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