Checkpoint Responses in Cancer Therapy
Checkpoint Responses in Cancer Therapy Cancer Drug Discovery and Development™ Beverly A. Teicher, Series Editor Checkpoint Responses in Cancer Therapy, edited by Wei Dai, 2008 Cancer Proteomics: From Bench to Bedside, edited by Sayed S. Daoud, 2008 Antiangiogenic Agents in Cancer Therapy, Second Edition, edited by Beverly A. Teicher and Lee M. Ellis, 2007 Apoptosis and Senescence in Cancer Chemotherapy and Radiotherapy, Second Edition, edited by David A. Gerwitz, Shawn Edan Holtz, and Steven Grant, 2007 Molecular Targeting in Oncology, edited by Howard L. Kaufman, Scott Wadler, and Karen Antman, 2007 In Vivo Imaging of Cancer Therapy, edited by Anthony F. Shields and Patricia Price, 2007 Transforming Growth Factor- in Cancer Therapy, Volume II: Cancer Treatment and Therapy, edited by Sonia Jakowlew, 2008 Transforming Growth Factor- in Cancer Therapy, Volume 1: Basic and Clinical Biology, edited by Sonia Jakowlew, 2008 Microtubule Targets in Cancer Therapy, edited by Antonio T. Fojo, 2007 Cytokines in the Genesis and Treatment of Cancer, edited by Michael A. Caligiuri, Michael T. Lotze, and Frances R. Balkwill, 2007 Regional Cancer Therapy, edited by Peter M. Schlag and Ulrike Stein, 2007 Gene Therapy for Cancer, edited by Kelly K. Hunt, Stephan A. Vorburger, and Stephen G. Swisher, 2007 Deoxynucleoside Analogs in Cancer Therapy, edited by Godefridus J. Peters, 2006 Cancer Drug Resistance, edited by Beverly A. Teicher, 2006 Histone Deacetylases: Transcriptional Regulation and Other Cellular Functions, edited by Eric Verdin, 2006 Immunotherapy of Cancer, edited by Mary L. Disis, 2006 Biomarkers in Breast Cancer: Molecular Diagnostics for Predicting and Monitoring Therapeutic Effect, edited by Giampietro Gasparini and Daniel F. Hayes, 2006 Protein Tyrosine Kinases: From Inhibitors to Useful Drugs, edited by Doriana Fabbro and Frank McCormick, 2005 Bone Metastasis: Experimental and Clinical Therapeutics, edited by Gurmit Singh and Shafaat A. Rabbani, 2005 The Oncogenomics Handbook, edited by William J. LaRochelle and Richard A. Shimkets, 2005 Camptothecins in Cancer Therapy, edited by Thomas G. Burke and Val R. Adams, 2005 Checkpoint Responses in Cancer Therapy Edited by Wei Dai, phd Department of Environmental Medicine, New York University School of Medicine, Tuxedo, NY Editor Wei Dai, PhD Department of Environmental Medicine New York University School of Medicine Tuxedo, NY Series Editor Beverly A. Teicher, PhD Genzyme Corporation Framingham, MA ISBN: 978-1-58829-930-7 e-ISBN: 978-1-59745-274-8 Library of Congress Control Number: 2007940763 ©2008 Humana Press, a part of Springer Science+Business Media, LLC All rights reserved. This work may not be translated or copied in whole or in part without the written permission of the publisher (Humana Press, 999 Riverview Drive, Suite 208, Totowa, NJ 07512 USA), except for brief excerpts in connection with reviews or scholarly analysis. Use in connection with any form of information storage and retrieval, electronic adaptation, computer software, or by similar or dissimilar methodology now known or hereafter developed is forbidden. The use in this publication of trade names, trademarks, service marks, and similar terms, even if they are not identified as such, is not to be taken as an expression of opinion as to whether or not they are subject to proprietary rights. While the advice and information in this book are believed to be true and accurate at the date of going to press, neither the authors nor the editors nor the publisher can accept any legal responsibility for any errors or omissions that may be made. The publisher makes no warranty, express or implied, with respect to the material contained herein. Cover illustration: Fig. 2A-C, Chapter 11, “Antiproliferation Inhibitors Targeting Aurora Kinases,” by Kishore Shakalya and Daruka Mahadevan. Printed on acid-free paper 987654321 springer.com Preface The cell cycle is tightly regulated by a number of molecular entities that maintain the genetic integrity of the cell and ensure that genetic information is passed correctly to the daughter cells. Starting in the 1980s, extensive research efforts have revealed the existence of evolutionarily conserved surveillance mechanisms—commonly referred to as checkpoints—that regulate the cell cycle by monitoring specific cellcycle processes and block progression through the cell cycle until these processes have been completed accurately. If any of the molecular events underlying these processes are impaired, the cell cycle stops to allow the cell time for repair. If the cell is damaged beyond repair, it will commit suicide by activating apoptotic processes. The loss of checkpoint function often results in infidelity of DNA replication, chromosome mis-segregation, or both, thereby predisposing the cell to genetic instability and neoplastic transformation. Cancer frequently results from damage to multiple genes controlling cell division. Existing evidence indicates that cancer cells are frequently defective in regulating one cell-cycle checkpoint, which makes them very susceptible to insults to a second checkpoint, resulting in apoptosis. Based on their mechanism of action, current antitumor drugs that target the cell cycle can generally be divided into three categories: inhibition of DNA synthesis, induction of DNA damage, and disruption of mitotic processes. Most cancer drugs used in clinical settings kill cancer cells by altering the DNA structure and inhibiting DNA replication. Such events inevitably lead to activation of the DNA replication or the DNA damage checkpoint. Since the 1980s, much effort has also been directed to the discovery of mitotic targets or processes, which, if altered, can lead to a mitotic catastrophe (a specialized case of apoptosis). It is known that mitotic processes are closely monitored by several surveillance mechanisms, including the G2/M transition checkpoint, the prophase stress checkpoint, and the spindle checkpoint. A defect in the regulation of any mitotic checkpoint often results in genomic instability, which predisposes the cell to malignant transformation. By characterizing the molecular components of cell-cycle checkpoint mechanisms and exploring differences in the checkpoint status between normal cells and malignant cells, we may be v vi Preface able to facilitate the discovery and development of chemotherapeutic compounds that are more effective and more specific for tumor cells. Checkpoint Responses in Cancer Therapy presents summaries of the advances made since the 1980s in identifying various components of cell-cycle checkpoints, elucidating their molecular regulation during checkpoint activation, and validating the use of checkpoint proteins as targets for the development of anticancer drugs. Proteins important for G1 progression are known to mediate cell-cycle checkpoint responses, especially after treatment with chemotherapeutic agents. Considerably less is known about the G1 or G1/S checkpoint than other cell-cycle checkpoints, in terms of their usefulness as targets for anticancer drug discovery. We do know that the Rb protein (pRb), a major tumor suppressor in the cells, plays a pivotal role in the negative control of the cell cycle. The pRb is responsible for regulating the G1 checkpoint, blocking the transition from G1 to S, thereby preventing cell proliferation. Our current knowledge of pRb is summarized in chapter 1. Many chemotherapeutic drugs used in clinical settings target key molecules regulating DNA synthesis or modulating the response to DNA damage. Great progress has been made recently in characterizing the structure and functions of proteins responsible for activating checkpoints that monitor DNA integrity, and many pharmaceutical companies have explored molecular targets that control the S phase checkpoint for therapeutic leads. Detailed discussions of the major molecular players affecting DNA synthesis and the response to DNA damage, from the viewpoint of biology and cancer drug development, are provided in chapters 2 through 6. Many checkpoints monitor the successful execution of the various stages of mitosis to ensure that the cell produces two daughter cells with identical genetic contents. Several protein kinase families are critical for the regulation of these checkpoints. Since the 1990s, intensive drug screening, coupled with studies of kinase structure and function, have led to the identification of a variety of chemical compounds that are capable of inhibiting individual mitotic kinases. Chapters 7 through 12 are devoted to advances in these areas. Posttranslational modifications of the histone tails are closely associated with regulation of the cell cycle as well as chromatin structure. Multiple histone tail acetylations result in the destabilization and decondensation of chromatin fibers. Given that histone acetylation and deacetylation play an important role in the regulation of gene expression and eventually the fate of the cell, much effort has been made to develop histone deacetylase inhibitors to serve as anticancer Preface vii agents. Some of these inhibitors may affect the regulation of cell-cycle checkpoints. Our current knowledge of histone deacetylase inhibitors is summarized in chapter 13. The overall goal of Checkpoint Responses in Cancer Therapy is to enhance our understanding of the many cell-cycle checkpoint molecules that have already been identified as effective targets for anticancer drug development, and to explore the possibility of developing a new generation of anticancer drugs with improved therapeutic indices based on their ability to target a number of checkpoint components that are yet to be fully studied. It is anticipated that this book will serve as a valuable source of information, not only for researchers in the pharmaceutical and biotechnology industries, but also for academic scientists studying cell-cycle regulation, signal transduction, and apoptosis, as well as those involved in cancer research. Wei Dai Tuxedo, NY Contents Preface ....................................................................................... v Contributors ............................................................................... xi 1 RB-Pathway: Cell Cycle Control and Cancer Therapy..... Erik S. Knudsen and Wesley A. Braden 1 2 Targeting the p53/MDM2 Pathway for Cancer Therapy ... 19 Christian Klein and Lyubomir T. Vassilev 3 DNA Topoisomerases as Targets for the Chemotherapeutic Treatment of Cancer .......................... 57 Ryan P. Bender and Neil Osheroff 4 Targeting ATM/ATR in the DNA Damage Checkpoint.... 93 Joseph M. Ackermann and Wafik S. El-Deiry 5 Compounds that Abrogate the G2 Checkpoint................... 117 Takumi Kawabe 6 CDK Inhibitors as Anticancer Agents ................................ 135 Timothy A. Yap, L. Rhoda Molife, and Johann S. de Bono 7 CHFR as a Potential Anticancer Target ............................. 163 Minoru Toyota, Lisa Kashima, and Takashi Tokino 8 Antimicrotubule Agents ...................................................... 177 Miguel A. Villalona-Calero, Larry Schaaf, and Robert Turowski 9 Kinesin Motor Inhibitors as Effective Anticancer Drugs.............................................................. 207 Vasiliki Sarli and Athanassios Giannis 10 Targeting the Spindle Checkpoint in Cancer Chemotherapy................................................................... 227 Jungseog Kang and Hongtao Yu ix x Contents 11 Antiproliferation Inhibitors Targeting Aurora Kinases ...... 243 Kishore Shakalya and Daruka Mahadevan 12 Plks as Novel Targets for Cancer Drug Design ................. 271 Wei Dai, Yali Yang, and Ning Jiang 13 Do Histone Deacetylase Inhibitors Target Cell Cycle Checkpoints that Monitor Heterochromatin Structure?... 291 Brian Gabrielli, Frankie Stevens, and Heather Beamish Index .......................................................................................... 311 Contributors Joseph M. Ackermann, PhD • Laboratory of Molecular Oncology and Cell Cycle Regulation, University of Pennsylvania School of Medicine, Philadelphia, PA Heather Beamish, PhD • Lions Research Fellow, Cancer Biology Program, Centre for Immunology and Cancer Research, University of Queensland, Brisbane, Queensland, Australia Ryan P. Bender, PhD • Department of Biochemistry, Vanderbilt University School of Medicine, Nashville, TN Wesley A. Braden, MD • Department of Cell and Cancer Biology, University of Cincinnati, Cincinnati, OH Wei Dai, PhD • Department of Environmental Medicine, New York University School of Medicine, Tuxedo, NY Johann S. de Bono, MD, FRCP, MSc, PhD • Senior Lecturer and Consultant, Section of Medicine, Institute of Cancer Research, Royal Marsden Hospital, Drug Development Unit, Sutton, Surrey, UK Wafik S. El-Deiry, MD, PhD • Radiation Biology Program, Abramson Comprehensive Cancer Center, Associate Director for Physician-Scientist Training, Hematology-Oncology Division, University of Pennsylvania School of Medicine, Philadelphia, PA Brian Gabrielli, PhD • NHMRC Senior Research Fellow, Head, Cell Cycle Group Diamantina Institute for Cancer Immunology and Metabolic Medicine, University of Queensland, R Wing, Princess Alexandra Hospital, Brisbane, Queensland, Australia Athanassios Giannis, PhD, MD • Institute of Organic Chemistry, Leipzig University, Leipzig, Germany Ning Jiang, PhD • Department of Pathology, New York Medical College, Valhalla, NY Jungseog Kang, PhD • Department of Pharmacology, University of Texas Southwestern Medical Center, Dallas, TX Lisa Kashima, MS • Department of Molecular Biology, Cancer Research Institute, Sapporo Medical University, Sapporo, Japan Takumi Kawabe, MD, PhD • President and CEO, CanBas Co. Ltd., Makiya, Numazu, Japan Christian Klein, PhD • Pharma Research, Roche Diagnostics, Penzberg, Germany xi xii Contributors Erik S. Knudsen, PhD • Department of Cell and Cancer Biology, University of Cincinnati, Cincinnati, OH Daruka Mahadevan, MD, PhD • Department of Medicine, Hematology/Oncology, Arizona Cancer Center, The University of Arizona College of Medicine, Tucson, AZ L. Rhoda Molife, MRCP, MSc, MD • Section of Medicine, Institute of Cancer Research, Royal Marsden Hospital, Drug Development Unit, Sutton, Surrey, UK Neil Osheroff, PhD • Professor of Biochemistry and Medicine, John G. Coniglio Chair in Biochemistry, Department of Biochemistry, Vanderbilt University School of Medicine, Nashville, TN Vasiliki Sarli, PhD • Institute for Organic Chemistry, University of Leipzig, Leipzig, Germany Larry Schaaf, PhD • Clinical Treatment Unit, The Ohio State University, Comprehensive Cancer Center, Columbus, OH Kishore Shakalya, BSc, MS • University of Arizona Cancer Center, Tucson, AZ Frankie Stevens, PhD • Cancer Biology Program, Centre for Immunology and Cancer Research, University of Queensland, Brisbane, Queensland, Australia Takashi Tokino, PhD • Department of Molecular Biology, Cancer Research Institute, Sapporo Medical University, Sapporo, Japan Minoru Toyota, MD, PhD • Department of Molecular Biology, Cancer Research Institute, Sapporo Medical University, Sapporo, Japan Robert Turowski, MBA, RPh • Departments of Internal Medicine and Pharmacology, The Ohio State University, Columbus, OH Lyubomir T. Vassilev, PhD • Discovery Oncology, Roche Research Center, Hoffmann-La Roche Inc., Nutley, NJ Miguel A. Villalona-Calero, MD, FACP • Associate Professor of Internal Medicine and Pharmacology, Division of Hematology and Oncology, The Ohio State University Medical Center, Columbus, OH Yali Yang, PhD • The Ohio State University Medical Center, Department of Environmental Medicine, New York University School of Medicine, Tuxedo, NY Contributors Timothy A. Yap, BSc, MBBS, MRCP • Section of Medicine, Institute of Cancer Research, Royal Marsden Hospital, Drug Development Unit, Sutton, Surrey, UK Hongtao Yu, PhD • Associate Professor, Department of Pharmacology, University of Texas Southwestern Medical Center, Dallas, TX xiii 1 RB-Pathway Cell Cycle Control and Cancer Therapy Erik S. Knudsen and Wesley A. Braden CONTENTS Dysregulation of the RB Pathway in cancer Cell Cycle Control Through the RB-Pathway Influence of the RB-Pathway Genotoxic Therapies Impact of RB Pathway on Antimetabolites Influence of RB on Antimicrotubule Agents Targeted Therapeutics and the Impact of RB Pathway Synopsis Abstract The retinoblastoma tumor suppressor (RB) is a negative regulator of cellular proliferation that impacts multiple facets of cell cycle control. To this end, RB function is commonly abrogated in tumorigenesis, thereby contributing to the uncontrolled proliferation of From: Cancer Drug Discovery and Development Checkpoint Responses in Cancer Therapy Edited by: W. Dai © Humana Press, Totowa, NJ, a part of Springer Science+Business Media 1 2 Knudsen and Braden cancer cells. However, it is becoming increasingly clear that RB is not only involved in the etiology and progression of cancer, but also modifies the response to specific therapeutic modalities. Here we discuss the role of RB in cell cycle control as it relates to the response to specific classes of therapeutic agents. Key Words: Tumor suppressor; retinoblastoma; cyclins; chemotherapy; molecularly targeted therapeutics; cell cycle; E2F; cyclins 1. DYSREGULATION OF THE RB PATHWAY IN CANCER The retinoblastoma tumor suppressor protein, RB, is subject to functional inactivation in a multitude of different tumor types. The Rb gene was identified based on bialleleic inactivation in the pediatric eye tumor, retinoblastoma (1–3). Subsequent analyses demonstrated that loss of heterozygosity of the Rb gene or histochemical deficiency of RB protein occurs in a wide fraction of human cancers and is heterogeneous in nature, depending on the specific tumor type (4–10). In addition to direct effects on Rb gene expression, the RB protein is subject to post-translational modifications that have a significant impact on its function. For example, viral oncoproteins (E1A, E7, T-Ag) can bind to and directly inactivate RB. Such proteins function by a combination of sequestration and targeting RB for degradation as key facets of their transforming activity (11–13). Additionally, RB activity is compromised as a consequence of CDK-mediated phosphorylation (14–16). These phosphorylation events modify RB conformation and disrupt virtually all of the biochemical activities associated with the tumor suppressor (17). Therefore, it is perhaps not surprising that deregulated phosphorylation of RB is a key factor in human cancer (15,18). Specifically, deregulation of CDK-activity occurs through the overexpression of cyclin D1 or loss of the CDK4/6-inhibitor, p16ink4a, and is believed to trigger the aberrant phosphorylation and hence inactivation of RB (14–16,19–21). Importantly, loss of RB function is found in many tumor types but typically in a nonredundant manner, such that deregulation of RB phosphorylation (e.g., p16ink4a loss), or RB loss, are mutually exclusive events (19–22). Thus, these studies have hastened the need to understand how RB functions, and correspondingly if defining RB status in a particular tumor can be used as a determinant for efficacious therapeutic intervention. Chapter 1 / RB-Pathway 3 2. CELL CYCLE CONTROL THROUGH THE RB-PATHWAY RB is a critical regulator of the G1/S transition of the cell cycle, which is responsive to CDK/cyclin activity. Conventionally, RB inhibits S-phase entry by binding to the E2F family of transcription factors and preventing transcription of genes required for DNA replication and productive passage through mitosis (23–26). When hypophosphorylated, RB is bound to the transactivation domain of multiple E2F proteins, eliciting transcriptional repression of E2F-regulated genes (27–29). To achieve promoter repression, RB recruits corepressors such as histone deacetylases (HDACs), histone methyltransferases, members of the SWI/SNF complex, and polycomb group proteins to E2F-regulated promoters (30–37). Upon mitogenic signaling, RB becomes hyperphosphorylated as a consequence of CDK-mediated phosphorylation, leading to release of E2F and transcription of downstream target genes (18). Through this action, it is generally believed that phosphorylation of RB by CDK/Cyclins is essential for entry into S-phase. Coordination between CDK/Cyclin kinase activity and E2F-transcription define the RB-pathway as a module that is functionally inactivated in most cancer (14–17,19–22,38). Antimitogenic signals, which can be broadly defined as those that impede cellular proliferation, often are dependent on RB to inhibit cell cycle progression (15,38). Such signals, are often targeted in existing cancer therapy or are under investigation in the context of new therapies (39,40). For example, DNA damage elicits cell cycle checkpoints that are dominant to the function of mitogenic signals (41), and DNA damaging agents represent a mainstay of current cancer therapy (42,43). Under conditions of such anti-mitogenic signals, RB phosphorylation is prevented, leading to subsequent reactivation of the protein (15,18,38). Strikingly, the mechanisms through which RB can become activated are quite varied, involving effects on both CDK/cyclin activity and potential dephosphorylation of RB by phosphatase activities (18,44,45). Such signaling pathways have led to a concerted analysis of how RB activation functions in discrete phases of the cell cycle. Additionally, RB status as a determinant of the response to each signaling pathway and impact on therapeutic signaling is currently being resolved. 4 Knudsen and Braden 2.1. The RB Pathway in S-Phase Control DNA replication is a very tightly regulated process. As cells exit mitosis and enter G1, origin recognition complexes (ORCs) are bound to sites of potential replication firing and recruit 2 proteins, cdc6 and cdt1, independently (46–50). Mini-chromosome maintenance proteins (MCM2-7), which are thought to function as the replicative helicases, are recruited by cdc6 and cdt1 to form the prereplicative complex (preRC) (48,51–53). As the cell progresses into S-phase, a multitude of other proteins necessary for DNA replication are subsequently recruited to the preRC, allowing for maturation of the replisome complex that facilitates duplication of the genome (48,49,54). The RB pathway is a critical molecular node that impinges upon the DNA replication machinery. More clearly, many of the genes necessary for DNA replication have been shown to be regulated by the E2F transcription factor (55,56). As such, loss or activation of RB activity significantly impacts RNA levels, protein levels and activity associated with discrete DNA replication factors. Several studies have dissected the impact of RB on replication control, and these studies have uniformly demonstrated that RB has the capacity to actively inhibit DNA replication (57–59). Functional analyses of the mechanism underlying this action have illustrated that RB has the capacity to disrupt PCNA chromatin association through a pathway associated with the down-regulation of CDK2 activity. Consistent with this finding, several laboratories have subsequently demonstrated that inhibition of CDK2 activity via distinct mechanisms, mediates similar influences on PCNA function (57,59,60). More recently, it was determined that p16ink4a, which signals upstream of RB to inhibit CDK4 activity, can inhibit MCM chromatin association in G1 (58). Perhaps even more surprising was that p16ink4a expression was able to inhibit total protein levels of cdc6 and cdt1, whereas RB was not (58). Although RB has no effect on replication control in G1, p16ink4a has no effect on replication in S-phase. This is most likely because of low CDK4 activity in S-phase, thus compromising p16ink4a signaling. These data suggest a fundamental difference by which RB and p16ink4a influence DNA replication and underscore that although these proteins exist within the same pathway, they have differential effects on cell cycle and replication control (61). In addition to these specific models of RB-mediated arrest, RB controls the expression of a wide spectrum of replication factors (e.g., MCM2, MCM5, cdc6, and PCNA) and thus, deficiency of RB has the capacity to dramatically influence the coordination of S-phase (57,62,63). Chapter 1 / RB-Pathway 5 2.2. Involvement of RB Pathway in G2/M Control Akin to the processes involved in DNA replication, progression through mitosis is achieved via a number of discrete steps associated with mitotic entry and subsequent exit from mitosis into G1. Mitotic entry is dependent on the activation of CDK1/cyclin B complexes that initiate nuclear envelope breakdown and chromosome condensation (64,65). However, mitotic progression from metaphase is dependent on passage through the spindle checkpoint. This checkpoint stalls progression to anaphase, and ensures that all chromosomes are associated with the mitotic spindle (66–68). Once all chromosomes are bound by the spindle, specific signals coalesce to promote the activation of the anaphase promoting complex (APC) to mediate the destruction of multiple substrates and allow progression through anaphase, and ultimately exit from mitosis (69). The extent to which the RB-pathway contributes to G2/M progression is less clearly defined than is the role of RB in other phases of the cell cycle. Activation of RB by multiple means has not demonstrated a pronounced effect on the progression through mitosis, as cells fail to accumulate in G2 or any mitotic stages (70). However, as is the case with DNA replication, multiple critical factors that are required both for mitotic entry (e.g., Cdk1 and Cyclin B1) and exit (e.g., Cdc20, Plk1, and Mad2) are regulated by RB (56,71–73). Thus, one would predict that the RB pathway impinges on the regulation of mitosis. Consistent with this supposition, loss of RB has recently been associated with delayed mitotic progression occurring as a consequence of MAD2 upregulation (71). 3. INFLUENCE OF THE RB-PATHWAY GENOTOXIC THERAPIES The majority of cytotoxic regimens utilized in the treatment of cancer cause DNA damage (74). For example, cisplatin, cyclophosphamide and ionizing radiation directly damage DNA, whereas agents such as the topoisomerase poisons, irinotecan, and doxorubicin induce DNA damage as a result of indirect effects (75–79). Following such treatment, S-phase is slowed or halted to allow for adequate repair of the lesions before progressing into mitosis (80–83). Combined, these controls limit the segregation of damaged DNA to daughter cells. It has been shown by multiple groups that RB is required for the solicitation of cell cycle arrest in the presence of DNA damage (17,45, 84,85). Initially, it was observed that RB-deficient embryonic fibroblasts failed to undergo the cessation of DNA replication following 6 Knudsen and Braden DNA damage (85,86). However, these cells still mount a G2/M checkpoint response, and as such fail to progress through mitosis and harbor a propensity to over-replicate their genomes (45,87). Using RNAi technology, RB was subsequently shown to play similar roles in response to cytotoxic therapy in tumor cells, whereas other components of the RB pathway (e.g., p16ink4a loss) did not influence the checkpoint response (84). The consequence of RB loss in both fibroblasts and tumor cell models is associated with increased sensitivity to DNA damaging agents (84,85,88). Thus, although RB loss enables checkpoint bypass, invariably this has been associated with enhanced cell killing. The mechanisms through which RB influences survival are not intrinsically clear, but could be dependent on additional secondary damage as a consequence of ongoing DNA replication in the presence of damage (89). Alternatively, it has been reported that RB loss deregulates the expression of many proapoptotic factors, thus shifting the balance toward cell death following DNA damage (90–92). Irrespective of the mechanism, these findings suggest that RB-deficient tumors may be particularly amenable to treatment with cytotoxic agents. However, an ongoing concern would be that tumors lacking checkpoint function would be genomically unstable, and thus although initial therapy may be quite effective, mutations afforded at the time of treatment could contribute to rendering the tumor more aggressive and ultimately therapy resistant. 4. IMPACT OF RB PATHWAY ON ANTIMETABOLITES Some of the most commonly utilized agents in the treatment of cancer are anti-metabolites that largely function by perturbing dNTP pools (93,94). For example, the thymidylate synthase inhibitors (5-fluorouracil, tomudex) are first line agents in the treatment of many cancers and have a pronounced deleterious effect on DNA replication (93). Interestingly, RB controls many facets of DNA replication, including the levels of many metabolic enzymes, such as thymidylate synthase, dihydrofolate reductase and ribonucleotide reductase subunits (60,95–99). As such, RB-deficient cells express highly elevated levels of these enzymes and RB has the capacity to dramatically alter dNTP pools (97). In cell culture models, RB loss enables ongoing DNA replication in the presence of such agents, suggesting that RB-deficiency negatively affects therapeutic response (97,98). Chapter 1 / RB-Pathway 7 5. INFLUENCE OF RB ON ANTIMICROTUBULE AGENTS A number of chemotherapeutic agents function by perturbing microtubule dynamics and correspondingly the function of the mitotic spindle. Specifically, taxanes and vinca-alkaloid agent are utilized clinically and prevent appropriate progression through mitosis (100–104). A consequence of this protracted mitotic-deficit is cell death, making these compounds very effective cytotoxic agents. As discussed above, there is evidence that RB-status impacts the regulation of mitotic progression. Taxanes promote microtubule stability and have been shown to influence RB phosphorylation (105–107). However, vinca-alkaloids, which destabilize microtubules, are one class of compounds for which the absence of RB has little effect on cell cycle response (86). In contrast, RB deficiency facilitates ongoing DNA replication and changes in cell ploidy following treatment with nocodazole that also destabilizes microtubule arrays (108,109). Strikingly, there is little indication of how RB loss ultimately modifies sensitivity to these clinically relevant agents. 6. TARGETED THERAPEUTICS AND THE IMPACT OF RB PATHWAY Recently, there has been a dramatic initiative to define anti-cancer agents that function upon specific molecular targets. Such therapeutics currently encompass a wide-range of targets (e.g., steroid hormone receptors, growth factor signaling pathways, cell cycle machinery, and chromatin). Many of these agents function by antagonizing mitogenic signaling cascades and thus inhibiting proliferation of tumor cells. As such, it may be suspected that loss of RB would have a significant impact on the response elicited downstream of such agents. Here we will briefly discuss several targeted therapeutics for which the role of RB has been investigated: 6.1. Staurosporine and 7-Hydoxystaurosporine Staurosporine and related analogs where classically believed to function as general PKC inhibitors. However, such agents impact a diverse range of targets including cyclin dependent kinases and Chk1 (110). Studies have demonstrated that cells containing functional RB respond to staurosporine treatment, resulting in diminished CDK4 activity and G1 arrest (110). Additionally, RB is required for 8 Knudsen and Braden staurosporine to elicit this G1 arrest (111). Similar data exists for 7-hydroxystaurosporine (UCN-01) implying RB-status is a key determinant of cell cycle response (112). In the case of UCN-01, RBdeficiency and corresponding cell cycle progression is associated with enhanced cell death (113). 7-Hydroxystaurosporine (UCN-01), which was originally identified as a protein kinase C selective inhibitor, is currently in clinical trials as an anticancer drug (114). It has been previously shown that UCN-01 induced preferential G1-phase accumulation in tumor cells and this effect was associated with the RB and its regulatory factors, such as CDK2 and CDK inhibitors p21Cip1 and p27Kip1 (115). However, recent evidence suggests UCN-01 arrests cells in G1 regardless of RB-status (113). Perhaps more striking was that UCN-01 induced apoptosis in RB-deficient cell lines, but not in RB-proficient cell lines (113). These observations suggest that G1-checkpoint function might be important for cell survival during UCN-01 treatment (116). Combined, the data implicate a functional RB pathway as a significant determinant of the sensitivity of tumor cells to UCN-01. 6.2. Geldanamycins Geldanamycin (GM) is an ansamycin antibiotic that functions to inhibit heat shock protein 90 (Hsp90) chaperone function and results in the subsequent degradation of important signaling molecules (e.g., cyclin D1). Although GM has been shown to diminish CDK2 and CDK4 activity resulting in G1 arrest in RB proficient backgrounds, GM had no effect on cell cycle kinetics in an RB negative background (117). Furthermore, release of RB negative cells from G2/M in the presence of GM continued through G1 and the subsequent cell cycle unperturbed (117). Thus, RB is necessary for effective GM treatment of human tumors. 6.3. CDK Inhibitors Flavopiridol, roscovitine, PD0332991 and other compounds in development inhibit CDK activity, preventing phosphorylation of various CDK target proteins and ultimately leading to cell cycle arrest (118). Although flavopiridol functions as a generic CDK inhibitor molecule, roscovitine can inhibit CDK1 and CDK2 function, and PD0332991 specifically inhibits CDK4 kinase activity. In xenograft models, these agents halt tumor growth and can reduce tumor volume (114,118,119). Although these results are promising, RB status has Chapter 1 / RB-Pathway 9 been shown to play a critical role in response to these agents. Particularly, the growth suppressive functions of PD0332991 are entirely dependent on RB, such that RB-deficient tumor models fail to respond to treatment. Therefore, understanding the functional status of RB will be an absolute requirement for the effective usage of such agents (58). 7. SYNOPSIS Combined, these studies indicate that RB status is a determinant of a variety of therapeutic approaches utilized in the treatment of cancer. In certain instances, RB-deficiency is associated with increased sensitivity to specific agents. This is most clearly established in the case of specific genotoxic agents. However, in other instances, RB-deficiency has an apparent deleterious impact on response, as exemplified with therapies directed at CDK activity. These studies provide an important basis for further investigation of RB action on therapeutic response and for devising methodologies to most efficaciously treat cancer rationally. However, to build upon these basic findings will require a number of further advances that will facilitate the utilization of RB-status as a determinant of therapeutic response. ACKNOWLEDGMENTS The authors regret any omissions in the preparation of this article and thank our colleagues for though-provoking discussion. 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INTRODUCTION Nearly 40,000 research and review articles published since 1980 have elevated the tumor suppressor p53 to the rank of the most studied protein in cancer research (1–5). However, the translation of this From: Cancer Drug Discovery and Development Checkpoint Responses in Cancer Therapy Edited by: W. Dai © Humana Press, Totowa, NJ, a part of Springer Science+Business Media 19
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