who received four administrations (50 or 200 mg) had stable disease at day 85 when taken off study. The trial again showed that the treatment was well tolerated and that no immune response to Vitaxin was elicited. During the same trial, the patients received a low dose of 1 mg of 99Tcm-radiolabeled Vitaxin prior to treatment in an attempt to visualize the distribution of the compound in the tumor vasculature. The 99 Tcm labeling appeared unstable in vivo and imaging of the tumor vasculature was unsuccessful, although a tumor was localized in a melanoma patient possibly because the tumor cells expressed the avb3 targeted integrin. Altogether, the anecdotal responses obtained with the Vitaxin antibody may reflect the use of a suboptimal regimen of administration or nonoptimal stability or affinity. MEDI-523 (Abegrin) is supposed to cope with the two latter issues. In an open-label phase I dose escalation study of 25 treatment-refractory patients with solid tumors, no significant toxicities was observed with MEDI-522 administered at 2–10 mg/kg/wk intravenously, only low-grade constitutional or gastrointestinal symptoms and infusion reactions [(50), see also preclinical data mentioned in this reference]. No maximum tolerated dose was identified. The half-life of MEDI-522 was similar to that of MEDI-523 (Vitaxin) and in the order of 59–106 hours. Also, no immune response to the compound was observed. Tumor blood flow was assessed at baseline and after eight weeks of treatment by dynamic computed tomography imaging on a limited number of patients. Of all the parameters studied (mean blood flow, blood volume, mean transit time, and permeability surface), only the mean transit time was significantly altered: according to the authors, the corresponding increase may be interpreted as a biological response in terms of impeding blood flow through small-caliber neovasculature. No complete or partial response was observed among the 25 patients enrolled, as defined by the RECIST criteria. Interestingly, three patients with metastatic renal cancer had prolonged stable disease for at least 34 weeks. Hypophosphatemia episodes were also observed in several patients. These observations point to a possible tropism of the molecule for renal tissues, normal or pathological. The avb3 integrin is expressed in renal tissues but, as commented by the authors of the phase I study, there are no report of integrin antagonists causing or exacerbating hypophosphatemia or other kidney diseases. Whereas the efficiency of MEDI-522 for metastatic renal cancer remains to be evaluated, a more tangible activity is suggested for patients with metastatic

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melanoma treated with the antibody administered in combination with dacarbazine (DTIC, the current standard of care in advanced melanoma). The phase II multicenter non-comparative trial involved 112 patients with stage IV metastatic melanoma randomized between September 2003 and May 2004 in two arms: MEDI-522 (8 mg/kg/wk) with (55 patients) and without (57 patients) DTIC (1000 mg/m2 once every three weeks). Preliminary observations published in abstract form for the 2005 ASCO meeting (66) hinted at efficacy in terms of overall survival. Adverse effects were mainly grade 1/2, with hematological grade 3/4 adverse events occurring in both arms (37% for MEDI-522 alone and 48% for the combination). Two MEDI-522-related deaths were reported, one in each arm (myocardial infarction and pulmonary embolism). Along with the results of this trial presented at ASCO, in a press release of May 14, 2005 (67) MedImmune announced a 12.7-month median survival for the patients treated with MEDI-522 alone, compared to 9.4-month median survival for the combination arm—the figures were compared to the 7.9-month median survival for DTIC alone in a former unrelated trial [Genasense
(Genta Inc., Berkeley Heights, New Jersey, U.S.A.) phase III trial, i.e., oblimersen sodium, an antisense therapy against the Bcl-2 antiapoptotic molecule]. Further data are expected after these promising interim results, as well as on a phase II trial conducted by MedImmune in patients with androgen-independent prostate cancer that has metastasized to bone (enrollment was closed in April 2005 and 126 patients are evaluated). As of August 2006, several phases I and II are ongoing with MEDI-522. A phase I translational study aims at identifying the dose for tumor saturation and biological activity in patients with advanced malignant melanoma. Preliminary data indicate that saturation occurs at 8 mg/kg, as revealed in a press release from MedImmune on April 10, 2006 (68). Phase I studies are in progress for patients with irinotecan-refractory advanced colorectal cancer, or with refractory advanced solid tumors or lymphoma. MEDI-522 is also the subject of phase I trials for adults with plaque psoriasis (subcutaneous injection) and to study the activity and progression of joint damage in patients with active rheumatoid arthritis suboptimally responding to methotrexate. Finally, according to the press release from MedImmune (68), preclinical data suggest that the majority of MEDI-522’s antitumor activity may be mediated through antibody-dependent cellular cytotoxicity or ADCC. While still targeted, the therapy with MEDI-522 may not necessarily relate to the blocking of the avb3 integrin function(s). The outcome with small integrin antagonists like Cilengitide may thus be totally different. Cilengitide The first phase I with the small cyclic peptidic integrin avb3 and avb5 antagonist Cilengitide recruited 37 patients with advanced solid tumors refractory to standard treatment (69). Twice-weekly treatment cycles (1-hour intravenous infusion at doses of 30–1600 mg/m2) were given and no dose-limiting toxicity was found. There was no hematological toxicity related to Cilengitide treatment and nonhematological toxicities were mild and limited to grade 2 (nausea, anorexia, vomiting, fatigue, and malaise). Consequently, a maximal tolerated dose could not be defined. Systemic exposure to Cilengitide increased in a dose-proportional manner. A dose of 120 mg/m2 enabled to reach the target plasma concentration (
10 mg/mL) inferred from animal studies for efficacy. No partial or complete response was observed and prolonged stable disease occurred in one patient with

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colorectal carcinoma (for 168 days) and two with renal carcinoma (for 164 days). The plasma half-life of Cilengitide was short (3–5 hours), hence, the treatment schedule may not be optimal (although the authors noted that similar schedules in mice elicited marked tumor responses). Preliminary data from another phase I trial presented at the ASCO 2006 annual meeting (70) revealed that continuous infusion was safe up to at least 18 mg/hr (one death of unknown cause occurred at the 27 mg/hr dose). Cilengitide may prove more efficient in combination therapy, an option considered for further development. Although Cilengitide entered clinical trial in 1998, extended publications on the clinical efficacy of Cilengitide are sparse, with the exception of a case study (71). When standard treatments and surgery failed after several phases of rapid and massive growth of a squamous cell carcinoma that had its origin in the left jaw, the highly vascularized tumor could not be resected due to extensive bleeding during the surgical attempt and the patient was assigned to best supportive care. A combination of Cilengitide (600 mg/m2 over 60 min intravenously) with a cytostatic agent with a mild toxicity profile (30 min infusion of gemcitabine at 1000 mg/m2 on days 1 and 8 every three weeks) for about six months led to a partial remission. Because of gemcitabine-linked hematological toxicity, the treatment was then switched to Cilengitide alone which led to stabilization for 12 months on maintenance therapy with no tendency toward spontaneous bleeding. This case report suggests that Cilengitide may be efficient for highly vascularized tumors. Several phase I, I/II, and II studies have been conducted or are ongoing with Cilengitide in various settings (72,73): in patients with unresectable stage III or stage IV melanoma; in patients with nonmetastatic or asymptomatic, metastatic androgen-independent prostate cancer (74); in patients with advanced solid tumors or lymphoma; as maintenance therapy in patients with acute myeloid leukemia in first complete remission; in patients with HIV-related Kaposi’s sarcoma; in children with refractory primary brain tumors; and in patients with recurrent or progressive malignant glioma or glioblastoma multiforme. The focus on brain tumors also stems from preclinical data strongly supporting the notion that, besides being effective on ECs during angiogenesis, Cilengitide may affect tumor cell survival directly (75), thus inducing a strong response in animal models (76,77). In their review of Cilengitide’s clinical trials referral resource (72), the authors mentioned—see also press release from Merck KGaA on November 22, 2004 (78)—that out of 51 malignant glioma patients enrolled in the multicenter dose-escalation study designed to determine the maximum tolerated dose of Cilengitide, two patients showed complete responses (twice weekly infusions at 360 and 2400 mg/m2) and three partial responses (two at 120 mg/m2 and one at 360 mg/m2). Four patients had stable disease for more than six months. A phase II study was subsequently initiated (October 2004) in patients with recurrent glioblastoma multiforme who are receiving Cilengitide after first line chemotherapy failed. Because the association with radiation therapy may also provide clinical benefit, it is investigated in a phase I/II randomized study of Cilengitide combined with radiation therapy and temozolomide in patients with newly diagnosed glioblastoma multiforme. Finally, Cilengitide has received a positive opinion for orphan designation for the treatment of glioma by the European Medicines Agency (79). It has now the orphan-drug status in both the European Union and in the United States, and could open a much needed new way for the treatment of glioblastoma.

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Less Advanced Integrin Antagonists (CNTO 95, Volociximab, ATN-161, E7820) By recognizing the av subunit, the human monoclonal antibody CNTO 95 targets the avb3 and avb5 integrins. A phase I dose-escalating study in patients with advanced refractory solid tumors (80) showed its good tolerance when administered from 0.1 up to 10 mg/kg (intravenous infusion on days 0, 28, 35 and 42), with manageable infusion-related fever. A partial response was observed in a patient with cutaneous angiosarcoma (ongoing after two months with prolonged treatment at 10 mg/kg) as well as a stable disease in a patient with ovarian carcinosarcoma (a lesion was undetectable on FDG-PET at day 49 after 10 mg/kg treatment). The initial rapid clearance observed at low doses and slower drug clearance at higher doses may be explained by tissue binding and saturation, respectively. However, preclinical studies did not reveal adverse effects due to widespread tissue binding (81). A phase I/II, multi-center, double-blinded, randomized, placebo-controlled study of the safety and efficacy of CNTO 95, alone (infusion at 3, 5 or 10 mg/kg) and in combination with dacarbazine, opened in April 2005, in patients with stage IV melanoma. Progression-free survival is the end-point of the phase II part. Volociximab is a chimeric humanized monoclonal antibody targeting the a5b1 integrin. A phase I study of the antibody (1-hour infusion at 0.5–15 mg/kg on days 1, 15, 22, 29, and 36) in patients with refractory solid tumors showed no dose-limiting toxicity up to 10 mg/kg, the highest dose evaluated in the report (82). Stable disease was observed in 9 patients out of 15 (with five of six patients receiving 10 mg/kg of antibody). Since monocytes express the target, monocyte saturation with the antibody could be assayed as a surrogate marker of integrin targeting and was achieved at 10 mg/kg. This dose was recommended for subsequent clinical trials. Results from several phase II trials were presented at the ASCO 2006 annual meeting (abstract in 83–85 and 86 for the corresponding posters). Volociximab (intravenous infusion at 10 mg/kg once every 2 weeks for up to 52 weeks) was well tolerated in conjunction with DTIC in the metastatic melanoma trial (83). Out of 37 patients, one had a partial response and 27 had stable disease at week 8. The median overall survival was 7.9 months in a population of which 55% were classified as poor prognosis stage M1c. In the phase II open-label study in patients with refractory metastatic clear cell renal cell cancer (84), Volociximab (intravenous infusion at 10 mg/kg once every 2 weeks for up to 52 weeks) was again well tolerated. Out of 40 patients, one partial response and 32 stable disease were observed. Overall survival was not reached after 11 months since the first patient on study. In the phase II study in patients with metastatic adenocarcinoma of the pancreas (85), Volociximab (intravenous infusion at 10 mg/kg once every 2 weeks for up to 2 years) was well tolerated when added to a conventional treatment with gemcitabine. Additional patients are being enrolled in the study at 15 mg/kg. Out of 19 patients, one partial response and 10 stable disease were observed. For patients who met the entry criteria (14 patients), a median overall survival of 8.2 months and a time to progression of 5.4 months have been observed. An open-label multi-center phase II study of Volociximab (infusion once every 2 weeks for up to 52 weeks) and erlotinib hydrochloride (oral Tarceva daily) in 40 previously treated patients with locally advanced (stage IIIb) or metastatic (stage IV) non–small cell lung cancer was launched in 2006. ATN-161, a small peptidic compound recognizing the a5b1 integrin, entered phase I in January 2003. Results from a phase I in patients with advanced solid

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tumors have been published recently (87). Because tumor growth inhibition by the peptide in preclinical studies was not dose-dependent—optimal response at 1–10 mg/kg and no response at 0.2 and 100 mg/kg (54 and introduction in 87)— the clinical doses were chosen to cover a similar range after interspecies dose conversion. Twenty-six patients were enrolled to receive thrice weekly 10-min intravenous infusion of 0.1–16 mg/kg of ATN-161. No dose-limiting toxicities occurred and treatment-related adverse effects were grade 2 or less. The observed half-life was 3–5 hours, but preclinical data indicated that intermittent dosing was efficient. There was no objective response and six patients experienced stable diseases of more than four months. Four patients (adenoid cystic, renal cell, prostate, and ovarian cancer) with prolonged stable disease had well-documented progressive disease prior to study entry. A phase II study is ongoing in advanced renal cell cancer. A phase I/II study combines ATN-161 with chemotherapy in patients with recurrent intracranial malignant glioma: patients will receive the peptide in combination with carboplatin. Finally, E7820 is a first-in-class oral antiangiogenic sulfonamide targeting the a2 integrin subunit at the expression level. Interim results of a phase I in patients with solid tumors have been presented at recent ASCO annual meetings (88,89). The compound was administered orally from 10 to 200 mg/day in a 28-day cycle. Administration revealed an excellent safety profile up to 100 mg/day, the maximum tolerated dose (MTD). At 200 mg/day, two patients experienced hematological toxicity (grade 4 thrombocytopenia and neutropenia). The half-life ranged from 6 to 12 hours. No changes in integrin levels (a2 integrin subunit expression in circulating platelets) were observed at doses less than 40 mg. Moderate decrease (less than 30%) occurred at 70 mg (2 out of 3 patients) and at 100 mg (3 out of 6); at 200 mg the decrease was more pronounced (50%) and lasted beyond day 28 in 3 out of 4 patients. Disease stabilization beyond the fourth cycle occurred in 6 patients out of 30, which lasted more than 6 months. A phase I/II study (not yet open for patient recruitment as of August 2006) will determine the safety and efficacy of E7820 plus cetuximab in colorectal cancer and explore the MTD of the combination in the first part of the study in patients with advanced solid tumors and then explore the efficacy of this combination in patients with colorectal cancer that is inoperable and/or metastatic. Future of Integrin Antagonists in the Clinic Our present knowledge on integrins suggests that they are not targets specific for ECs during tumor progression. Their concomitant presence on tumour cells, and also on other stromal cells besides the endothelium, complicates the mechanistic interpretation of the action of integrin antagonists seen in the clinic. To illustrate the point with nonendothelial stromal cells, it was recently shown that Volociximab can alter the pattern of cytokine secretion by macrophages (90). It could thus exert indirect antiangiogenic effects by modulating the availability of pro-angiogenic cytokines through a5b1 integrin ligation on stromal cells. Monocytes indeed release pro-angiogenic CXC cytokines when plated on fibronectin, the ligand recognized by a5b1. ATN-161 may exert similar effects since it is homologous to the PHSCN sequence that blocks this release (91). Clearly, the mechanism of action of some of the compounds presented may not be restricted to endothelial targeting. The presence of integrins on ECs and tumor cells is also exploited for other clinical applications: to deliver conjugated cytotoxic compounds and as radioactive and imaging agents for diagnosis or treatment. Exploiting the relatively specific

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presence of avb3 integrins on tumors has been the focus of intense research for several years in these fields, especially with RGD monomers or multimers (92,93). Noninvasive selection of patients with avb3-expressing primary or metastatic tumors is now feasible (94,95) and will undoubtedly help identifying patients more likely to respond to anti-avb3 integrin treatment. OTHER ENDOTHELIAL CELL PROPAGATION INHIBITORS The integrins mentioned above, avb3, avb5, and a5b1, have been clearly identified—although not necessarily fully validated, especially in the clinic—as targets for blocking tumor progression. They are also receptors for pathophysiological ligands shown to block angiogenesis in preclinical settings. The corresponding molecules have in common to be fragments of proteins not necessarily associated with angiogenesis or tumor progression. Angiostatin is thus a fragment of plasminogen (9), endostatin is derived from collagen type XVIII (10), and tumstatin is a noncollagenous fragment of collagen IV (96). They can interact with various receptors—usually distinct from those of the parental proteins. In fact, there is now a long list of protein fragments susceptible to block angiogenesis (8). They do not necessarily interact with integrins as their main receptors and their action may not be limited to targeting the ECs or their propagation. Only two tumor-derived antiangiogenic fragments have entered clinical trials: angiostatin and endostatin. Tumstatin, another likely candidate, has not yet entered phase I trials. Thrombospondin also deserves a mention as the first physiological protein identified to inhibit angiogenesis (97). Its antiangiogenic activity was confirmed in knock-out animals and by transfection experiments for overexpression [reviewed in (8,11)]. This large extracellular matrix component could in fact also play a role in promoting angiogenesis depending on its proteolysis; hence, the interest of evaluating fragments. Dissecting the role of thrombospondin in adhesive events and endothelial tube formation led to an interest in its type I repeat domains shown to interact with its receptors, including the cell surface antiangiogenic receptor CD36. A short sequence from the second type I repeat was optimized for antiangiogenic activity. It led to the nonapeptide analogue called ABT-510 which is a potent antiangiogenic agent in vitro and in vivo mimicking the corresponding inhibitory activity of thrombospondin on EC migration, proliferation and tube formation (98,99). It can also promote apoptosis of ECs, a process improved by concomitant administration of metronomic low-dose chemotherapy (100). It is currently in phase II. Tumor-Derived Protein Fragments (Angiostatin and Endostatin) Angiostatin Angiostatin was discovered in 1994 in Judah Folkman’s laboratory (9). A phase I trial of the compound administered daily intravenously showed its safety without limiting toxicity (101). At room temperature, the activity of the compound in solution is rapidly lost and prevents continuous infusion. The feasibility and safety of subcutaneous administration were shown later (102). Twice daily subcutaneous administration in a small number of patients with advanced non–small cell lung cancer and in combination with paclitaxel and carboplatin yielded results similar to those seen with carboplatin/paclitaxel/bevacizumab in a phase III study (103).

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However, financial issues at EntreMed, the company in charge for the development, cast a shadow on the future of the development of this molecule as a recombinant protein (see below with endostatin). Other scenarios are investigated to bathe the body with the protein: producing angiostatin by direct proteolysis of the plasminogen precursor protein (104) or through the administration of adenoviral constructs encoding the gene sequence of angiostatin as a fusion with the sequence of endostatin (105). A phase I trial for evaluating the first scenario validated the approach although no clinical objective responses were observed. Also, antibodies against one of the receptors of angiostatin on ECs, angiomotin (106–108), are presently generated and may mimic the activity of angiostatin. It is hoped that such antibodies would be easier to produce than angiostatin itself (109). A vaccine strategy has also been tried with success against angiomotin (110). Endostatin Endostatin was discovered in 1997, again in Judah Folkman’s laboratory (10). Daily intravenous administration was found to be safe in the first phase I trials in 1999–2001 without redhibitory toxicity (111–113). A combination of two ways of administration—intravenous for 28 days followed by daily subcutaneous dosing— was found to be safe in human (114). In 2002–2003, phase II trials with the compound administered subcutaneously twice a day showed only two minor responses in 41 patients with advanced neuroendocrine tumors (115). Endostatin was associated with minimal toxicity but overall did not result in significant tumor regression in this indication (116). Endostatin development had to face challenges in terms of protein production and administration. The protein is insoluble in physiological fluids and the production in the bacteria Escherichia coli is associated with problems of stability and proper refolding, not to mention reproducibility issues. Production in the yeast, although feasible, led to a soluble form but with a very low yield, hence requiring high costs for development. In 2003, because of high production costs, EntreMed announced discontinuation of the production of endostatin. The stocks in the United States lasted until 2005, and clinical trials had to be stopped, much to the disappointment of clinical investigators (117). Following the creation of the Yantai Medgenn joint-venture between Dr Luo et Yantai R.C. Pharmaceutical Co. (a Chinese company involved in traditional medicine), a new form of endostatin was produced. Nine amino acids were added at the N-terminal to stabilize the protein and confer a correct folding. Between 2001 and 2004, 493 patients with lung carcinoma received the corresponding protein, Endostar, as part of their chemotherapy in a clinical trial. The results were presented at ASCO 2005 (118): 35.4% of patients in the combination arm exhibited reduced tumor size versus 19.5% in the arm receiving chemotherapy alone. Survival at one year was also enhanced (60% surviving patients in the combination arm vs. 30%). Endostar was approved by the Chinese State Food and Drug Administration in October 2005 for the treatment of non–small cell lung cancer in combination with chemotherapy. This event ranked by the Ministry of Science and Technology of the People’s Republic of China as number 7—just after the re-evaluation of the height of mount Everest, i.e., 8844.43 meters instead of the preceding 8848.13 meters announced in 1975—in the top 10 domestic Science and Technology events for 2005 (119). As with angiostatin and to circumvent the difficulties in manufacturing the recombinant protein, attempts are also being made to administer endostatin

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through gene therapy. A phase I trial of an adenovirus vector for endostatin gene transfer, Ad-rhEndo or E10A (120) has been completed in China in patients with advanced solid tumours and the construct appears to be well tolerated (121). Thrombospondin Fragment (ABT-510) ABT-510 has been evaluated as subcutaneous administrations (cycles of continuous infusion or once- or twice-daily administration as a bolus for 28 days) in a phase I in 39 patients with advanced cancer (122). Continuous infusion was inconvenient since it necessitated daily change of the infusion site to prevent skin reactions. The product showed linear, time-dependent pharmacokinetics and a favorable toxicity profile. No tumor regressions were observed, but several cases of prolonged stable disease occurred. A dose of 100 mg twice daily subcutaneously was retained for phase II studies. A phase II study evaluating the activity of ABT510 alone in the first-line treatment of patients with advanced renal cell carcinoma showed that the treatment was well tolerated but it did not provide obvious improvement in efficacy compared to historical controls (123). On the other hand, progression-free survival in patients with advanced soft tissue sarcoma and treated with ABT-510 exceeded the 14% rate reported by the EORTC for active drugs in this pathology (124). Single-agent therapy or combination treatment may thus be beneficial in this pathology. ABT-510 was granted an Orphan Drug designation by the FDA for soft tissue sarcoma—press release from Abbott May 9, 2005 (125). Since combination studies are expected to yield improved effects, several phase I trials of ABT-510 in combination with chemotherapy have been conducted without additional toxicity. A combination with 5-fluorouracil and leucovorin (5FU/LV) was evaluated in 12 patients with advanced solid malignancies, but the authors (126) cautioned that a larger study may reveal unexpected major toxicities, as seen with the tyrosine kinase VEGF receptor inhibitor SU5416 or with the antiVEGF antibody bevacizumab (occurrence of thromboembolic events in combination with 5FU/LV). In combination with gemcitabine and cisplatin in 13 patients with advanced solid tumors, three partial responses were observed (out of 12 patients evaluable) in patients with head and neck cancer, melanoma, or NSCLC (127). A phase I study in combination with bevacizumab is in progress in patients with advanced solid tumors, as well as combination therapy (carboplatin/taxol) in subjects with NSCLC. A phase II study of ABT-510 alone for the treatment of previously treated metastatic melanoma did not show significant clinical efficacy (128). ABT-510 is currently evaluated in phase II studies for the following pathologies: advanced head and neck cancer, locally recurrent or metastatic renal cell cancer, refractory lymphoma and locally advanced or metastatic soft tissue sarcoma. CONCLUSION It is too early to conclude on a possible benefit in a given niche for integrin inhibitors as a class, even with the anti-avb3 compounds that are more advanced in clinical development, such as Abegrin (MEDI-522) and Cilengitide. As with any new targeted therapy, conducting trials with only the agents under investigation in patients with advanced disease may fail to reveal the potential of integrin antagonists. Their evaluation as adjuvant therapy or in combination with cytotoxic

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agents may prove beneficial. Therefore, the outcome of ongoing and additional combination trials of integrin antagonists is eagerly awaited, since the results with monotherapy treatments were, overall, somewhat anecdotal, with no objective responses in clinical trials. Integrins are believed to be good targets for fighting tumor progression, but the precise mechanisms of their participation in this process is not entirely clear. This may not prevent clinical development of the so-called integrin antagonists, but comes as a warning: in some situations—yet to be discovered—the balance may shift from antiangiogenic to protumorigenic activity, as seen with the first generation of matrix metalloproteinases inhibitors. Large, tumor-derived, antiangiogenic protein fragments, such as angiostatin and endostatin, may fare better than integrin antagonists in the clinic because of their multiple cell receptor targets—including integrins. Again, the mechanism of action of such compounds is not entirely elucidated. The possibility that a common cross-b structure present in many antiangiogenic fragments exerts amyloid-induced cell toxicity has been raised (129). A correct folding of these molecules is indeed necessary for activity, something not always achieved during some processes of recombinant protein production. This may be at the heart of the difficulties encountered at the production level for this class of EC propagation inhibitors, a difficulty potentially resolved by turning to gene therapy—although other burdens in clinical development may occur. The list of endogenous inhibitors of angiogenesis has been steadily growing, recently with about 27 candidates (8), including thrombospondin. Determining the minimally active sequence(s) in these molecules is feasible—as exemplified for endostatin (130) or thrombospondin with ABT-510—and may facilitate development, although the full spectrum of activity of the parental protein may be lost if several receptors or targets participate in the activity (131,132). A large variety of protein fragments is thus likely to enter clinical trials in the future as antiangiogenic compounds. REFERENCES 1. Frisch SM, Screaton RA. Anoikis mechanisms. Curr Opin Cell Biol 2001; 13:555–62. 2. Grossmann J. Molecular mechanisms of “detachment-induced apoptosis—anoikis”. Apoptosis 2002; 7:247–60. 3. Hynes RO. Integrins: bidirectional, allosteric signalling machines. Cell 2002; 110:673–87. 4. Rundhaug JE. Matrix metalloproteinases and angiogenesis. J Cell Mol Med 2005; 9:267–85. 5. Vlodavsky I, Friedmann Y. Molecular properties and involvement of heparanase in cancer metastasis and angiogenesis. J Clin Invest 2001; 108:341–7. 6. Miao HQ, Liu H, Navarro E, et al. Development of heparanase inhibitors for anticancer therapy. Curr Med Chem 2006; 13:2101–11. 7. Clamp AR, Jayson GC. The clinical potential of antiangiogenic fragments of extracellular matrix proteins. Br J Cancer 2005; 93:967–72. 8. Nyberg P, Xie L, Kalluri R. Endogenous inhibitors of angiogenesis. Cancer Res 2005; 65:3967–79. 9. O’Reilly MS, Holmgren L, Shing Y, et al. Angiostatin: a novel angiogenesis inhibitor that mediates the suppression of metastases by a Lewis lung carcinoma. Cell 1994; 79:315–28. 10. O’Reilly MS, Boehm T, Shing Y, et al. Endostatin: an endogenous inhibitor of angiogenesis and tumor growth. Cell 1997; 88:277–85. 11. Ruegg C, Hasmim M, Lejeune FJ, Alghisi GC. Antiangiogenic peptides and proteins: from experimental tools to clinical drugs. Biochim Biophys Acta 2006; 1765:155–77.

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HIF-1 Inhibitors Giovanni Melillo Developmental Therapeutics Program, SAIC Frederick, Inc., National Cancer Institute at Frederick, Frederick, Maryland, U.S.A.

INTRODUCTION Hypoxia inducible factor 1 (HIF-1) is a transcription factor that controls the response of mammalian cells to oxygen deprivation. HIF-1 has been implicated in a variety of pathophysiological conditions, including development, inflammation and cancer. Thus, its role as a mediator of fundamental biological processes and the potential modulation of its activity for therapeutic purposes have attracted considerable interest (1,2). Several aspects of the involvement of HIF-1 in human cancer should be emphasized: (1) The regulation of HIF-1a by hypoxia, a common feature of solid tumors known to profoundly affect tumor biology, response to treatment, and patients' prognosis; (2) the influence of genetic alterations, e.g., Von Hippel-Lindau tumor supressor (VHL), PTEN, and p53, frequently detected in human cancers, on HIF-1a expression and function; (3) the induction of HIF-1a accumulation by RTK signaling pathways frequently dysregulated in human cancers; (4) the expression of HIF-1 in multiple cellular components that are present in the tumor microenvironment, including stromal infiltrating cells and endothelial cells. These features legitimate HIF-1 as a crucial player in cancer development and progression and as a potential target for the development of novel therapeutics. Indeed, the interest in HIF-1 is documented by the exponentially increasing number of papers published on this topic over the past decade and by the growing number of academic groups and pharmaceutical industries actively engaged in the identification of novel strategies aimed to inhibit HIF-1 in human cancer. However, many questions still remain unanswered regarding the distinct role of HIF in different tumor types and the best way to achieve HIF inhibition in cancer patients. It can be anticipated that over the next few years more inhibitors will be identified and will approach the preclinical and clinical arena for further testing. A rational plan to validate HIF-1 inhibitors in preclinical models and test them in early clinical trials is warranted, so that this exciting and promising avenue for cancer therapy may yield positive results. HIF-1 AS A TARGET FOR CANCER THERAPY Regulation of HIF-1 Expression HIF-1 is a basic helix–loop–helix PAS transcription factor composed of two subunits, a and b. The b-subunit, also known as aryl hydrocarbon receptor nuclear translocator (ARNT), is constitutively expressed in an oxygen independent fashion, and is also involved in other transcriptional pathways, e.g., by dimerizing with the dioxin receptor, AhR [reviewed in (3,4)]. In contrast, the a-subunit, of which two, HIF-1a and HIF-2a, are best characterized, is rapidly degraded under normoxic conditions but accumulates under low oxygen levels. The mechanism by 283

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which the a-subunit is degraded has been elegantly elucidated over the past few years. A family of enzymes, called PHDs, mediates hydroxylation of two proline residues of HIF-a in a reaction that requires O2, Fe2þ, and 2-oxyglutarate. Upon hydroxylation, the a-subunit is recognized by the product of VHL, pVHL, which functions as the recognition component of an E3 ligase that mediates ubiquitylation and proteasomal degradation of HIF-a. As mentioned above, mutations of pVHL, which are frequently detected in patients with clear cell renal carcinoma, cause an accumulation of HIF-a under normoxic conditions due to impairment of its degradation. However, an increasing number of genetic alterations frequently implicated in human cancers have been associated with dysregulation of HIF-a. In addition to gain-of-function mutations, such as v-src and Ras, loss-of-function alterations, including PTEN, p53, TSC, succinate dehydrogenase, fumarate hydratase, and PML, have been implicated in the accumulation of HIF-a under nonhypoxic conditions (3). In addition, growth factor-dependent signaling pathways frequently dysregulated in human cancers, including EGF, IGF, and Her2/ Neu, have also been implicated in the induction of HIF-1a under normoxic conditions by activation of the PI3K/AKT/mTOR and MAPK pathways, further emphasizing the complexity of HIF-1a regulation and its involvement in fundamental processes of cancer progression. HIF-1 and Gene Expression The list of genes and functions that are controlled by HIF is constantly expanding. The impact that HIF may have in human cancer is highlighted by the function of genes that are controlled by HIF and that profoundly affect the behavior of cancer cells. HIF-inducible genes control tumor metabolism, angiogenesis, cell survival, and migration/invasion, all of which are hallmarks of cancer progression (5). HIF plays a crucial role in the induction of angiogenesis, a feature that may have important therapeutic implications for HIF inhibitors. Vascular endothelial growth factor (VEGF), the best characterized angiogenic factor, is transcriptionally induced by HIF via an HRE present in its promoter, although hypoxia may also control VEGF mRNA stability and/or its translation, and HIF-independent pathways have also been identified (6). A critical pathway controlled by HIF is aerobic glycolysis, a key feature of cancer cells, which have high level of glycolysis even in the presence of oxygen. Indeed, HIF-1 induces a coordinate upregulation of genes involved in glucose metabolism and glycolysis. Finally, in the past few years, a critical role of HIF in the control of cell migration and invasion has also been elucidated by induction of genes such as CXCR4 (7) and lysyl oxidase (8), implicated to a different extent in invasion and metastasis. Each of these genes could represent a viable therapeutic target as well as be affected by strategies targeting HIF-1. HIF-1 Expression in Human Cancer A number of experimental models have confirmed that HIF-1 plays a critical role in tumor formation. However, controversial evidence has also been generated depending on the tumor model used, which has led to some early skepticism as to whether HIF is a good target for therapy (9,10). HIF is overexpressed in a variety of human cancers and its expression is associated with poor prognosis and poor response to treatment (9,11). The pattern of HIF-1 staining that is detected in tumor tissue highlights the involvement of different pathways of HIF activation

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in cancer patients. Indeed, expression of HIF can be detected in perinecrotic areas of hypoxia, where the role of oxygen in its regulation is predominant. However, HIF has also been detected in well-oxygenated areas, consistent with its regulation by growth factors and genetic alterations, as well as in stromal infiltrating cells, which raises the question of the contribution of this cellular component to tumor growth and response to therapy. HIF INHIBITORS: MECHANISMS OF ACTION The majority of HIF-1 inhibitors identified so far can be classified as }nonselective,} as they target signaling molecules or pathways that affect multiple cellular functions (9,10,12). With this caveat in mind, in the next sections of this chapter, information is provided regarding the mechanism of action of some of the HIF inhibitors identified so far. In particular, emphasis is placed on compounds that are relevant to the clinical setting, either because they are in clinical development or because they target pathways for which inhibitors are available (Fig. 1). Inhibitors of Signaling Pathways Consistent with the redundant involvement of HIF-1 in multiples RTK-mediated signaling pathways that are dysregulated in human cancers, several novel inhibitors that have approached the preclinical and clinical arena also have the potential or have indeed been shown to inhibit HIF-1 or HIF-dependent functions. This finding raises the question as to how, if at all, RTK inhibitors (RTKI) can be used in the clinic as HIF-1 inhibitors. There are at least two implications of HIF-1 inhibition by RTKI.

FIGURE 1 Potential mechanisms of action of HIF-1 inhibitors. Abbreviations: EGFR, epidermal growth factor receptor; HDAC, histone deacetylase; HIF, hypoxia inducible factor; VHL, Von-Hippel Lidau tumor supressor gene; 2ME2, 2-methoxyestradiol.

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The first is that inhibition of HIF-1 may play a role in the response of patients to therapy with RTKI. This possibility is supported by findings indicating that HIF-1 is downstream of a number of signaling pathways targeted by RTKI, and, in a cell-type specific fashion, HIF-1 may be a critical mediator of these dysregulated pathways. The second implication is that inhibition of HIF-1 may become a valuable biomarker of activity of RTKI, which can be validated in relevant preclinical models to be then incorporated in early clinical trials. Inhibitors of the mTOR Pathway The mTOR pathway has been implicated in the growth factor-dependent induction of HIF-1a translation (13) as well as in HIF-1a degradation (14). It is then conceivable that mTOR inhibitors currently in clinical development might inhibit HIF-1 and have an impact on downstream pathways, including angiogenesis. Indeed, evidence has been provided that tirosel/temsirolimus (Wyeth, Madison, New Jersey, U.S.A.), a novel mTOR inhibitor in clinical development, inhibited hypoxic dependent induction of HIF-1 and VEGF production (15) and rhabdomyosarcoma xenograft growth by an antiangiogenic mechanism dependent on mTOR/HIF-1a/VEGF signaling (16). Importantly, in a mouse model of AKT1-dependent prostate intraepithelial neoplasia HIF-1a targets, including genes encoding most glycolytic enzymes, constituted the dominant transcriptional response to AKT activation and mTOR inhibition (17) and loss of VHL sensitized kidney cancer cells to the mTOR inhibitor tirosel/temsirolimus in vitro and in mouse models (18). Thus, HIF-1a might be a biomarker of response in cancers in which the mTOR pathway is dysregulated and may also represent an attractive biomarker that could facilitate preclinical and early clinical development of mTOR inhibitors. EGFR Inhibitors HIF-1a is induced upon stimulation of the epidermal growth factor receptor (EGFR) pathway (13). Accordingly, EGFR tyrosine kinase inhibitors, including Tarceva and Iressa currently used in the clinic, inhibit VEGF expression by both HIF-1-dependent and independent mechanisms (19), which may also have implications for the induction of apoptosis by these agents (20). Cetuximab, a monoclonal antibody targeting the epidermal growth factor receptor, also inhibits HIF-1a levels in A431 epidermoid carcinoma cells (21). Inhibitors of AKT AKT is a critical signaling molecule, mediating RTK-dependent pathways that may ultimately affect HIF-1 activity. Thus, AKT has been implicated in the mechanism of action of many small molecule inhibitors of HIF-1 described in the literature (22–26). Little evidence has been provided so far that this is a feasible approach in preclinical models, but obviously AKT is an attractive target for cancer therapy and HIF-1 may represent one of many downstream targets affected by AKT inhibition. Other signaling pathways that are frequently dysregulated in human cancers have been implicated in HIF-1a regulation, and inhibitors of these pathways may potentially block HIF-1a accumulation. In particular, evidence has been provided that the Her2/Neu (27), c-KIT (28), and BCR/ABL (29) pathways are implicated in the induction of HIF-1a and VEGF expression in breast cancer, lung cancer, and leukemic cell lines, respectively.

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Inhibitors of Protein Accumulation Inhibition of HIF-1a by the agents described in this section has been associated with biochemical inhibition of targets that are known to be affected by these compounds. Topoisomerase I Poisons Topotecan, a topoisomerase I poison, is a small molecule inhibitor of HIF-1 identified in a cell-based reporter screen of the NCI Diversity set using U251-HRE cells, which express a luciferase reporter gene under control of three copies of a canonical hypoxia responsive element (30). Several camptothecin analogs have since then been identified as HIF-1 inhibitors, and HIF-1 inhibition seems to be a common property of this class of compounds. Topotecan inhibits HIF-1a translation by a mechanism independent from DNA replication–dependent DNA damage and proteasome function (31). Importantly, daily administration of topotecan inhibited HIF-1a expression in xenografts experiments, which was associated with the inhibition of angiogenesis and tumor growth (32). These results have led to the implementation of a clinical trail that is currently ongoing at the NCI (http://www.clinicaltrials.gov/ct/show/NCT00182676), where the effect of topotecan on HIF-1a expression in tumor tissue is being evaluated in patients with metastatic cancers. This pilot study will provide useful information regarding the potential to inhibit HIF-1a expression in tumor tissue using small molecules. Microtubule-Targeting Agents 2-Methoxyestradiol (2ME2), a novel antitumor and antiangiogenic agent, which is currently in clinical development stage, was found to inhibit tumor growth and angiogenesis at concentrations that efficiently disrupt tumor microtubules (MTs) in vivo (33). In addition, 2ME2 downregulated HIF-1a by inhibiting its translation and blocked HIF-1-induced transcriptional activation of VEGF expression. 2ME2/ tubulin interaction was required for HIF-a downregulation. Interestingly, early clinical trials of this compound have shown that 2ME2 is not associated with common toxicities observed with other microtubule-targeting agents; thus, inhibition of HIF-1 and angiogenesis may be an important mechanism contributing to its biological activity. Hsp90 Inhibitors The benzoquinone ansamycin geldanamycin, an Hsp90-specific inhibitor, was found to inhibit HIF-1a protein accumulation by a mechanism involving its degradation in a proteasome-dependent but VHL-independent fashion (34,35). HIF-1a is one of many Hsp90 client proteins and it is unclear how much inhibition of Hsp90 may be associated with downregulation of HIF-1-target functions. However, analogs of geldanamycin, including 17-AAG and 17-DMAG, are currently in clinical trials for cancer therapy and HIF-1 inhibition may be potentially contributing to the therapeutic activity and/or may represent a valuable biomarker of activity of these compounds. Hsp90 has also been implicated in the mechanism of action of other HIF-1 inhibitors, including radicicol (36) and the farnesyltransferase inhibitor SCH66336 (37). Histone Deacetylase Inhibitors Several mechanisms of action have been suggested for the activity of histone deacetylase (HDAC) inhibitors, a class of compounds in clinical development, on

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HIF-1a degradation. In particular, HDACIs may induce the proteasomal degradation of HIF-1a by a mechanism that is independent of VHL and is secondary to a disruption of the HSP70/HSP90 axis function (38). Alternatively, it has been shown that class II HDAC4 and HDAC6 were associated with the HIF-1a protein and may directly affect its degradation (39). These results suggest that inhibitors of class II HDACs might be used to target HIF-1a in human cancers. Whether or not HIF-1a inhibition contributes to the therapeutic activity observed with the administration of HDAC inhibitors remains to be determined. Inhibitors of DNA Binding An attractive strategy for the inhibition of transcription factors is blocking the DNA binding to specific recognition sequences. Pioneer work in this area has been conducted by Peter Dervan and his group, who have designed synthetic polyamides that can specifically target consensus sequences recognized by transcription factors. Indeed, a synthetic polyamide that specifically inhibits HIF-1 DNA binding has been designed and found to inhibit, as postulated, HIF-1 transcriptional activity (40). A limitation of polyamides as therapeutic reagents may be their poor cellular permeability and diffusion in tumor tissue, although they offer significant advantages for their potential increased specificity. Echinomycin, a small molecule that binds DNA in a sequence-specific fashion, has been identified in a cell-free screen aimed to identify small molecule inhibitors of HIF-1 DNA binding. Echinomycin inhibited HIF-DNA binding but not the binding of AP1 or NF-kB to cognate DNA-binding sites, suggesting a relative degree of sequence specificity (41). Since the HRE-binding site may also overlap with an E-box sequence (CACGTG), echinomycin was also found to inhibit binding of myc to the E-box, a feature that might have potential therapeutic implications. Inhibitors of HIF-1 Transcriptional Activity The transcriptional activity of HIF-1 is mediated by two domains, N-TAD and C-TAD. The C-TAD binds to CBP/p300 for maximal transcriptional activity, and is modulated by posttranslational modifications, including hydroxylation of Asn 803. Chetomin, a small molecule that inhibits HIF-1 binding to CBP, was identified in a screen aimed to identify inhibitors of HIF-1 transcriptional activity (42). This molecule was found to be active in in vitro and in vivo models, which provided proof of principle that HIF inhibition is a viable therapeutic strategy. However, the clinical development of chetomin for cancer therapy appears to be hampered by poor pharmacological properties. Interestingly, a recent evidence has emphasized that inhibition of the proteasome function, which blocks HIF-1a protein degradation, is also associated with inhibition of HIF-1 transcriptional activity by a mechanism that involves the TAD of HIF-1 (43). Velcade, an inhibitor of proteasome function, which is approved for therapy of myeloma, is currently being tested in several tumor types, and it will be interesting to see whether HIF-1 inhibition may be part of its therapeutic activity in tumors overexpressing HIF-1a. Miscellaneous HIF-1 inhibitors are continuously discovered and reported in the literature. However, in many cases, a clear mechanism of action of HIF-1 inhibition is neither reported nor identified.

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PX-478 is a potent antitumor agent and is found to be active in many xenograft models (44). PX-478 inhibits HIF-1a protein accumulation by an unknown mechanism of action. Interestingly, its activity in tumor xenografts seems to be associated with HIF-1a levels (44). Inhibition of HIF-1a protein expression following treatment with PX-478 has been demonstrated in animal models, and this agent will soon be tested in clinical trials as HIF-1 inhibitor in solid tumors. YC-1 is a cyclic GMP activator, known for its antiplatelets and vasodilatory effects. YC-1 inhibits HIF-1a protein accumulation by a mechanism that appears to be independent from the activation of cGMP (45). YC-1 was active in animal models and inhibited HIF-1a expression in tumor tissue; thus, it may be soon tested as an anticancer agent. Many different agents have been implicated in HIF-1 inhibition. Among these, thioredoxin inhibitors were originally found to inhibit HIF-1 protein accumulation (46), although recent evidence indicates that these agents may also inhibit transcriptional activity (47). Curcumin, a component of the yellow spice turmeric, inhibits HIF-1a protein accumulation by several mechanisms, including degradation of HIF-1b, which may be potentially associated with inhibition of HIF-1 activity (48). Gene Therapy Genetic approaches to target HIF-1a expression and function are an attractive strategy to inhibit HIF-1 in human cancers and have been tested in animal models with promising activity. In particular, expression of therapeutic genes under control of HRE, adenoviruses engineered to be expressed under hypoxic conditions, and antisense and siRNA approaches have all been tested and found to be somewhat active in different tumor models (49–54). Although targeting the hypoxic tumor is an attractive therapeutic strategy, the issue of delivery is still largely unresolved and currently hampers the potential application of this approach. Natural Products Many natural products have been identified and found to inhibit HIF-1 protein expression and function (55). In most instances, the exact mechanism of action of these compounds has not been elucidated and the activity has only been shown in cell culture and has not been validated in vivo. Natural products may have novel and interesting mechanisms of action in inhibiting HIF-1. Further studies will be required to determine if any of the agents identified so far has the potential to be used as therapeutic agent for cancer therapy. PRECLINICAL DEVELOPMENT AND TRANSLATIONAL END POINTS The development of molecular targeted agents requires a rationally designed plan to validate the activity on the intended target and to implicate this effect in a meaningful therapeutic activity. Unlike cytotoxic agents, whose development has been largely based on efficacy studies in multiple xenograft models, the development of molecular targeted agents requires preclinical models tailored to the specific agent under investigation. Several approaches have been described to validate the activity of HIF-1 inhibitors. Human cancer cell lines engineered to express the luciferase reporter

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gene under control of hypoxia response elements have been established (32). These cell lines have been used in xenograft and orthotopic models to monitor the activity of HIF-1 inhibitors on their target. The advantage of this approach is that luciferase can be measured in a noninvasive fashion giving the opportunity to serially monitor the effect of an agent on a functional basis. More elegant models based on noninvasive imaging of reporter genes may be anticipated in the future, and they should provide a valuable tool for validating the activity of HIF inhibitors. Since inhibition of HIF-1 may be associated with inhibition of angiogenesis and tumor metabolism, tissue endpoints reflecting these activities have been developed to monitor the effect of HIF inhibitors. Evaluation of tissue endpoints, documenting the functional inhibition of the HIF pathway, is essential to validate the activity of HIF inhibitors and to better understand how to use these agents in the clinic. Tissue endpoints can be easily measured in animal models, and careful analysis of these endpoints should be encouraged and warranted for the development of HIF inhibitors. Analysis of HIF-1a protein levels by IHC or western blot, mRNA expression of HIF-1 target genes by real-time PCR, and surrogate markers such as MVD or CAIX, have all been proposed and applied (32,44,45). A potential limitation of translating these pharmacodynamic endpoints to the clinic is that tissue must be acquired from patients, which is not always feasible or applicable. An alternative approach that could overcome these limitations and find a broader application is the use of imaging techniques assessing functional inhibition of HIF-1. Two main strategies have been used so far: 18FDG-PET, which provides an indication of tumor metabolism, and DCE-MRI, which reflects blood flow and angiogenesis (56–58). The rationale for using tumor metabolism as a readout of HIF-1 activity is based on the coordinate transcriptional regulation of glycolytic enzymes by HIF-1, which is consistent with the possibility that inhibition of HIF is associated with a decrease of PET signal. The application of DCEMRI relies on the assumption that inhibition of HIF may be associated with meaningful inhibition of angiogenesis. Again this is largely supported by the direct induction of a number of angiogenic factors by HIF, including, but not limited to, VEGF. However, these techniques are not widely available for application in preclinical models, and further studies are required to fully elucidate the association between inhibition of HIF and functional results. EARLY CLINICAL DEVELOPMENT Many agents have been shown to inhibit HIF-1a protein expression and function in cell culture models. Several of the agents identified have also been shown to inhibit HIF-1 in animal models, which has been, in turn, associated with antitumor and antiangiogenic activities. However, many questions remain to be answered as to the potential application of these results to the clinical setting. As discussed in the previous paragraph, validation of these agents in relevant preclinical models is essential for further clinical development. As therapeutic efficacy cannot be used as a reliable readout of HIF inhibition, more appropriate translational endpoints should be defined and used in preclinical and early clinical trials to validate the activity of HIF-1 inhibitors. Indeed, early clinical trials of HIF-1 inhibitors should emphasize the activity of the investigational agent on HIF-1 expression and/or function according to the known or proposed mechanism of action of the compound. Imaging techniques should also be developed to measure inhibition of

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HIF-1 or HIF-1-target functions, including, but not limited to, angiogenesis and tumor metabolism. Studies on tumor tissue are warranted to validate the activity of HIF-1 inhibitors on meaningful biological endpoints that may then be correlated with clinical benefit. Although the use of HIF-1 inhibitors in the clinical setting is still in its early phase of development, a strong scientific rationale has been provided for testing these agents in clinical trails aimed to validate the activity on HIF-1 and HIF-1target functions. However, it is plausible that HIF-1 inhibitors may have limited activity when used as single agents (59). Importantly, evidence has been provided that HIF-1 may contribute to resistance to chemotherapy (60) and radiation therapy (61,62), further suggesting that HIF-1 inhibitors may find a valuable application in combination with currently available therapeutic strategies. Indeed, combination therapies should be tested in preclinical models and rapidly translated to relevant clinical models. The rational development of combination strategies with conventional therapeutic approaches, i.e., chemotherapy and radiation therapy, as well as with novel molecular targeted therapies is warranted to fully exploit the potential of this novel and exciting area of developmental therapeutics. ACKNOWLEDGMENTS I would like to thank all the members of my laboratory for helpful discussions. My apologies to the many authors whose work describing HIF-1 inhibitors is not referenced, which is solely due to space limitations. REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13.

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Zhang X, Kon T, Wang H, et al. Enhancement of hypoxia-induced tumor cell death in vitro and radiation therapy in vivo by use of small interfering RNA targeted to hypoxia-inducible factor-1a. Cancer Res 2004; 64:8139–42. Chang Q, Qin R, Huang T, Gao J, Feng Y. Effect of antisense hypoxia-inducible factor 1a on progression, metastasis, and chemosensitivity of pancreatic cancer. Pancreas 2006; 32:297–305. Nagle DG, Zhou YD. Natural product-based inhibitors of hypoxia-inducible factor-1 (HIF-1). Curr Drug Targets 2006; 7:355–69. Jordan BF, Runquist M, Raghunand N, et al. Dynamic contrast-enhanced and diffusion MRI show rapid and dramatic changes in tumor microenvironment in response to inhibition of HIF-1a using PX-478. Neoplasia 2005; 7:475–85. Robey IF, Lien AD, Welsh SJ, Baggett BK, Gillies RJ. Hypoxia-inducible factor-1a and the glycolytic phenotype in tumors. Neoplasia 2005; 7:324–30. Jordan BF, Black K, Robey IF, Runquist M, Powis G, Gillies RJ. Metabolite changes in HT-29 xenograft tumors following HIF-1a inhibition with PX-478 as studied by MR spectroscopy in vivo and ex vivo. NMR Biomed 2005; 18:430–9. Li L, Lin X, Shoemaker AR, Albert DH, Fesik SW, Shen Y. Hypoxia-inducible factor-1 inhibition in combination with temozolomide treatment exhibits robust antitumor efficacy in vivo. Clin Cancer Res 2006; 12:4747–54. Brown LM, Cowen RL, Debray C, et al. Reversing hypoxic cell chemoresistance in vitro using genetic and small molecule approaches targeting hypoxia inducible factor. Mol Pharmacol 2005; 69:411–18. Moeller BJ, Cao Y, Li CY, Dewhirst MW. Radiation activates HIF-1 to regulate vascular radiosensitivity in tumors: role of reoxygenation, free radicals, and stress granules. Cancer Cell 2004; 5:429–41. Moeller BJ, Dreher MR, Rabbani ZN, et al. Pleiotropic effects of HIF-1 blockade on tumor radiosensitivity. Cancer Cell 2005; 8:99–110.

18

Antivascular Agents Jane Robertson Global Oncology Research and Development, AstraZeneca Pharmaceuticals, Aderley Park, Macclesfield, U.K.

INTRODUCTION All tumors need to generate their own blood supply in order to obtain sufficient oxygen and nutrients to grow beyond a volume of approximately 1 mm3 (1). They achieve this through the complex processes of angiogenesis, whereby endothelial cells proliferate in response to growth factors and invade the basal lamina, resulting in the budding of new vessels from the existing vasculature (2–4). The endothelial cells of tumor blood vessels are attractive targets for drug development because of their pivotal role in cancer cell survival, growth, and metastasis, and because they are more genetically stable, and potentially less likely to develop resistance to therapeutic agents than the tumor cells themselves (4). Furthermore, because endothelial cell targets are different from those targeted by chemotherapy agents, the potential for combination treatment can be exploited. A number of compounds that target components of the angiogenesis pathways are in development [currently, the most important of these is the vascular endothelial growth factor (VEGF) signaling pathway], and these are described in Chapter 6. The present chapter focuses solely on the development of compounds that target and occlude the established tumor vasculature to interrupt the blood supply. Such compounds have previously been termed “antivascular agents” or “vascular targeting agents,” but it has recently been proposed that the most useful term to distinguish their mechanism of action from antiangiogenic compounds is “vascular disrupting agents” (VDAs) (5), and this term is used hereafter in this chapter. Tumor vasculature differs from normal vasculature in a number of ways: tumor vessels are immature, with incomplete pericyte coverage and increased tortuosity, permeability, and fragility compared to normal blood vessels (6,7). The high proliferative rate of tumor vascular endothelial cells relative to normal endothelial cells was initially identified as a possible target for selective cancer therapies over 20 years ago (8–11). Such targeted approaches are needed because cells in areas of tumors that are relatively distant from the supplying blood vessels are subject to a hypoxic and acidic microenvironment. In these conditions, there are local areas of necrosis, and increased likelihood of cellular resistance to radiotherapy and chemotherapy agents. The delivery of anticancer agents is suboptimal, which also renders conventional treatment approaches less effective (12). VDAs act rapidly after administration to initially occlude and then collapse the existing tumor blood vessels, effectively starving areas of tumors of their blood supply. The result is marked ischemia, necrosis, and hemorrhage in tumors (13–17). These effects are more marked in the central areas of tumors. In contrast, a thin, viable rim of tumor cells characteristically remains at the periphery, where the cells are nourished either by diffusion of nutrients from normal adjacent tissue or from host blood vessels in close proximity that are less susceptible to the agents than the tumor vessels (Fig. 1). 295

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Tumor pre-treatment Necrotic tumor cells

Viable tumor cells

VDA

Endothelial cells

Endothelial cells change shape and detach

Loss of endothelial cells from substratum leads to congestion of blood vessel

Tumor necrosis due to lack of nutrient supply

FIGURE 1 Mechanism of action of vascular disrupting agents (VDAs).

The clinical development program for VDAs has focused on combining these compounds with a variety of anticancer therapies including chemotherapy and radiotherapy, since the latter are more effective in the relatively well-oxygenated and nourished periphery of the tumor where cells are dividing quickly (17). For the same reason, combinations of VDAs with antiangiogenic agents are attractive treatment options since the majority of endothelial cell proliferation and angiogenesis occurs in these peripheral areas. The scheduling of the different modalities appears to be important; VDAs appear to be more effective if given following chemotherapy or radiotherapy than if given beforehand (14,18), possibly because if given beforehand, they may impair the delivery of chemotherapy agents, or because they remove some of the most chemo/radioresistant cells from the hypoxic center of the tumor. In preclinical studies, combinations of VDAs and chemotherapy/radiotherapy agents have not been shown to increase the toxicities of either regimen (14,18). Whereas antiangiogenesis agents may be expected to be most effective when started in the early stages of cancer, VDAs may be applicable to later stages and larger sized tumors (Fig. 2). Also, while chronic dosing of antiangiogenic agents may be required for a sustained antiangiogenic effect, VDAs have an acute effect, and intermittent dosing schedules appear effective. The two approaches may therefore be complementary, and combinations of antiangiogenic agents with VDAs are under investigation. APPROACHES TO THE DEVELOPMENT OF VASCULAR DISRUPTING AGENTS There have been two main approaches to the development of VDAs: a surface ligand targeted approach and the development of small molecules of different classes that enter cells to exert their effects. The compounds in these classes that are currently in clinical development are summarized in Figure 3. The surface-ligand targeted approach links molecules that induce endothelial cell damage or thrombosis to specific antigens on the luminal surface of tumor vessel endothelial cells by means of a monoclonal antibody. Selectivity for tumor endothelial cells is achieved by choosing antigens that are overexpressed on

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Inhibition of angiogenesis

Vascular disruption

Blood vessel

Angiogenesis inhibitor

Inhibit tumor growth

Blood vessel

Vascular disrupting agent

Tumor necrosis

FIGURE 2 Differences in the mechanism of action and antitumor effects between antiangiogenesis agents and vascular disrupting agents.

tumor vessels with respect to normal vessels, e.g., VEGF receptors (19), CD105 (endoglin) (20), ab integrins (21) or the fibronectin ED-B domain (22). Most of these agents have proven effective in preclinical models, but have yet to enter clinical trials. More recently, the cell adhesion molecule N-cadherin has been confirmed as a promising target for VDAs, as the antagonist compound ADH-1 (Adherex Technologies, Durham, North Carolina, U.S.A.) has reported positive Phase I results in the clinic (23,24). There are two major classes of small molecule VDAs: tubulin binding agents and flavonoids. The former bind to b-tubulin subunits to prevent polymerization and microtubule formation, thereby destabilizing the microtubule cytoskeleton, causing a change of shape in endothelial cells and their subsequent detachment from the basal lamina and apoptosis (25–27). Flavonoids cause cytokine release that induces vessel collapse. This chapter focuses specifically on the VDAs that are currently in clinical trials with reference to the development challenges they pose, and their emerging safety, tolerability and efficacy profiles. Tubulin-Binding Agents Microtubules are key components of the cytoskeleton and are vital for a number of essential cellular functions; they form the cytoskeleton of endothelial cells and are required for the maintenance of cellular shape, the formation of the mitotic spindle, and movement of organelles, receptors and transporters through the cytoplasm (28). Microtubules are composed of a- and b-tubulin heterodimers and are dynamic structures, growing by polymerization. Inhibition of microtubular function results in poor alignment of chromosomes during mitosis, mitotic arrest and apoptosis (29), and this forms the basis of the mechanism of action of a number of chemotherapy drugs, specifically the vinca alkaloids and taxanes that can be classified into three groups based on their tubulin-binding domains: the Vinca domain, the taxane domain and the colchicine site. At high doses, vinca

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Small molecules Tubulin binding agents Anti-mitotic and anti-vascular effects • TZT-1027 • ABT-751 Primary anti-vascular effect Combretastatin derivatives • CA4DP • AVE-8062 N-acetylcholinol prodrug • ZD6126 Second generation agents • MN-029 • NPI-2358 • Oxi-4503

Surface-ligand targeted agents Flavonoids DMXAA

Liposomes encapsulating cytotoxic effectors • EndoTAGTM -1 Anti N-cadherin • ADH-1 Exherin™

FIGURE 3 Vascular disrupting agents in clinical development.

alkaloids, taxanes and colchicine have all demonstrated antivascular effects in preclinical models because binding to tubulin alters the cytoskeleton and shape of endothelial cells. This shape change increases vascular permeability and ultimately leads to their detachment from the basal lamina, vessel wall collapse and occlusion of tumor blood flow (25–27). Because these compounds have a narrow therapeutic window with associated toxicities, especially in the gastrointestinal tract and peripheral nervous system, the antivascular effect is not achieved at doses that are tolerable for man. Successful attempts have been made to increase the therapeutic window either by enveloping taxanes within lipid complexes that target endothelial cells in order to deliver high doses to the tumor without high systemic exposure, or by generating novel tubulin-binding agents with a similar but more favorable safety profile compared to taxanes and vinca-alkaloids. MBT-1 (EndoTAG
; MediGene, San Diego, California, U.S.A.) is an agent that envelopes paclitaxel within a cationic liposome with a high affinity for tumor vascular endothelial cells. Phase I trials are complete (30,31) and the compound is currently in phase II clinical development as a monotherapy in advanced breast cancer and in combination with gemcitabine in pancreatic cancer. Examples of the second approach are the intravenous (i.v.) dolastatin analogue TZT-1027 (Daiichi Sankyo, Ltd., Tokyo, Japan) and the oral sulfonamide ABT-751 (Abbott Laboratories, Abbott Park, Illinois, U.S.A.), both of which have a similar side effect profile to taxanes in terms of ileus and constipation, but ABT-751 caused markedly less leucopenia and TZT-1027 reported less neurotoxicity. TZT-1027 is currently in phase II development in patients with metastatic soft tissue sarcomas having completed a phase I program designed to identify the optimum dose and schedule for use as monotherapy and as a combination agent (32–36). ABT-751 underwent a similar phase I monotherapy and chemotherapycombination development program (37–42) but in its phase II studies the compound is predominantly used as a monotherapy agent in patients with refractory colorectal cancer, renal cancer, breast cancer and non–small cell lung cancer (NSCLC) (43–46). While the mechanism of action of these agents may be considered

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primarily as antimitotic rather than antivascular, antivascular effects have been demonstrated preclinically (47). The remainder of this section focuses on the tubulin-binding agents whose primary effects are antivascular rather than antimitotic. The first agent to exhibit antivascular effects at tolerable doses was combretastatin (48), a natural derivative of the bark of the South African willow tree (Combretum caffrum) that binds to the colchicine site of b-tubulin. A number of synthetic prodrugs of combretastatin were developed to overcome solubility issues. The most advanced in clinical development is Combretastatin A4 di-phosphate [CA4P (Zybrestatin
; OxiGENE, Inc., Waltham, Massachusetts, U.S.A.). Others include Combretastatin A1 di-phosphate (Oxi4503; OxiGENE, Inc.) and AVE-8062 (Sanofi-Aventis, Paris, France). ZD6126 (Angiogene Pharmaceuticals, Ltd., Oxford, U.K.), a water-soluble pro-drug of N-acetylcolchinol, also binds to the colchicine site and has a similar mechanism of action but different pharmacokinetic properties than Combretastatin. Second-generation tubulin binding VDAs such as MN-029 (MediciNova, Inc., San Diego, California, U.S.A.), Oxi4503 (OxiGENE, Inc.), and NPI-2358 (Nereus Pharmaceuticals, San Diego, California, U.S.A.) have more recently moved into clinical development. Preclinical Studies In preclinical assays, CA4P, ZD6126 and AVE-8062 have demonstrated very similar antivascular and antitumor effects at doses that are less than one-tenth of the maximum tolerated dose (49). Demonstration of “proof of principle” for such compounds includes the evidence that they disrupt the tubulin cytoskeleton of tumor vascular endothelial cells leading to endothelial detachment and exposure of the basal lamina and extensive endothelial cell apoptosis (25–27). These compounds have also been shown to exhibit antitumor activity in a broad range of tumors in a manner in keeping with their proposed mechanism of action: reduction in blood flow and tumor vascular volume followed by widespread necrosis (50–53). In vitro, these effects occur selectively in tumors; treatment with CA4P or ZD6126 results in rapid changes in the three-dimensional shape of proliferating endothelial cells, but not of resting endothelial cells, and either no or limited effects are seen in normal tissues (50–53). Preclinically, the compounds have been shown to enhance the antitumor effects of chemotherapy agents, including cisplatin (51), paclitaxel (53), doxorubicin (54) and 5-FU (55), as well as radiotherapy (56–58), radioimmunotherapy (59); hyperthermia (60) and antiangiogenic agents (61). Clinical Studies Table 1 summarizes the main Phase I development programs of CA4P, ZD6126 and AVE-8062, the three tubulin-binding agents whose mechanism of action is primarily antivascular and are most advanced in terms of clinical development. Combretastatin A4 Diphosphate The initial phase I development of CA4P consisted of three monotherapy studies to investigate the safety, tolerability and pharmacokinetic profile of the compound and identify an optimum treatment schedule. The first study (62,63) investigated single doses given at 3 weekly intervals in 25 patients with advanced cancers. The study monitored effects on tumor blood flow using dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI) and changes in plasma levels of cell

10 min i.v

10 min i.v

30 min i.v

20 min i.v

CA4P

ZD6126

AVE-8062

DMXAA

3 weekly Weekly Weekly (lower doses up to 3000 mg/m2 explored)

Every 3 wk Weekly for 3 wk out of 4 5 days every 3 wk Every 2 or 3 wk Weekly 5 days every 3 wk Weekly for 3 out of 4 wk 5 days every 3 wk 3 weekly

Schedule 2

3700 mg/m2 3700 mg/m2 1200 mg/m2

60 mg/m 68 mg/m2 52 mg/m2 80 mg/m2 20 mg/m2 1 g/m2/day 20 mg/m2 22 mg/m2 22 mg/m2

MTD

DLTs Tumor pain, acute coronary syndrome Ataxia, syncope, neuropathy Dyspnea, syncope Tumor/abdominal pain Pulmonary embolism, Reduced LVEF QTc prolonged, hepatic enzymes increased hypotension Cerebral ischemia, myocardial ischemia Myocardial ischemia, asymptomatic hypotension, transient cerebral ischemia Confusion, anxiety, visual disturbance, reduced LVEF, urinary incontinence, QTc prolonged Transient, moderate increases in QTc, increases in blood pressure, visual disturbances

Abbreviations: LVEF, left ventricular ejection fraction; NSCLC, non–small cell lung cancer.

Infusion

Compound

1200 mg/m23 weekly þ chemo NSCLC Ovary Prostate

Ovary NSCLC Anaplastic Thyroid 45–63 mg/m2 weekly for 3 wk out of 4 Renal colorectal NSCLC program suspended pending further preclinical evaluation Colorectal Dose range not defined Program temporarily suspended but restarted

Phase II/III dose range and tumors

TABLE 1 Summary of Early Phase I Clinical Development Program for Small Molecule VDAs and Plans for Further Development

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adhesion molecules in patients at the higher-dose levels. Four dose levels were examined from 18–90 mg/m2. The maximum tolerated dose (MTD) was defined as 60 mg/m2. Dose limiting toxicities (DLTs) above this dose were tumor site pain and two episodes of reversible acute coronary syndrome. Other “vascular” adverse effects included hot flushing, headache, abdominal cramping, and changes in blood pressure and heart rate. There were no typical cytotoxic adverse effects similar to those seen with taxanes. Dose and pharmacokinetic related increases in heart rate (HR) and QTc interval were observed in 15 patients. These effects were reversible by 24 hours. None of the patients was considered to have clinically significant increases in QTc interval, defined as any QTc interval >500 ms, a >25% increase in QTc over baseline, or ventricular arrhythmias. Pharmacokinetic analysis showed a short plasma half-life of approximately 30 min. Significant reductions in tumor blood flow by DCE-MRI were observed in six out of seven patients treated at 60 mg/m2. Three patients had prolonged disease stabilization and one patient with anaplastic thyroid cancer had a complete response that was durable for at least 3 years. In another study, 37 patients with solid malignancies received CA4P daily for 5 days repeated every 3 weeks (64). Again, the unique dose limiting toxicity was tumor pain, observed at a dose of 75 mg/m2. Cardiovascular DLTs (syncope and dyspnoea or hypoxia) were observed at this dose and the MTD in this study was defined as 52 mg/m2, a dose at which decreased tumor perfusion was observed by DCE-MRI. Other adverse events included hypotension, ataxia, headache and transient sensory neuropathy. A partial response was observed in a patient with metastatic soft tissue sarcoma, and prolonged disease stabilization was demonstrated in a number of patients. In the third phase I study, CA4P was given weekly for 3 weeks out of four (65). The only drug-related toxicity up to 40 mg/m2 was tumor pain. Dose-limiting toxicities at higher doses were variable: reversible ataxia at 114 mg/m2, vasovagal syncope and motor neuropathy at 88 mg/m2, and fatal ischemia in previously irradiated bowel at 52 mg/m2. Other adverse events included hypertension, hypotension, visual disturbance and dyspnea. Overall, CA4P was considered to be tolerable at doses of 52 or 68 mg/m2 at this dose schedule. Further phase I studies of CA4P focused on combination therapy with a number of agents including radiotherapy, chemotherapy and radioimmunotherapy. In a phase Ib trial, CA4P at a dose of 50 mg/m2 was given in combination with different radiotherapy schedules (66). The most common drug related toxicities were grade 1 hypertension, mild bradycardia and QTc prolongation (13 ms), lymphopenia, and severe tumor pain that often required opioids. One patient had grade 3 postural syncope. There was no increase in the number or severity of reactions to radiotherapy, and no accumulation of toxicity with repeated doses. Tumor blood flow was assessed using perfusion CT and demonstrated a sustained reduction in tumor blood volume. Following a phase I study of CA4P in combination with carboplatin (67), a three-center phase Ib/II trial of CA4P in combination with carboplatin and paclitaxel, both alone and together, was carried out in patients with advanced ovarian cancer and other solid tumors (68,69). The adverse effects in the Phase Ib portion of the study were mild and self-limiting with no cardiac toxicity, and CA4P added no additional toxicity to the chemotherapy alone. Dose-limiting toxicities of hypertension and ataxia occurred at a CA4P dose of 72 mg/m2. Objective tumor responses, defined according to the response evaluation criteria

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for solid tumors (RECIST) (70) were observed in a number of patients. In a subset of patients with advanced ovarian cancer, RECIST or CA-125 responses were observed in 10 out of 15 patients and four others had prolonged disease stabilization. On the basis of these data, the FDA granted orphan drug status for CA4P for the treatment of this disease in May 2006. The Phase II study in patients with platinum-resistant ovarian cancer (CA4P at 63mg/m2, combined with 175 mg/m2 of paclitaxel and carboplatin at AUC 5) is ongoing. Two studies in NSCLC are planned for 2006. The first, a Phase Ib/II study of CA4P plus chemoradiotherapy for the treatment of stage IIIa/IIIb unresectable NSCLC, will initially evaluate the tolerability of the regimen and identify a recommended Phase II dose for CA4P, then proceed as a randomized, open label, study to determine the RECIST response rate and one-year survival benefit in approximately 80 previously untreated patients. The second study is a randomized, double blind, placebo controlled Phase III study of CA4P in combination with carboplatin and paclitaxel in patients with newly diagnosed unresectable Stage IIIb/IV NSCLC, with the primary endpoint of overall survival. Because of the responses seen in advanced anaplastic thyroid cancer in the monotherapy studies, a trial program in this disease setting was also initiated. Results of the first Phase II study were presented at ASCO annual meeting 2006 (71). In this study, patients with metastatic anaplastic thyroid cancer were treated with CA4P as a single infusion at a dose of 45 mg/m2 on days 1, 8 and 15 of each 28-day cycle until disease progression. The treatment was well tolerated with adverse events of tumor pain but no significant cardiac toxicity. No objective responses were observed but approximately one-quarter of patients experienced prolonged disease stabilization (>3 months). A second phase II study of induction chemotherapy (doxorubicin and cisplatin) followed by CA4P and radiotherapy has been initiated in patients with newly diagnosed regionally advanced anaplastic thyroid cancer with primary objectives of objective response rate and overall survival and secondary endpoints of defining clinical predictors of response (e.g., pretreatment tumor microvessel density, changes in sICAM-1 levels, and tumor blood flow). ZD6126 A number of single agent Phase I studies of ZD6126 were carried out to identify an optimum schedule and define the tolerability and PK profile. In the first study, patients with advanced solid tumors received single doses of ZD6126 either once every 21 days or once every 14 days. DCE-MRI was used to assess tumor blood flow before and 6 hours after dosing. Significant reductions in tumor blood flow were documented at doses of 80 mg/m2 and higher (72). For patients who received ZD6126 every 3 weeks, additional DCE-MRI assessments were carried out 24 hours and 21 days after dosing and, although some recovery in tumor blood flow was apparent at 24 hours, reduced blood flow was maintained for up to 3 weeks in some patients (73). The terminal phase half-life was approximately 2 hours. The maximum tolerated dose of ZD6126 was defined as 80 mg/m2 for either dosing schedule. The major dose-limiting toxicity was abdominal pain. ZD6126 was associated with asymptomatic, reversible ECG changes and increased troponin concentrations suggestive of ischemia in some patients (74). In a second study 32 patients received weekly infusions of ZD6126 at doses of 5–28 mg/m2 (75). The pharmacokinetics appeared linear across this dose range. The terminal phase half-life was 1–3 hours. The MTD was identified as 20 mg/m2 with dose-limiting toxicities above this level of pulmonary embolism and reduced

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left ventricular ejection fraction. During the study, three patients with progressive disease experienced grade 3 and 4 thromboembolic events (pulmonary embolism and inferior vena cava thrombosis). Other adverse events included grade 1 and 2 reductions in LVEF, asymptomatic increases in CK-MB, and one patient with a history of ischemic heart disease had an acute myocardial infarct 2 weeks after discontinuing ZD6126. A broad Phase II program was initiated to assess the efficacy and tolerability of ZD6126 administered every 2–3 weeks, in monotherapy and combination regimens. The development of ZD6126 was suspended due to toxicity seen early in the Phase II program, and preclinical studies to understand the cardiovascular effects of the drug were initiated. AVE-8062 AVE-8062, a water-soluble analogue of Combretastatin A4, underwent a similar Phase I development program to the other VDAs described previously. The first Phase I study of AVE-8062 in patients with advanced cancer investigated a weekly schedule for 3 weeks out of 4 (76). Patients received doses ranging from 4.5 to 30 mg/m2 and the 30 mg/m2 cohort was expanded due to a DLT of asymptomatic systolic hypotension without evidence of CPK, troponin I, or ECG changes. Reductions in tumor vascular flow by DCE-MRI were observed at and above the 15.5 mg/m2 dose level. AVE8062 was rapidly eliminated with a t1/2 of 15 minutes although an active metabolite was identified with a t1/2 of 7 hours. In a second Phase I study AVE-8062 was given for five consecutive days every 3 weeks, and a third investigated the 30 min i.v. infusion given every 3 weeks in patients with advanced solid tumors (77). Because of the occurrence of four potentially drug-related vascular events (myocardial ischemia, transient asymptomatic hypotension, transient cerebral ischemia, asymptomatic ventricular tachycardia) in the 5-day and weekly schedule studies without residual clinical deficits, all trials were voluntarily and temporarily interrupted. However, since no vascular events have been observed in the three weekly schedule study up to a dose of 22 mg/m2, this trial was later resumed at that dose, with restricted eligibility criteria and increased cardiovascular monitoring (continuous 24-hours ECG, continual ambulatory blood pressure monitoring, serial CPK, troponin, ECG, ventriculographies and echocardiograms) and the 22 mg/m2 dose was found to be well tolerated (77). Two combination studies are currently open: a phase I study of AVE-8062 in combination with oxaliplatin every 3 weeks, and a study in combination with cisplatin 70 mg/m2 every 3 weeks both in patients with advanced solid tumors. Exclusion criteria for both studies are strict with respect to cardiovascular risk factors and patients are required to remain in the hospital for intensive cardiac monitoring during the study. Second Generation Tubulin Depolymerizing Agents MN-029 (denibulin hydrochloride) was designed to be more potent and with possibly less central nervous system toxicity than first generation VDAs. Preliminary results of the first phase I study of MN-029 given every 3 weeks have been reported (78). At the time of the presentation, the maximum tolerated dose had not been reached but the 180 mg/m2 cohort was expanded to six patients because of a dose-limiting toxicity (DLT) of acute reversible myocardial ischemia in one patient. Adverse events included nausea, vomiting, hypotension, fatigue and diarrhea.

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Tumor blood flow reduction assessed by DCE-MRI was recorded at doses of 120 mg/m2 and higher. A second Phase I study of MN-029 given every 7 days (days 1, 8 and 15), followed by a 2-week recovery period over two treatment cycles is currently open. OXi4503 (combretastatin-1 phosphate) is a second-generation combretastatin analogue that has recently entered clinical development. In the first Phase I dose escalation trial it was given as a weekly i.v. infusion for 3 weeks to identify the maximum tolerated dose and effects on tumor blood flow by DCE-MRI and PET scanning in patients with advanced solid tumors. NPI-2358 is a novel VDA tubulin-binding agent derived from a marine fungus. The first phase I study commenced in April 2006. N-Cadherin as a Target for VDAs N-cadherin (N-cad) is a cell adhesion molecule expressed by both the vascular endothelial cells and tumor cells of invasive tumors. The switching of cadherin between its E- and N-forms appears to increase tumor cell survival and their invasive and metastatic potential. ADH-1 Exherin
(Adherex Technologies) is an antagonist of N-cad that has shown tumor vascular disruption and apoptosis in preclinical models. Results from two phase I studies of ADH-1 were reported in 2006 (23,24). The first was a phase I study of weekly doses of i.v. ADH-1 given to patients with N-cad positive solid tumors, to evaluate safety, pharmacokinetics, antitumor activity and effect on tumor vasculature assessed by DCE-MRI. Of the 55 patients screened, 56% were N-cad positive. ADH-1 was given weekly for 3 weeks out of 4 at but the schedule was later amended to weekly ADH-1 without interruption. No patients had experienced greater than grade 2 study drug related AEs at the time of the report. Preliminary evidence of antitumor activity, reductions in tumor blood flow and tumor pain, was reported. The second Phase I study was in patients with refractory solid tumors stratified according to their N-cad expression. ADH-1 was well tolerated and the maximum tolerated dose was not defined. The most commonly reported adverse events were grade 1–2 fatigue, nausea, dysgeusia, and flushing. Four of the 28 subjects in the N-cad positive group demonstrated antitumor activity (one PR, one MR and two patients with prolonged (>7 months) SD, while no antitumor activity was noted in the N-cad negative group (n ¼ 18). The mean terminal Phase t1/2 was 2.2 hours. Further development has concentrated on N-cad positive patients. Two Phase II studies are ongoing in subjects with recurrent N-Cad positive advanced solid tumors. In the first, ADH-1 is administered as a single agent at 600mg/m2 once every 3 weeks to patients with selected solid tumors (renal cell carcinoma, hepatocellular carcinoma, adrenocortical carcinoma, head and neck squamous cell cancer, gastro-oesophageal carcinoma, breast carcinoma or NSCLC). A second study is examining a weekly dosing schedule of 600 mg/m2 of ADH-1 in patients with similar tumor types. Three combination studies have also been initiated with docetaxel, carboplatin, and capetitabine. Flavonoids Flavonoids such as flavone-8-acetic acid (FAA) and its derivative 5,6 dimethylxanthenone-4-acetic acid (DMXAA [AS-1404]; Antisoma, plc, London, U.K.) do not directly bind to tubulin but cause similar levels of tumor endothelial cell damage as tubulin-binding agents (79,80). They induce a number of plasma cytokines including serotonin, nitric oxide, interferon and tumor necrosis factor a (TNF-a), and

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cause the local release of TNF-a from activated macrophages within the tumor itself (81–83). TNF-a has been shown to cause hemorrhagic necrosis in tumors (84) and it appears to be a necessary component for the response to flavonoids since antibodies against TNF-a inhibit FAA-induced vascular collapse in preclinical models (81,82). DMXAA has a higher potency than FAA in animal models and, unlike FAA, has been shown to stimulate human macrophages to produce TNF-a (85,86). In preclinical models, DMXAA has been shown to act synergistically with a number of treatment modalities including radiotherapy (87), chemotherapy (88), bio reductive agents (89), radioimmunotherapy (90), and immunotherapy (91). DMXAA (AS1404)—Clinical Studies The Phase I program consisted of three studies designed to investigate the safety and pharmacokinetic profile of DMXAA, and to identify an optimum dose and schedule for Phase II development. The first study investigated a three weekly schedule (92). Dose-limiting toxicities occurred at 4900 mg/m2 and included confusion, tremor, slurred speech, visual disturbance, anxiety, urinary incontinence and possible left ventricular failure. These were all rapidly reversible on stopping the drug. Transient asymptomatic prolongation of the QTc interval was seen at doses of 2000 mg/m2 and above. Tumor blood flow reduction was demonstrated by DCE-MRI and increases in plasma 5-hydroxyindoleacetic acid (5-HIAA) and plasma nitrate occurred in most patients treated at doses above 850 mg/m2. The second study evaluated a weekly dosing schedule (93). Dose-dependent increases in 5-HIAA were observed at doses of 650 mg/m2 and above. There was one unconfirmed partial response at 1300 mg/m2. The terminal half-life was approximately 8 hours. In both studies the pharmacokinetic profile was non-linear due to saturation of protein binding at higher doses. The maximum tolerated dose from the two studies was established at 3700 mg/m2 due to dose-limiting toxicities of urinary incontinence, visual disturbance and anxiety occurring at 4900 mg/m2. The visual disturbance was transient, rapid in onset and consisted of blurring, flickering and alteration of color discrimination associated with electroretinogram (ERG) abnormalities. A third study (94) was initiated to further investigate the cardiac and ophthalmic adverse events, to define a range of doses that produced an acceptably small effect on the QTc interval, and to identify an optimal dose for combination studies. Patients were allocated randomly to receive one of six sequential doses of DMXAA (300–3000 mg/m2), each given once weekly. Transient, moderate increases in QTc, increases in blood pressure and visual disturbances were observed at the two highest doses. Plasma levels of 5-HIAA increased acutely after treatment, dose dependently, up to 1200 mg/m2, and then plateaued. Doses in the range of 1200 mg/m2 were well tolerated and selected for further studies. Subsequent studies have concentrated on combination therapy. Preliminary results of a phase I/II study in combination with carboplatin/paclitaxel in patients with stage IIIb or IV previously untreated NSCLC have been reported (95). The safety of the combination was initially assessed in one patient who received standard doses of carboplatin and paclitaxel with DMXAA at 600 mg/m2. No toxicities were observed and the patient had a partial response to treatment. Patients were then randomized to receive up to six cycles of carboplatin and paclitaxel alone or with DMXAA at 1200 mg/m2. In a further study of six patients, a dose of 1800 mg/m2 was investigated. The safety profile in the control and DMXAA arms was comparable. No pharmacokinetic interactions were observed. Seventy patients were evaluable for efficacy, of whom 34 received DMXAA plus

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standard chemotherapy while 36 received standard chemotherapy alone. There was an improvement in time-to-tumor progression (132 vs. 115 days for the standard chemotherapy group) and objective tumor response (31.2% vs. 22.2% with standard chemotherapy) assessed by central independent review. The projected 6-month survival rate was 82.0% for patients receiving DMXAA and 54.8% for patients receiving standard chemotherapy, and the projected median survival is currently 12.0 months with DMXAA and 7.6 months for the standard chemotherapy group. An extension study in which additional NSCLC patients are treated at 1800 mg/m2 is ongoing and a Phase III study is planned. A second randomized Phase II study has been carried out in combination with carboplatin and paclitaxel in 70 patients with recurrent platinum-sensitive ovarian cancer with a progression-free interval of more than 6 months after initial response to platinum-based chemotherapy (96). Patients were randomized to receive carboplatin and paclitaxel with or without DMXAA at 1200 mg/m2. The combination appeared tolerable in this disease setting and efficacy assessments are ongoing. A third randomized Phase II study has completed recruitment of patients with hormone-refractory metastatic prostate cancer. Patients were randomized to receive three weekly docetaxel doses with or without DMXAA for up to 10 cycles in the absence of disease progression, and following progression patients will receive weekly DMXAA alone. Preliminary data from the first 64 of 74 patients in the trial have recently been reported (97). There was a PSA response rate of 57% in those receiving AS1404 plus docetaxel chemotherapy compared with 35% in those receiving docetaxel alone, while the proportion of men showing disease progression by PSA was 17% in the AS1404 group compared with 29% with docetaxel alone. The combination of AS1404 with docetaxel was well tolerated, without exacerbation of chemotherapy side effects. OPPORTUNITIES AND CHALLENGES IN THE DEVELOPMENT OF VDAS The development of VDAs has provided an exciting new approach to the treatment of cancer. The preclinical and clinical studies outlined in this chapter illustrate the effectiveness of these agents in multiple tumor types. Since no single approach is likely to be effective alone in the treatment of such a complex disease as cancer, the potential of VDAs to be synergistic with a number of other anticancer measures, including chemotherapy, radiotherapy, immunotherapy, and antiangiogenic agents, is of primary importance. However, the description of the clinical development programs of the most advanced agents also serves to illustrate the new challenges posed by these compounds, some of which are shared by the antiangiogenic compounds and other novel targeted therapies currently in development. The first challenge has been that VDAs, while causing extensive necrosis and hemorrhage within tumors, may not themselves cause tumor shrinkage as single agents, making assessment of objective tumor responses difficult by conventional methods. This has resulted in the implementation of alternative imaging methods, such as DCE-MRI or dynamic contrast CT (DC-CT), to assess tumor blood flow as evidence that the compounds are exerting an antivascular effect in tumors. While such methods provide convincing and often dramatic evidence of tumor blood flow reduction following administration of single doses, there are currently limitations to the applicability of the techniques. In particular, the high degree of intra- and

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inter-patient variability makes interpretation and quantification of absolute levels of reduction difficult, and because the relationship between tumor blood flow reduction and efficacy in terms of progression-free survival is not yet clear, the value of such imaging techniques in guiding the choice of dose for Phase II development is limited. There are also technical challenges, such as identification of the optimum contrast agents suitable for use in humans, and the current limited reproducibility of results between different centers. In most early clinical trials, the advanced imaging techniques have been carried out at only one center to minimize variability, which is an approach possible in Phase I development. However, in order to determine whether demonstration of early tumor blood flow reduction is a true biomarker of efficacy, DCE-MRI changes will need to be evaluated in larger efficacy studies in single tumor types, which will raise the issue of inter-center variability. Another major challenge is the choice of dose and schedule for the VDAs. The development of any i.v. agent requires multiple Phase I studies to define the optimum schedule, but, since the side effect profile for the majority of cytotoxic agents is well understood and clearly dose-related, the strategy has generally been to define the maximum tolerated dose to take into the next phase of development. In contrast, there are significant challenges to defining an optimum dose and schedule for an agent with an unknown adverse event profile, and where the goal is to define an optimum biologically active dose rather than a maximum tolerated dose. None of the agents in development has taken the maximum tolerated dose forward to further studies, but some have identified a lower biologically active dose based on a combination of safety, a pharmacokinetic profile in the range predicted to be effective from preclinical experiments, and evidence of pharmacodynamic effects on tumor blood flow and, in some cases, soluble biomarkers, e.g., HIAA for DMXAA, circulating endothelial cells for ZD6126, and sICAM for CA4P and ADH-1. In most of the clinical studies of individual VDAs, the adverse event profile has not been clearly dose-related or consistent between Phase I studies. VDAs cause a unique adverse event of tumor pain, presumably due to ischemia and necrosis, but this has not been sufficient to guide dosing. These agents are associated with a different spectrum of adverse events compared to conventional cytotoxic agents, with the major class effect occurring in the cardiovascular system, possibly because of drug-induced microvessel vasoconstriction and/or endothelial cell activation leading to the formation of micro-thrombi in the coronary circulation and compromising the blood supply to the cardiac muscle. Most clinical studies of all the VDAs in development have reported adverse events of asymptomatic elevations in troponin or CK-MB indicative of myocardial damage, acute coronary syndrome, QTc prolongation, and reduction in left ventricular ejection fraction. In most cases, the agents have a short half-life, and the effects are quickly reversible; however, a number of cases of pulmonary embolism and myocardial infarction have been reported. For three agents (AVE-8062, ZD6126, and DMXAA) this led to re-evaluation of the clinical programs. In the case of DMXAA, a study was specifically designed to further evaluate the observed cardiac and visual effects; in the case of ZD6126, further preclinical studies have been performed to define ways of preventing and managing the cardiac effects; and for AVE-8062, clinical development was restarted at a lower dose, with a less frequent treatment schedule, and with a number of strict restrictions on cardiovascular eligibility criteria and extensive monitoring of cardiac function and blood pressure. While this is possible in a clinical trial setting, it may prove challenging to provide the necessary close cardiac monitoring for patients once such agents are approved and widely prescribed.

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The choice of dose and schedule for a VDA faces a further challenge when combining these novel agents with conventional chemotherapy agents; in this case the choice of schedule may be limited, for practical as well as scientific reasons, by the need to align with the chemotherapy cycles. The possibility of drug interactions must be taken into account, e.g., the potential of the combination to cause or exacerbate QTc prolongation, the potential for a pharmacokinetic interaction, or the potential for overlapping cardiac toxicities, which may be the case with high cumulative doses of anthracyclines. In summary, this is an exciting new class of agents, some of which are at an advanced stage of clinical development. The results of the Phase III studies of CA4P in ovarian cancer and DMXAA in NSCLC will clarify the potential of VDAs to improve response rates, progression free survival, and overall survival in these diseases compared to conventional chemotherapy alone, and will further define the risk to patients in terms of cardiovascular side effects. Future challenges for the class will be to define which patients are most likely to benefit from the addition of such agents, and which of the current chemotherapies and novel targeted agents in development are most suitable for combination to maximize their antitumor effects. REVIEWS Thorpe PE. Vascular targeting agents as cancer therapeutics. Clin Cancer Res 2004; 10:415–27. Chaplin DJ, Dougherty GJ. Tumour vasculature as a target for cancer therapy. Br J Cancer 1999; 80:57–64. Siemann DW, Chaplin DJ, Horsman MR. Vascular-targeting therapies for treatment of malignant disease. Cancer 2004; 100:2491–9.

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19

Matrix Metalloproteinases Stéphane Vignot Service d’Oncologie Médicale, Groupe Hospitalier Diaconesses Croix Saint Simon, Paris, France

Jean-Philippe Spano Service d’Oncologie Médicale, Groupe Hospitalier Pitié Salpétrière, Paris, France

INTRODUCTION Basic research about cancer was initially focused on tumor cells, and carcinogenesis was then considered as the result of serial mutations (activation of oncogenes and loss of expression of tumor suppressor genes) modifying their phenotype (1). A good understanding of tumor progression led afterwards to a growing interest in stroma (2). Stroma is composed of nontransformed cells in the host and an extracellular matrix (ECM) that supports adhesion of cells and transmits signals through cellsurface adhesion receptors. The ECM contains collagens, noncollageneous glycoproteins, and proteoglycans. The basement membrane is a specialized compound of ECM that separates the epithelial cells from the underlying stroma, providing the first barrier against invasion of carcinomas cells (3). Modifications of stroma are mainly assured by matrix metalloproteinases (MMPs). These proteolytic enzymes are involved in many physiological processes, including ovulation, embryogenesis, and immune regulation. Disruptions in MMPs regulation leads to the pathogenic mechanism of cancer and also other diseases, such as rheumatoid arthritis, multiple sclerosis, aortic aneurysms, arterial restenosis lesions, and bullous skin disorders. This chapter will expose recent data on MMPs, focusing on their role in carcinogenesis, and presenting main clinical implications. THE MMP FAMILY Classification and Structure The MMPs comprise a large family with more than 21 enzymes. The MMPs are endopeptidases that can cleave virtually any component of the ECM. These enzymes were initially named according to their substrate and then divided into collagenases, gelatinases, stromelysins and matrilysins. Actual classification is based on their structure and a sequential numbering system has been adapted (Table 1) (4). All MMPs have a similar domain structure with a “pre” region to target for secretion, a “pro” region to maintain latency and an active catalytic region that contains the zinc-binding active site. The majority of MMPs have additional domains that are important in substrate recognition and in inhibitor binding (5). A subset of MMPs, formally known as membrane type MMPs (MT-MMPs) also contains a transmembrane domain (6). Unlike other MMPs, they are not 315

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TABLE 1 The Matrix Metalloproteinases (MMP) Family MMP designationa MMP-1 MMP-2 MMP-3 MMP-7 MMP-8 MMP-9 MMP-10 MMP-11 MMP-12 MMP-13 MMP-14 MMP-15 MMP-16 MMP-17 MMP-18 MMP-19 MMP-20 MMP-21 MMP-22 MMP-23 MMP-24 MMP-25 MMP-26 MMP-27 MMP-28

Common name(s) Collagenase-1, interstitial collagenase, fibroblast collagenase, tissue collagenase Gelatin-binding Gelatinase A, 72-kDa gelatinase, 72-kDa type IV collagenase, neutrophil gelatinase Stromelysin-1, transin-1, proteoglycanase, procollagenase activating protein Matrilysin, matrin, PUMP1, small uterine metalloproteinase Collagenase-2, neutrophil collagenase, PMN collagenase, granulocyte collagenase Gelatinase B, 92-kDa gelatinase, 92-kDa type IV collagenase Stromelysin-2, transin-2 Stromelysin-3 Metalloelastase, macrophage elastase, macrophage metalloelastase Collagenase-3 MT1-MMP, MT-MMP1 MT2-MMP, MT-MMP2 MT3-MMP, MT-MMP3 MT4-MMP, MT-MMP4 Collagenase-4 (Xenopus; no human homologue known) RASI-1, MMP-18b Enamelysin Homologue of Xenopus XMMP CMMP (chicken; no human homologue known) Cysteine array MMP (CA-MMP), femalysin, MIFR MT5-MMP, MT-MMP5 MT6-MMP, MT-MMP6, leukolysin Endometase, matrilysin-2 Epilysin

a

MMP-4, -5, and -6 have been abandoned. b MMP-19 was initially named MMP-18 but, since this designation was already used for a Xenopus homologue the classification was modified.

secreted but remain attached to cell membrane, which allows control of proteolytic activity in the direct neighborhood of the cell. The secreted MMPs can also localize to the cell surface by binding to integrins or to CD44, or through interactions with cell-surface–associated heparan sulphate proteoglycans collagen type IV or the ECM metalloproteinase inducer (EMMPRIN) (7–9). In addition to the MMPs, a second family of proteins with metalloproteinase activity has been described and named ADAMs (a disintegrin and metalloproteinase) or adamlysin metalloproteinases. These proteins are characterized by a disintegrin region that can mediate cell adhesion and fusion events. Among 30 known ADAMs, 16 have an additional metalloproteinase activity (10,11). In addition, adamlysin metalloproteinases with thrombospondin motifs (ADAM-TS) have been described in the ECM (12,13). Physiological Functions The main activity of MMPs was thought to participate to degradation of structural components of the ECM in order to facilitate cell migration. Cleavage by MMPs of ECM proteins like integrins also affects direct cellular signaling and functions (14) and can generate fragments with new functions [cleavage of laminin-5 and collagen type IV results in exposure of cryptic sites that promote migration

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(15–17)], and cleavage of cell-adhesion molecules allows cell migration and releases other factors that enhance mobility (18,19). MMPs and ADAMs participate in the activation of precursor forms of many growth factors, including transforming growth factor-a (TGFa) (cell-membrane– bound precursor) and TGFb (inactive extracellular complex) (20,21). Growth factor receptors are also MMP substrates such as FGFR 1, c-MET, HER2 and HER4 (22–24). Finally, the MMPs participate in the activation of their own zymogen and of other MMPs, as described below. Regulation MMP activity is regulated at multiple levels. The MMP genes are transcriptionally responsive to many growth factors, cytokines and hormones (25). MMPs are secreted as inactive zymogens (pro-MMPs). They are kept inactive by an interaction between a cysteine-sulfhydryl group in the propeptide domain and the zinc ion bound to the catalytic domain. Activation requires proteolytic removal of the propeptide prodomain to release the catalytically active enzyme. This processing can be achieved outside the cell by other MMPs or by other proteases, especially plasmin (5). However, MMP-11, MMP-28 and the MT-MMPs can also be activated by intracellular furin-like serine proteinases before they reach the cell surface (7). MMP activity is tightly controlled by endogenous inhibitors. The main inhibitor of MMPs in tissue fluids is a2-macroglobulin, an abundant plasma protein that binds to MMPs (26). The a2-macroglobulin–MMP complex that binds to a Scavenger receptor is irreversibly cleared by endocytosis. Thrombospondin-2 acts in a similar way by forming a complex with MMP-2 and facilitating scavengerreceptor–mediated endocytosis (27). Thrombospondin-1 binds to pro-MMP-2 and -9 and directly inhibits their activation (28). Other known molecules involved in the regulation of MMP activity are tissue-inhibitors of metalloproteinases (TIMPs 1–4) which can reversibly inhibit specific MMPs (levels of expression of TIMPs are tissue specific); RECK (REversion-inducing Cysteine-rich protein with Kazal motifs, the only known membrane-bound MMP inhibitor) and a1-PDX (29). IMPLICATION OF MMP IN CARCINOGENESIS: FUNDAMENTAL DATA Mechanisms of Action Originally, MMPs were considered to be important almost exclusively in invasion and metastasis. However, they are involved in other steps of carcinogenesis: regulation of cell growth, apoptosis, angiogenesis or immune response. Although MMPs favor tumor progression according to most data, some results suggest, on the contrary, that MMPs could somehow inhibit tumor progression. This dual regulation emphasizes the complexity of MMPs network in this setting. The main involved mechanisms are summarized in Table 2 and detailed more precisely below. Invasion and Metastasis Metastasis formation requires multiple steps. First, cancer cells have to cross the epithelial basement membrane and invade the surrounding stroma. They enter blood or lymphatic vessels, extravasate and finally establish new proliferating colonies. Experimental evidence for the role of MMPs in metastasis is based on in vitro invasion assays: TIMPs inhibits, and MMP-2, -3, -13 and -14 promote invasion of cell lines through collagen type I or matrigel (experimental ECM secreted by the

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TABLE 2 MMPs and Carcinogenesis Favor carcinogenesis Invasion/ metastasis Angiogenesis Proliferation

Apoptose Immune response

Disfavor carcinogenesis

Destruction of ECM Disruption in chemokine/ Remodeling of ECM chemokine Cleavage of cell receptor receptor signal? Remodeling of ECM Direct action on endothelial cells? Release of growth factors Activation of TGFb Release of (membrane bounded or ECM FasL, TNFa trapped) Disruption in integrins regulation Release of both anti-and proapoptotic factors Cleavage of IL2-R Chemokines disruption Activation IGFb

Abbreviations: ECM, extracellular matrix; FasL, Fas ligand; ILZ-1, interleukin Z-R; MMP, matrix metalloproteinase; TGF, transforming growth factor; TNF, tumor necrosis factor.

Engelbrecht-Holm-Swarm mouse sarcoma cell line) (30,31). In vivo xenograft metastasis assays confirm this result: downregulation of MMP-9 in cancer cells is associated with reduction of the number of metastases formed in the lungs of mice after cancer cell injection (32), and Mmp2- and Mmp9-null mice exhibit less lung metastasis as compared with wild-type mice (33). During the first step of migration, cancer cells must detach from both neighboring cells and the surrounding matrix. MMPs are involved in this process by cleaving CD44 (main receptor of hyaluronan) (19) or E-cadherin. Cleavage of E-cadherin releases in addition a fragment that promotes cell invasion in a paracrine manner in vitro, possibly by binding to and interfering with the function of other full-length E-cadherin molecules (18). This event is also thought to participate in the epithelial to mesenchymal transition, a phenotypic change of cancer cell associated with invasion potency (34–36). Activation of PAR1 by MMP-1 has also been shown to be linked to invasion and tumorigenesis (37). MMPs participate in the late events in the metastatic process, when the cancer cells must enter, survive and exit the blood or lymphatic vessels. For example, MMP-9 seems to be necessary for intravasation (38) and overexpression of MMP-14 (MT-MMP1) increases the number of cancer cells that survive intravenous injection in an experimental metastasis assay (39). On the other hand, implication of MMP activity in extravasation is less clear. Experimentally, TIMP1-overexpressing cancer cells exit the vasculature equally as well as control cells (40). However, metastases are fewer and smaller due to diminished cancer cell growth after extravasation (40). The proliferation at the secondary site, the final step in the establishment of secondary tumors, then probably also involves MMP activity. Regulation of Angiogenesis Angiogenesis is actually known as a crucial aspect of carcinogenesis. The role of MMPs in this process is accessed by animal models where endogenous and synthetic MMP inhibitors reduce tumor angiogenesis (41–43). The first hypothesis would be that MMPs simply degrade ECM and then favor endothelial cell invasion during formation of new vessels (44), but data suggest that MMPs are also involved in direct regulation of endothelial cell migration (45) or regulation of VEGF circulating level (46).

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On the other hand, MMPs may inhibit angiogenesis by releasing fragments with reduce proliferation and/or invasion of endothelial cell (angiostatin and endostatin) (47–50). MMPs and Growth Factors Three mechanisms have been described to explain the impact of MMPs on growthpromoting signals. First, MMPs or ADAMs release the cell-membrane–bound precursors of some growth factors (e.g., TGF-a) (20). Second, some growth factors are sequestered by ECM proteins (e.g., IGFs) and can be released when ECM is degraded by MMPs (51,52). Third, MMPs could disrupt antiproliferation mediated by integrins (53). On the contrary, cancer-cell growth might be negatively regulated by MMPs, for example, by activation of TGF-b (54) or generation of proapoptotic molecules such as Fas ligand (FasL) or TNF-a. Modulation of Apoptosis MMPs or ADAMs actively participate in the apoptotic process by cleaving VEcadherin, PECAM-1 and E-cadherin during apoptosis of endothelial or epithelial cells (55–57). In fact, MMPs have both pro- and antiapoptotic actions. Whereas overexpression of MMP-3 seems to favorite apoptosis in mammary epithelial cells (58), MMP-7 is able to promote cell survival by cleaving proheparin–binding epidermal growth factor (pro-HB-EGF) to generate mature HB-EGF, which stimulates HER4 and then inhibits apoptosis (59). In addition, MMP-7 is involved in the release of membrane-bound FASL, a transmembrane stimulator of the death receptor FAS. Released FASL induces apoptosis of neighboring cells, or decreases cancer-cell apoptosis, depending on the system (60,61). Moreover, the release of IGFs mediated by MMPs could act as a survival factor. This mechanism is thought to explain the results of tumor assay models where overexpression of MMP11 decreases spontaneous apoptosis and of xenograft experiments on Mmp11-null mice where cancer cell injection results in a higher rate of spontaneous apoptosis than in wild-type hosts (62). MMPs and Immune Response MMPs are implicated in the escape mechanisms that are observed in tumors that prevent the immune system from recognizing and attacking cancer cells. IL-2Ra can be cleaved by MMPs (especially MMP-9) that suppress the proliferation of T lymphocytes (63). MMPs also activate TGF-b, an important inhibitor of the T-lymphocyte response against tumors (21,64). MMPs are additionally involved in the regulation of chemoattractant factors (activation of CXCL8, inactivation of CXCL-1, -4, -7, -12, or CCL7). This action leads to a lower infiltration of tumors by neutrophil and macrophages (62,65,66). Interestingly, chemokines and chemokine receptors are thought to be involved in the metastasis process as the biological support of the “seed and soil” theory (67,68). MMPs activity on this system may then inhibit metastasis. Tumor–Stroma Interactions Cancer cells were initially considered the only source of MMPs during previously described phenomena (69). This hypothesis is correct for MMP-7 but most MMPs are in fact synthesized by stromal cells. In situ hybridization experiments

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demonstrate that mRNA of MMP-1, -2, -3, -14 are primarily localized in stromal fibroblasts, especially in proximity to invading cancer cells but not in carcinoma cells in various human tumors (70–72). Cancer cells might stimulate tumor stromal cells to synthesize MMPs in a paracrine manner (7). MMPs that are secreted by stromal cells can still be recruited to the cancer-cell membrane. Indeed, MMP-2 mRNA is expressed by stromal cells of human breast tumors, whereas MMP-2 protein is found on both stromal and cancer-cell membranes (73). Besides interleukins and growth factors, EMMPRIN (CD147) might be a key molecule for the stimulation of MMPs, secretion by stromal cells. This 58-kDa glycoprotein is highly expressed during embryogenesis and was originally designated tumor collagenase stimulating factor (TCSF) after isolation and purification from the plasma membrane of cancer cells and demonstration of its function in stimulating fibroblast synthesis of MMP-1 (74). This action was also observed for fibroblast synthesis of MMP-2 and MMP-3 (75). In vitro, recombinant EMMPRIN or EMMPRIN purified from cancer cells to stimulate fibroblast production of MMPs (75,76) and cancer cell lines transfected with EMMPRIN cDNA exhibits more secondary tumors in xenograft metastasis assays (77). Regulation of EMMPRIN synthesis by cancer cells might be controlled by HER1 pathways (78). Note that this glycoprotein is also thought to be involved in HIV internalization (79,80). After secretion from fibroblasts, MMP-1 is able to bind to EMMPRIN on the tumor cell surface, forming a highly active proteolytic system involved in ECM degradation and activation of other MMPs (81). CLINICAL ASPECTS MMP and Prognosis The expression and activity of MMPs are increased in almost every type of human cancer, and multiple studies show a negative association between MMP levels and prognosis. This correlates with advanced tumor stage, increased invasion and metastasis, and shortened survival. For example, expression of MMP-11 in breast cancer has been associated with malignant disease only, and it is not expressed in normal breast tissue or benign fibroadenomas (82). MMP-11 expression was observed not only in invasive breast carcinomas but also in some in situ carcinomas where other factors indicated a high risk for the development of an invasive phenotype. Several studies have reported an association between MMP-11 expression, lymph node metastasis, and/or shorter disease-free survival in patients with infiltrating ductal carcinoma of the breast (83–86). In addition, MMP-14 was identified as an independent factor of poor overall survival for nonmetastatic breast cancer when adjusted for clinical prognostic factors (87). In addition, increased tumoral expression of MMP-3, -11, and -14 could be independent negative prognostic factors for survival in small cell cancer while decreased tumoral expression of TIMP-1 is significant for response (88). In colon cancer samples, immunohistochemical detection of interstitial MMP-1 and -7 expression are associated with a poor prognosis independent of Dukes’ stage (89). MMP-1 and MMP-7 expression have also been suggested to be of prognostic value in esophageal cancer (90,91). Pancreas cancer patients with MMP-7 carcinoma had a significantly shorter overall survival time than did those with MMP-7-negative carcinoma and MMP-7 was a significant independent prognostic factor for overall survival in multivariate analysis (92).

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Concerning prostate cancer, serum levels of MMP-2 were found to be significantly higher in men with prostate cancer than in those with benign prostate hyperplasia or with no disease (93). Plasma TIMP-1 is also associated with prostate cancer but not with benign prostate hyperplasia or normal prostate tissue (94). Another study reports that tissue expression of activated MMP-2 was associated with Gleason score, with the highest levels found in tumors with the highest Gleason score and in lymph node metastases (95). Downregulation of E-cadherin and upregulation of MMP-9 and MMP-2 in localized pancreatic tumors had significant prognostic value. The ratio of the mean of the expression of MMP-2 and MMP-9 to E-cadherin expression (MMP: E-cadherin ratio) was found higher in patients with recurrent disease compared with patients remaining in remission (96). Expression of MMP-2 is similarly associated with poor prognosis for ovarian cancer (97), and elevation of serum levels of either MMP-2 or MMP-3 or both could be new predictors of recurrence in patients with advanced urothelial carcinoma after complete resection (98). There are few cases in which increased expression of specific MMPs is correlated with a favorable prognosis. In colon cancer, MMP-9 expression by infiltrating macrophages is associated with reduced metastases (99), although MMP-9 expression in tumors was more recently shown as a negative prognostic factor (100). MMP-12 expression by carcinoma cells is associated with increased survival (101). The mechanisms underlined by this observation are not clear. MMP activity may be associated with the generation of antitumor molecules such as proapoptotic factors or to the recruitment of cytotoxic T-cells. On the other hand, MMP activity in these cases may be irrelevant and the MMPs would just be markers of differentiation of the cancer cells or of an immune reaction. Another hypothesis could derive from inappropriate tests to evaluate MMP levels that are usually defined according to immunochemistry or molecular biology (in situ hybridization or RT–PCR) on tumor samples. As for MMPs being secreted as pro-enzymes, the level of synthesis is, however, not correlated with level of activity, which would be best estimated by zymography (but fresh tissue is needed). Some studies found that high levels of TIMP-1 and -2 also correlate with a poor prognosis. This might reflect the fact that the balance between expression of MMPs and TIMPs, although still favoring the MMPs, is at a higher overall level during the increased matrix remodeling that occurs in tumor progression. High-TIMP levels would therefore be associated with tumor progression and a worse prognosis, but would not cause it. Prognostic value of MMP-2/TIMP-2 imbalance has, for example, been proven for hepatocarcinoma (high level of TIMP-2 and low level of MMP-2 correlated with better relapse-free survival) (102). Targeting MMP in Anticancer Therapy Several agents have been developed in this field. Inhibition of MMP synthesis could be the consequence of other therapies, such as tyrosine kinase receptor signaling inhibition, but we will focus here on compounds that block MMPs activity (MMPI: MMP inhibitors). MMPIs have been developed and have been shown in preclinical systems to inhibit local tumor growth and metastasis (70,103). Because of their noncytotoxic effects on the tumor, phase I studies with MMPIs have sought to establish tolerable doses of drug, suitable for protracted administration, that produce serum levels that exceed the inhibitory concentration of targeted MMPs without causing

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unacceptable normal tissue toxicity. Although preclinical models have demonstrated the antitumoral activity of MMPIs, phase II clinical trials, where objective response was primary end point, failed to demonstrate substantial regression of large primary tumors. The development of MMPIs has proceeded rapidly to phase III trial design with the end point of survival. Disappointing results now lead to reconsidering MPPI development. Three main categories of synthetic MMPI have been developed: the collagen peptidomimetics, the collagen nonpeptidomimetics and the tetracycline derivates. Alternative molecules can also be considered. This classification is summarized in Table 3. Collagen Peptidomimetics MMP Inhibitors The peptidomimetic MMP inhibitors mimic the cleavage sites of MMP substrates and include batimastat and marimastat. Batimistat is an inhibitor of MMP-1, MMP-2, MMP-3, MMP-7, and MMP-9, and is effective in reducing the frequency of metastases and improving survival for the treated animals (104), but cannot be administered orally and is no longer tested for the treatment of human cancer. Marimastat, similar to batimistat, is a broad-spectrum peptidomimetic MMPI with inhibitory activity against MMP-1, MMP-2, MMP-3, MMP-7, and MMP-9 (103,105). The principal effect of marimastat is to retard tumor growth and metastatic spread. It does not display cytotoxic activity in cell cultures, and no tumor regression is observed in animal models but a benefit in survival was observed in treated animals (106,107). The molecules have been combined with cytotoxic agents with evidence of activity in animal models (108). Unlike batimistat, marimastat is orally bioavailable (recommended administration twice a day). In clinical trials, musculoskeletal symptoms consisting of pain and tenderness in muscles and joints is the most common toxicity, develops in approximately 30% of patients treated at 10 mg b.i.d. after a median of 3–6 months. Symptoms diminished when the drug was no longer administrated (109,110). This toxicity was observed with other MMPIs and may be mediated by inhibition of the tumor necrosis factor alpha converting enzyme (111). Phase II trials may have exhibited encouraging results in terms of progression-free survival (gliobastoma, pancreas cancer, lung cancer) (112,113), but phase III trials in patients with advanced malignancies have yielded modest results. For example, a trial for advanced pancreatic cancer intended to detect differences in survival between patients treated with various doses of marimastat and gemcitabine failed to detect

TABLE 3 Matrix Metalloproteinase Inhibitors (MMPIs) Category Collagen peptidomimetics MMPI Collagen non-peptidomimetics MMPI

Tetracycline derivates Other molecules

Compound Batimistat Marimastat Prinomastat/AG3340 BAY 12-9566/tanomastat BMS 275291 COL-3 Bisphosphonates AE-941 ( )Epigallocatechin-3-gallate Urokinase-activated recombinant anthrax toxin

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increased survival for the marimastat-treated groups even if the highest dose of marimastat was as effective as the conventional therapy (114). The combination of marimastat with gemcitabine provided no benefit in survival compared to gemcitabine alone (115). Another trial evaluated marimastat against placebo in 369 patients with inoperable gastric cancer who had received no more than one prior chemotherapy regimen. No improvement was detected in the primary end point of the trial, overall survival. Nevertheless, subset analysis suggested an improvement in survival in patients without distant metastases at the time of enrollment, raising the question of the use of MMPIs in order to prevent metastatic spread (116). Collagen Nonpeptidomimetics MMP Inhibitors The nonpeptidomimetic MMPI are synthesized on the basis of the conformation of the MMP active site. This group mainly includes Prinomastat/AG3340, BAY 12-9566/tanomastat and BMS 275291. Preclinical studies of prinomastat have demonstrated reduction in the rate of primary tumor growth and in the number and size of distant metastases in animal tumor models. Furthermore, when prinomastat was administered in combination with a variety of cytotoxic chemotherapeutic agents in these models, antitumor effects were enhanced without an increase in chemotherapy-related toxicity (117,118). Early clinical trials reported dose- and time-dependent musculoskeletal complaints. Prinomastat has been studied in advanced non–small cell lung cancer in combination with platinum-based chemotherapy (gemcitabine and cisplatin) in previously untreated patients and there was no improvement in survival seen with the addition of the MMPI (119). The phase I trial with BAY 12-9566 showed relatively mild adverse effects, and in some cases, disease was stabilized (120). However, all studies with BAY 129566 were terminated when preliminary results from Phase III trials on advanced pancreatic or small cell lung cancers showed significantly poorer survival for groups treated with the drug than for placebo-treated groups (121). In a phase I study, BMS-275291 was well tolerated and dose-limiting toxicities included hypersensitivity (characterized by rash, fever, and dyspnea) and elevations of serum ALT, but not musculoskeletal toxicity (122). BMS-275291 was then thought to be suitable for a prolonged period, during chemotherapy and following completion of treatment without severe musculoskeletal toxicity (123). A randomized phase II/III study was initiated to determine the additional value of BMS-275291 to paclitaxel and carboplatin for advanced non–small cell lung cancer. Final results on 774 patients showed that BMS-275291 added to chemotherapy increases toxicity and does not improve survival in this setting (124). Tetracycline Derivates COL-3, a chemically modified tetracycline is a MMPI that is distinct in its ability to inhibit the activity, activation, and production of MMPs, whereas other MMPIs target only the active enzyme (103). COL-3 inhibits MMP-2 and MMP-9 in vitro and the expression of MMPs in human colon and breast carcinoma cells. It inhibits invasion of various cancer cell lines in vitro, and inhibits tumor growth and metastasis in a rat model (125,126). In a recent phase II trial, COL-3, when administered at 50 mg/day, is both active and reasonably well tolerated (skin toxicity) in the treatment of AIDS-related Kaposi sarcoma, with significant declines

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in MMP-2 and MMP-9 plasma levels from baseline to minimum value with treatment (127). Although formal conclusion is not possible due to the limited nature of a phase II trial, COL-3 warranted further investigation. Other Molecules Another group of drugs might also possess unrecognized activity against the MMPs. The bisphosphonates were originally developed for the treatment of disturbances in calcium homeostasis and for the prevention and palliation of bone metastasis. Their mechanism of action has not been completely elucidated, but they inhibit the enzymatic activity of MMPs (128). Unconventional MMPIs are also being tested in clinical trials: AE-941 (Neovastat), an extract from shark cartilage, inhibits MMPs (129), shows antiangiogenic effects in animal experiments and is now in Phase III clinical trials for the treatment of metastatic non–small cell lung cancer (130). (–)Epigallocatechin3-gallate, a component of green tea, also acts as MMP-2 and -9 inhibitors in vitro (131,132). Finally, cytotoxic agents that are activated by MMPs have been proposed to take advantage of the increased MMP activity in tumors. These include recombinant proteins containing anthrax toxin fused with an MMP cleavage site. These recombinant toxin proteins are activated by MMP cleavage at the cell surface and are internalized by the cell, leading to cell death on in vitro models (133,134). PERSPECTIVES Disappointing results of clinical trials involving MMPIs led to reconsidering the way to use these molecules. The hypothesis was raised that MMPIs would not be expected to have a beneficial effect in patients with active metastatic disease, but they could have a potential role as a component of adjuvant therapy in patients with a carcinoma that has never responded to chemotherapy or ever been completely resected but who are at high risk for relapse. A phase III trial for glioblastoma after completion of surgery and radiotherapy found no improvement in time to progression or overall survival with single agent marimastat or placebo (135). Similarly, marimastat did not prolong survival in patients with small cell cancer who achieved a complete or partial response to initial chemotherapy (136) or in patients with metastatic breast cancer who have responding or stable disease after first line chemotherapy (137). In those studies, marimastat had a negative impact on quality of life due to toxicity, raising the problem of the use of MMPIs as adjuvant therapy for early stage cancer. Phase II studies of MMPIs as adjuvant therapy for operable breast cancer confirm this concern, with high rates of treatment discontinuation (marimastat or BMS-275291) (138,139). Patients developing side effects may, however, have a benefit in survival according to a subset analysis from a double-blind placebo trial of adjuvant treatment by marimastat in patients with unresectable colorectal liver metastases, where no significant effect on survival was observed in all patients (140). BAY 129566 was also evaluated in a randomized phase III as maintenance therapy in patients with advanced ovarian cancer responsive to primary surgery and paclitaxel/platinum containing chemotherapy. The treatment was generally well tolerated and, at the time of the final analysis, there was no evidence of an impact of BAY 12-9566 on progression-free survival or global survival (141). The reasons

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for the disappointing results observed for MMPIs as cancer therapy are not definitively clear. Preclinical data supported this approach, but it is now established that the presence of the target does not mean that the target is relevant. These data suggest that preclinical models may be insufficient. First of all, the complexity of a metastatic process requires targeting multiple steps and biological processes rather than only MMPs. In addition, MMP inhibition may have paradoxical effects that promote rather than inhibit tumor growth and angiogenesis. This dual activity, which has been exposed previously in this chapter and summarized in Table 2, should lead us to consider MMP as a complex network. It has become clear that MMPs do more than degrade structural ECM proteins to promote invasion and metastasis, and a better understanding of the functions of the MMPs would be helpful to determined when and how they could be targeted. Future studies may, for example, focus on downstream effectors of MMPs that are shown to be involved in invasion, such as EMMPRIN or PAR1 (37,77). REFERENCES 1. Hanahan D, Weinberg RA. The hallmarks of cancer. Cell 2000; 100:57–70. 2. Bissell MJ, Radisky D. Putting tumours in context. Nat Rev Cancer 2001; 1:46–54. 3. Yurchenco PD, Schittny JC. Molecular architecture of basement membranes. Faseb J 1990;4:1577–90. 4. Egeblad M, Werb Z. New functions for the matrix metalloproteinases in cancer progression. Nat Rev Cancer 2002; 2:61–74. 5. Woessner JF. The matrix metalloproteinase family, In: Parks WC, Mecham RP, eds. Matrix Metalloproteinases. San Diego, CA: Academic Press, 1998:1–14. 6. Sato H, Okada Y, Seiki M. Membrane-type matrix metalloproteinases (MT-MMPs) in cell invasion. Thromb Haemost 1997; 78:497–500. 7. Sternlicht MD, Werb Z. How matrix metalloproteinases regulate cell behavior. Annu Rev Cell Dev Biol 2001; 17:463–516. 8. Yu Q. Stamenkovic I. Localization of matrix metalloproteinase 9 to the cell surface provides a mechanism for CD44-mediated tumor invasion. Genes Dev 1999; 13: 35–48. 9. Brooks PC, Stromblad S, Sanders LC, et al. Localization of matrix metalloproteinase MMP-2 to the surface of invasive cells by interaction with integrin alpha v beta 3. Cell 1996; 85:683–93. 10. Loechel, F., Gilpin BJ, Engvall E, Albrechtsen R, Wewer UM. Human ADAM 12 (meltrin alpha) is an active metalloprotease. J Biol Chem 1998; 273: 16993–7. 11. Wolfsberg TG, Primakoff P, Myles DG, White JM. ADAM, a novel family of membrane proteins containing a Disintegrin and Metalloprotease domain: multipotential functions in cell-cell and cell-matrix interactions. J Cell Biol 1995; 131:275–8. 12. Kuno K, Kanada N, Nakashima E, Fujiki F, Ichimura F, Matsushima K. Molecular cloning of a gene encoding a new type of metalloproteinase-disintegrin family protein with thrombospondin motifs as an inflammation associated gene. J Biol Chem 1997; 272:556–62. 13. Kuno K, Terashima Y, Matsushima K. ADAMTS-1 is an active metalloproteinase associated with the extracellular matrix. J Biol Chem 1999; 274:18821–6. 14. Streuli C. Extracellular matrix remodelling and cellular differentiation. Curr Opin Cell Biol 1999; 11:634–40. 15. Giannelli G, Falk-Marzillier J, Schiraldi O, Stetler-Stevenson WG, Quaranta V. Induction of cell migration by matrix metalloprotease-2 cleavage of laminin-5. Science 1997; 277:225–8. 16. Xu J, Rodriguez D, Petitclerc E, et al. Proteolytic exposure of a cryptic site within collagen type IV is required for angiogenesis and tumor growth in vivo. J Cell Biol 2001; 154:1069–79.

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20

Src Inhibitors Francisco Cruzalegui Division of Cancer Research and Drug Discovery, Institut de Recherches Servier, Croissy-sur-Seine, France

INTRODUCTION Many excellent reviews have been published describing in great detail the history of the discovery of the Src family kinases (SFKs), their molecular structure, their, biochemical properties, and their functions in normal tissues. In addition, many papers have described the effects of exogenous expression of viral or mutated forms of c-Src (v-Src) that may or may not represent the biology of deregulated c-Src found in cancer cells. In this review, emphasis is given to evidence found in cancer tissue or cell lines derived from tumors. In addition, small molecule inhibitors targeting Src family kinases are reviewed, from the first broad-spectrum compounds described in the early 1990s to the present day drugs used in the clinic. Src FAMILY KINASES AS TARGETS Link Between Src Family Kinases and Cancer c-Src was the first protein kinase to be identified as selective for tyrosine residues. Its tyrosine kinase activity was initially discovered by studying the 60 kDa protein encoded by the v-Src gene in the chicken Rous sarcoma virus (1,2). The presence of a similar protein in vertebrate cells had already been described (3) and the cellular chicken gene was soon characterized (4). Together, these discoveries led to the awarding of the 1989 Nobel Prize in Physiology and Medicine to Harold Varmus and Michael Bishop. According to the official nomenclature for viral and cellular counterparts, the viral form is known as v-Src and the cellular gene as c-Src (5). The domain structure of c-Src is very highly conserved across species and throughout all the 8 SFKs (c-Src, c-Yes, Fyn, Lyn, Fgr, Lck, Hck, Blk) known today in humans. The C-terminal half is a consensus tyrosine kinase domain containing an autophosphorylation site at Tyr419 (Tyr416 in the chicken sequence). The N-terminal half is more divergent and contains a membrane docking sequence that is myristoylated in all SFKs and additionally palmitoylated in all SFKs except c-Src and Blk (6,7), followed by an SH3 domain and an SH2 domain. In all SFKs, the SH2 domain interacts with a phosphorylated tyrosine at position 530 in the C-terminal end (human c-Src nomenclature). The SH2-P-Tyr530 interaction maintains the kinase in a closed conformation preventing autophosphorylation and substrate phosphorylation (8–10). The kinase responsible for this phosphorylation is called Csk (C-terminal Src kinase) (11). Contrary to c-Src, Csk is a cytoplasmic protein that is recruited to the membrane by docking proteins in order to phosphorylate c-Src. Since c-Src is found to be overactivated in tumors, it is likely that Csk is unable to downregulate c-Src activity in cancer cells. Surprisingly, Csk levels were found to be up to 10 times higher in tumors compared with healthy tissue (12). Whether Csk is inactive or simply uncoupled from c-Src inhibition is unclear. 333

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In the chicken viral forms of c-Src and c-Yes (v-Src and v-Yes), the amino acid sequence is truncated and lacks the tyrosine normally phosphorylated by Csk. Consequently, the mutant v-Src and v-Yes are constitutively active and highly oncogenic. However, since its discovery, mammalian c-Src has very rarely been found mutated in tumors or in healthy tissue. In one case, 12% of analyzed advanced colon carcinomas presented a Gln to stop mutation at codon 531 (13). Since that publication, several groups around the world have tested tumor samples looking for activating mutations with little success (14–18). A group in Japan found that 1 out of 68 endometrial carcinomas contained such a mutation (19). In China, a study reported 1 sample out of 110 examined contained the 531 mutation (20). Consequently, it is now accepted that activating mutations are very rare and that the main mechanisms for elevation of c-Src activity in cancers are overexpression at the RNA level and deregulation of c-Src kinase activity by deficient Tyr530 phosphorylation. Another possibility is that a phosphatase that regulates Tyr530 dephosphorylation is absent in tumors (11). A very large proportion of studies have focused on c-Src, it being usually the most abundant SFK in solid tumors and the one for which most tools have been developed. Consequently, despite nearly 30 years since the discovery of SFKs, little has been done to elucidate selective functions for each of them. The lack of inhibitors targeting a particular SFK has hindered the dissection of any possible selective roles of these kinases. In colon cancer, only c-Src and c-Yes appear to be highy activated compared to healthy tissue (21). c-Yes is strongly activated in colon cancers and in colon cancer cell lines, its activity correlating strongly with worse prognosis of colon cancer (22,23). In melanomas, c-Yes activity is 5–10 times higher than in nontransformed melanocytes although it is not found mutated (24). Furthermore, c-Yes found in melanoma cell lines appears to be further activated by neurotrophins and this activation correlates with increased invasive potential of these cell lines (25). Recently, Lyn was identified as the most prevalent SFK in glioblastomas (26). In B cell chronic lymphocytic leukemia (B-CLL), while all other SFKs are expressed normally, Lyn is overexpressed and constitutively activated. In addition, Lyn is found uniformly distributed all over the cell surface of leukemic lymphocytes, unlike normal B cells where Lyn is localized to lipid rafts. Finally, treatment of B-CLL cells from patients with SFK inhibitors induces apoptosis, suggesting that Lyn activity is necessary for driving survival in this form of leukemia (27). In conclusion, it is possible that in some cancer types, SFKs other than c-Src are critical for tumor survival, growth, or metastasis. Inhibitors that target individual SFKs in such pathologies could help reduce potential side effects of broad-spectrum Src inhibitors, due to inhibition of SFKs involved in lymphocyte functions such as Lck (8). Experimental Validation of SFKs as Targets In the case of the Src family, the transforming ability of v-Src or artificially activated mutants introduced in mammalian cells has often been considered as evidence of an important role of c-Src in cancer. In fact, after all these years, relatively little has been published on validation of endogenous c-Src or other SFK as targets in cancer cells. The induction of DNA synthesis in cultured mouse NIH3T3 mouse fibroblasts following PDGF addition has been a paradigm widely used to examine the role of c-Src downstream of growth factor receptors. Using this model, Sara Courtneidge’s group showed in the 1990s that injection of

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dominant-negative forms of c-Src and Fyn or neutralizing antibodies against c-Src, Fyn and Yes into serum-starved fibroblasts could inhibit PDGF-induced DNA synthesis (28), and that this inhibition could be rescued by c-myc overexpression (29). Later on, using the same system, further papers suggested a pathway leading to c-myc expression, requiring Src family kinases and phosphorylation of the adapter protein Shc and potentially Stat3 (30,31). Finally, further work by Serge Roche’s group revealed a tyrosine kinase cascade in which c-Src phosphorylation of cytoplasmic cAbl leads to the removal of a p53-mediated block for entry into S phase following PDGF stimulation (32). Although these experiments enlightened us on the position of c-Src in a signaling cascade triggered by PDGF, the relevance of PDGF-induced DNA synthesis on starved fibroblasts to cancer is unclear, given that these experiments appear not to have been performed using cultured cancer cells. Still using fibroblasts, Roche et al. also explored a potential role for Src family kinases during mitosis (33). In this case, microinjection of antibodies inactivating c-Src, c-Yes, and Fyn into fibroblasts was carried out in cells syncronized to be in G2. This treatment led to a block of cells at the entry to mitosis. Some years later, these experiments were expanded by microinjection of the same antibodies in cells already in mitosis, resulting in binucleate cells (34). These experiments suggest that Src family kinases play a role in cytokinesis and, although indirectly, suggest that deregulated c-Src in tumor cells could also contribute to progression through mitosis. Experimental evidence of a requirement for c-Src in cell growth and survival of a tumor comes from expression of antisense and dominant-negative constructs in colon carcinoma cells. In colon carcinoma HCT15 cells expression of a dominantnegative c-Src construct inhibits migration of cells in vitro and strongly inhibits peritoneal dissemination in SCID mice. The antimigration effect of DN-Src was shown by a reduced invasion of peritoneal tumors into surrounding tissue and their higher fibrotic nature (35). The same group also showed that treatment of mice with PP2, a selective SFK inhibitor, after intrasplenic injection of HT29 cells, reduced the size of liver metastasis obtained (36). Also in colon carcinoma cells, antisense constructs directed to c-Src reduce the ability of these cells to survive to anoikis in vitro (37). Recently, Gallick et al. used c-Src shRNA expression in the metastatic L3.6pl pancreatic tumor cell line to reduce endogenous c-Src levels. In this context, c-Src knock-down had a clear effect on the metastasis potential of these cells when implanted orthotopically but had no impact on growth of the primary tumor. In contrast, treatment of L3.6pl tumors with Dasatinib, a potent broad Src family inhibitor, had an effect both on primary tumor growth and on metastasis (38). This suggests that in the context of this particular pancreatic cell line, inhibition of c-Src alone is sufficient to stop metastasis, but it may not be sufficient in vivo to trigger tumor regression. Finally, a recent paper reports the effect of inducible overexpression of dominant-negative c-Src in mammary cancer cells. This resulted in a reduction of migration and proliferation rate with an accumulation of cells in G1 and tumor growth in vivo (39). As illustrated by the variety of results obtained in different cell lines, the difficulty of validating endogenous c-Src or other SFKs as targets in preclinical studies is that the signaling context of a particular tumor cell line could determine the response obtained. Selecting a tumor cell line based only on its c-Src activity to test the effect of its inhibition could lead to wrong conclusions. This was shown recently by Johnson et al. in a variety of cell NSCLC and H&N cell lines. The authors showed that although the SFK inhibitor Dasatinib inhibited migration in all

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ReceptorTyr Kinase

Adhesion proteins c-Src

c-Src

Shc rasGAP Integrins

ras

Adherens junctions

c-Raf Akt

STAT3

FAK

E-cadherin

ERK Bad Cyclin D Proliferation

Mcl-1 Bim

Bcl-XL

Survival

Tumor growth

VEGF

Angiogenesis

Modulation of matrix adhesion

Reduced cellcell adhesion

Migration, Invasion

FIGURE 1 Signaling pathways downstream of c-Src. c-Src activity, associated to receptors with tyrosine kinase activity (e.g., EGFR, PDGFR, VEGFR), regulates pathways known to be important in cell proliferation, cancer cell survival, and angiogenesis. The resulting effect is overall tumor growth. The pool of c-Src that associates to adhesion proteins regulates the plasticity of focal adhesions via its regulation of FAK activity and the strength of cell–cell adhesions via the downregulation of E-cadherin at cell–cell junctions. This results in an increased propensity to migrate and invade other tissues. Activation or increased expression of proteins is indicated by an ascending arrow. Inhibition or down-regulation are marked with a descending arrow.

these cell lines, it triggered apoptosis in only few. It was revealed that only in those sensitive cells lines, SFK inhibition lead to Mcl-1 and Bcl-XL degradation (40). The lesson from this paper is that given the variety of signaling pathways in which c-Src is involved, each tumor type will have evolved to position c-Src at different nodes of this network (Fig. 1). The role of c-Src in migration, adhesion and metastasis may be generally maintained, independent of other changes in signaling. c-Src, however, becomes essential for pathways important in growth and survival only in particular contexts. For this reason, caution must be used in drawing conclusions from validation experiments that may be carried out in a “wrong” cellular context in which c-Src only has its “generic” function in promoting migration. While the experiments described above addressed the issue of whether c-Src is required for tumor growth or survival, a different question is whether c-Src can be sufficient to generate tumors. This has been addressed using transgenic expression of wild-type or activated forms of c-Src in mice. Webster et al. (41) expressed activated c-Src (mutation F530) in breast under the control of the MMTV promoter leading to hyperplasias but variable progression to real carcinomas. In skin, however, expression of activated c-Src has different effects depending on the exact cellular layer. Expression in the epidermal spinous layer under the control of the keratin 1 promoter, c-SrcF529 (mouse sequence), only led to hyperplasias that showed enhanced sensitivity to skin tumor promotion in a two-stage tumor

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337

promotion model (DMBA/TPA treatment) (42). Expression of the wild-type human sequence in the epidermal basal cells, driven by the keratin 5 promoter, led to squamous cell carcinomas, although with slow kinetics (70% of transgenic mice after 1 year) and showed enhanced responsiveness to DMBA/TPA treatment. In the same experiments, expression of activated human c-Src (c-Src530F) led to severe skin defects and death soon after birth (43). Later on, the same group reported that inducible expression of human c-Src530F under the control of an inducible promoter provoked hyperplasias and hyperkeratosis. Remarkably, in this inducible skin model, real squamous cell carcinomas were induced along the periphery of the area of punch biopsies, suggesting that secondary insults may cooperate with Src for triggering carcinogenesis (44). These results suggest that elevated c-Src activity may be sufficient to induce the whole process of carcinogenesis depending on the tissue and the signaling context of the cell. Another issue not addressed in transgenic models yet is the possible need for other Src-family kinases to cooperate with c-Src. As described previously, c-Yes is also highly deregulated in melanoma, colon, and maybe other cancers. It is possible that concomitant activation of c-Src, c-Yes or another signaling pathway may be required for full carcinogenesis. DOWNSTREAM SIGNALING CONSEQUENCES OF SFK DEREGULATION As reviewed recently by Gallick (45), c-Src and other SFKs are connected to a large network of signaling pathways. Understanding the signaling context for c-Src in a particular type of cancer cell not only will allow us to define a functional readout that would reflect Src inhibition (migration, proliferation, apoptosis, etc.) but also it will help us to identify potential biomarkers to monitor a pharmacological response to Src inhibitors in preclinical and clinical studies. However, observations made in a particular tumoral cell line about c-Src’s influence on a signaling cascade may not be generalized to all cell types or pathologies. c-Src associates with receptor tyrosine kinases (RTKs) via an interaction between its SH2 domain and phosphorylated tyrosines on the intracellular domain of the receptor. Downstream of an RTK, c-Src can be linked to the Raf-MEK-ERK cascade either by phosphorylating c-Raf and increasing its activity (46) or via phosphorylation of RasGAP, this reducing its GTPase-enhancing activity and increasing the levels of GTP-bound Ras. A third mechanism by which c-Src can activate the ERK cascade is via its phosphorylation of the adaptor protein Shc, which serves as docking point for the Grb2/SOS resulting in Ras activation (30). Increased ERK activity activates transcription of genes involved in cell proliferation such as c-fos and Cyclin D. ERK activity downstream of c-Src may also regulate the protein level of Mcl-1, an antiapoptosis Bcl-2 family member that can be regulated by ERK. Conversely, the proapoptotic protein Bim is destablized by ERK phosphorylation. High activity of the ERK pathway would then lead to higher turnover of Bim and lower apoptosis (47,48). In response to EGF stimulation, c-Src recruitment by the phosphorylated receptor is rapidly increased. This results in a synergistic phosphorylation of STAT3 on Tyr705, allowing it to dimerize and translocate to the nucleus. STAT3 will then bind to the promoter of genes such as myc, VEGF, and Bcl-XL leading to their increased transcription (49,50). In cancer cells, this pathway can be constitutively activated. In breast cancer cells, in particular, STAT3 is highly phosphorylated in a c-Src-dependent manner (51). Another target of STAT3 is the proto-oncogene c-myc.

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Activation of c-myc expression has mainly been described downstream of PDGF stimulation of serum-starved cells (31). Akt is a Ser/Thr protein kinase activated by lipids and by phosphorylation via PDK1. The lipid ligand involved is generated by PI3K which in turn is activated by survival signals. One of the most important substrates for Akt is the proapoptotic protein Bad. When phosphorylated by Akt, Bad is recruited by 14-3-3 proteins and is prevented from triggering apoptosis. c-Src can increase the activity of PDK1 by direct phosphorylation of tyrosines Y373 and Y376, thus increasing survival signaling (52). In cells in which v-Src or mutated forms have been introduced (such as mouse fibroblasts), v-Src is constitutively found associated with focal adhesions and structures known as podosomes (30). In these cells, the prevailing model is that activated c-Src phosphorylates and activates RhoGAP. This in turn reduces the levels of GTP-loaded Rho and results in actin fiber dissassembly. Endogenous c-Src found in cancer cells, in contrast, is localized to endosomal membranes and very little colocalized with the actin cystoskeleton. In nontransformed cells, c-Src participates in fibronectin receptor signaling since c-Src / fibroblasts show reduced adherence to fibronectin and reduced phosphorylation of proteins involved in cellmatrix adhesion. Upon attachment of cells to fibronectin, c-Src is transiently activated and is localized to focal adhesions. Following this, FAK is autophosphorylated on Tyr397, creating a pTyr attachment point for c-Src. c-Src is then able to phosphorylate FAK on Tyr861 and Tyr925 and increase FAK’s kinase activity. These c-Src-mediated phosphorylated tyrosines allow recruitement of Grb2 and activation of the Ras-ERK cascade. The complex formed by FAK and c-Src will also serve as a scaffold for other signaling proteins such as PI3K. The latter will associate with the SH3 domain of c-Src, rendered available by the activation by FAK. Unfortunately, such a detailed molecular dissection of the role of c-Src in cell-matrix adhesion and migration is lacking in cancer cells where nonmutated but deregulated c-Src exists. Indirect evidence for such c-Src–FAK interaction in cancer cell lines is a constitutive level of SFK-dependent phosphorylation of Tyr861 end Tyr925, and high paxillin phosphorylation (30,53). Src activity has also been shown to be associated with reduced cell–cell adhesions and increased dispersion of tumor cells. E-cadherin, the major adhesion molecule in epithelia, is often expressed at lower levels in metastatic tumors. Elevated c-Src activity correlates inversely with the amount of E-cadherin found at cell junctions (54). Inhibition of SFK activity by the selective inhibitor PP2 leads to E-cadherin accumulation at cell–cell junctions (36,55). The effect of Src on cell–cell adhesion is also present in endothelial cells. In this case, VE-cadherin is directly phosphorylated by c-Src (56,57), leading to dissociation of the cadherin–120 catenin–beta catenin complex. This dissociation could induce an increase in vascular permeability and tumor cell extravasation. Finally, c-Src is also involved in angiogenesis in two ways: first, Src can induce transcription of VEGF in tumor cells (58). Second, VEGFR downstream signaling in endothelial cells is also regulated by c-Src (59). Src INHIBITORS Historical Overview Some of the first reported broad-spectrum ATP-competitor kinase inhibitors were reported to inhibit c-Src, such as Tyrphostins, Genistein, Quercetin, and

339

Src Inhibitors

Staurosporin. Given the poor selectivity and high toxicity of these molecules, it was assumed that selectivity could not possibly be achieved by targeting the ATP pocket. For most of the 1980s and early 1990s, little progress was made in the pharmaceutical industry toward selective kinase inhibitors. One of the first compounds to be reported were Pfizer’s pyrazolo-pyrimidines PP1 and related compound PP2 (Fig. 2 and Table 1). These compounds were synthesized with the aim of inhibiting T-cell activation via Src family kinases Lck and FynT. PP1 and PP2 showed potency against Lck (IC50 of 4 nM) and a surprising selectivity for the Src family (60). These days, although less potent against c-Src itself (IC50enz 170 nM), PP2 remains one of the most selective Src inhibitors available and a very useful laboratory tool. For example, in vivo intraperitoneal injection of PP2 reduces invasion of HT29 cells into the liver following intrasplenic injection (36). The next generation of SFK inhibitors appeared in the late 1990s. PD173955 and later PD173956 were shown to inhibit c-Src with an IC50 of 20 nM on pure enzyme assays and at 5 mM in cells. Selectivity was lower than that of PP2, since these compounds are inhibitors of c-Abl, Csk, PDGFR and EGFR. Used at 5 mM, PD173955 was shown to block several cell lines in the G2/M phase of the cell cycle, although an effect on apoptosis was not clearly demonstrated (61). Later, at 1 mM, PD173955 was shown to produce accumulation of cells in G1 and increased the proapoptotic effect of detaching cells from matrix (37). So far, no data have been published on in vivo studies using PD173955 or PD173956. At around the same time, Novartis developed pyrrolo-pyrimidine (ex. CGP77675) and olomouCl

Cl

O O

H N

Cl

O N

N

O

N OH

N H

S

HN

O

N N N

N

N N

O

O

FDA approved for Glivec-resistant CML and B-ALL, Phase Il solid tumors

O

HN Cl

N

Phase I/II solid tumors

AZD0530

BMS 354825 (Dasatinib), Sprycel®

N

O

N

N

Phase I CML and solid tumors

SKI606 (Bosutinib) O

O

NH2

P Cl

N HN

N

N

N

N

NH2

N N N

N

N

N O O N H

Preclinical

Preclinical

AP23994

CGP76030

Preclinical

PP2

FIGURE 2 Molecular structure of Src family kinase inhibitors. While Dasatinib has recently been approved for use against chronic myelogenic leukemia (CML), clinical trials evaluating its effectiveness against solid tumors are still in progress. In constrast, no development of AZD0530 as CML therapy has been reported.

AstraZeneca, Macclesfield, U.K.

Wyeth, Pearl River, New York, U.S.A. Ariad, Cambridge, Massachusetts, U.S.A. Novartis, Basel, Switzerland Exelixis, South San Francisco, California, U.S.A. Pfizer, Groton, Connecticut, U.S.A. Pfizer Pfizer

AZD0530

SKI606 (Bosutinib) AP23994

280 nM 250 nM

22 nM

No available data

c-Abl, Csk Lck

c-Abl

c-Abl, EGFR, Met, c-KIT IGF1R, c-Abl

c-Abl, Flt1, Flt3

0.5 nM

5 nM

c-Abl, Csk

c-Abl, EphA2

c-Abl, c-KIT, PDGFR, Csk, EphA2

Non-SFK targets with IC50 <100 · IC50Src

1.2 nM

4 nM

0.6 nM

IC50c-Src pure enzyme

No data No data

No data

300 mg/kg po q3d

100 mg/kg po

100 mg/kg BID po 50 mg/kg po

50 mg/kg po

30 mg/kg po

Active dose (mouse model) Animal model used (Ref.)

No data HT29 liver invasion (36)

No data

MDA-231 orthotopic metastasis (64) Colo205, MCF7 subcutaneous growth (85)

L3.6pl pancreas metastasisa CSrcF530-NIH3T3 (84) and NBT11 subcutaneous growth (76) HT29, HCT116, colo205 subcutaneous growth HeyA8, SKOV31p1 tumor burden (83)

L3.6pl pancreas metastasis (38)

No development No development

No development

IND filed for CML

Preclinical

Preclinical

Phase I

FDA approved for CML and B-ALL Phase I and II on solid tumours Phase I/II

Stage of development

a Only published for AZM475271. Abbreviations: B-ALL, b-cell acute lymphoblastic leukemia; CML, Chronic myelogenic leukemia; FDA, U.S. Food and Drug Administration; PDGFR, platelet-derived growth factor receptor.

SU6656 PP2

PD173955/56

XL228

CGP76030

Bristol-MyersSquibb,New York, New York, U.S.A.

Company

BMS354825 (Dasatinib,
Sprycel )

Compound

TABLE 1 Main Src Family Kinase (SFK) Inhibitors

340 Cruzalegui

Src Inhibitors

341

cine-derived (ex. NVPAAK980) compounds as Src inhibitors targeted both to cancer and osteoporosis (62). Amongst these compounds, CGP77675 showed activity in vivo in bone resorption models. CGP76030, a second compound of the same type, was later shown to inhibit migration and DNA synthesis of PC3 prostate cancer cells in vitro (63). Very recently, CGP76030 has been shown to be active in vivo, orally in a bone metastasis mouse model using MDA-231 breast cancer cells (64). In this paper, no effect of CGP76030 on primary tumor growth was reported, although expression of dominant-negative c-Src in the same cellular context led to reduced primary growth and bone metastasis. Whether CGP76030 will be developed in clinical trials against solid tumors remains to be seen. In 2000, Sugen reported the effects of SU6656, an indolinone typical of Sugen’s library, showing high selectivity but relatively low potency against c-Src (280 nM) (30), on Src-induced podosomes in fibroblasts. This was followed closely by two successors SU11333 and SU11336, which showed target hitting in vivo in breast and colon tumor xenografts after intraperitoneal injection (51). These compounds also showed inhibition of VEGF-induced vascular permeability in vivo. Since 2001 a third generation of Src inhibitors has appeared. This time, molecules are characterized by their higher potency in enzyme assays and their dual inhibition of c-Abl and a variety of Glivec-resistant cAbl mutations. The strong activity against c-Abl accompanying potent anti-Src activity can be explained by the strong structural similarity of the ATP-binding sites in both kinases (10,65). In particular, SFKs and and c-Abl share a deep hydrophobic pocket that allows docking of a phenyl ring present in all these molecules. This dual selectivity has allowed some of the pharmaceutical companies developing these compounds to take advantage of the anti-c-Abl activity to accelerate the development of their anti-Src compounds, using very sensitive cell and animal models driven by the BCR-Abl translocation. However, given the existence of Glivec as standard therapy against chronic myelogenous leukemia (CML), inhibition of Glivec-resistant mutant BCR-Abl forms by these new SFK inhibitors needs to be proven. Although inhibition of BCR-Abl is clearly beneficial for CML patients, there is evidence that the strong activity of these compounds against endogenous, nonmutated c-Abl could lead to side effects in healthy tissues. A recent paper describes cardiotoxicity of Imatinib linked to its strong inhibition of c-Abl in the heart (66). Furthermore, c-Abl has been known for many years to participate in cell death caused by DNA damage. Inibition of c-Abl could then lead to survival of cells containing DNA damage and potentially to new tumorigenic events (67). Consequently, association of a dual c-Src/c-Abl inhibitor with a DNA damaging agent may not be a rational strategy to follow. Molecules in Clinical Trials SKI-606 Wyeth reported a quinoline-carbonitrile series in 2002, which included SKI-606 (Fig. 2 and Table 1) (68). This compound is a dual c-Src-Abl inhibitor with an IC50 of 1.2 nM against c-Src. It can inhibit c-Src in cultured cancer cells at 1 mM doses in vivo; oral administration of this compound at 100 mg/kg to nude mice leads to complete inhibition of c-Src in implanted colon carcinoma tumors (69). Due to its anti-c-Abl activity, SKI-606 causes regression of K562 tumors grown subcutaneously in nude mice (70). However, and despite this efficient target hitting activity, oral treatment with SKI-606 results only in a delay in growth of colon carcinoma tumors at 100 mg/kg, its maximal tolerated dose (69). As mentioned

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Cruzalegui

above, it is entirely possible that the cell lines used in these studies contain a signaling network in which c-Src is not essential for tumor survival. As a result, based on its activity against c-Abl, Wyeth initiated CML clinical trials but results have not yet been reported. A phase I on patients carrying solid tumors was also announced in 2005 but no further reports have emerged. Very recently (71), a new compound, SKI785, was presented as a potential successor of SKI-606 for clinical trials. SKI-606 has also been used in pre-clinical studies to evaluate the role of SFKs in vascular permeablity. SKI-606 was tested in mice in which c-Src or c-Yes had been deleted, characterized by a decrease in vascular permeability. These mice present less damage in the heart and brain following episodes of ischemia. Treatment of WT mice with SKI-606 also led to a decrease in vascular permeability, prevention of tumor cell extravasation, and protection from ischemiainduced cell death in the myocardium (72,73). More significanltly, decreased VP also prevents extravasation of circulating tumor cells and formation of metastasis. SKI-606 was shown to reduce extravasation and formation of liver and lung metastasis. These data suggest that SFK inhibition can have an impact both on the ability of tumor cells to grow and survive and also a separate impact on the vasculature, preventing exit of metastasizing cells and invasion. AZD0530 This anilino-quinazoline (Fig. 2 and Table 1)) and its related compounds are also mixed c-Src/Abl compounds [IC50 against c-Src of 4nM for ADZ0530 (84)]. Published data on the in vivo activity of AZD0530 are sketchy. Most of the preclinical data published relate to another related compound AZM475271. In particular, AZM475271 showed activity in an orthotopic metastasis model in which L3.6pl pancreatic carcinoma cells are injected into the pancreas. Daily oral treatment with 50 mg/kg AZM475271 for 2 weeks only led to 40% inhibition of primary tumor growth. Furthermore, a complete inhibition of liver metastasis was observed (74). In the same study, a combination of AZM475271 with gemcitabine led to 90% inhibition of primary tumor growth. In a paper published around the same time, gemcitabine association with PP2 had shown similar results on a variant of the PANC1 cell line made resistant to gemcitabine. Interestingly, this cell line was characterized by its elevated c-Src activity compared with the parental line (75). AZD0530 is orally active and was shown to delay growth of NBTII tumors and of c-SrcF530-transformed fibroblasts in nude mice (76). Inhibition of c-Src in implanted tumors has not been shown. AstraZeneca initiated phase I clinical trials on healthy volunteers to evaluate toxicity, PK and the use of the bone resorption markers CTX and NTX. In these studies, 70% inhibition of CTX accumulation was observed at the dose of 180 mg/day which, based on a their Phase I data, is the maximal tolerated dose in man (77,78). Plasma concentrations at this dose were calculated around 0.6 mM. Given that AZD0530 inhibits c-Src in cells at 1 mM, it is unclear whether the maximal administered dose will be sufficient to inhibit c-Src in tumors. On the basis of the reports showing effects on pancreatic models in combination with gemcitabine, a phase I/II is in progress on pancreatic cancer patients in combination with gemcitabine. In this study, patient survival will be evaluated as well as objective responses. BMS-354825 BMS-354825 (Dasatinib, Sprycel
) (Fig. 2 and Table 1) is the most potent Src inhibitor in clinical development. Its IC50 on purified enzyme is 0.6 nM and it

Src Inhibitors

343

inhibits c-Src efficiently in cultured cells at doses between 0.1 and 0.3 mM. This compound emerged from a series of Lck inhibitors originally synthesized as antiinflammatory compounds. As a consequence, Dasatinib is also a potent inhibitor of Lck with a IC50 of approximately 4 nM. BMS-354825 is potent against cAbl and BCR-Abl, inhibiting the pure enzyme with an IC50 of 0.05 nM. Dasatinib is also strongly active against EGFR, PDGFR, cKit, EphA2 and Csk with IC50s below 200 nM. Due to this activity against BCR-Abl but especially due to its activity against mutant forms of BCR-abl that resist Glivec treatment, Bristol-Myers developed this compound as therapy against CML and B-ALL in clinical trials. As expected, Dasatinib inhibited BCR-Abl in CML cells implanted in mice (K562 cell line) and caused complete regressions of subcutaneous K562 tumors (79). In clinical trials, Dasatinib treatment of chronic phase and accelerated phase CML patients led to impressive results. These results led to FDA approval for Glivecresistant CML in June 2006 at a recommended dose for CML of 70 mg/day. As shown in vitro, Dastinib was effective on patients harboring mutations resistant to Glivec but not in those patients whose CML cells contained mutation T315I. This mutant proved to be resistant to Dasatinib in vitro. Although Dasatinib is generally well tolerated at the doses effective on CML, episodes of pleural effusions have been observed, and because of the hERG inhibition observed in preclinical development, patients are monitored at every dose increase. Despite this success on CML, the possibility of using Dasatinib as therapy on solid tumors in which cAbl is not a target and where BCR-Abl does not exist is unclear. Unlike CML, which is a clonal blood disease based on the activity of BCR-Abl, metastasizing solid tumors present a more complex challenge, with less clearly defined targets and higher barriers. Although Dasatinib is active against growth of many cancer cell types in vitro, very little data are available on the activity of BMS354825 on tumor models other than the CML cell line K562. As discussed above, testing the activity of a Src inhibitor using a mouse xenograft model in which the only readout is the size of the subcutaneous mass following treatment is highly debatable. One presentation by BMS described 60% growth delay of PC-3 tumors in nude mice at the dose of 30 mg/kg (80) but no tumor regressions. The effect of inhibiting c-Src on this type of model will be highly dependent on the cell line and on the particular link of c-Src to survival signals. Perhaps the most adequate model tested in order to evaluate the efficacy of Dasatinib as an anti-Src drug is an orthotopic, metastasizing mouse model. Along this line, Gallick et al. recently described a 50% growth delay of pancreatic L3pl tumors grown orthotopically and a complete inhibition of metastasis (38). So far it is unclear whether Dasatinib will be able to inhibit c-Src inside solid tumors at doses compatible with safety observed in the clinic. The only biomarker data made available so far (from mice or humans) are based on inhibition of SFKs in peripheral blood cells. Partial results of a phase I trial carried out in Glasgow on a variety of solid tumors were made available in 2005 (80). In this study, two GIST patients presented stable disease. This could be explained by its activity against c-KIT, the kinase thought to be responsible for GIST. In this phase I trial, in which 88% of patients showed grade 1–3 anemia, the safe dose was fixed at 90 mg twice a day. Whether this dose will be sufficient to achieve therapeutic plasma concentrations against solid tumors remains to be seen, since no biopsy c-Src inhibition has been shown. Dasatinib has a relatively short half-life in man (<5 hours) and, as mentioned above, has been shown to have hERG inhibitory activity. Increasing doses of Dasatinib may be problematic due to its toxicity.

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Compounds in Preclinical Development Ariad Pharmaceuticals has reported a succession of potent dual SFK/cAbl inhibitors. Initially, AP23464 (IC50 below 1 nM) was shown to inhibit FAK phosphorylation at c-Src–dependent sites in KM12C cells (53). This dual c-Src/Abl inhibitor also blocked production of the proangiogenic factors IL-8 and VEGF (81,82). Later on, AP23994 (Fig. 2 and Table 1), an orally available compound with similar potency, was reported as able to reduce the weight of HeyA8 and SKOV3ip1 ovarian tumors in mice by 70% when given daily at 50 mg/kg. In this report, a combination with docetaxel proved to be even more effective, with 95% tumor volume reduction (83). Given its pharmacokinetic qualities, it is possible that AP23994 could enter clinical trials soon. Other companies besides those described so far in this review have active Src inhibitor programs (Table 1). They have reported potent compounds, including TG100435 (Targegen), XL-228 (or EXEL2280, Exelixis) (85), as well as yet unnamed compounds from Hoffman La Roche. Of these, XL-228 is particulary interesting because, as a dual inhibitor of IGF1R and SFKs, it may have a different profile from other compounds in developement. A recent press release by Exelexis revealed that this compound is also active against the BCR-Abl mutant T315I. Exelixis has filed for IND to initiate clinical trials on CML. CONCLUSIONS Inhibition of Src family kinases in solid tumors is likely to lead to objective responses in a subset of patients. In these patients, signaling defects may have generated both c-Src deregulation and dependence on its activity for tumor survival. Identifying these patients by the use of reliable selection biomarkers is a challenge. Despite the selective nature of the effect on the primary tumor, a more general effect on reducing the metastatic potential of many, if not all, tumors will be observed. However, this beneficial effect may be difficult to measure in clinical trials and practically impossible to detect in a classical phase I trial. Because of the lack of an appropriate c-Src–dependent animal tumor growth model, none of the Src inhibitors being developed in the clinic at present has shown significant tumor regressions in non-BCR-Abl mouse xenograft models. On the other hand, both BMS354825 and AZM475271 (and presumably AZD0530) have shown activity in a L3.6pl pancreatic orthotopic model for metastasis. Finally, the success of mixed c-Src/Abl drugs, such as Dasatinib on CML, should not distract us from the more ambitious challenge of sufficiently inhibiting Src in solid tumors to obtain a clinical response. Phamacokinetics needed for a solid tumor may be different from those required for a blood disease such as CML. POST SCRIPTUM Since drafting of this review, preliminary results of AZD0530 Phase I trials have been reported at the 2007 ASCO annual meeting in Chicago, Illinois (abstract 3520). Inhibition of c-Src pharmacodynamic markers in tumor samples was observed at the maximal tolerated dose of 175 mg QD. Toxicities observed at higher doses included grade 3 leukopenia, asthenia, grade 4 renal failure, grade 5 septic shock, and respiratory failure. No objective responses were reported but some patients are still receiving treatment. Results of a Phase I SKI-606 trial on solid tumors were also reported (abstract 3552). This compound was well tolerated at 400 mg QD. Toxicity at higher

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doses were grade 3 diarrhoea and skin rash. Although no pharmacodynamics were shown, treatment led to stable disease (>15 weeks) in 10% of patients. Phase I results for Dasatinib on patients with solid tumors have been presented at two meetings since ASCO 2005 (80). Dasatinib treatment at the MTD (100 mg BID daily) led to no objective responses, but >3 month stable disease in 21% of patients (abstract 383, AACR-NCI-EORTC, Prague 2006), and 2–10 month stable disease in 23% of patients (abstract 14042, ASCO 2007, Chicago). ACKNOWLEDGMENTS I would like to thank Dr. John Hickman (Servier) and Dr. Serge Roche (CNRS, Montpellier, France) for helpful comments in the preparation of this manuscript. REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18.

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Telomerase and Telomere Interacting Agents Jean-Fran¸c ois Riou Laboratoire d’Onco-Pharmacologie, UFR de Pharmacie, Université de Reims Champagne Ardenne, Reims, France

Anne de Cian and Lionel Guittat Laboratoire de Biophysique, Muséum National d’Histoire Naturelle, Paris, France

Dennis Gomez and Céline Douarre Laboratoire d’Onco-Pharmacologie, UFR de Pharmacie, Université de Reims Champagne Ardenne, Reims, France

Laurent Lacroix Laboratoire de Biophysique, Muséum National d’Histoire Naturelle, Paris, France

Chantal Trentesaux Laboratoire d’Onco-Pharmacologie, UFR de Pharmacie, Université de Reims Champagne Ardenne, Reims, France

Jean-Louis Mergny Laboratoire de Biophysique, Muséum National d’Histoire Naturelle, Paris, France

INTRODUCTION Researchers have long been investigating novel targets for anticancer drugs. Telomeres and telomerase represent, at least in theory, extremely attractive targets in oncology. However, no such molecule has currently obtained approval, and the most advanced compounds are currently in clinical trials. The next few years will be critical for target validation in the clinic. Independent of this outcome, the extreme ends of chromosomes represent a fascinating field of investigation for cell biologists and oncologists. At their normal state, telomeres protect chromosomal ends from fusion events and provide a means for complete replication of the chromosome. Telomere repeats are added by a specialized enzyme, telomerase, which is overexpressed in most tumor cells. In contrast, telomerase gene expression is repressed in most somatic cells, although limited expression associated with the S phase has been observed in normal cycling cells (1). This differential explains the rationale for telomerase inhibitors. Not only could telomerase become a reliable cancer marker in some pathologies, but it could also be a target for inhibitors, as long-term proliferation of cancer cells requires a telomere-maintenance mechanism. Unlimited proliferative potential, which depends on telomere maintenance, is one of six properties considered hallmarks of cancer cells (2). The validation of telomerase as a potential target for oncology was obtained by the expression of a

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dominant-negative mutant of the catalytic subunit in tumor cells, which results in the inhibition of telomerase activity and reduction in telomere length, as well as cell death and abrogation of tumorigenicity in vivo (3). Less straightforward is the rationale for the development of telomere-interacting agents. Normal cells also have telomeres, thus this target cannot be considered cancer-specific. Nevertheless, it may be proposed that telomeres from normal and cancer cells exhibit differences in structure or accessibility, explaining how a telomere ligand may exhibit selective toxicity. Recent results demonstrated that alterations of the telomere structure is responsible for the genomic instability associated with the tumoral transformation. Some of the initial predictions concerning the mechanism of action of telomere and telomerase agents have been validated, and other new prediction have emerged. Several classes of telomerase inhibitors have been developed and inhibit this enzyme through the targeting of its RNA or catalytic components as well as its DNA substrate, the single-stranded 30 -telomeric overhang. Telomerase inhibitors are chemically diverse and include modified oligonucleotides as well as small molecules, from natural to synthetic origin. This chapter presents the latest view on the mechanism(s) of action of these inhibitors, with an emphasis on a specific class of telomere ligands called Gquadruplex ligands, and discusses their potential use in oncology. Owing to space limitations, we will not consider the use of telomerase as a cancer marker or a prognosis factor [for recent reviews, see (4,5)], and we will restrict our discussion to the therapeutic target aspect of telomerase. Recent excellent reviews (6–10) cover in greater detail some aspects that cannot be detailed here [e.g., Telomerase promoter–mediated suicide gene therapy or telomeric PARP, tankyrases as targets for cancer therapy (11)]. Finally, one should note that telomerase may be involved in other aspects of human health (12), such as aging (13), or genetic diseases such as Dyskeratosis congenita, a rare, inherited bone marrow failure syndrome (14–16). PRESENTATION OF THE TELOMERE The telomeres of human cells range in size from 3 to 15 kb and are composed of tandem repeats of the sequence (50 -TTAGGG-30 ) with a 30 overhang of the G-strand extending 150–400 bases beyond the C-strand (Fig. 1A) (17). Telomere length and structure are critical to cellular proliferation. Normal human cells proliferate for a limited period of time and eventually undergo senescence, a state where cells cease to divide but remain viable. Inactivation of the p53 and Rb tumor suppressor pathways allow cells to bypass senescence, resulting in continued cellular division and further telomere shortening. These cells eventually reach a second proliferative block referred to as crisis, which is characterized by telomere dysfunction and cell death. Cells emerging from crisis maintain stable telomere lengths through the activation of a telomere maintenance mechanism (see below). Since DNA replication machinery is unable to completely replicate the extreme end of linear DNA molecules, telomere sequences are shortened at each round of normal cell division, a situation that leads to the proliferation arrest of normal somatic cells when a critical size is reached. Two mechanisms have been described in immortal and tumor cells to maintain the telomere length:

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FIGURE 1 Chemical formula of some telomerase inhibitors. (A) Various catalytic inhibitors. (B) Left: Structure of a G-quartet involving four coplanar guanines. Right: Possible conformation of the intramolecular G-quadruplex formed by human telomeric DNA. (C) Chemical formula of some G-quadruplex ligands.

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1. A specialized enzyme called telomerase is able to copy, as a reverse transcriptase, the short GGTTAG motif at the end of the telomere. Telomerase was first identified in ciliates (18). The catalytic subunit of this enzyme, hTERT, uses its RNA subunit (hTR) as a template for adding GGTTAG repeats to the ends of chromosomes [for a recent review, see (19)]. Telomerase is overexpressed in a large number of tumors (about 85%) and is involved in the capping of the telomere end (20) and in the DNA-damage response (21). 2. Telomerase activity is absent in about 15% of tumors and the telomere lengthening is obtained by recombination events between telomeres, known as alternative lengthening of telomere (ALT) (22). Telomere length is heterogeneous, varying from long telomeres (>20 kb) in ALT cells to a shorter size (3–15 kb) in telomerase positive cells. Telomere length reflects a homeostasis between the telomere lengthening mechanisms and the replicating degradation at each round of division, and is controlled by a complex association of telomere-binding proteins that tightly regulates the accessibility of telomerase to the G-overhang. Although identical telomere-binding proteins are qualitatively found in ALT cells, the exact mechanisms involved in the recombination at the telomere are poorly understood. In ALT cells, telomeric sequences and telomeric proteins are associated with large nuclear complexes to form APBs (ALT associated PML bodies), which also contain recombination factors.

OLIGONUCLEOTIDIC INHIBITORS Targeting the RNA Component (hTR) The RNA component of telomerase hTR is absolutely required for telomerase reverse transcription and is therefore a natural target for antitelomerase agents. Unlike hTERT, hTR is present in most normal tissues that do not express telomerase activity. Thus, targeting hTR in normal human somatic cells is not thought to generate toxicity, as these cells are mostly telomerase-negative. Different strategies and chemical modifications (Fig. 1) have successfully been developed to target hTR, starting with antisense oligomers. hTR has several features that makes it a good target for oligonucleotidic inhibitors: (i) hTR is not translated and should remain unprotected by ribosomal machinery, so that RNAs H-independent strategies for targeting hTR should be possible; and (ii) hTR provides a template (nucleotides 46–56; 50 -CUAACCCUAAC-30 ) for reverse transcription that is expected to be highly accessible. These oligonucleotides should be considered as “template agonists” rather than true antisense agents, as their targets are not translated but reverse transcribed. Nevertheless, this class of agents faces the same challenges as antisense agents: cell penetration and chemical stability. Regular DNA is probably too unstable toward nucleases to be considered for in vivo applications, explaining why different groups have tried several modifications to enhance uptake and/or stability. PNAs directed against the template region of hTR were among the first oligomers tested that could efficiently inhibit telomerase (23). Using a scanning approach, the binding determinants within hTR that are needed for the potent inhibition of telomerase by PNAs have been delineated (24). 20 -O-methyl-RNA (20 O-MeRNA) and 20 -O-(2-methoxyethyl) (20 -MOE) RNAs directed against the template region of hTR possess favorable pharmacokinetic properties and inhibit human telomerase with IC50 values of 2–10 nM (25,26). 20 ,50 -Oligoadenylate

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antisense oligomers directed against hTR have been described that could efficiently inhibit telomerase (27) as well as the growth of xenografted tumors (28,29). The most promising analogs may be thiophosphoramidates, which are potent inhibitors (IC50 ¼ 1 nM) that can induce senescence and telomere shortening (30). Two such oligomers, called GRN163 and GRN163L (a lipid palmidate conjugated to GRN163), were tested in vivo against a large panel of xenografted tumors. They exhibited strong antitumor activities on prostate, lymphoma, and myeloma models with short telomeres (31–33). GRN163L effectively inhibited telomerase activity in a dose-dependent fashion in breast cancer cell lines, independently of their genetic background (ERþ, ER
, HER2þ, BRCA1 mutants, etc.) (34). Breast cancer cells that exhibited telomerase inhibition also exhibited significant reduction in colony formation and tumorigenicity. Furthermore, GRN163L suppressed tumor growth and lung metastases of MDA-MB-231 cells in vivo (34). GRN163 and GRN163L were also tested in preclinical studies using systemic administration to treat flank xenografts of different human hepatoma cell lines in nude mice. In vivo treatment with GRN163L is also effective in preventing lung metastases in xenograft animal models (35). Breast cancer cells that were treated with GRN163L prior to plating in invasion chambers exhibit significantly diminished invasive potential (36). These assays revealed that GRN163L was superior to the nonlipidated parent compound, GRN163 (37,38): it does not require a lipid carrier to facilitate cellular uptake. GRN163L has recently entered into Phase I/II clinical trials for chronic lymphocytic leukemia. Silencing hTR/hTERT RNA interference (RNAi) has been shown to be an effective method for inhibiting the expression of a given gene in human cells. siRNAs against hTERT have been shown to inhibit telomerase activity in the HT29 immortal human colorectal adenocarcinoma cell line. Moreover, telomere lengths were reduced in cells stably expressing this particular RNA sequence, ultimately leading to a proliferation arrest (39). CATALYTIC INHIBITORS Nucleoside Analogs Nucleoside analogs acting as chain-terminating inhibitors of reverse transcriptases were among the first drugs to be tested for their ability to inhibit telomerase (40). In two immortalized human cell lines, dideoxyguanosine (ddG) caused reproducible, progressive telomere shortening over several weeks of culture, after which point telomeres became stable but remained short (41). Enduring AZT treatment of T-cell leukemia virus I-infected cells, in vitro and in vivo in patients, results in inhibition of telomerase activity, progressive telomere shortening, and increased p14(ARF) expression (42). Azidothymidine also induces apoptosis and inhibits cell growth of human parathyroid cancer cells (43). When tested in combination with 5-fluorouracil (5-FU), the presence of AZT increased 5-FU cytotoxicity, suggesting that the effects of these two drugs are synergistic (44). A very potent and specific nucleoside telomerase inhibitor, 6-thio-20 -deoxyguanosine 50 -triphosphate (TDGTP; IC50 ¼ 0.06 mM), has also been described (45) (Fig. 1A). However, despite these early advances, no clinical or pre-clinical trial is, to our knowledge, scheduled for nucleoside telomerase inhibitors.

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Nonnucleoside Inhibitors A variety of nonnucleoside drugs have also been shown to inhibit telomerase. Examples are epicatechin derivatives, such as epigallocatechin gallate (EGCG), which strongly and directly inhibit telomerase (46–49). The screening of chemical libraries allowed the identification of various molecules as potent inhibitors of telomerase such as DPNS and TNQX (50,51) (Fig. 1A). Long-term cultivation of breast cancer cell line MCF7 with a TNQX concentration that did not cause acute cytotoxicity resulted in progressive telomere attrition followed by an increased incidence of chromosome abnormalities as well as the induction of a senescence phenotype. An independent screen led to the identification of isothiazolone derivatives (52) (IC50 ¼ 1 mM for TMPI). Inhibition of telomerase by TMPI was quenched by dithiothreitol or glutathione, suggesting that these inhibitors act on a cysteine residue. Bisindole derivatives with IC50 values in the submicromolar range were also described (53). Finally, with an IC50 of 93 nM, BIBR1532 (Fig. 1A) is one of the most potent nonnucleoside inhibitors of telomerase (54). BIBR1532 is a mixed-type noncompetitive inhibitor (55). With no evidence of acute cytotoxicity, treatment of cancer cells with this compound led to progressive telomere shortening and, after a characteristic lag, to a proliferation block displaying hallmarks of senescence, which included morphological and proliferative changes, chromosomal aberrations, and altered patterns of gene expression (54). In a mouse xenograft model, pretreatment of tumor cells with this inhibitor led to telomerase inhibition, telomere shortening, and a marked reduction in tumorigenic potential. At higher concentrations, this class of telomerase inhibitor exerts a direct cytotoxic effect on malignant cells of the hematopoietic system, which appears to derive from direct damage of the structure of individual telomeres and is not related to overall telomere shortening (56). Pharmacological telomerase inhibition by BIBR1532 can sensitize drug-resistant and drug-sensitive cells to chemotherapeutic treatment (57). This is an important observation, as BIBR alone has a relatively weak in vivo antitumor activity. Miscellaneous A number of compounds have an effect on telomerase activity or expression of the subunits. Helenalin, a natural sesquiterpene lactone, is a potent and selective inhibitor for human telomerase. In vitro studies indicate that the inhibitory action of this drug on telomerase is selective and direct (58). Using a forward chemical genetics approach, Nakai et al. screened a microbial products library and identified three structurally unrelated antibiotics, chrolactomycin, UCS1025A, and radicicol, as active compounds. Chrolactomycin inhibited human telomerase in a cell-free assay, induced telomere shortening, and a population-doubling–dependent antiproliferative activity (59). On the other hand, a number of molecules, including candidate or validated anticancer drugs, have an effect on telomerase via an indirect mechanism. Telomerase regulation is complex, and a number of pathways may act on telomerase. Here are a few examples: 1. Heat shock protein 90 (Hsp90) is a molecular chaperone whose association is required for stability and function of multiple proteins that promote cancer cell growth and/or survival [for a review, see (60)]. Hsp90 client proteins include telomerase: Hsp90 facilitates the assembly of telomerase and remains

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associated with the functional complex. It is therefore not surprising that Hsp90 inhibitors such as novobiocin or radicicol reduced telomerase activity (61,62). One Hsp90 inhibitor, 17-AAG, is currently in Phase II clinical trial. Telomerase activity has also been reported to be upregulated by protein kinase C (PKC). A PKC inhibitor, bisindolylmaleimide I, inhibited telomerase activity but had no effect on the expressions of telomerase core subunits (61). Imatinib mesylate (Gleevec), a tyrosine kinase inhibitor, demonstrates activity against malignant cells expressing BCR-ABL, c-KIT, and platelet-derived growth factor receptor (PDGF-R). Imatinib mesylate also causes a dosedependent inhibition of telomerase activity in c-KIT-expressing SK-N-MC (Ewing sarcoma), SK-MEL-28 (melanoma), RPMI 8226 (myeloma), MCF-7 (breast cancer), and HSC 536/N (Fanconi anemia) cells. The inhibition of proliferation was associated with a decrease in the S-phase of the cell cycle and an accumulation of cells in the G2/M phase (63). U-73122, an amphiphilic alkylating agent that is commonly used as an inhibitor for phospholipase C, is also a potent and selective inhibitor of human telomerase (64). Interferon-gamma signaling induces growth arrest in many tumors. This compound leads to a posttranscriptional upregulation of the p27 tumor suppressor protein, which inhibits hTERT mRNA expression and telomerase activity (65). This inhibition of the hTERT expression and telomerase activity may be a novel tumor suppressor function of p27 (66). Trichostatin A, a histone deacetylase (HDAC) inhibitor, induces apoptosis in human leukemia cell line U937. The increase in apoptosis was associated with the upregulation in proapoptotic Bax expression and downregulation of antiapoptotic Bcl-2 and Bcl-XL. TSA treatment also markedly inhibited the activity of telomerase in a dose-dependent fashion (67). Cells transfected with dominant-negative hTERT were more likely to undergo apoptosis induced by Trichostatin A than cells transfected with wild-type hTERT (68). Telomerase activity and hTERT expression may also be inhibited by etodolac, a selective COX-2 inhibitor, leading to the conclusion that antitumor effects of etodolac on TMG-L cells are due to inhibition of both angiogenesis and telomerase activity (69). The antiproliferative effects of the tyrosine kinase inhibitor genistein seem to be mediated, at least in part, by its action on hTERT transcriptional activity (70). Pharmacological agents that act on hormonal pathways may interfere with telomerase expression. Estrogens regulate telomerase (71,72) and tamoxifen negatively controls telomerase activity (73). Telomerase has also been proposed as a target for retinoid therapy (74–76). Finally, c-myc seems to be a key factor in the expression level of the catalytic subunit (77). It is therefore not surprising to observe that molecules that inhibit this oncogene also inhibit telomerase (78).

Furthermore, increasing evidence suggests that the role of telomerase is not restricted to a simple lengthening of telomeres. This enzyme, or some of its subunits, could be involved in cell signaling and cell proliferation (the so-called telomerase extracurricular activities). For example, hTERT seems to be involved in mitochondrial apoptosis (79,80). hTERT depletion facilitated the induction of apoptotic cell death by cisplatin, etoposide, mitomycin C, and reactive oxygen

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species, and facilitated the conformational activation of Bax induced by genotoxic agents (80). Therefore, telomerase inhibition could be a promising approach in association with drugs promoting mitochondrial apoptosis.

IMMUNIZATION WITH TELOMERASE Immunotherapeutic strategies that can selectively target tumor cells are actively being sought. The catalytic subunit of telomerase (hTERT) is considered a universal tumor-associated antigen (TAA) due to its higher rate of expression by cancers [for a recent review, see (9)]. It might even be described as a candidate “universal cancer vaccine” (81) and this strategy is currently being tested in clinical trials (phases I/II; a phase III trial is scheduled this year). The rationale of using such vaccines is that TAA will be taken up and processed by antigen presenting cells, loaded onto major histocompatibility complexes (MHC), and recognized by T-lymphocytes (9). Efforts are being made to develop a strategy to elicit a telomerase-specific T-lymphocyte immune response in order to target hTERTþ cancer cells. The telomerase catalytic subunit is capable of triggering antitumor cytotoxic T lymphocyte (CTL) responses. The first 9 amino acid-long hTERT peptide used was p540 (82), also called HR2822. Its sequence is ILAKFLHWL; several other hTERT peptides have been described such as GV1001 (EARPALLTSRLRFIPK); their complete sequences and references may be found in (9). Clinical applications of telomerase vaccinations have been tested recently. A phase I clinical trial was performed with p540; p540-specific CTLs were induced in four out of seven patients and were able to kill tumor cells and partial tumor regression was obtained for one patient (83). Significant results were obtained in the case of non–small cell lung cancer, using a combination of telomerase peptides (GV1001 and HR2822) (84). Immune responses against GV1001 were detected in 13 of 24 evaluable patients, while two patients responded to HR2822. A complete tumor response was observed in one patient. A phase III assay in pancreatic adenocarcinoma is scheduled this year with GV1001. This peptide will be developed as a combination or add-on treatment to gemcitabine, which is currently used as a standard in pancreatic cancer treatment. In theory, hTERT vaccination could harm normal cells expressing the enzyme, especially stem cells and germ cells. Fortunately, hTERT vaccination did not result in a detectable decline in hematopoietic potential despite the expression of hTERT and MHC class I in bone marrow progenitors and stem cells (85). No CTL effect was found in normal telomerase-positive CD34þ hematopoietic cells. Another study did not reveal autoimmune manifestations resulting from vaccination with hTERT (86). In another study, no bone marrow toxicities were observed in long-time survivors with immune responses (84). Not all immunization attempts with telomerase fragments have been successful: recent results suggest that hTERT:p540 is not presented on the surfaces of tumor cells and will not be useful for the immunotherapy of patients with cancer (87). A lack of tumor recognition by hTERT peptide p540-specific CD8þ T cells from melanoma patients has been reported, raising questions as to the use of this peptide for cancer-specific immunotherapy. This reveals inefficient antigen processing by the proteasome (88). Finally, the efficient expansion of hTERT-specific CTLs from donor peripheral blood T lymphocytes remains a major challenge.

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TELOMERE LIGANDS Telomeres and Quadruplexes Telomeric DNA consists of highly repetitive but short sequences characterized by an asymmetry of guanines and cytosines, which are placed on two separate strands. The 30 -terminal region of the G-rich strand of human telomeres is single-stranded and may adopt particular conformations such as T-loops or G-quadruplexes. G-quadruplexes are formed in the presence of monovalent cations and consist of four-stranded structures stabilized by G-quartets. Self-association is favored by self-complementary hydrogen bond donors and acceptors present on both sides of the base, leading to the formation of a cyclic arrangement held by eight hydrogen bonds per quartet (Fig. 1B, left). The presence of a central cation helps maintain the stability of the structure. Potassium, which is very abundant in the intracellular medium, is a very favorable cation for quadruplex formation, explaining why this structure may be very stable under physiological conditions. Different G-quadruplex structures exist, depending on the orientation of the DNA strands and the syn/anti conformation of the guanines. The telomeric G-overhang can fold in several intramolecular quadruplexes that differ by the position of the adjacent loop regions (Fig. 1B, right). Recent data suggest a predominant conformation under physiological conditions (89–91). The existence of G-quadruplexes in vivo was initially established in ciliates using specific antibodies (92,93). Quadruplexes have now been studied in a variety of organisms from Escherichia coli (94) to humans. Concerning human cells, the in vivo existence of G-quadruplexes has been proposed or demonstrated for oncogene promoters and telomeres through the use of specific ligands (see below) (95,96). Optimal telomerase activity requires an unfolded single-stranded substrate, because G-quadruplex formation directly inhibits telomerase elongation in vitro (97). Therefore, ligands that selectively bind to G-quadruplex structures may interfere with telomere conformation and telomere elongation. The number of identified G4 ligands has grown rapidly over a few years. A range of G-quadruplex ligands has been shown to bond quadruplexes in vitro, such as TmPYP4 (98), PIPER (99), amidoanthracene-9,10-diones (such as BSU1051) (100), 2,7-disubstituted amidofluorenones (101), trisubstituted acridines (BRACO19) (102–105), ethidium derivatives (106,107), triazine derivatives (12459) (108), fluoroquinoanthroxazines (109), indoloquinolines (110), dibenzophenanthrolines (MMQ1) (111), bisquinacridines (112), pentacyclic acridinium (RHPS4) (113,114), and 2,6-pyridin-dicarboxamide derivatives (307A and 360A) (96,115). Natural compounds such as telomestatin (116), cryptolepine (117), and meridine (118) also recognize quadruplex DNA and inhibit telomerase activity in the TRAP assay [for a review, see (119)]. The chemical structure of some of these ligands is presented in Figure 1C. Features shared by many of these ligands include a large flat aromatic surface, presence of cationic charges, and ability to adopt a terminal stacking mode. Some of these molecules have also been shown to induce telomere shortening and/or telomere instability triggering apoptosis and/or senescence programs in various cell lines (see below). One of the most interesting demonstrations that telomere is a significant target for these ligands has been provided by a study using a radiolabeled Gquadruplex ligand. The localization of the chromosomal-binding sites of the tritiated pyridine dicarboxamide derivative 360A has been performed in metaphase spreads from cell lines bearing different telomere lengths. The result showed that 360A preferentially binds to the terminal parts of chromosomes in mitosis (96). Binding sites in internal chromosomal regions were also observed but were significantly fewer than those at the end of chromosomes.

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Other Types of Telomere Ligands Minor groove binders may also be used to target double-stranded telomeric repeats (120). A hairpin polyamide–cyclopropanepyrroloindole (CPI) conjugate alkylates its target adenine in the telomere repeats, 50 -CCCTAA-30 , and inhibits the growth of a variety of cancer cell lines (121). The anticancer drug cisplatin, aside from its possible recognition of G-quadruplexes (see above), may also recognize duplex telomeric DNA, as these long-tandem repeats are potential targets for cisplatin and other platinum compounds. Platinum derivatives, including 2,3-dibromosuccinato (2-(methylaminomethyl)pyridine)platinum (II) (122) and cis-dichloropyridine-5-isoquinolinesulfonic acid Pt(II) (123), have been found to represent strong telomerase inhibitors, with IC50 in the mM range. CONSEQUENCES OF TARGETING THE TELOMERE RATHER THAN TELOMERASE Are Telomere Ligands “Simple” Telomerase Inhibitors? There are fundamental differences between the targeting of the telomeric G-overhang using specific ligands and the inhibition of the catalytic activity of telomerase. Telomeres exist in the absence of telomerase activity, and G-quadruplex ligands are expected not only to have an effect against ALT cells but also on normal dividing cells. In contrast, catalytic inhibitors will take the advantage of the very low expression of telomerase activity in normal cells and should not dramatically affect their growth. According to the initial paradigm of the lifespan control by telomerase activity and telomere length, G-quadruplex ligands were first evaluated as telomerase inhibitors to induce telomere shortening and replicative senescence (3). For G-quadruplex ligands, this paradigm is partially true since a functional telomerase inhibition was observed in cell lines treated for several weeks with subtoxic dosages of certain compounds. For example, long-term treatment of the human cancer cells with subtoxic doses of disubstituted triazines or telomestatin induces telomere shortening that correlates with the induction of senescence (108,124–126). However, in some cases, no shortening was observed with G-quadruplex ligands (103,108,127,128). Interestingly, these derivatives were able to downregulate telomerase activity (103,108,113,124). In some cases, this effect may be related to c-myc repression (129,130) or to a modification of hTERT splicing (131). For BRACO19, an important decrease in the nuclear hTERT, together with the formation of cytoplasmic hTERT bound to ubiquitin, may explain the telomerase activity downregulation (132). Other antitumor agents have also been found to downregulate the telomerase activity and hTERT expression has been reported as a marker of the cell proliferation. Thus, the simplest explanation for the ability of the G-quadruplex ligands to downregulate telomerase activity is related to their antiproliferative activity. Direct Effects of G-quadruplex Ligands on Telomeres: Induction of Telomere Dysfunction It was also early observed that G-quadruplex ligands induced a short-term response (apoptosis) that cannot be explained by the sole telomerase inhibition (108,124,128). Telomestatin induced the activation of ATM and Chk2 corresponding to an activation of the DNA damage (124). Subtoxic concentrations of the

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acridine G-quadruplex ligands RHPS4 or BRACO19 could trigger growth arrest in tumor cells after just 15 days of exposure, before any detectable telomere shortening (103,113). 12459 induced apoptosis through the mitochondrial pathway and also provoked the early activation of P53 (133). Short-term and massive apoptosis were also observed from the interference of the telomere capping function of telomerase when hTERT or hTR were modified by mutations. The observation that BRACO19 causes chromosome end-to-end fusion associated with the appearance of p16-associated senescence led researchers to propose that G-quadruplex ligands mostly act to disrupt the telomere structure (134). Such telomeric dysfunction was also observed in cell lines treated with other quadruplex ligands and in cell lines resistant to a triazine derivative with typical images of telophase bridges (125,127,128). These studies suggest that the direct target of these ligands is telomere rather than telomerase activity. The evidence that the antiproliferative effect of G-quadruplex ligands is independent of the presence of telomerase activity also comes from a series of observations: 1. Overexpression of hTERT or a dominant-negative of hTERT in a telomerase positive cell line did not modify the antiproliferative effect of the triazine derivative 12459 (135). 2. All described ligands were also found active in blocking the proliferation of ALT cell lines (108,113,134). 3. On the other hand, the reintroduction of hTERT in ALT cell lines only produces a partial protection against the antiproliferative effects of telomestatin and 307A. In that case, we propose that hTERT acts to protect the integrity of telomeres.

RESISTANCE MECHANISMS; ILLUSTRATION WITH QUADRUPLEX LIGANDS Isolation of Resistant Clones As for most therapeutic agents, the biological effect of G-quadruplex ligands is susceptible to being overcome by the appearance of acquired resistance phenotypes. Cell lines resistant to the triazine derivative 12459 have been selected from parental A549 human lung adenocarcinoma by either progressive adaptation (JFA2) or by EMS mutagenesis (JFD clones) (125,135). Interestingly, these cell lines presented a resistance to the short-term activity of the ligand. However, resistance to the long-term effects of 12459 was only observed in JFA2 cells. The cross resistance to other G4 ligands (telomestatin, BRACO19) is absent for short-term treatment and is only observed for the longterm senescence induction (125,135). These results point out interesting differences between the short- and long-term effects of 12459. Since telomerase was initially established as one of the potential targets of these ligands, we have determined whether hTERT expression, telomerase activity, and telomere length are modified in these resistant models. Both transcriptional enzymatic activity and telomere length were increased in JFA2 and in the majority of the JFD clones (8/15), suggesting that the upregulation of telomerase activity and the telomere length increase could play a role in the resistance to this ligand. Important alterations of hTERT splicing were also observed in these resistant cells, in order to overcome a downregulation of hTERT active transcript induced by 12459 (131). The interpretations of these initial findings become more complex in light of experiments in which an increased telomerase activity was reintroduced into

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the parental cell line (135). In this case, no resistance to 12459 was found, using either short- or long-term treatment by the ligand, in A549 cells overexpressing hTERT. Therefore, the increased telomerase activity appears to be insufficient per se to confer a resistance phenotype. Since these resistant cell lines displayed an increased number of mitotic alterations (anaphase bridges) that transduced important telomere capping alterations, we propose that hTERT overexpression may serve to stabilize or protect the telomere extremities following changes induced by the resistance acquisition. Indeed, the transfection of a dominant-negative hTERT in JFD18 cells partially restores the sensitivity to the short-term treatment with 12459 (135). The upregulation of hTERT expression and the increase of telomere length were also reported in a HCT116 resistant cell line established against the cyclin kinase inhibitor flavopiridol (136). In addition, an overexpression of POT1 mRNA was also described for this cell line, suggesting that an alteration of the shelterin complex (see below) might be associated with the resistance phenotype. Although the potential link between these telomere alterations and the mechanisms underlying the flavopiridol resistance remains unclear, the G-quadruplex ligand BRACO19 presents a synergistic long-term effect with flavopiridol to overcome the resistance. The flavopiridol-resistant cell line is also hypersensitive to BRACO19 alone. However, the very poor permeability of BRACO19 currently limits its biopharmaceutical potential (137). Altogether, these studies, together with the observation that G4 ligands are also active against telomerase negative cell lines, strongly suggest that other factors than telomerase are involved in the mechanism of action of these ligands. Resistance and Apoptosis For most of the G4 ligands studied so far, an apoptotic cell death could be achieved after several cell cycles in tumor-derived cell lines. The triazine ligand 12459 activates the mitochondrial cell death pathway through an alteration of the Bax/Bcl-2 balance, which leads to caspase 3 activation. At short-term, it could be noticed that apoptosis predominates over the appearance of senescent cells for this ligand (133). Some of the JFD clones selected for resistance to 12459 also present an overexpression of the Bcl-2 protein. In addition, A549 cells transfected by Bcl-2 display a resistance to the apoptotic action of 12459 (133). However, the Bcl-2 overexpression is not sufficient to confer resistance to the long-term effect of 12459. Thus, 12459-directed senescence is uncoupled from apoptosis, a result that fits well with the differences observed between JFA2 and JFD clones for long-term and short-term resistance studies. CONSEQUENCES OF TELOMERE TARGETING The Single-Strand G-Overhang is Altered and Degraded Telomestatin induces an important telomere degradation in some cell lines that arises earlier than expected as a result of telomerase inhibition (124,135). These data, together with reports that G-quadruplex ligands also impaired the growth of ALT cell lines lacking telomerase activity, have suggested that additional mechanisms may explain the biological activity of the ligands in tumor cell lines. The G-rich 30 extension (G-overhang) has been implicated in the structure of the telomere extremities to create the T-loop that protects chromosome ends from fusion and their degradation has been associated with the onset of the replicative senescence and more recently to the deprotection of telomeres though inactivation

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of proteins from the shelterin complex (138) (Fig. 2). The hypothesis that G-quadruplex ligands preferentially act to modify the G-overhang conformation or induce its degradation has emerged and has been experimentally evaluated. By using two techniques [T-OLA (139) and a hybridization assay (135,139)], in vitro experiments concluded that telomestatin remains tightly and specifically attached to the G-overhang from treated cells and prevents hybridization of the oligonucleotide probe (140). These observations, together with in vivo DMS protection experiments, represent good evidence for the existence of G-quadruplexes at the telomeric G-overhang when treated by a G-quadruplex ligand. Further experiments established that a real (but partial) degradation of the G-overhang occurred after a longer telomestatin treatment in A549 cells (8–12 days); it was associated with the growth arrest of the cells. Interestingly, in other cell lines models (EcR293 and HT1080), telomestatin is able to induce at short-term (48–72 h) a rapid degradation of the G-overhang, suggesting that an active nucleolytic process is triggered by the ligand. This is also the case for 12459 in A549 cells (128,133) but not for 307A or 360A in A549 and T98G cells, where only a limited G-overhang degradation occurs (128). This suggests that the cellular response to the Gquadruplex stabilization at the telomeric G-overhang varies greatly, depending on the nature of the ligands or the cell line, possibly through a different activation of the DNA damage machinery. Telomere-Binding Proteins are Deregulated The proteins that protect telomeres were identified during the past decade and compose a complex now called shelterin (138). This complex is composed of six proteins, three of which bind directly to the telomeric repeats: TRF1, TRF2, and POT1. TRF1 and TRF2 present a Myb-type DNA-binding domain that allowed the

FIGURE 2 The shelterin complex. (A) Schematic organization of the telomere with the 30 G-overhang. (B) Representation of the shelterin complex that binds and protects the telomere ends. Source: From Ref. 138.

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recognition of 50 -YTAGGGTTR-30 in duplex DNA (141), while POT1 presents two OB-folded domains and displays a strong preference for the single-stranded 50 -(T)TAGGGTTAG-30 sequence (142). The three other proteins are TIN2 (that binds TRF1 and TRF2), TPP1 (that binds TIN2 and POT1), and Rap1 that binds TRF2. Thus, shelterin appears to make the connection between the duplex telomeric DNA and the 30 -G-overhang. Shelterin also associates with several proteins involved in recombination alrepair (Mre11/Rad50/Nbs1,ERCC1/ XPF, WRN, BLM, DNA-PK, PARP-2, and TANK). When either TRF2 or POT1 are inactivated (143,144), the overall amount of the single-stranded G-overhang is diminished by 30–50% and a specific DNA damage response is induced at most telomere ends (145). After TRF2, TIN2, or POT1 inactivation or when telomeres become critically short, 53BP1, g-H2AX, the Mre11 complex, and phosporylated ATM accumulate at chromosome ends (143,146,147). The structures formed by these DNA damage factors are referred to as telomere dysfunction induced foci (TIFs) (148). These studies are consistent with the view that telomere ends are arranged in a peculiar structure in order to protect their integrity. The protein complex shelterin is able to actively change its architecture and to control the detection by DNA damage factors. Interestingly, the inactivation of TRF2 and POT1 cause cellular effects analogous to those reported with G-quadruplex ligands, such as chromosomal instability and loss of the telomeric G-overhang, followed by the appearance of apoptotic and/or senescent cells. Thus, the effect of G-quadruplex ligands has been investigated on the binding of POT1 to the telomeric G-overhang in vitro and in human cells using a GFPPOT1 fusion protein. G-quadruplex stabilization by telomestatin dramatically impairs the binding of POT1 to the telomeric G-overhang in vitro and in some cell lines (HT1080) (149). Interestingly, telomestatin also displaces the telomere localization of TRF2 in tumor cell lines (including HT1080), but not in normal or immortalized cell lines (including EcR293) (149,150). An extensive telomeric repeat fragment (TRF) decrease is also observed in telomestatin-treated tumor cells-explaining the TRF2 decrease at telomeric ends. Accordingly, telomestatin treatment (24-hour) also induced the formation of telomeric phosphorylated g-H2AX foci, which corresponds to TIFs in HT1080 cells, suggesting the induction of an early DNA damage response associated with telomeres triggered by the ligand (Gomez and Riou, unpublished data; Fig. 3). The antitumor response to the G-quadruplex ligand BRACO19 paralleled the loss of the nuclear hTERT protein expression (132). A cytoplasmic hTERT expression that colocalized with ubiquitin was observed in immunostaining of xenograft tissues, suggesting an enhanced destruction of hTERT due to BRACO19 treatment. Since the telomerase complex binds and acts at the telomeric G-overhang, these results are in good agreement with the notion that G-quadruplex ligands will impair or inactivate the function of single-stranded telomere-binding proteins. Efforts are now being made to determine the effect of G-quadruplex ligands on other components of the shelterin complex and associated factors, such as WRN or BLM helicases, that cooperate with POT1 for telomere sequence unwinding (151). QUADRUPLEX RESOLVASES Several proteins have been described to act as G-quadruplex resolvases. Human RecQ helicases, Bloom’s and Werner’s syndrome DNA helicases (152,153,154),

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FIGURE 3 G-quadruplex ligands induce telomere dysfunction. Model to explain the differential effects of G-quadruplex ligands in normal and tumor cells to induce G-overhang degradation and/ or TRF loss. G-quadruplex stabilization using a ligand is expected to induce a G-overhang degradation that might be processed after signalization of damage. G4 resolvase might modulate the effect of the ligand. As a consequence, G-overhang degradation induces the release of POT1 from telomeres (normal or immortalized cells). In tumor cells, G-overhang degradation induces a further t-loop instability followed by an important TRF loss associated with the release of TRF2 from telomeres. In ALT cells, the consequences of the G-overhang degradation are unknown.

have been shown to unwind intra- and intermolecular G-quadruplex formed by telomeric sequences or other G-rich sequences. BLM has been proposed to maintain telomeres in ALT cells through recombination mechanisms (155). WRN belongs to the DNA polymerase d replication complex and defective WRN cells are unable to fully replicate telomeres due to a defect in lagging strand synthesis (156). The DEXH helicase encoded by DHX36 was also identified as the major source of G-quadruplex resolvase in HeLa extracts (157). Furthermore, Rtel, a murine gene encoding a DNA helicase homologue to dog-1 is required for telomere elongation (158). Dog-1 was initially identified in the nematode Caenorhabditis elegans for maintaining the stability of long poly dG–poly dC runs (159). Its putative role might be to resolve G-quadruplex associated with the poly dG

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repeats formed during replication, in order to maintain genome integrity. More recently, a study in ciliates has demonstrated that the G-quadruplex formation at telomeres is regulated through the cell cycle. TEBP a–b proteins tether the telomere to the nuclear matrix and stabilize G-quadruplex formation (93). During S phase, the phosphorylation of TEBPb results in the release of the telomeric DNA and the dissociation of G-quartets. Then, the chromosome ends are accessible for replication and extension of the G-overhang by telomerase. These observations and the redundancy of G-quadruplex resolvases indicate that mammalian cells require mechanisms for the removal of G-quadruplex during replication. G-quadruplex ligands from the trisubstituted acridine series were also found to inhibit in vitro unwinding by the RecQ helicases, BLM and WRN (160). Therefore, these ligands should also disrupt telomere synthesis. In addition, their action against other G-quadruplex resolvases during replication might provoke DNA synthesis defects in other G-rich regions of the genome. It is not clear whether such effect would contribute to an antitumor activity. A possible way to determine this is to examine the effect of the ligands in cells defective for these resolvases. CONCLUDING REMARKS Because of the unique character of the telomerase target, in vivo assays and clinical trials involving telomerase inhibitors will require careful consideration compared to those looking at conventional anticancer cytotoxic drugs. Some authors actually consider that telomerase inhibitors should only be used in complement to (or in combination with) a direct cytotoxic agent. According to the initial paradigm for telomerase inhibitors (161), a long delay is expected between the start of the treatment and the proliferation arrest, making these agents alone inefficient against tumors with long telomeres. This drawback has been verified for some inhibitors such as BIBR1532 (54). However, this limitation has been challenged by the observation that telomerase also plays an important role in capping or cell survival, making it an attractive target for immediate effects, even if the initial telomere length is long. Pharmacological strategies that aim at inhibition of telomerase in cancer cells should take into account not only overall telomere shortening, but also rapid induction of a high level telomere dysfunction (162). Another current field of interest is in relation to the possible existence of cancer stem cells. At least in breast cancer, these stem cells are telomerase positive (163): telomerase inhibitors could target these rare cells as, the rest of the tumor. Apart from these “classical” telomerase inhibitors, at least two strategies involving telomerase have been tested or will be tested soon in clinical trial. The telomerase immunization approach is by far the most advanced, since phase III trials are scheduled for 2006. Concerning G-quadruplex ligands, in order to design effective therapies, the end point is (1) to achieve antitumor activity and (2) to keep the toxicity at a low value. The first criterion was recently obtained for the BRACO19 derivative that showed antitumor activity as a single agent in human xenografts (132). Since several other G-quadruplex ligands were reported to exhibit selectivity for tumor cell lines instead of normal progenitors, primary astrocytes, or normal cell lines in culture (124,134), the second criterion to achieve a therapeutic index seems possible. Owing to the presence of G-quadruplexes in other parts of the genome, including oncogene’s promoters, a strategy to develop G4 promoter ligands was

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initiated (130,164,165) and led to the selection of a ligand called CX-3543 (Cylene Pharmaceuticals) that entered a Phase I clinical trial in 2005. Some ligands display interesting in vitro selectivity for the mixed parallel/antiparallel G-quadruplex structure adopted by the c-myc G-quadruplex, as compared to the telomeric Gquadruplex (166). Recent refinements of the structure adopted in solution by the human G-quadruplex raised the possibility that this agent could also target telomeres and thus transform the initial “telomere targeted strategy” to a “genome targeted G-quadruplex strategy.” ACKNOWLEDGMENTS We wish to thank all the members of the laboratories for fruitful discussions to conceive this chapter, E. Mandine, F. Boussin, A. Londono-Vallejo, K. Shin-ya, M.P. Teulade-Fichou, and M.F. O’Donohue for scientific collaborations. This work was supported by the “Association pour la Recherche contre le Cancer”, grant nos 3365 (to J.L.M.) and 3644 (to J.F.R.), by an European Union FP6 grant “MolCancerMed” (LSHC-CT-2004-502943; to J.L.M.) and by the Ligue Nationale Contre le Cancer, Equipe labellisée 2006 (to J.F.R.). REFERENCES 1. Masutomi K, Yu EY, Khurts S, et al. Telomerase maintains telomere structure in normal human cells. Cell 2003; 114(2):241–53. 2. Hanahan D, Weinberg RA. The hallmarks of cancer. Cell 2000; 100(1):57–70. 3. Hahn WC, Stewart SA, Brooks MW, et al. Inhibition of telomerase limits the growth of human cancer cells. Nat Med 1999; 5(10):1164–70. 4. Weise JM, Gunes, C. Telomeres and telomerase. A survey about methods and recent advances in cancer diagnostic and therapy. Histol Histopathol 2006; 21(11):1249–61. 5. Ulaner GA. Telomere maintenance in clinical medicine. Am J Med 2004; 117(4):262–9. 6. Shay JW, Wright WE. Telomerase therapeutics for cancer: challenges and new directions. Nat Rev Drug Discov 2006; 5(7):577–84. 7. Olaussen KA, Dubrana K, Domont J, Spano JP, Sabatier L, Soria JC. Telomeres and telomerase as targets for anticancer drug development. Crit Rev Oncol Hematol 2006; 57(3):191–214. 8. Pendino F, Tarkanyi I, Dudognon C, et al. Telomeres and telomerase: pharmacological targets for new anticancer strategies? Curr Cancer Drug Targets 2006; 6(2):147–80. 9. Huo LF, Tang JW, Huang JJ, et al. Cancer immunotherapy targeting the telomerase reverse transcriptase. Cell Mol Immunol 2006; 3(1):1–11. 10. Kelland LR. Overcoming the immortality of tumour cells by telomere and telomerase based cancer therapeutics—current status and future prospects. Eur J Cancer 2005; 41(7):971–9. 11. Seimiya H. The telomeric PARP, tankyrases, as targets for cancer therapy. Br J Cancer 2006; 94(3):341–5. 12. Blasco MA. Telomeres and human disease: ageing, cancer and beyond. Nat Rev 2005; 6(8):611–22. 13. von Zglinicki T, Martin-Ruiz CM. Telomeres as biomarkers for ageing and age-related diseases. Curr Mol Med 2005; 5(2):197–203. 14. Mitchell JR, Wood E, Collins K. A telomerase component is defective in the human disease dyskeratosis congenita. Nature 1999; 402(6761):551–5. 15. Marrone A, Walne A, Dokal I. Dyskeratosis congenita: telomerase, telomeres and anticipation. Curr Opin Genet Develop 2005; 15(3):249–57. 16. Goldman F, Bouarich R, Kulkarni S, et al. The effect of TERC haploinsufficiency on the inheritance of telomere length. Proc Natl Acad Sci USA 2005; 102(47):17119–24.

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141. Court R, Chapman L, Fairall L, Rhodes D. How the human telomeric proteins TRF1 and TRF2 recognize telomeric DNA: a view from high-resolution crystal structures. EMBO Rep 2005; 6(1):39–45. 142. Lei M, Podell ER, Cech TR. Structure of human POT1 bound to telomeric singlestranded DNA provides a model for chromosome end-protection. Nat Struct Mol Biol 2004; 11(12):1223–9. 143. Hockemeyer D, Sfeir AJ, Shay JW, Wright WE, deLange T. POT1 protects telomeres from a transient DNA damage response and determines how human chromosomes end. EMBO J 2005; 24(14):2667–78. 144. Zhu XD, Niedernhofer L, Kuster B, Mann M, Hoeijmakers JHJ, de Lange T. ERCC1/ XPF removes the 3' overhang from uncapped telomeres and represses formation of telomeric DNA-containing double minute chromosomes. Mol Cell 2003; 12(6):1489–98. 145. Celli GB, deLange T. DNA processing is not required for ATM-mediated telomere damage response after TRF2 deletion. Nat Cell Biol 2005; 7(7):712–8. 146. Takai H, Smogorzewska A, de Lange T. DNA damage foci at dysfunctional telomeres. Curr Biol 2003; 13(17):1549–56. 147. Kim S, Beausejour C, Davalos AR, Kaminker P, Heo SJ, Campisi J. TIN2 mediates functions of TRF2 at human telomeres. J Biol Chem 2004; 279(42):43799–804. 148. d’Adda di Fagagna F, Reaper PM, Clay-Farrace L, et al. A DNA damage checkpoint response in telomere-initiated senescence. Nature 2003; 426(6963):194–8. 149. Gomez D, O’Donohue MF, Wenner T, et al. The G-quadruplex ligand telomestatin inhibits POT1 binding to telomeric sequences in vitro and induces GFP-POT1 dissociation from telomeres in human cells. Cancer Res 2006; 66(14):6908–12. 150. Tahara H, Shinya K, Seimiya H, Yamada H, Tsuruo T, Ide T. G-quadruplex stabilization by telomestatin induces TRF2 protein dissociation from telomeres and anaphase bridge formation accompanied by loss of the 3' telomeric overhang in cancer cells. Oncogene 2006; 25(13):1955–66. 151. Opresko PL, Mason PA, Podell ER, et al. POT1 stimulates RecQ helicases WRN and BLM to unwind telomeric DNA substrates. J Biol Chem 2005; 280(37):32069–80. 152. Sun H, Karow JK, Hickson ID, Maizels N. The Bloom's syndrome helicase unwinds G4 DNA. J Biol Chem 1998; 273(42):27587–92. 153. Mohaghegh P, Karow JK, Brosh RM, Jr, Bohr VA, Hickson ID. The Bloom's and Werner's syndrome proteins are DNA structure-specific helicases. Nucleic Acids Res 2001; 29(13):2843–9. 154. Fry M, Loeb LA. Human Werner syndrome DNA helicase unwinds tetrahelical structures of the fragile X syndrome repeat sequence d(CGG)n. J Biol Chem 1999; 274:12797–802. 155. Stavropoulos DJ, Bradshaw PS, Li X, et al. The Bloom syndrome helicase BLM interacts with TRF2 in ALT cells and promotes telomeric DNA synthesis. Hum Mol Genet 2002; 11(25):3135–44. 156. Crabbe L, Verdun RE, Haggblom CI, Karlseder J. Defective telomere lagging strand synthesis in cells lacking WRN helicase activity. Science 2004; 306(5703):1951–3. 157. Vaughn JP, Creacy SD, Routh ED, et al. The DEXH protein product of the DHX36 gene is the major source of tetramolecular quadruplex G4-DNA resolving activity in HeLa cell lysates. J Biol Chem 2005; 280(46):38117–20. 158. Ding H, Schertzer M, Wu XL, et al. Regulation of murine telomere length by Rtel: An essential gene encoding a helicase-like protein. Cell 2004; 117(7):873–86. 159. Cheung I, Schertzer M, Rose A, Lansdorp PM. Disruption of dog-1 in Caenorhabditis elegans triggers deletions uptstream of guanine-rich DNA. Nat Genet 2002; 31:405–9. 160. Li J-L, Harrison RJ, Reszka AP, et al. Inhibition of the Bloom's and Werner's Syndrome Helicases by G-Quadruplex Interacting Ligands. J Med Chem 2001; 40:15194–202. 161. Herbert BS, Pitts AE, Baker SI, et al. Inhibition of human telomerase in immortal human cells leads to progressive telomere shortening and cell death. Proc Natl Acad Sci USA 1999; 96(25):14276–81. 162. Pantic M, Zimmerman S, Waller CF, Martens UM. The level of telomere dysfunction determines the efficacy of telomerase-based therapeutics in a lung cancer cell line. Int J Oncol 2005; 26(5):1227–32.

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163. Ponti D, Costa A, Zaffaroni N, et al. Isolation and in vitro propagation of tumorigenic breast cancer cells with stem/progenitor cell properties. Cancer Res 2005; 65(13):5506– 11. 164. Siddiqui-Jain A, Grand CL, Bearss DJ, Hurley LH. Direct evidence for a G-quadruplex in a promoter region and its targeting with a small molecule to repress c-MYC transcription. Proc Natl Acad Sci USA 2002; 99(18):11593–8. 165. Sun DY, Guo KX, Rusche JJ, Hurley LH. Facilitation of a structural transition in the polypurine/polypyrimidine tract within the proximal promoter region of the human VEGF gene by the presence of potassium and G-quadruplex-interactive agents. Nucleic Acids Res 2005; 33(18):6070–80. 166. Rezler EM, Seenisamy J, Bashyam S, et al. Telomestatin and diseleno sapphyrin bind selectively to two different forms of the human telomeric G-quadruplex structure. J Med Chem Soc 2005; 127(26):9439–47.

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Targeting Hsp90: The Cancer Super-Chaperone Paul Workman and Swee Sharp Cancer Research U.K. Centre for Cancer Therapeutics, The Institute of Cancer Research, Haddow Laboratories, Sutton, Surrey, U.K.

INTRODUCTION: SETTING THE CONTEXT The dramatic increase in our understanding of how cancer cells subvert normal cellular signaling systems to drive malignant progression (1) has led to the equally dramatic switch in drug discovery and development from the dominance of cytotoxic agents to a new era of targeted molecular cancer therapeutics (2). Our ability to discover and develop new drugs targeted to the underpinning molecular pathology of particular cancers has been accelerated by the integrated application of a powerful toolkit of new technologies and approaches, including genomics and proteomics; high-throughput screening (HTS) of chemical libraries; structure-based design; chemical biology; animal models; and molecular biomarkers for selecting patients, demonstrating proof of concept for target modulation, and enabling rational decisions in clinical trials (3). The selection of new drug targets (4) is frequently based on our increasing identification and understanding of cancer genes (5–8). These can be categorized broadly into oncogenes, tumor suppressor genes, and genome fidelity genes (1) which generally conspire together to hijack biochemical pathways and biological networks to produce the hallmark traits of malignancy (9). Clearly, the products of individual genetically or epigenetically deregulated cancer genes, or other proteins in the pathway that they subvert, can be targeted for cancer therapy (10). However, it is becoming clear that the elements of certain }cancer support systems,} which are not themselves bonafide oncogenes in the classical sense, can also be targeted for effective cancer treatment. Examples of these are the chromatin modifying enzymes, particularly histone deacetylases (11), and components of the protein quality control machinery, specifically the proteasome (12) and the molecular chaperone Hsp90 (13–15), which is the subject of this present chapter. Hsp90: AN UNUSUAL DRUG TARGET Until relatively recently, Hsp90 would not have been near the top of anyone's list as an obvious cancer drug target. As mentioned above, it is not a cancer gene, per se, and is not mutated in malignancy. Moreover, it is widely expressed in healthy cells and plays and important role within them, particularly under stressful conditions (15). Thus, interference with this protective molecular chaperone might have been expected to result in considerable toxicity. However, just as interest in the biology of Hsp90 has grown impressively over the past few years, so has the enthusiasm to develop inhibitors of the chaperone as molecular cancer

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therapeutics (14,16–18). Indeed, research into the translational and pharmacological aspects of Hsp90 have not only developed in parallel, but in fact have been very closely interactive with, and mutually beneficial to, the more fundamental investigations, as this review illustrates. A Pubmed search on }Hsp90} carried out in February 2007 yielded nearly 3300 hits—a remarkable tally for a protein with fairly humble origins. ORIGINS Heat shock proteins, known as Hsps, were first discovered over 40 years ago as proteins that exhibited increased expression in cells in response to elevated temperature (19,20). Many Hsps are molecular chaperones that have an important function in the correct folding of polypeptide chains, a process which can be challenging in the crowded molecular environment of the cell (21–23). Chaperones, including Hsp90, protect the cell against the effects of protein misfolding and aggregation. They carry out this role under normal conditions and also in reaction to cellular stresses, particularly in the adaptive response (24–26). Hsps are numbered according to their respective apparent molecular weights (26). The family of related Hsp90 proteins are a group of abundant 90KDa molecular chaperones that contribute as much as 1–2% of cellular protein, even under normal conditions (15,27–29). The family is made up of Hsp90a and Hsp90b, which predominate in the cytoplasm, Grp94, located in the endoplasmic reticulum, and TRAP1, in the mitochondria. An additional related protein, Hsp90N, has been identified, but its significance and precise role have not been elucidated in detail (30). STRUCTURE AND FUNCTION OF THE Hsp90 SUPER-CHAPERONE The function of Hsp90 is to regulate the conformation, activation, stability, and function of so-called }client} proteins (14–16). All four of the best known Hsp90 family members mentioned above are closely related in sequence and function. However, Hsp90a and Hsp90b are the most studied and best understood, and this review focuses mainly on them. They are present as functional dimers in the cell. The molecular structure of Hsp90 has been elucidated in considerable detail, with X-ray crystallographic studies being particularly informative (31–33). Individual subunits contain three functional domains. These are the N-terminal adenosine triphosphate (ATP)-binding domain, a middle domain that is implicated in client protein binding, and a C-terminal dimerization domain containing the tetratricopeptide repeat (TPR) binding motifs (31). A variety of evidence has shown that ATP binding, ATP hydrolysis, and ATP/adenosine diphosphate (ADP) nucleotide exchange are essential for Hsp90 function (34–38). Together with a highly integrated and orchestrated set of interactions with co-chaperones and other accessory proteins that join with Hsp90 to form a highly dynamic super-chaperone complex, the processes of nucleotide exchange and ATP hydrolysis drive a cycle of conformational changes, which in turn modulate the further interactions with client proteins (31). The recent X-ray crystal structure of the full-length yeast Hsp90 dimer in a complex with a nonhydrolyzable ATP analogue and the co-chaperone p23/Sba1 has been extremely important in supporting what was previously a controversial }molecular clamp} mechanism that is coupled to the ATPase cycle and which involves the closure of

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the so-called }lid} segment and transient dimerisation of the N-terminal domain in the ATP-bound conformational state (39,40). The new structure has revealed the intricate architecture of the }closed} conformational state of the molecular chaperone (32). The structure also sheds light on the extensive series of interactions between the various protein chains. The detailed conformational changes that occur in the N-terminal domain upon ATP binding are defined, as is the structural basis for how the closed state is stabilized by p23/Sba1. Very surprisingly, the closed Hsp90 conformation would not allow client proteins to be enclosed within the Hsp90 dimer in the expected fashion, but rather a bipartite binding surface is provided, the formation and disruption of which is coupled to the ATPase cycle of the chaperone. The structure clearly confirms that the ATPase-coupled molecular clamp model is correct and demonstrates that the proposed dimerization-coupled }split} ATPase mechanism (39,40) that is seen with other members of the GHKL class of ATPases, including the MutL mismatch repair proteins and type II topoisomerases (41, see later), also operates in the case of Hsp90. In addition, the recent X-ray crystallography work gives us a structural basis upon which we can begin to answer the critical question of how client protein activation is achieved. Some insight into this question has been provided by the recent electron microscopic reconstruction of the complex between yeast Hsp90, the kinase cochaperone Cdc37, and the kinase client Cdk4 (33). From this it appears that the two lobes of the Cdk4 kinase are in fact interacting with different domains of the Hsp90. In this way, the conformation of the kinase client can be coupled directly to changes in the relative positioning of the chaperone domains with which it interacts. The precise details of how the coupled chaperone-client interactions result in client protein activation remain to be defined, and this is now a key challenge for the field. The individual roles of the various co-chaperones in the ATPase-driven chaperone cycle are becoming clearer. Client proteins interact first off with the Hsp70/Hsp40/Hip complex (13,31). Hsp70 is then linked to Hsp90 by the adapter Hop/p60, which binds to its respective C-terminal domain via its TPR domain. The binding of Hop/p60 is restricted to ADP-bound Hsp90 in the open conformation, which has a high affinity for hydrophobic substrates. When Hsp90 exchanges ADP for ATP, this brings about the conformational change and transient dimerisation of the N-terminal domains, as discussed above. Hsp70/Hsp40/Hip and Hop then dissociate from the complex, facilitating ATPdependent association with other co-chaperones, such as Cdc37, p23, and immunophilins, thus forming the so-called }mature} complex. Cdc37 loads kinase clients, whereas p23 stabilizes the ATP-bound form of Hsp90, thereby prolonging the period in which the client protein activation can take place (32). Examples of client protein activation are the changes that allow steroid hormone receptors to bind ligand or kinases like AKT to be phosphorylated as part of signal transduction cascades. Prevention of ATP binding to N-terminal Hsp90 (e.g., by inhibitors, see later) blocks the mature complex formation and, instead of client protein activation, promotes degradation of the client via the ubiquitin proteasome pathway (see later). In some instances this has been shown to involve recruitment to the immature Hsp90 complex of the ubiquitin ligase CHIP, another TPR domain protein that can bind to both Hsp90 and Hsp70 (42,43). The action of the Hsp90 activating protein AHA1 (44) is mediated via binding to the middle domain of the chaperone; this then promotes a

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conformational switch in the middle segment catalytic loop that releases the catalytic Arg380 residue (yeast Hsp90 numbering) and facilitates its interaction with ATP in the N-terminal domain (45). In addition to the precise mechanisms by which Hsp90 brings about client protein activation (see above), another outstanding conundrum is how particular clients are recognized while other related proteins are not. Little is understood about the determinants of Hsp90 interaction with its client proteins. In one study, a loop within the N lobe of the kinase domain of ERBB2 was identified as important for Hsp90 binding (46). The amino acid sequence of this loop affects the electrostatic and hydrophobic nature of the surface of the kinase, which appears in turn to influence the interaction with Hsp90. A point mutation in the ERBB2 loop region was found to disrupt Hsp90 binding. These considerations appear to explain the differences in the interaction with Hsp90 of different members the ERBB2 family. Also of interest, the nascent, immature forms of ERBB2 and ERBB1 (EGFR) are both dependent on Hsp90 chaperoning, but whereas the mature ERBB2 retains this dependence, the mature ERBB1 becomes Hsp90-independent. Another recent study of a large number of kinases has again indicated that surface features, rather than the contiguous amino acid sequence, are important in defining recognition of kinases by Hsp90 (46). Furthermore, the authors suggested, based on a comparison of the mitogen-activated protein kinase and the phosphatidylinositol 3-kinase pathways, that the selectivity of Hsp90 towards particular kinases has a functional basis, with Hsp90 regulating kinases that act as hubs that integrate signals from multiple inputs (47). There is much more work to do to understand the rules that govern what makes a client protein for Hsp90. Hsp90 CLIENTS AND OTHER INTERACTIONS To be accepted as a bonafide Hsp90 client, a protein is generally expected to fulfil the following criteria: & & &

Bind to Hsp90 Be depleted in cells after treatment with an Hsp90 inhibitor Undergo degradation by the ubiquitin-proteasome pathway upon Hsp90 inhibition

Although Hsp90 is generally considered to be a relatively selective molecular chaperone, acting as it does at a relatively late stage in the maturation and activation of a relatively small group of client proteins, its customer list nevertheless continues to grow. The current client count is in excess of 100 proteins (15,28). An updated list of }Hsp90 interactors,} for which inclusion requires biochemical evidence of binding, is maintained as part of a very useful website (48). Featuring strongly among the clientele are protein kinases and transcription factors. Greater numbers of proteins have been shown to exhibit evidence of some type of interaction with Hsp90 based on global profiling or genetic approaches (47,49–52). For example, one study identified a network of 198 putative physical interactions and 451 genetic and chemical genetic interactions (49). On the basis of this study, as well as being labeled with terms such as the }super-chaperone} and }cancer chaperone,} Hsp90 has also been referred to as a }master regulator} (16,49). This is not only because of the sheer number of interactions, above and beyond those physical and functional interactions with bona fide clients and co-chaperones, but also in view of the diverse range of biological processes that Hsp90 influences. These involve not only linking a large

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array of homeostatic control mechanisms to the status of the external environment of the cell organism, for example integrating signal transduction pathways with stress responses, but also impacting on chromatin remodeling and gene transcription (15,49,52,53).

Hsp90 INHIBITORY NATURAL PRODUCTS: CHEMICAL BIOLOGY IN ACTION The natural products geldanamycin (Fig. 1) and radicicol (Fig. 2) were the first Hsp90 inhibitors to be discovered and have proved to be extraordinarily invaluable as chemical tools for probing the biology of Hsp90 and demonstrating proof of concept for the molecular chaperone as a therapeutic target. Furthermore, geldanamycin analogues were the first Hsp90 inhibitors to progress to clinical trials (see later). In 1992, Whitesell et al. (54) reported that the benzoquinone ansamycins related to geldanamycin and herbimycin A showed potent antitumor activity that was distinct from their reported inhibition of oncogenic kinases such as SRC. Having defined the 17-position (located on the quinone ring) as one that allows chemical manipulation without compromising antitumor activity, Whitesell et al. carried out a seminal experiment in which they coupled geldanamycin to beads via a linker/spacer group and used this to fish out the main binding protein from cell extracts (55). The main binding protein was identified as Hsp90 by the use of specific antibodies (55) and subsequently by micro-sequencing of protein bound to radio-labeled affinity-tagged geldanamycin (56). As part of the early series of experiments, geldanamycin was shown to inhibit the association of Hsp90 with the v-SRC tyrosine kinase oncoprotein (55). This association between the chaperone and the viral oncogene product had been demonstrated using virally transformed cells as early as 1981 (57–59). Subsequent reports that geldanamycin associated with kinases such as C-RAF (60) and other signaling proteins such as steroid hormone receptors (59) stimulated interest in Hsp90 as a potentially important drug target. This was supported by the recognition that

O R

O O

Cl H H OH N

O N H OH

O

N OH H

O NH2

O

O

OH

O

O

NH2 O

17-DMAG R = NHCH2 CH2N (CH3)2 17-AAG R = NHCH2CH = CH2

O

IPI-504

Geldanamycin R = OCH3 FIGURE 1 Chemical structures of the benzoquinone ansamycin class of Hsp90 inhibitors.

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O

O

O

H

O

O

O

H

HO

O

H

HO Cl

H

H

O

Cl OH

Radicicol

H

HO

O

OH

O

X= N

O

Cl

O

OH

N

Cycloproparadicicol

KF58333 (E-isomer) H HO

OH O

O

Cl HN

O HO Cl

O O

HO O Pochonin D

O OMe

O Radester

FIGURE 2 Chemical structures of the radicicol class of Hsp90 inhibitors.

Hsp90 was expressed in much higher levels in many tumor cells as compared to their normal counterparts, and in some cases this expression is of prognostic significance (28,60,61,62). In other important experiments, treatment of cells with geldanamycin and the related natural product herbimycin A was shown to cause client protein destabilization and degradation, usually via the ubiquitin-proteasome pathway (63). Also of tremendous importance was the use of a combination of biochemical methods and X-ray crystallography to demonstrate that geldanamycin binds at an N-terminal ATP site on Hsp90 (34,35,56). Previously, Csermely et al. had suggested that Hsp90 bound ATP, leading to a conformational change in the chaperone (64). A similar series of experiments to those done with geldanamycin was also carried out with the chemically different natural product radicicol (65–67). Following multiple demonstrations of the bioactivity of geldanamycin and radicicol using in vitro screens, derivatives of the agents have shown anticancer activity in a variety of animal models (14,17). Subsequently, the geldanamycin analogue 17-AAG (tanespimycin, KOS953) (Fig. 1) was progressed to clinical trials, followed by other analogues (68,69; see next section). Radicicol derivatives have not progressed to the clinic but their mode of binding to Hsp90 is very similar to the pyrazole/isoxazole class of inhibitors which are now undergoing development (70; see later). Thus it can be seen that the natural products geldanamycin, radicicol and related compounds have been invaluation both as chemical tools to interrogate Hsp90 biology and also to demonstrate proof of concept in cancer models, leading to clinical trials of 17-AAG. In the following sections, we provide concise summaries of the different classes of Hsp90 inhibitory chemotypes, starting with further information on the natural products and moving onto the more recent synthetic small molecule classes. The current status of the various Hsp90 inhibitors is listed in Tables 1 and 2.

Peptidomimetic Non-peptidic

Interaction between Hsp90 and survivin

Shepherdin 5-aminoimidazole4-carboxamide

SAHA and others

Abbreviations: NECA, 5-N-ethylcarboxamideadenosine; SAHA, suberoylanilide hydroxamic acid.

Various

Bristol-Myers Squibb (New York, New York, U.S.A.) Merck (Whitehouse Station, New Jersey, U.S.A.) Ref (103) Ref (105)

Paclitaxel (Taxol
)

Taxane

Geldanamycin dimer

Geldanamycin dimer

Ref (100) Ref (104) NeuTec (Manchester, U.K.)

Radicicol oxime derivatives

Radicicol

Vernalis (Great Abington, Cambridge, U.K.); Novartis (Hanover, New Jersey, U.S.A.); The Institute of Cancer Research (Sutton, Surrey, U.K.) Kyowa Hakko Kogyo (Chiyoda-ku, Tokyo, Japan) Conforma Therapeutics

Novobiocin analogues NECA Mycograb

CCT018159, VER-49009

Pyrazole

Coumarin Adenosine analogue Antibody

CNF2024

Infinity Pharmaceuticals (Cambridge, Massachusetts, U.S.A.) Conforma Therapeutics

Kosan Biosciences; National Cancer Institute

17-DMAG (alvespimycin, KOS1022) IP-504

Company/institution Kosan Biosciences; (Hayward, California, U.S.A.); National Cancer Institute; (Bethesada, Maryland, U.S.A.); Conforma Therapeutics (San Diego, California, U.S.A.)

17-AAG (tanespimycin, KOS953, CNF1010)

Drug or lead compound

Purine

Hydroquinone form of 17-AAG

Benzoquinone ansamycin

Chemical class

Histone deacetylase

C-terminal Grp94 Other inhibitors

N-terminal ATPbinding pocket

Mode of Hsp90 binding

TABLE 1 The Current Status of Hsp90 Inhibitors Current status

Preclinical research Preclinical research

Preclinical research Preclinical research Phase II clinical trial for invasive candidiasis Phase I combination clinical trial with 17-AAG Phase I clinical trials

Preclinical research

Preclinical research

Phase I clinical trials in multiple myeloma, with or without bortezomib (Velcade
) Phase II in melanoma and breast cancer with trastuzumab (Herceptin
) CNF1010 is in Phase I clinical trials in solid tumors Phase I clinical trials in hematologic cancers and solid tumors/breast cancer with trastuzumab Phase I clinical trials in multiple myeloma and gastrointestinal stromal tumors Phase I clinical trials in solid tumors Preclinical discovery

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TABLE 2 Other Hsp90 Inhibitors that are Being Developed by Pharmaceutical Companies Pharmaceutical company

Compound

Development stage/current status

Astex Therapeutics (Milton Road, Cambridge, U.K.) Serenex (Durham, North Carolina, U.S.A.)

AT-13387

Lead compound selected; preclinical development Preclinical development Lead compound; target client specific Preclinical research Phase I clinical trials in solid tumors Lead optimization Preclinical research Preclinical research

ArQule (Woburn, Massachusetts, U.S.A.) Abraxis (New York, New York, U.S.A.) Biotica (Saffron Walden, Essex, U.K.) TopoTarget (Kobenhavn, Denmark) Locus (Blue Bell, Pennsylvania, U.S.A.)

SNX-5422 SNX-4862 ARQ-250RP ABI-010 – – –

GELDANAMYCIN ANALOGUES Early work focused mainly on geldanamycin (Fig. 1) and the related benzoquinone-ansamycin natural product, herbimycin A. In addition to work reviewed in the previous section, early studies in Japan revealed the bioactivity of these agents in several systems (71). Natural product inhibitors of Hsp90 are now well known to show up as hits in a variety of cellular screening assays. Some of these early screens demonstrated that the benzoquinone ansamycins could reverse oncogenic transformation by v-SRC (72). Though the effects were originally attributed to the relative inhibitory activity on tyrosine kinases like SRC, the observations did support the exploration of the benzoquinone ansamycins as potential antitumor agents. The activity in v-SRC transformation reversion assays was subsequently reinterpreted in the light of the knowledge that v-SRC is a client protein that is highly dependent on Hsp90, and which is rapidly degraded in cells exposed to Hsp90 inhibitors. As mentioned above, Hsp90 is a member of the small group of GHKL-type ATPases, including the MutL mismatch repair proteins and type II topoisomerases (41). X-ray crystallography studies revealed that the N-terminal domain adopts a so-called Bergerat fold. This is characterized by a two-layer a/b sandwich, in which the a helices define a deep nucleotide-binding pocket that extends all the way from the external protein surface to the buried face of the highly twisted b-sheet (31). The shape of the nucleotide binding site is very unusual compared to other ATP-binding proteins, including other classes of ATPases and kinases (41). As a result, the bound nucleotide is forced to adopt a bent topology (35). X-ray crystallography studies of geldanamycin bound to the N-terminal domain of Hsp90 (Fig. 3) showed, importantly, that the antibiotic adopts a folded or C-shape conformation as a result of the switch to a cis-amide bond, as distinct from the trans-amide bond that is adopted in solution, resulting in accurate mimicry of the unusual bent configuration of the natural nucleotide (67). The unusual ATP site topography forms the basis of the high selectivity of Hsp90 inhibitors. Much of the bound water found in Mg2þ-ADP complexes with Hsp90 is displaced by geldanamycin, although key water molecules are retained and participate in the anchoring network of hydrogen bonding interactions in the ATP pocket (67). The benzoquinone ring of geldanamycin is located at the top of the deep nucleotide binding pocket, whereas the ansa ring is oriented deep in the bottom of the ATP site. The X-ray co-crystal structure provides a basis for the rational design of inhibitory analogues based of geldanamycin. Certain features

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FIGURE 3 Pymol diagram showing the binding interactions between the adenosine triphosphate (ATP) binding site of yeast N-terminal domain of Hsp90 and geldanamycin. Hydrogen bonds are shown as dotted lines, water molecules are black colored balls, nitrogen atoms are in black, and oxygen atoms are in light grey. This representation is based on the structure published by Roe et al. Source: From Ref. 67.

are essential for activity, including the 7-carbamate group. Geldanamycin proved to be too toxic to give a therapeutic index in animal models (73). From a series of analogues, 17-AAG was identified as promising (74,75). 17-AAG has similar activity to geldanamycin against cancer cells in vitro (76) and causes cell cycle arrest and apoptosis in human colon cancer cell lines (77). It has also shown good antitumor effect at well tolerated doses in animal models (78). In addition, it exhibits strong anti-angiogenic properties (79). Complications with 17-AAG include its limited water solubility, leading to the use of a cumbersome formulation in the clinic, as well as variable metabolism by polymorphic cytochrome P450 CYP3A4 and also by NQO1/ DT-diaphorase, together with susceptibility to efflux by P-glycoprotein and lack of oral bioavailability (80,81). The analogue 17-DMAG (alvespimycin, KOS1022) (Fig. 1) is more water soluble and orally bioavailable while retaining antitumor activity (82). This agent has followed 17-AAG into the clinic. A remaining concern about the geldanamycin series is the presence of the quinone that participates in the target binding (Fig. 3) (69). Quinones are known to undergo redox cycling and to cause hepatotoxicity, which is seen with 17-AAG in the clinic (see later). Our discovery that 17-AAG is much more potent in tumor cells containing high levels of NQO1 led us to suggest that this reductase may metabolize 17-AAG to a more potent Hsp90-inhibitory reduction product, most likely the hydroquinone (80). Subsequently, Guo et al. confirmed that the 17-AAG hydroquinone is a somewhat more potent Hsp90 inhibitor (83). Recognizing that the hydroquinone would be more soluble than 17-AAG, this agent, which is known as IPI-504 (Fig. 1), is now being developed as a drug in its own right for intravenous administration (84) and has recently entered clinical trials.

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RADICICOL ANALOGUES As referred to above, radicicol (Fig. 2) behaves in many ways very similarly to the geldanamycins. However, radicicol achieves its nucleotide mimicry in a different way (69). It is oriented in the opposite sense to the benzoquinone ansamycin in the N-terminal nucleotide pocket. Thus, the aromatic resorcinol ring points down into the base of the deep pocket, whereas the macrocycle containing the conjugated bond system and the epoxide group binds at the top near the surface. Of note is the network of hydrogen bonds, including key water molecules, that anchor the resorcinol unit at the bottom of the ATP site. As with geldanamycin, radicicol adopts a folded structure, but this is less pronounced than with the benzoquinone ansamycin, being more of an L-shape than a C-shape, and is the conformation also adopted by the unliganded radicicol. Radicicol has a more potent binding affinity for Hsp90 (i.e., it has a lower dissociation constant Kd), at least under the conditions and short term incubations used in the experimental determination (69). This is apparently due to differences in enthalpy/entropy factors. Lacking the quinone, radicicol is not susceptible to metabolism to NQO1. It does, however, still contain structural features that are not desirable in a drug, including the presence of potentially reactive electrophilic sites. Presumably because of stability, metabolic liability, and potential toxicity issues, radicicol itself did not show promising activity at non-toxic doses in animal models. On the other hand, radicicol oximes (e.g., KF58333) (Fig. 2) do show activity in human tumor xenografts in vivo (85). Despite this animal model activity, no radicicol analogue has progressed to clinical trial, possibly due to toxicity to the eye (17,18). However, the key resorcinol unit present in radicicol features as an important structural element within the pyrazole/isoxazole scaffold class of Hsp90 inhibitors (70; see later). Given its modest complexity and amenability to fairly short syntheses, a range of variants have been prepared, including cycloproparadicicol, pochonin D, and also radester (Fig. 2), which is a hybrid comprising part structures of radicicol (resorcinol unit) and geldanamycin (benzoquinone). (For more details, see references 16 and 86.) A series of simplified ring and conformational analogues of radicicol were synthesized and showed a degree tolerance for different macrocyclic ring sizes and quite a high level of potency in certain examples (87). PURINE-SCAFFOLD INHIBITORS Given, on the one hand, the combined promise and limitations exhibited by the natural product-based Hsp90 inhibitors and, on the other hand, the technologies that are available for HTS and structure-based design, it was logical that projects would be initiated to discover synthetic small molecule classes. The first of these series to emerge have been the purine-scaffold inhibitors designed by Chiosis et al. on the basis of molecular modeling (88) using the published crystal structures of Hsp90 (34,35,67). The initial agents were exemplified by the first compound PU3 (89) and the more potent analogue PU24FCl (90) (for chemical structures, see Fig. 4). Contrary to the rigid docking predictions, X-ray co-crystal structures showed that the trimethoxyphenyl ring did not bind in the expected fashion but induces a conformational change that opens up a new hydrophobic pocket in Hsp90a (91).

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FIGURE 4 Chemical structures of purine-scaffold Hsp90 inhibitors.

Analogue PU24FCl showed activity in a human tumor xenograft at quite high doses (90). A more recent report has disclosed a number of water soluble 8-arylsulfanyl, 8-arylsulfoxyl, and 8-arylsulfonyl adenyl derivatives based on the purine-scaffold; PU-H58 (Fig. 4) has emerged to be the most potent in vitro and showed good activity in animal models (92). Optimization based on detailed structure-activity relationships has resulted in compounds that show therapeutic activity by the oral route at fairly high doses (93), which may be needed to overcome rapid clearance. The purine analog CNF2024 (structure not disclosed) is now in Phase I clinical trials (Table 1) (94). PYRAZOLE-SCAFFOLD INHIBITORS The fertile pyrazole-scaffold compounds (70) were discovered by HTS using the yeast Hsp90 enzyme and malachite green as a readout for inorganic phosphate release upon ATP hydrolysis (95,96). This screen identified the 3,4-diarylpyrazole resorcinol hit CCT018159 (Fig. 5), which had similar single digit micromolar potency on the Hsp90 protein target as the clinical agent 17-AAG, although the cellular activity was lower (97). More detailed biological evaluation showed that the activity of CCT018159 on human cancer cells was not affected by NQO1 or P-glycoprotein, representing significant advantages over 17-AAG (97). Protein crystallography studies were initiated very soon after hit identification, defining the principal ligand-protein interactions, thus allowing a structurebased approach to be taken. As referred to earlier, the resorcinol unit in the pyrazole series binds in the identical way to the equivalent structural motif in radicicol (96,97). In the same way as with radicicol, the resorcinol unit anchors the compounds into the base of the nucleotide binding pocket via a network of HO

O

HO

Cl

O

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H N

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HO

HO N

N H

CCT018159

N H

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VER-49009 CCT0129397

FIGURE 5 Chemical structures of pyrazole-scaffold Hsp90 inhibitors.

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hydrogen bonds with the protein, both direct and water-mediated. The ethyl group on the resorcinol ring of CCT018159 binds into a hydrophobic pocket, as do other lipophilic substituents in that position, such as chloro (96). Structure-based design, carried out in collaboration between our own group and Vernalis Ltd, led to a major gain in potency by the addition of a hydrogen bond donor at C5 of the pyrazole ring (98). A series of potent C5 amides were generated that benefited from the predicted additional protein-ligand interaction. These are exemplified by VER-49009 (CCT0129397) (Fig. 5). Structurally related compounds were found in an independent screen, but the particular compounds disclosed had little activity in cells, probably due to poor membrane penetration as a result of the presence of a carboxylate residue (99). Other studies have shown that pyrazole-scaffold can be replaced by alternative heterocyclic rings, such as isoxazole and triazole (70). The progress with the pyrazole resorcinols has nicely illustrated the combined power of HTS coupled with X-ray crystallography-driven structure-based design. NOVOBIOCIN ANALOGUES The coumarin-based antibiotics bind to bacterial DNA gyrase, an ATPase closely related to Hsp90. The coumarin antibiotic novobiocin (Fig. 6) was reported to bind to a proposed ATP site in the C-terminus of Hsp90 (100,101). X-ray crystallography studies are not informative on the C-terminus. More potent analogues have been identified from a library of novobiocin derivatives, of which compound A4 (Fig. 6) was the most active (102). OTHER Hsp90 INHIBITORS Various companies and academic groups are now developing a range of small molecule Hsp90 inhibitors (Tables 1 and 2). The peptidomimetic shepherdin was designed by modeling the interface between Hsp90 and the antiapoptotic and mitotic regulator client protein survivin (103). It has been reported to interact with the ATP pocket of Hsp90, to destabilize client proteins and to induce massive apoptosis, with antitumor activity also seen in vivo at well tolerated doses (103). Anti-leukemic activity has been described (104). The non-peptidic small molecule 5-aminoimidazole-4-carboxamide was designed using a combined structure-activity and dynamics-based computational chemistry strategy and shown to be active (105).

OH

O

O

OH

O

NH O

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MeO

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OH Novobiocin

FIGURE 6 Chemical structures of novobiocin and A4.

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Based on experience with other drug target classes and the potential for distinct effects on different client proteins, the identification of isoform-selective inhibitors of particular Hsp90 family members is of interest. The adenosine analogue 5-N-ethylcarboxamideadenosine (NECA) (Fig. 7) is reported to be selective for Grp94 over Hsp90 (106). A cell-impermeable geldanamycin derivative was reported to exhibit antiinvasive activity, potentially via effects on Hsp90a, which can be secreted extracellularly where it interacts with matrix metalloproteinase 2 (107,108). A human recombinant antibody directed against fungal Hsp90 has been developed as an antifungal agent and shows promising activity in that setting (Table 1) (109). It has also been considered as a potential cancer treatment (110). The cytotoxic anticancer agents cisplatin and paclitaxel (Taxol
; BristolMeyers Squibb, New York, New York, U.S.A.) have been reported to bind weakly to Hsp90, but the significance of this is unclear (see ref 14). Histone deacetylase (HDAC) inhibitors such as depsipeptide FK228 and LAQ824 (Fig. 8) have been reported to inhibit Hsp90 based on the molecular signature of client protein depletion and induction of Hsp70 (see later) in cancer models and patients (111–113). HDAC inhibitors increase the acetylation of Hsp90 and an acetylation site in the middle domain of Hsp90 has been identified which regulates chaperone function (114). This appears to be mediated by HDAC6. Other post-translational modifications are known to occur on Hsp90 that may also affect its activity. A synergistic interaction between HDAC and Hsp90 inhibitors (LBH589 and 17-AAG, respectively) was reported in leukemic cells (115). However, we have observed an antagonistic interaction between the HDAC inhibitor trichostatin A and 17-AAG in a human ovarian cancer cell line; this antagonism was consistent with effects on total cellular acetylation, which was reduced by 17AAG, and also with changes in the expression of chromatin-modifying enzymes and other chromatin-associated proteins (116). These interactions are complex and potentially context-dependent. They likely relate to the functional link, mentioned earlier in this chapter, between Hsp90, chromatin regulation, and gene transcription. THERAPEUTIC SELECTIVITY It is important to consider how the therapeutic selectivity of Hsp90 inhibitors for tumor versus normal cells may be achieved. This may be related to one or more of the following factors: & &

Simultaneous combinatorial depletion of multiple oncogenic client proteins (117) Combinatorial effects on all of the hallmark traits of cancer (9,117) NH2 N

N N

O

N O

N H HO NECA

OH FIGURE 7 Chemical structure of an inhibitor of the Hsp90 isoform Grp94.

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Me H N

O OH

O N H

N

OH

HN

S

S

NH Me

O Me

HN

O

HN Me

LAQ824

O O

Me

O

FK228

FIGURE 8 Chemical structures of inhibitors of histone deacetylase. & & &

&

&

&

Exploitation of }oncogene addiction} (117), whereby cancer cells are more dependent upon, or addicted to, the primary oncogenic drivers of the disease The greater dependence of mutated oncogenic client proteins on Hsp90, as compared to the wild type counterpart (e.g., as in B-RAF) (118,119) The stressed state of malignant cells due to the expression of overexpressed and mutated oncogenes and microenvironmental factors such as hypoxia, nutrient deprivation, and so on Related to the stressed state, the predominance within cancer cells of Hsp90 in a super-chaperone complex that is hypersensitive to Hsp90 inhibition, as distinct from the uncomplexed, more resistant form that is seen in normal cells (120) The triggering of toxicity via the unfolded protein response, especially in those cancers where protein secretion is important, or simply due to the high load of overexpressed and mutated kinases in malignant cells The accumulation of Hsp90 inhibitors in malignant versus normal cells, perhaps related to the super-chaperone complex (120)

Combinatorial activity on multiple oncogenic proteins is believed by the present authors to be very important for the anticancer activity and selectivity of Hsp90 inhibitors (117). Examples of Hsp90 client proteins that regulate the cancer hallmark traits and that are depleted by Hsp90 inhibitors include several kinases (ERBB2, B-RAF, C-RAF, CDK4), hormone receptors (androgen and Oestrogen receptors), other transcription factors (p53 and HIF1a), and additional proteins (catalytic subunit of telomerase hTERT) (Fig. 9). It is clear that further studies are required to elucidate the mechanistic basis for the selectivity of Hsp90 drugs. This may also help to develop biomarkers that would predict for sensitivity of individual patients (121). CLINICAL TRIALS The first-in-class Hsp90 inhibitor 17-AAG has undergone a series of Phase I clinical trials (68,69) and is now in Phase II evaluation as a single agent, as well as in combination studies (Table 1). In the Phase I trials, 17-AAG was shown to give good pharmacokinetic exposures, consistent with activity in preclinical models, and the molecular signature of Hsp90 inhibition was demonstrated in peripheral blood mononuclear cells

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FIGURE 9 Examples of Hsp90-dependent client proteins involved in the six hallmark traits of malignancy. As a result of combinatorial depletion of multiple Hsp90 client proteins, all of the hallmark traits are modulated, leading to a powerful anticancer effect.

(PBMCs) and tumor biopsies (122–125). In the Phase I study at our own institution, we used the molecular signature of target modulation that we validated in a human tumor xenograft model (126). This comprised depletion of the kinase client proteins C-RAF and CDK4 and induction of the co-chaperone Hsp70. Toxicities observed consisted of liver transaminitis, diarrhea, nausea, vomiting, anorexia, fatigue, and anemia. There may have been some contribution to the side-effects from the rather cumbersome vehicle that contained egg phospholipid and dimethyl sulphoxide (DMSO). The DMSO gave rise to an unpleasant odor. Various schedules were evaluated, including weekly, twice weekly (days 1, 4), daily · 5 (21 day cycle), and daily · 3 (14 day cycle). The toxicity seen was both dose- and schedule-dependent, with more hepatotoxicity seen upon daily administration. Pharmacokinetic-pharmacodynamic (PK-PD) data obtained from PBMCs and a limited number of tumor biopsies (122) were compatible with the preclinical PK-PD data from many human cancer cell lines in vitro and from human tumor xenografts in immunosuppressed mice (122). In our own trial, the weekly dose of 450 mg/m2 was limited by the formulation. The PK-PD data indicated that optimal Hsp90 inhibition may have been obtained with a twice weekly schedule, but this was not practical (122). The PK-PD data also suggested that antitumor activity may have been expected (122). During the initial Phase I studies, there were no complete or partial responses as defined by the Response Evaluation Criteria in Solid Tumors (RECIST) system. This was probably not surprising, given that 17-AAG is more cytostatic than pro-apoptotic when tested against cancer cells in culture, probably related to the induction of antiapoptotic co-chaperones such as Hsp70 and Hsp27 (77). In our study, we saw two cases of prolonged stable disease in patients with heavily pretreated, drug resistant, metastatic, malignant melanoma (122). Activity was also seen in renal cancer and in prostate cancer, as determined by decreases in circulating prostate tumor antigen (PSA) and computed tomography (CT) scans in patients with androgenindependent disease (69). Phase I studies have been initiated in pediatric cancer (127). Various Phase II studies are now underway using once weekly (300–450 mg/m2) or twice weekly (220 mg/m2) schedules in patients with melanoma,

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prostate, breast, thyroid, renal, and ovarian cancers, together with mantle cell lymphoma and malignant mast cell neoplasm (69,127). Improved formulations of 17-AAG are being evaluated in the clinic. A cremphor-based formulation has been developed by Kosan Pharmaceuticals (KOS953) (Table 1). Following a Phase I trial of KOS953 across tumor types, a Phase II study in trastuzumab (Herceptin
; Genentech, Inc., South San Francisco, California, U.S.A.) refractory ERBB2 amplified breast cancer is underway (69,128). The trastuzumab was continued with the 17-AAG. The more frequent side-effects were fatigue and gastrointestinal toxicities, with minimal liver toxicity, and no bone marrow suppression. An ERBB2-positive breast cancer patient had a confirmed partial response according to RECIST criteria and three further breast cancer patients had tumor regressions in the range of 21–25%. A further Phase I trial of KOS953 in combination with the proteasome inhibitor bortezomib (Velcade
) is ongoing in multiple myeloma patients. Initial results appear promising with 6 of 12 patients with bortezomib-refractory cancer showing a response to the combined treatment (69). A second improved formulation of 17-AAG involving an oil-in-water emulsion has been developed by Conforma Therapeutics (now Biogen Idec) (129). The pharmacokinetic properties were similar to those in the egg phospholipid/ DMSO formulation initially developed by the U.S. National Cancer Institute, and no toxicities appeared to be associated with the new formulation. A decrease in the expression of the ERBB2 extracellular domain in plasma, determined as a pharmacodynamic endpoint, was observed at doses >83 mg/m2. Three minor responses were recorded at these dose levels in melanoma, gastric, and duodenal cancers. A series of Phase Ib combination studies are underway with the original formulation of 17-AAG and involving either solid tumors or hematological malignancies (69,127). Drugs that are being combined with 17-AAG include docetaxel (Taxotere
), paclitaxel (Taxol), irinotecan, cytarabine, cisplatin/gemcitabine (Gemzar
), bortezomib (Velcade), and imitanib (Glivec
). The combination of 17-AAG and taxanes has appeared very promising in preclinical studies (130,131). In a Phase I study of 17-AAG plus docetaxel, patients were treated using a weekly or three weekly schedule (69). Toxicity to date has involved fatigue and neutropenia. A partial response by RECIST criteria was observed, with minor responses in prostate, lung, melanoma, and urethral cancers. There does not appear to be any pharmacokinetic interaction. The more water soluble 17-AAG analogue, 17-DMAG has followed the parent drug into the clinic (Table 1). 17-DMAG has the advantages of greater aqueous solubility and oral bioavailability. Both intravenous and oral studies are underway (69,127). The highly water soluble hydroquinone derivative of 17-AAG, IPI-504, is being developed by Infinity Pharmaceuticals and has entered Phase I studies (132).The first non-ansamycin Hsp90 inhibitor to enter clinical trials is the purine-scaffold compound CNF2024 from Conforma Therapeutics. It will be interesting to see whether the non-benzoquinone ansamycins are devoid of liver toxicity, as metabolism of the quinone is one hypothesis to explain the hepatotoxicity. Hsp90 inhibitors from a variety of other companies are in preclinical development (Tables 1 and 2) and it can be anticipated that a number of these will enter clinical trials in the coming months and years. Biomarkers are essential for rational modern drug development (133). A variety of pharmacodynamic biomarkers have been or can be explored in the

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clinical trials. The initial studies mainly looked at the expression of the kinase clients C-RAF and CDK4 in PBMCs in leukocytes. In addition, induction of Hsp70 was also measured. Western immunoblotting was generally used. Although quantitation is difficult with western blotting, the use of the molecular biomarker signature proved very informative (122). ELISA methodology has been developed, allowing accurate quantitation in PBMCs and tumor tissue (134). In addition, it may be possible to monitor Hsp70 in the plasma (134). ERBB2 extracellular domain and insulin-like growth factor binding protein 2 show promise as biomarkers that can be monitored in the circulation by ELISA (135). In addition to the range of client proteins and co-chaperones that are candidate biomarkers for normal tissue and tumor studies based on a large literature of preclinical work, we have used non-biased global profiling methods successfully to identify mRNAs and proteins that exhibit increased or decreased expression in cells following treatment with Hsp90 inhibitors (44,116,136). Of particular potential interest was our identification of elevations in the expression of Hsp90 isoforms—the targets of the drug treatment—as well as the induction of the Hsp90-activating protein AHA1 (44,136). Induction of these proteins could impact sensitivity as well as provide pharmacodynamic biomarkers. Similarly, induction of the anti-apoptotic chaperones, such as Hsp70 isoforms and Hsp27, may also influence drug sensitivity. Our recent proteomic analysis identified interesting protein expression changes that may be of biological significance or provide further potential biomarkers (116). Of note was the altered expression of a group of chromatin-modifying enzymes and other chromatin-associated proteins. One of these was the protein arginine methyl transferase 5 (PRMT5), which exhibited decreased expression in human cancer cells after Hsp90 inhibition. Follow up studies showed that PRMT5 was bound to Hsp90 and is a potential client protein (116). The alteration in chromatin-modifying enzyme levels may be biologically relevant with respect to histone modification and the control of gene expression. Indeed, as mentioned earlier, treatment of cells with 17-AAG caused a decrease in protein acetylation in human ovarian cancer cells and antagonized the increase in protein acetylation caused by the HDAC inhibitor trichostatin A (116). Furthermore, these agents exhibited an antagonistic effect on proliferation. In contrast, however, a synergistic interaction has been seen with the combination of HDAC and Hsp90 inhibitors in leukemic cells, suggesting context-dependence that requires further analysis. Minimally invasive methods are of great interest as an alternative source of biomarker endpoints (137). In collaborative studies we have identified an unusual metabolite signature by magnetic resonance spectroscopy (MRS) in cell and tumors following treatment with 17-AAG (138). This signature involves choline metabolism in the cell. Preliminary studies have been carried out to determine the feasibility of monitoring changes in choline uptake and metabolism by positron emission tomography (PET) (139). Also of potential clinical application is the use of a 68Ga-labelled F(ab')2 fragment of trastuzumab to detect the rapid degradation of ERBB2 using PET (140). WHAT CLIENT PROTEINS ARE IMPORTANT IN WHICH DISEASES? It is a considerable advantage of Hsp90 inhibitors that they do exert combinatorial effects on numerous oncogenic client proteins, signaling pathways and hallmark traits (117). This results in a powerful all out attack on cancer cells on many fronts.

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In addition, it is likely that the simultaneous combinatorial effects will decrease the likelihood of drug resistance developing. At the same time, however, the fact that so many oncogenic players are affected makes it difficult to ascertain how any particular group of patients will respond or whether any particular client proteins will dominant the response in a particular tumor type or patient. Some of the main reasons for expecting that differential effects may occur are the observations that: (i) some client proteins are more important than others in driving particular cancers; and (ii) various client proteins show differential responses to Hsp90 inhibition in terms of both dose-response and time-dependence. For example, ERBB2 is depleted at much lower Hsp90 inhibitor concentrations and after much shorter exposures compared to most other kinases. So, how may we expect these client protein differences to play out in terms of tumor response in the clinic? &

&

&

&

& &

&

&

Clearly, in those patients with ERBB2-positive breast cancers, the depletion of this receptor tyrosine kinase is likely to dominate the response. This likely explains the activity that is being seen in the clinic in this setting. In hormone-refractory prostate cancer, effects on both ERBB2 and the androgen receptor are likely to be very important and this is a disease setting in which Hsp90 inhibitors should be evaluated. The RAS-RAF-MEK-ERK pathway is very important in melanoma. Effects on mutant B-RAF and wild type C-RAF may well be important in the responses seen in this cancer. Since mutant EGFR receptors are very sensitive to Hsp90 inhibitors, activity in non–small cell lung cancers (NSCLCs) harboring these mutations may be anticipated. Mutations of KIT in gastrointestinal stromal tumors (GIST) may lead to activity in these sarcomas. In cancers where the PI3 kinase pathway is important, effects on players in this pathway, such as phospho-AKT, may play a key role, as in ovarian cancer, glioblastoma, and other malignancies. Renal cell cancers are driven by loss of VHL, which causes stabilization of HIF1a, leading to increased expression of VEGF and other key downstream gene products. Depletion of HIF1a, which is a client protein for Hsp90, is likely to be important in this disease. In addition, HIF1a is stabilized by hypoxia in many cancers, and depletion of this could contribute to the powerful anti-angiogenic effects of Hsp90 inhibitors. In cancers where protein trafficking and the unfolded protein response are especially important, as in multiple myeloma, we can expect the effects of Hsp90 inhibitors of this pathway to the very important (141).

The above are just a few examples of tumor types in which therapeutic activity may be mediated by effects on particular client proteins. It is likely that other examples will emerge as we gain greater knowledge of the molecular pathology of human cancers and of the molecular pharmacology of Hsp90 inhibitors. CONCLUSIONS AND FUTURE PERSPECTIVES The Hsp90 story is a fascinating one and remains incomplete. There is no doubt that our knowledge of the complex and multi-faceted role of Hsp90 in cancer

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and normal cells will continue to grow very rapidly over the next few years. Equally well, as many Hsp90 inhibitors enter the clinic over the same time frame, we will learn more about how best to use these intriguing agents, and, through them, we will discover much about the role of Hsp90 in human biology and pathology. The tolerability and early signs of clinical activity with the geldanamycinbased agents is encouraging many academic investigators and companies to get involved in this area. It should be emphasized, however, that, until an Hsp90 inhibitor gains regulatory approval in a human cancer, the ultimate proof of concept for Hsp90 as an important therapeutic target will remain unproven. It will be interesting to see whether such therapeutic activity will be revealed with single agent Hsp90 inhibitors or will require these drugs to be used in combination with other targeted molecular cancer therapeutics or cytotoxic agents. What is clear is that the natural product Hsp90 inhibitors have been extraordinarily valuable in helping to probe the biology of Hsp90 in normal and tumor cells and to pave the way for the development of synthetic small molecule inhibitors. Complementing this chemical biology approach, various other drug discovery technologies are also proving important, particularly the combinations of HTS and structural-based design. In addition, the discovery and application of various molecular biomarkers of drug effects has also been extremely valuable. Pharmacodynamic biomarkers are now readily available and the increasing emphasis will be on non-invasive methodologies, such as PET and MRS/MRI, as well as the discovery and validation of biomarkers that will be predictive of which patients will respond. As both intravenous and oral Hsp90 inhibitors of various chemotypes enter the clinic, there are some key questions that need to be addressed. For example, which Hsp90 isoforms should be inhibited and which ones should be spared? Will a particular isoform be more important for anticancer activity and others for toxic side-effects? These questions can be addressed using both chemical tools and RNA interference. Based on experience with kinases and other drug targets, it might be envisioned that the initial drugs will be pan-Hsp90, whereas follow up drugs might explore the impact of isoform selectivity with respect to activity and toxicity. Drugs that interfere with features of Hsp90 other than nucleotide binding and hydrolysis, such as co-chaperone interactions, may provide differing biological effects. It is clear that the next few years will be exciting as the role of Hsp90 inhibitors in cancer treatment is defined. But, in addition, Hsp90 inhibitors are likely to be evaluated in diseases other than cancer. Clearly there is potential in conditions where protein folding defects are involved in the disease pathology, including Huntingdon's, Alzheimer's, and prion-related diseases (142). Recent studies have shown that the downregulation of the Hsp90 co-chaperone AHA1 rescues the misfolding of the common disease variant of the cystic fibrosis (CF) transmembrane conductance regulator (CFTR) (143). The results suggest AHA1 as a potential target in the treatment of cystic fibrosis. Thus targeting Hsp90 and other components of the chaperone may provide a general framework for correction of misfolding disease. The activity of the proteasome inhibitor bortezomib and of Hsp90 inhibitors in cancer provides validation for the modulation of protein quality control and protein folding as a viable therapeutic approach. Success will stimulate interest in modulating other targets in these pathways for cancer and other conditions.

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ACKNOWLEDGMENTS The work of the authors' laboratory was funded by Cancer Research U.K. grant numbers CA309/A2187 and C309/A8274. Paul Workman is a Cancer Research U.K. Life Fellow. We thank our colleagues in the Signal Transduction and Molecular Pharmacology Team and Chaperone Project Team and also our many collaborators for valuable discussion. We also thank Dr. Chrisostomos Prodromou for Figure 3 and Pam Stevens for help with preparation of the manuscript. REFERENCES 1. Vogelstein B, Kinzler KW. Cancer genes and the pathways they control. Nature Med 2005; 11:261–3. 2. Workman P. Genomics and the second golden era of cancer drug development. Mol BioSyst 2005; 1:17–26. 3. Collins I, Workman P. New approaches to molecular cancer therapeutics. Nature Chem Biol 2006; 2:689–700. 4. Workman P. Drugging the cancer kinome: progress and challenges in developing personalized molecular cancer therapeutics. Cold Spring Harbor Symp Quant Biol 2005; 70:1–18. 5. Futreal PA, Coin L, Marshall M, et al. A concensus of human cancer genes. Nat Rev Cancer 2004; 4:177–83. 6. Futreal PA, Wooster R, Stratton MR. Somatic mutations in human cancer: insights from resequencing the protein kinase gene family. Cold Spring Harb Symp Quant Biol 2005; 70:43–9. 7. http://www.sanger.ac.uk/genetics/cgp/census (accessed 11 February 2007). 8. Sjoblom T, Jones S, Wood LD, et al. The consensus coding sequences of human breast and colorectal cancers. Science 2006; 314(5797):268–74. 9. Hanahan D, Weinberg RQA. The hallmarks of cancer. Cell 2000; 100:57–70. 10. Benson JD, Chen YN, Cornell-Kennon SA, et al. Validating cancer drug targets. Nature 2006; 441:451–6. 11. Marks PA, Breslow R. Dimethyl sulfoxide to vorinostate: development of this histone deacteylase inhibitor as an anticancer drug. Nat Biotechnol 2007; 25:84–90. 12. Adams J, Kauffman M. Development of the proteasome inhibitors Velcade (Bortezomib). Cancer Inve 2004; 22:304–11. 13. Powers M, Workman P. Targeting of multiple signalling pathways by heat shock protein 90 molecular chaperone inhibitors. Endo Rel Cancer 2006; Suppl 1:S125–35. 14. Sharp S, Workman P. Inhibitors of Hsp90 molecular chaperone: Current status. Adv Cancer Res 2006; 95:323–48. 15. Whitesell L, Lindquist SL. HSP90 and the chaperoning of cancer. Nat Rev Cancer 2005; 5(10):761–72. 16. McDonald E, Workman P, Jones K. Inhibitors of the Hsp90 molecular chaperons: Attacking the master regulator in cancer. Curr Top Med Chem 2006; 17:1091–109. 17. Chiosis G, Radina A, Moulick K. Emerging Hsp90 inhibitors: from discovery to clinic. Anticancer Agents Med Chem 2006; 6:1–8. 18. Janin YL. Heat shock protein inhibitors. A text book example of medicinal chemistry? J Med Chem 2005; 48:7503–12. 19. Ritossa F. A new puffing pattern induced by temperature shock and DNP in Drosophila. Experientia 1962; 19:571–73. 20. Ritossa F. Discovery of the heat shock response. Cell Stress Chaperones 1996; 1:97–8. 21. Bukau B, Deuerling E, Pfund C, et al. Getting newly synthesised proteins into shape. Cell 2000; 101:119–22. 22. Hartl U, Hayer-Hartl M. Molecular chaperones in the cytosol: from nascent chain to folded proteins. Science 2002; 295:1852–8. 23. Young JC, Agashe VR, Siegers K, et al. Pathways of chaperone-mediated protein folding in the cytosis. Nat Rev Mol Cell Biol 2004; 5:781–91.

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Index

Aggressive fibromatosis, 132–133 Agonistic antibodies, 211–214 AKT, inhibitors of, 72–73, 286 Alkylphospholipid (APL), 72 AMG706, 126–127 Amphiregulin, 19 Angiogenesis, 258 regulation of, 318–319 survivin regulation of, 198, 200 targeting with oral agents, 241–255. See also under Targeting tumor angiogenesis, integrins role in, 260–266 Angiopoietin-1 (Ang-1), 198 Angiostatin, 258, 265, 272–273 Aniliquinazoline, 26 Antiangiogenic agents, 11–13 endothelial cell propagation targeting, 12 hypoxia inducing factor inhibition, 12 VEGF targeting, 11–12 VEGFR targeting, 12 tyrosine kinase inhibitory activity, 12 Antiangiogenic tyrosine kinase inhibitors in clinical trials, 241–249 Axitinib (AG 013736), 248 AZD2171, 243, 248–249 CP-547,632, 248 Sorafenib (BAY 43-9006), 241–244 SU5416 and SU6668, 247 Sunitinib, 243–246 Vatalanib, 243, 247–248 Antibody-dependent cellular cytotoxicity (ADCC), 46 Anticancer therapy, targeting MMP in, 321–322 Anti-invasive agents, 13–14 SRC inhibitors, 13–14 targeting metalloproteinase, 13 Antisense survivin oligonucleotide (ASO), 11 Antisense therapy, 199–200, 214 Antivascular agents, 13–14, 295–313 Anti-VEGF therapies, 226–232 adverse effects of, 234–236 disease specific toxicities, 235–236 hypertension, 234–235

12459 G-quadruplex ligand, 351 17-AAG, 383, 388–390 17-Allylamino, 17-demethoxygeldanamycin (17-AAG), 215 2C3 Antibody, 226 2-Cyano-3, 12-dioxoolean-1,9-dien-28-oic acid (CDDO), 215 2-Methoxyestradiol (2ME2), 287 307A G-quadruplex ligand, 351 5,6 Dimethylxanthenone- 4-acetic acid. See DMXAA 5-Fluorouracil (5FU), 227, 353 5-Hydroxyindoleacetic acid (5-HIAA), 305 7-Hydroxystaurosporine, 106

A4 inhibitors, 386 AbegrinTM (MEDI-522, etaracizumab), 264, 266–272 ABT-510, thrombospondin fragment, 265, 274 ABT-751, 298 Adamlysin metalloproteinases with thrombospondin motifs (ADAMTS), 316 Adenoid cystic carcinomas, 133 Adenomatous polyposis coli (APC) protein, 197 Adenovirus, 150, 184, 274, 289 serotype 5, 179 Advexin animal models, toxicity studies on, 183–184 clinical experience with, 184–189 in combination therapy, 186: locally advanced breast cancer treatment, 187–188; NSCLC treatment, 186–187, 189; and radiation therapy, 189 as monotherapy, 184–185 genome map, 178 p53 targeting, 179–181 in combination therapy, 180–181 effects on normal cells, 182–183 as monotherapy, 179–180 AEE788, 57–58

401

402 [Anti-VEGF therapies adverse effects of] proteinuria, 235 pivotal trials evaluating, 226–232 AP23994, 339, 340 Apoptosis, 214, 360 apoptosis modulators, 10–11, 177–190, 319 Bcl-2, 11 p53 targeting, 10. See also p53 survivin, 11 toxicity preclinical studies, 182–184: advexin effects. See Advexin TRAIL, 11 Aprinocarsen, 109 Aryl hydrocarbon receptor nuclear translocator (ARNT), 283 ATN–161, 264, 270–271 Aurora A, 157–158 biological functions, 157–158 in cancer, 159–161 as therapeutic targets, 161 Aurora B, 158–159 biological functions, 158–159 in cancer, 161 as therapeutic targets, 162 Aurora C, 159 biological functions, 159 Aurora kinase inhibitors, 10, 157–169. See also individual entries biological functions, 157–159 aurora A, 157–158 aurora B, 158–159 aurora C, 159 in cancer, 159–161 clinical data, 162–166 AT9283, 166 AZD1152, 165 MK-0457, 164 MLN8054, 165–166 PHA739358, 166 SU6668, 166 discovery, 157 future clinical development, 166–169 clinical indications, 166 combination with other agents, 168 dose escalation schemes, 167 drug development, 166–167 heavily pretreated patients,168 imaging, use of, 169 novel clinical trial designs, 167–168 novel pharmacodynamic assays, 168–169 as therapeutic targets, 161–162

Index AVE-8062, 298–299, 303 Axitinib (AG 013736), 248 AZD0530, 339, 340, 342 AZD2171, 243, 248–249 Azidothymidine, 353

B cell chronic lymphocytic leukemia (B-CLL), 334 Batimistat, 322 BAY 12-9566/tanomastat, 322 B-cell non-hodgkin’s lymphoma, 108 Bcl-2 protein, 214 Bcl-XLprotein, 214, 336 Benzoquinone ansamycin class of Hsp90 inhibitors. chemical structures of, 379 Bergerat fold, 382 Betacellulin, 19 Bevacizumab, 5, 11, 225 action of, mechanisms, 232–236 tumor cells, effects on, 234 vessel function, effects on, 233–234 vessel numbers, effects on, 232–233 for advanced stage disease, 228 anti-VEGF therapy, adverse effects of, 234 Capecitabine þ Bevacizumab, 231 and chemotherapy for non–small cell lung cancer, 230–231 in combination with chemotherapy for mCRC, 227–230 in metastatic breast carcinoma, 231–232 Paclitaxel
Bevacizumab, 231 BIBR1532, 351 BMS 275291, 322 BMS-214662 in hematologic malignancies, 93, 97 in solid tumors, 91, 94–95 BMS-354825 (Dasatinib, Sprycel
), 339, 340, 342 BMS-599626, 60–61 chemical structure, 61 Bortezomib, 215 BRACO19, 351 Breast cancer gefitinib, 33 HER2 in, 45–46 HER2 measurement, 45–46 pathogenesis, 45 pathophysiology of, 45 trastuzumab, 47–51

Index Bryostatin, 108 BSU1051, 351 Bugula neritina, 108

CA4DP, 298–299 Cancer therapy cancer vaccine/immunotherapy, survivin in, 200–201 HIF-1 inhibitors as target for, 283–285. See also HIF-1 inhibitors Carboplatin, 301 Carcinogenesis, fundamental mechanisms, 3 Catalytic inhibitors, 353–356 chrolactomycin, 354 Imatinib mesylate (Gleevec), 355 nonnucleoside inhibitors, 354 nucleoside analogs, 353 radicicol, 354 Tamoxifen, 355 Trichostatin A, 355 U-73122, 355 UCS1025A, 354 CCT018159, 385 Celecoxib, 72 Cell signaling process, 4 potential pharmaceutical intervention, 4 Cell-cycle inhibitors, 9–10 aurora kinase inhibitors, 10. See also separate entry Cellular FLICE-like inhibitory protein (cFLIP), 207 Cetuximab, 5 colorectal cancer, 28 head and neck cancer, 30–31 CGP76030, 339, 340–341 Chemotherapy, 25 locally advanced breast cancer treatment, 187–188 NSCLC treatment, 186–187 Chordomas, 133 Chrolactomycin, 354 Chronic lymphocytic leucemia (CLL), 11 Chronic myelogenous leukemia (CML), 132, 162 CI-1033, 58–60 chemical structure, 59 Cilengitide, 268–269 Cilengitide EMD 121974, 264 in combination therapy, 269 Cisplatin, 111

403 Classic chemotherapy, 1–2 Client proteins, 389, 391–392 CNTO 95, 264, 270–271 COL-3, 322 Collagen non-peptidomimetics MMPI, 322, 323 BAY 12-9566/tanomastat, 322 BMS 275291, 322 prinomastat/AG3340, 322 Collagen peptidomimetics MMP inhibitors, 322–323 Batimistat, 322 Marimastat, 322 Colorectal cancer (CRC), 23, 28–29 cetuximab trials, 28 Combretastatin A4 diphosphate (CA4DP), 299–302 Cowden’s syndrome, 69 CP-547,632, 248 Cross talk, 3 c-Src protein, 333–334 c-Src shRNA expression, 335 c-SrcF529, 336 Cyclin-dependent kinase inhibitors (CDKIs), 15 Cyclin-dependent kinases (CDKs), 9 Cyclo-oxygenase-2 inhibitors, 14 Cytochrome c, 209 Cytoplasmic signal transduction, 5–9 Cytotoxic T lymphocytes (CTL), 209, 356

Darier–Ferrand dermatofibrosarcoma protuberans, 132 Dasatinib, 14, 339, 342–343 Death receptors DR4, 208–210 DR5, 208–210 Death-inducing signaling complex (DISC), 208–209 Denosumab, 210 Deschloroflavopiridol, 9 Desmoid tumors, 132–133 Dideoxyguanosine (ddG), 353 Dimethyl sulphoxide (DMSO), 389 DMXAA (AS1404), 304 clinical studies, 305–306 DNA binding, inhibitors of, 288 Docetaxel, 59 Doppler ultrasonography, 242 DPNS, 351

404 DR4 (death receptor 4), 208–213 DR5 (death receptor 5), 208–213 Dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI), 299

E7820, 265, 270–271 Echinomycin, 288 ECM metalloproteinase inducer (EMMPRIN), 316, 320 ECOG 3200 trial, 227 EGCG, 351 EGFR (Epidermal growth factor receptor), 19–36, 286 EGFR signaling pathways, 19–20 major components representation, 20 inhibitors, 20–23 clinical trials, 24–34: breast cancer, 33; colorectal cancer, 28–29; esophageal cancer, 32; glioblastoma, 32; head and neck cancer, 29–32; non–small cell lung cancer, 24–28; pancreatic cancer, 32 response predictors, 34–36: biological features, 34–36; clinical features, 34 small molecules, 20–23: EGFR TKIs classes, 20–23; mimotopes, 23; monoclonal antibodies, 23 ligands of, 19 signal transduction inhibitors, 19–36 Endostar, 265 Endostatin gene therapy, 265, 272, 273–274 Endothelial cell propagation targets integrins as, 259–266. See also Integrins Enzastaurin, 110–112 Epidermal growth factor receptor. See EGFR Epigallocatechin gallate (EGCG), 354 Epiregulin, 19 ErbB receptor, 55–56 human solid tumors, expression in, 56 Erlotinib, 5, 20 glioblastoma, 32 head and neck cancer, 31 non–small cell lung cancer, 25 Esophageal cancer, 32 Everolimus, 74 Extracellular regulated kinase (ERK), 5, 19

Index Farnesyl transferase inhibitors, 85–98 hematologic malignancies clinical data, 95–97 preclinical data, 91–93 mechanisms, 89–90 Ras. See separate entry solid tumors clinical data, 93–95 preclinical data, 90–91 Fas-associated death domain (FADD), 208 Flavone-8-acetic acid (FAA), 304 Flavonoids, 297, 304–306 DMXAA, 304–306. See also separate entry Flavopiridol, 9 Fluorescence in situ hybridization (FISH), 35–36 Fluorouracil, 227 FOLFOX [Folinic Acid (leucovorin), 5-FU, Oxaliplatin], 227 Fyn, 335

Gastrointestinal stromal tumors. See GIST Gefitinib, 5, 20 breast cancer, 33 colorectal cancer, 29 esophageal cancer, 32 glioblastoma, 32 head and neck cancer, 31 non–small cell lung cancer, 25 Geldanamycin, 379, 380–383 Gemcitabine, 111 Gene therapy/expression, 289 HIF-1 and, 284 to target survivin, 201 Genistein, 338 GIST (Gastrointestinal stromal tumors), 107, 245, 392 chronic myelomonocytic leukemia, 132 Darier–Ferrand dermatofibrosarcoma protuberans, 132 hypereosinophilic syndromes, 132 molecular alterations, 128 sunitinib and resistance management, 129 multiple lines of therapy in, 131 mutations of KIT, PDGFR, PDGF, 127–134 imatinib, role of, 127–128 therapeutic agents, 130–131 AMG706, 130 masatinib, 131

Index [GIST (Gastrointestinal stromal tumors) therapeutic agents] nilotinib, 130–131 valatinib, 131 Glioblastoma, 32 Gliomas, 133 G-quadruplexes, 357 effects on telemeres, 358–359 inducing telomere dysfunction, 363 G-quartet, 351 G-rich 3’extension (G-overhang), 360 GRN163 oligomer, 353 GRN163L oligomer, 353 Growth factors, MMPs and, 319 GSK3 (Glycogen synthase kinase-3 beta), 66, 143 GTPase-activating protein (GAP), 68, 86

hAGP (Human alpha-1 acid glycoprotein), 72 Head and neck cancer, 29–32 recurrent/metastatic (R/M) disease, 29–30 squamous cell carcinoma, 29 Heat shock protein 90. See Hsp90 Helenalin, 354 Hematologic malignancies BMS-214662, 93, 97 lonafarnib, 93, 97 tipifarnib, 91–93, 95–97 Heparin binding EGF, 19 HER2 inhibition, 45–51 action mechanism, 46–47 breast cancer, 45–46 clinical achievement in, 47–51: lapatinib, 51; trastuzumab, 47–51. See also separate entry measurement, 45–46 pathogenesis, 45 pathophysiology in, 45 resistance mechanism, 47 Herbimycin A, 379 Heterogeneous tumors, 197 HGS-ETR1, 212 HGS-ETR2, 212–213 HIF inhibitors. See also HIF-1 inhibitors 2-Methoxyestradiol (2ME2), 287 of AKT, 286 of DNA binding, 288 early clinical development, 290–291 EGFR inhibitors, 286

405 [HIF inhibitors] gene therapy, 289 HDAC inhibitors, 287–288 of HIF-1 transcriptional activity, 288 Hsp90 inhibitors, 287 mechanisms of action, 285–289 microtubule-targeting agents, 287 of mTOR pathway, 286 natural products, 289 of protein accumulation, 287 of signaling pathways, 285–286 preclinical development and translational end points, 289–290 topoisomerase I poisons, 287 HIF-1 (Hypoxiainducible factor ), 12 HIF-1 inhibitors, 283–294 gene expression and, 284 as a target for cancer therapy, 283–285 HIF-1 expression: in human cancer, 284–285; regulation, 283–284 HIF-1a (Hypoxia-inducible factor-1a), 143 Histone deacetylase (HDAC) inhibitors, 15, 214, 287–288, 387 chemical structures of inhibitors of, 388 HKI-272, 61 HR2822, 356 Hsp90 inhibitors, 202, 215, 287, 354 benzoquinone ansamycin class of, 379 current status of, 381 Hsp90 isoform Grp94 chemical structure, 387 radicicol class of, 380 Hsp90 super-chaperone, 375–394 A4 inhibitors, 386 clinical trials, 388–391 co-chaperones, 377 function of, 376–378 ADP nucleotide exchange in, 376 ATP binding in, 376 ATPase hydrolysis in, 376 geldanamycin analogues, 382–383 Hsp90 clients and other interactions, 378–379 Hsp90 inhibitory natural products, 379–382 novobiocin analogues, 386 origins, 376 purine-scaffold inhibitors, 384–385 pyrazole-scaffold inhibitors, 385–386 radicicol analogues, 384 structure of, 376–378

406 [Hsp90 super-chaperone] therapeutic selectivity, 387–388 hTR telomerase, RNA component of, 352–353 Human alpha-1 acid glycoprotein (hAGP), 72 Human epidermal growth factor receptor (HER), 19–36, 216. See also EGFR HER2 inhibition. See also separate entry pan-HER inhibitors. See separate entry HuMV833 antibody, 226 Hypereosinophilic syndromes, 132 Hypertension, 234–235 Hypoxia inducible factor. See HIF inhibitors

IGF-1R, 141–152 Imatinib mesylate (Gleevec), 355 Imatinib, 125 Immune response and MMPs, 319 Immunization with telomerase, 356 Immunotherapy, survivin-directed, 200–201 Inhibitors, EGFR, 21–22. See also EGFR inhibitor of apoptosis (IAP) gene family, 11, 197 Insulin-like growth factor 1 receptor (IGF1R), 141–152 activated pathways, 141–143 circulating levels, polymorphisms and cancer, 145–148 targeted therapies, resistance to, 147–148 tyrosine kinase receptors, 146–147 expression in tumors, 144–145 inhibition, 150–151 involvement in cancer, evidence for, 143–144 ionizing radiation, 148–149 radio-sensitivity and inhibition, 149–150 targeting compounds, 150 Integrins, 258 a1b1, 260 a2b1, 260 a3b1, 260 a4, 261 a5b1, 260, 272 a5, 261 a6b1, 260 a6b4, 260 b8, 261 a9, 261

Index [Integrins] avb3, 260, 272 avb5, 260, 272 av, 261 Cilengitide, 268–269 clinical trial status, 264–265 AbegrinTM (MEDI-522, etaracizumab), 264 ABT-510, 265 Angiostatin, 265 ATN–161, 264 Cilengitide EMD 121974, 264 CNTO 95, 264 E7820, 265 Endostar, 265 Endostatin gene therapy, 265 Vitaxin
(MEDI-523), 264 Volociximab Eos-200-4 M-200, 264 endostatin, 272 as endothelial cell propagation targets, 259–266 rationale for, 262–263: a2 subunit, 263; a5b1, 263; avb3, 262; avb5, 262 integrin antagonists, future, 271–272 integrin family, 259–260 less advanced integrin antagonists ATN-161, 270–271 CNTO 95, 270–271 E7820, 270–271 Volociximab, 270–271 plasminogen, 272 thrombospondin, 272 thrombospondin fragment (ABT-510), 274 in tumor angiogenesis, 260–266 Irinotecan, 28, 227

KAI-/CS-9803, 109–110 Karnofsky performance status (KPS), 185 Kinase inhibitors targeting (KIT), 123 empirical use, 132–133 adenoid cystic carcinomas, 133 aggressive fibromatosis, 132–133 chordomas, 133 gliomas, 133 prostate cancer, 133 and stem-cell factor, 124 tumors with mutations, 127–130 GIST. See separate entry tyrosine kinase inhibitors, 125–127

Index [Kinase inhibitors targeting (KIT) tyrosine kinase inhibitors] AMG706, 126–127 imatinib, 125 nilotinib, 125–126 sunitinib, 125 valatinib, 127

Lapatinib, 51 dual-HER inhibitors, 56–57 for trastuzumab resistance, 51 Leishmaniasis, 72 Lesions, premalignant, 211 Leucovorin (LCV), 227 Locally advanced breast cancer (LABC), 187 neoadjuvant advexin and chemotherapy, 187–188 Lonafarnib in hematologic malignancies, 93, 97 in solid tumors, 90–91, 94 Lyn, 334

M(4)N, 201 mAb (Monoclonal antibodies), 23 Marimastat, 322 Matrix metalloproteinases (MMPs) in carcinogenesis, implication of, 315–332 angiogenesis regulation, 318–319 fundamental data, 317–320 invasion and metastasis, 317–318 mechanisms of action, 317 clinical aspects, 320–324 collagen non-peptidomimetics MMPI, 322–323. See also separate entry collagen peptidomimetics MMP inhibitors, 322–323. See also separate entry ECM metalloproteinase inducer (EMMPRIN), 316 and growth factors, 319 immune response and, 319 membrane type MMPs (MT-MMPs), 315 MMP family, 315–317 classification and structure, 315–316 MMP-1, 320 MMP-11 expression, 320 MMP-2, 321 MMP-7, 320

407 [Matrix metalloproteinases (MMPs)] physiological functions, 316–317 plasma TIMP-1, 321 and prognosis, 320–321 regulation, 317 targeting MMP in anticancer therapy, 321–322 tetracycline derivates, 322, 323–324 tumor–stroma interactions, 319–320 MBT-1, 298 Mcl-1, 336 mCRC chemotherapy, 227–230 Metastasis, 317–318 metastatic breast carcinoma, Bevacizumab in, 231–232 metastatic colorectal cancer (mCRC), 226 Microtubule-targeting agents, 287 Miltefosine, 72 Mimotopes, 23 Mitogen-activated protein kinase (MAPK), 19, 73, 142 Mitogen-activated protein kinase-kinase (MAPKK), 5 Mitotic centromere-associated kinesin (MCAK), 158 MMQ1, 351 MN-029 (denibulin hydrochloride), 298, 303 mTOR (mammalian targetof rapamycin) inhibitors, 8, 73–76 mTOR pathway, inhibitors of, 286 Myelodysplastic syndrome, 164

Natural products, inhibiting HIF-1, 289 N-cadherin (N-cad) as a target for VDAs, 304 Neoangiogenesis, 11 Neovascularization, 257 Neuropilin-1 (NRP-1), 224–225 Neuropilin-2 (NRP-2), 224–225 Nilotinib, 125 Nonnucleoside inhibitors, 354 Non–small cell lung cancer (NSCLC), 20, 298, 392 advexin and chemotherapy, 186–187 advexin and radiation therapy, 189 chemotherapy for, 230–231 protein kinase C inhibitors. See separate entry Novobiocin analogues, 386 NPI-2358, 299

408 Oblimersen, 214 Oligonucleotidic inhibitors, 352–353 hTR telomerase, RNA component of, 352–353 silencing hTR/hTERT, 353 Oncogenes, 70 addiction, 177 Oral agents angiogenesis targeting with, 241–255. See also under Targeting Osteoprotegerin (OPG), 208, 210 Oxi4503, 299

p53 targeting, 10 gene therapy in animal models, 181–182 in vitro, 179–181 advexin monotherapy, 179–180 advexin with other agents, 180–181 regulatory pathway, 178 Paclitaxel, 301 Pan-HER inhibitors, 55–62 dual-HER inhibitors, 56 AEE788, 57–58 lapatinib, 56–57 ErbB receptors and ligands, 55–56 limits and prospects, 61–62 natural inhibitors, 58 BMS-599626, 60–61: chemical structure, 61 CI-1033, 58–60: chemical structure, 59 HKI-272, 61 PD173955/56, 340 Perifosine, 72 Peripheral blood mononuclear cells (PBMCs), 388–389 Peroxisome proliferator-activated receptor g(PPARg), 215 Peutz-Jeghers syndrome, 69 Phosphatidylinositol 3-kinase (PI3K), 19 inhibitors, 70–71 Phosphoinositide 3-kinase/Akt/ mTOR pathway, 65–77 in normal and cancer cells, 65–70 oncogenes and tumor suppressor genes, 70 targeting agents, 70–76 AKT Inhibitors, 72–73: Perifosine, 72–73 mTOR inhibitors, 73–76 PDK1 Inhibitors, 71–72 PI3K Inhibitors, 70–71

Index Picropodophyllin, 151 PIPER, 351 Placenta growth factor (PlGF), 223 Plasminogen, 272 Platelet-derived growth factor receptor (PDGFR), 123–134 empirical use, 132–133 adenoid cystic carcinomas, 133 aggressive fibromatosis, 132–133 chordomas, 133 gliomas, 133 prostate cancer, 133 and PDGFs, 124–125 tyrosine kinase inhibitors, 125–127 AMG706, 126–127 imatinib, 125 nilotinib, 125–126 sunitinib, 125 valatinib, 127 PP2, 339, 340 Premalignant lesions, TRAIL pathway in, 211 Prinomastat/AG3340, 322 Prognosis, MMPs and, 320–321 Progression-free survival (PFS), 242 Proheparin–binding epidermal growth factor (pro-HB-EGF), 319 Prostate cancer, 133 Protein accumulation, inhibitors of, 287 Protein arginine methyl transferase 5 (PRMT5), 391 Protein kinase C inhibitors non–small cell lung cancer treatment, 103–114 future development considerations, 112–114 nonspecific PKC inhibitors, 104–109: bryostatin, 108–109; PKC412, 107; staurosporin, 104–106; UCN-01, 106–107 selective inhibitors, 109: aprinocarsen, 109; KAI-/CS-9803, 109–110; enzastaurin, 110–112 Protein kinase C isoforms (PKC), 71, 107 Proteinuria, 235 Purine-scaffold Hsp90 inhibitors, 384–386 CCT018159, 385 VER-49009 (CCT0129397), 385 PX-478, 289 PX-866, 71

Index Quadruplex resolvases, 362–364 Quadruplexes, telomeres and, 357 G-quadruplexes, 357 Quercetin, 338

Radicicol analogues, 354, 379, 384 of Hsp90 inhibitors, 380 Rapamycin, 143 Ras, 85–88 biology, 85–87 in human tumors, 87–88 K-Ras mutations, 87 signaling pathways, 86 as therapeutic target, 88 Receptor tyrosine kinases (RTKs), 337 Recombinant human (rh) TRAIL, 207, 211–214 safety, in vivo, 211 Renal cell carcinoma (RCC), 241–244 Response evaluation criteria for solid tumors (RECIST), 232, 301, 389 RHPS4, 351 RhTRAIL. See Recombinant human (rh) TRAIL Rituximab, 108

SCCHN advexin, 184–186 Serine, 473, 67 Shelterin, 361 Shepherdin, 202 Signal transduction inhibitors, 2–9 categories, 5 cytoplasmic signal transduction inhibition, 5–9 C-kit inhibitors, 9 farnesyl transferase inhibitors, 7 MEK inhibitors, 7 PI3K and AKT inhibitors, 7–8 protein-kinase C (PKC) inhibitors, 8 rapamycin and m-TOR inhibitors, 8 rapamycin pathway, 7 Ras and Raf kinase inhibitors, 6–7 STAT inhibitors, 9 transcription pathway activation, 8–9 PDGFR. See Platelet derived growth factor receptor receptor function inhibition, 5 Single-strand G-overhang, 360–361

409 Sirolimus, 73–74 SKI606 (Bosutinib), 339, 340, 341–342 Small interfering RNA (siRNA), 200 Small molecules, to target survivin, 201–202 Solid tumors BMS-214662, 91 lonafarnib, 90–91, 94 tipifarnib, 90, 93–94 Sorafenib (BAY 43-9006), 241–244 Sprycel
, 339, 340, 342 Squamous cell carcinoma, 29 Src family kinases (SFKs) cancer and, link between, 333–334 c-Src protein, 333 c-Src, 334–338 deregulation, downstream signaling consequences, 337–338 as targets, 333–337 experimental validation of, 334–337 v-Src, 334–338 Src inhibitors, 338–344. See also Src family kinases (SFKs) AP23994, 339 AZD0530, 339 BMS 354825 (Dasatinib), 339 CGP76030, 339 compounds in preclinical development, 344 historical overview, 338–341 molecular structure of, 339 molecules in clinical trials, 341–343 AZD0530, 342 BMS-354825 (Dasatinib, Sprycel
), 342–343 SKI-606, 341–342 PD173955/56, 340 PP2, 339 PP2, 340 SKI606 (Bosutinib), 339 Sprycel
, 339 SU6656, 340 STAT (Signal transducer and activator of transcription), 8, 19 Staurosporin, 9, 71, 104–106, 339 STK11 (Serine threonine kinase 11), 66 SU5416 and SU6668, 247 SU6656, 340–341 Sunitinib (SU11248), 125, 244–246 in GIST, 245 resistance management in GIST, 129 Surface-ligand targeted approach, to VDAs development, 296–298

410 Survivin, 11, 197–205 in angiogenesis regulation, 198, 200 biology of, 197–198 in ancillary aspects of tumor, 198 as an apoptosis inhibitor, 198 in mitosis of tumoe cells, 198 future prospects, 202–203 survivin-based therapeutics, 199–202 antisense, 199–200 as anticancer agents, 198 cancer vaccine/immunotherapy, 200–201 conceptual advantages, 198 gene therapy, 201 rationale of, 198–199 shepherdin, 202 small molecules, 201–202 targeting effect on mitosis, 199 tumor-specific expression of, 197–198 unique features, 197

Tamoxifen, 355 Targeted cancer therapy, 197 Targeted molecular treatment, 123 Targeting angiogenesis with oral agents, 241–255 antiangiogenic tyrosine kinase inhibitors in clinical trials, 241–249. See also separate entry future development, 249–250 TDG-TP, 351 Telomerase and telomere interacting agents, 349–365 12459, 351 307A, 351 and quadruplexes, 357 BIBR1532, 351 BRACO19, 351 BSU1051, 351 catalytic inhibitors, 353–356. See also separate entry DPNS, 351 EGCG, 351 G-quartet, 351 immunization with, 356 MMQ1, 351 oligonucleotidic inhibitors, 352–353. See also separate entry PIPER, 351 presentation, 350–352

Index [Telomerase and telomere interacting agents] quadruplex resolvases, 362–364. See also separate entry resistance mechanisms, 359–360 apoptosis and, 360 isolation of resistant clones, 359–360 RHPS4, 351 TDG-TP, 351 telomerase vaccinations, 356 telomere ligands, 357–358 telomere targeting versus telomerase targeting, 358–359 G-quadruplex ligands effects on, 358–359 telomere targeting, consequences of, 360–362 single-strand G-overhang: alteration and degradation, 360–361 telomere-binding proteins: deregulation, 361–362 telomestatin, 351 therapeutic strategies, 14 TMPI, 351 TmPyP4, 351 TNQX, 351 Telomestatin, 351 Tirosel/temsirolimus, 8, 73–76, 143, 242, 243, 286 Tetracycline derivates, 322, 323–324 COL-3, 322 Tetra-O-methyl nordihydroguaiaretic acid (M(4)N), 201 TGF-a (Transforming growth factor alpha), 19 Therapies, existing, 1–15 antiangiogenic agents, 11–13. See also separate entry anti-invasive agents, 13–14 antivascular agents, 13 apoptosis modulators, 10–11. See also Apoptosis cell-cycle inhibitors, 9–10. See also separate entry classic chemotherapy, 1–2 classisfication, 2 definitions, 1 molecular targeted therapy, 1–2 signal transduction inhibitors, 2–9. See also separate entry telomerase and telomere interacting agents, 14 transversal mechanisms, 14

411

Index [Therapies, existing transversal mechanisms] cyclo-oxygenase-2 inhibitors, 14 heat shock proteins, 15 histone deacetylase inhibitors, 15 proteasome inhibition, 14–15 Threonine 308, 67 Thrombospondin, 272 ABT-510, 274 Tipifarnib in hematologic malignancies, 91–93, 95–97 in solid tumors, 90, 93–94 Tissue-inhibitors of metalloproteinases (TIMPs), 317 TMPI, 351 TmPyP4, 351 TNQX, 351 Topoisomerase I poisons, 287 TRAIL (Tumor necrosis factor-related apoptosis inducing ligand) modulators, 11, 207–221 in lymphocyte proliferation, 209 in premalignant lesions, 211 physiological role, 209–210 signaling pathway, 207–209 targeting of, 211–214 therapeutic implications, 211–216. See also Recombinant human (rh) TRAIL potential of combination therapies, 214–216 TRAIL death receptors agents targeting, 212 TRAIL receptors, role of, 210–211 Trastuzumab, 5 for adjuvant breast cancer, 48–50 in metastatic breast cancer, 47–48 in neoadjuvant therapy, 50–51 Trichostatin A, 355 Tuberous sclerosis complex (TSC), 67–68 Tubulin binding agents, 297–303 ABT-751, 298 AVE-8062, 298, 303 CA4DP, 298 clinical studies, 299–303 MN-029, 298 NPI-2358, 298 Oxi-4503, 298 preclinical studies, 299 combretastatin A4 diphosphate, 299–302 second generation tubulin depolymerizing agents, 303–304 MN-029 (denibulin hydrochloride), 303

[Tubulin binding agents second generation tubulin depolymerizing agents] N-cadherin as target for VDAs, 304 TZT-1027, 298 ZD6126, 298, 302–303 Tumor necrosis factor-related apoptosis inducing ligand. See TRAIL Tumor suppressor genes, 70 Tumor-derived protein fragments angiostatin, 272–273 endostatin, 272–274 Tumor–stroma interactions, 319–320 Tyr419, 333–334 Tyrosine kinase inhibitors (TKIs), 20 Tyrphostins, 338 TZT-1027, 298

U-73122, 355 UCN-01, 71, 106–107 UCS1025A, 354

Vaccination, survivin-based, 201 Valatinib, 127 Vandetanib (ZD6474), 243, 247–248 Vascular disrupting agents (VDAs), 295 in clinical development, 298 development approaches to, 296–306: surface-ligand targeted approach, 296 opportunities and challenges in, 306–308: choice of dose and schedule, 307; tumor shrinkage, 306 flavonoids, 297, 304–306. See also separate entry mechanism of action of, 266 tubulin binding agents, 297–303. See also separate entry Vascular endothelial growth factor. See VEGF Vasculature, tumor normal vasculature versus, 295 Vasoconstriction, 233–234 Vatalanib (PTK787/ZK 222584), 243, 246–247 VEGF receptors, 5, 11–12, 26, 198, 223–224, 284 VEGF targeting, 223–240. See also Bevacizumab

412 [VEGF targeting] anti-VEGF therapies. See Anti-VEGF therapies functions of, 225 NRP-1 and NRP-2, 224–225 VEGF ligand family, 223 VEGF ligand, agents targeting, 225–226 2C3, 226 HuMV833, 226 VEGF-Trap, 226 VEGF receptors, 223–224 VEGFR1 (Flt-1), 223–224 VEGFR2, 224, 235 VEGFR3, 224 VER-49009 (CCT0129397), 385 Vessel numbers, 232–233 Vitaxin
(MEDI-523), 264, 266–272 99 Tcm labeling, 267 Volociximab, 270–271

Index [Volociximab] Volociximab Eos-200-4 M-200, 264 Von Hippel-Lindau tumor supressor (VHL), 283

Wortmannin, 71

YC-1, 289 YM155, 201

ZD6126, 298–299, 302–303 ZD6474, 243, 247–248