DNA Repair in Cancer Therapy: Molecular Targets and Clinical Applications

Cancer therapeutics include an ever-increasing array of tools at the disposal of clinicians in their treatment of this disease. However, cancer is a tough opponent in this battle, and current treatments, which typically include radiotherapy, chemotherapy and surgery, are not often enough to rid the patient of his or her cancer. Cancer cells can become resistant to the treatments directed at them, and overcoming this drug resistance is an important research focus. Additionally, increasing discussion and research is centering on targeted and individualized therapy. While a number of approaches have undergone intensive and close scrutiny as potential approaches to treat and kill cancer (signaling pathways, multidrug resistance, cell cycle checkpoints, anti-angiogenesis, etc.), other approaches have focused on blocking the ability of a cancer cell to recognize and repair the damaged DNA that primarily results from the front-line cancer treatments; chemotherapy and radiation.

This comprehensive and timely reference focuses on the translational and clinical use of DNA repair as a target area for the development of diagnostic biomarkers and the enhancement of cancer treatment.

• Saves academic, medical, and pharmaceutical researchers time in quickly accessing the very latest details on DNA repair and cancer therapy, as opposed to searching through thousands of journal articles
• Provides a common language for cancer researchers, oncologists, and radiation oncologists to discuss their understanding of new molecular pathways, clinical targets, and anti-cancer drug development
• Provides content for researchers and research clinicians to understand the importance of the breakthroughs that are contributing to advances in disease-specific research

• Language: English
• Publisher: Academic Press
• Release date: Oct 20, 2011
• ISBN: 9780123850003

Book preview

Table of Contents

Cover image

Front Matter

Copyright

Contributors

Foreword

Preface

Acknowledgments

Chapter 1. Introduction and Overview of DNA Repair Targets

Chapter 2. MGMT

Chapter 3. Blockade of Base Excision Repair

Chapter 4. The Role of PARP in DNA Repair and its Therapeutic Exploitation

Chapter 5. Chemotherapeutic Intervention by Inhibiting DNA Polymerases

Chapter 6. Targeting the Nucleotide Excision Repair Pathway for Therapeutic Applications

Chapter 7. Targeting Homologous Recombination Repair in Cancer

Chapter 8. DNA Double-Strand Break Repair by Non-homologous End Joining and Its Clinical Relevance

Chapter 9. Defective DNA Mismatch Repair-dependent c-Abl-p73-GADD45α Expression Confers Cancer Chemoresistance

Chapter 10. Checkpoint Kinase and Wee1 Inhibitors as Anticancer Therapeutics

Chapter 11. Apurinic/Apyrimindinic Endonuclease in Redox Regulation and Oxidative Stress

Chapter 12. Personalized Cancer Medicine

Chapter 13. The Role of DNA Damage and Repair in Neurotoxicity Caused by Cancer Therapies

Chapter 14. Future Directions with DNA Repair Inhibitors

Color Plates

Index

Front Matter

DNA Repair in Cancer Therapy

Molecular Targets and Clinical Applications

Edited by

M ark R. K elley

Betty and Earl Herr Professor in Pediatric Oncology; Research Professor, Departments of Pediatrics, Biochemistry & Molecular Biology and Pharmacology & Toxicology; Director, Program in Pediatric Molecular Oncology; Associate Director, Herman B Wells Center for Pediatric Research; Associate Director of Basic Science Research, Indiana University Simon Cancer Center; Indiana University School of Medicine, Indianapolis, IN

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Contributors

Susan Ashwell, PhD

AstraZeneca R&D Boston, Waltham, MA

Anthony J. Berdis, PhD

Department of Pharmacology, Ireland Comprehensive Cancer Center, Case Western Reserve University, Cleveland, OH

David A. Boothman, PhD

Laboratory of Molecular Stress Responses, Program in Cell Stress and Cancer Nanomedicine, Simmons Comprehensive Cancer Center, University of Texas Southwestern Medical Center at Dallas, Dallas, TX

Djane Braz Duarte, PhD

Department of Pharmacology and Toxicology, Indiana University School of Medicine, Indianapolis, IN

Nicola J. Curtin, PhD

Northern Institute for Cancer Research, Newcastle University, Newcastle upon Tyne, UK

Yvette Drew, MD

Northern Institute for Cancer Research, Newcastle University, Newcastle upon Tyne, UK

Melissa L. Fishel, PhD

Herman B Wells Center for Pediatric Research, Indiana University School of Medicine, Indianapolis, IN

Millie M. Georgiadis, PhD

Department of Biochemistry and Molecular Biology, Department of Chemistry and Chemical Biology, Indiana University–Purdue University Indianapolis, Indiana University School of Medicine, Indianapolis, IN

Stanton L. Gerson, MD

Department of Medicine, Case Western Reserve University, Cleveland, OH

Mark R. Kelley, PhD

Departments of Pediatrics, Biochemistry & Molecular Biology and Pharmacology & Toxicology; Program in Pediatric Molecular Oncology; Herman B Wells Center for Pediatric Research; Indiana University Simon Cancer Center; Indiana University School of Medicine, Indianapolis, IN

Susan P. Lees-Miller, PhD

Department of Biochemistry and Molecular Biology, and Southern Albert Cancer Research Institute, University of Calgary, Calgary, Alberta, Canada

Long Shan Li, MD, PhD

Laboratory of Molecular Stress Responses, Program in Cell Stress and Cancer Nanomedicine, Simmons Comprehensive Cancer Center, University of Texas Southwestern Medical Center at Dallas, Dallas, TX

Yuan Lin, PhD

Department of Medicine, Case Western Reserve University, Cleveland, OH

Lili Liu, MD, PhD

Department of Medicine, Case Western Reserve University, Cleveland, OH

Srinivasan Madhusudan, MBBS, FRCP, PhD

Academic Unit of Oncology, School of Molecular Medical Sciences, University of Nottingham, UK

Mark Meyers, PhD

Laboratory of Molecular Stress Responses, Program in Cell Stress and Cancer Nanomedicine, Simmons Comprehensive Cancer Center, University of Texas Southwestern Medical Center at Dallas, Dallas, TX

Asima Mukhopadhyay, MD

Northern Institute for Cancer Research, Newcastle University, Newcastle upon Tyne, UK

Steve M. Patrick, PhD

Department of Biochemistry and Cancer Biology, University of Toledo, Toledo, OH

Christina Perry, MSc, MRCP

Academic Unit of Oncology, School of Molecular Medical Sciences, University of Nottingham, UK

Heike N. Pfäffle, PharmD

Department of Radiation Oncology, Massachusetts General Hospital, Boston, MA

Ruth Plummer, MD, PhD

Northern Institute for Cancer Research, Newcastle University, Newcastle upon Tyne, UK

Rebeka Sultana, MPharm

Academic Unit of Oncology, School of Molecular Medical Sciences, University of Nottingham, UK

John J. Turchi, PhD

Department of Medicine, Indiana University School of Medicine, Indianapolis, IN

Carlo Vascotto, PhD

Department of Medical and Biological Sciences, University of Udine, Udine, Italy

Michael R. Vasko, PhD

Department of Pharmacology and Toxicology and Department of Anesthesiology, Indiana University School of Medicine, Indianapolis, IN

Mark Wagner, PhD

Laboratory of Molecular Stress Responses, Program in Cell Stress and Cancer Nanomedicine, Simmons Comprehensive Cancer Center, University of Texas Southwestern Medical Center at Dallas, Dallas, TX

Michael Weinfeld, PhD

Department of Oncology, University of Alberta, and Cross Cancer Institute, Edmonton, Alberta, Canada

Henning Willers, MD

Department of Radiation Oncology, Massachusetts General Hospital, Boston, MA

Lee Zou, PhD

Center for Cancer Research, Massachusetts General Hospital, Boston, MA

Foreword

The vast literature on biological responses to DNA damage includes an increasing proliferation of textbooks on this topic. These weighty tomes (one is over 1100 pages) comprehensively address multiple aspects of biological responsiveness to genomic insult. The relevance of DNA repair in mitigating the potential for mutations that may predispose to cancer has been extensively explored. However, relationships between perturbations of DNA repair and cancer have received much less attention. Such relationships may transpire in two diametrically opposite ways. Improving the efficacy of repair by one means or another may lower the mutational burden of non-neoplastic cells that have sustained repairable DNA damage, thereby offering the potential of cancer prevention. On the other hand, interfering with the efficacy of DNA repair mechanisms may improve the outcome of cancer therapy by interfering with the removal of deliberately inflicted DNA damage intended to cripple rapidly dividing cells. This volume, orchestrated by Mark Kelley from Indiana University, offers 14 chapters by acknowledged experts that address this particular relationship between DNA repair and cancer.

The book opens with a well-considered introduction and overview of DNA repair targets for cancer therapy. Subsequent chapters consider specific DNA repair pathways that may be profitably targeted to enhance the eradication of neoplastic cells. These chapters hone in on a DNA repair enzyme called O ⁶-alkylguanine transferase, which selectively removes alkyl groups from the O ⁶ position of guanine in DNA, base excision repair (BER) that removes non-DNA distorting lesions from the genome, poly(ADP-ribose) polymerase, an enzyme that over the years has been implicated in a variety of cellular functions in addition to DNA repair, mismatch repair, and an apurinic/apyrimidinic (AP) endonuclease that selectively attacks sites of base loss (AP sites) in DNA. The last mentioned enzyme has also been implicated in regulating a switch between oxidation and reduction of key transcription factors, a function that has been questioned by some.

The repair mechanisms just mentioned are all relatively specific for the repair of particular types or classes of DNA damage. Nucleotide excision repair (NER) is a much more general DNA repair mechanism that is able to rid the genome of a multitude of different types of base damage caused by physical or chemical agents. For this reason alone targeting NER has long been a dream of cancer therapeutists. Other more general responses to DNA damage consider the notion of interfering with DNA replication by inhibiting various DNA polymerases, and the utility of inhibitors of proteins required for normal checkpoint control during the cell cycle. Two chapters are also devoted to the notion of targeting homologous recombination in order to promote cell killing.

The content of the book is considerably broadened and enhanced by addressing topics such as the possible use of alterations in DNA as predictive biomarkers (measures that help determine which patients do well with particular types of treatment) and the role of DNA damage and its repair in neurotoxicity associated with cancer therapy. Kelley appropriately concludes the volume with a thoughtful exploration of future directions in the use of inhibitors of the DNA damage response.

Writers of forewords to books can be adept at conveying the impression that they have read the book under consideration from cover to cover. I make no such pretense. But I have read enough to draw the unequivocal conclusion that this volume is a most welcome addition to the literature on biological responsiveness to DNA damage, since most (if not all) books on this ever enlarging field fail to address the known and potential effects (both benefits and risks) of perturbing these responses when devising cancer treatments.

Errol C. Friedberg

University of Texas Southwestern Medical Center at Dallas

Preface

The field of DNA repair is enjoying a remarkable time of interest, with the recent use of specific DNA repair inhibitors in cancer clinical trials as well as the development of additional molecules that either are being tested or are rapidly moving through the preclinical developmental stage. This recent focus has not decreased the basic science interest in pursuing research in the still-unknown mechanisms involving the various DNA repair pathways, though. Particularly interesting is the continuing discovery of interactions between the various pathways, which will afford opportunities for future translational and clinical efforts. As with any book, we have tried to include the most current information possible in the contents; however, as the field is rapidly accelerating, we acknowledge that some information will not be current by publication time, particularly the information concerning specific inhibitor molecules and clinical or preclinical successes and failures. In spite of this, we anticipate that this book will be a strong reference for those who want to delve into DNA repair, understand pathways and their basic mechanisms, and understand its relevance to human cancer. This background information will not be eclipsed by future discoveries, but will serve as the foundation for future studies. We also see this book as a complement to the outstanding book by Dr Errol Friedberg and colleagues, DNA Repair and Mutagenesis, but our book extends the contents of that work into the translational and clinical realm with a clear focus on cancer. In the end, the real purpose of this book is to give an overview of where those who study DNA repair stand, today, in our understanding and development of agents to fight against cancer. This area has been highly underappreciated and is finally gaining the recognition it so richly deserves.

Acknowledgments

I would like to thank all of the members of the scientific community and particularly the scientists who have worked in the area of DNA repair for decades to bring us to this point in time when a large amount of the fruits of their labor is now being implemented in human cancer diagnoses and treatments. Also, thanks to all who have contributed chapters in this book, as such undertakings are time-consuming on already stressed schedules. Additional gratitude goes out to my laboratory staff, and special thanks go to Lana Christian who helped me significantly on the two chapters I contributed to this volume. This project was also spearheaded and supported by the excellent people at Elsevier, particularly Mara Conner and Megan Wickline, as well as their devoted staff, to bring this book to fruition in a timely manner. Support for me on this effort came from the Herman B Wells Center for Pediatric Research, Indiana University Simon Cancer Center, and the Riley Children’s Foundation through the Betty and Earl Herr Chair in Pediatric Oncology Research. This support was instrumental in the completion of this project. Finally, I would like to personally thank my family and particularly my wife, Sue, who has always encouraged me in my work and career even as far back as our college days at DePauw University.

Mark R. Kelley

Chapter 1. Introduction and Overview of DNA Repair Targets

From Bench to Clinic

Mark R. Kelley

Indiana University School of Medicine, Indianapolis, IN

Scientists have long known that DNA can be damaged by many endogenous and exogenous agents, and that general knowledge was used in developing the earliest anticancer agents. However, the knowledge of how DNA repairs itself was largely an academic pursuit until the last two decades. The ongoing molecular characterization of DNA damage response and repair pathways, as well as pathway crosstalk, is helping scientists see how DNA repair activities influence chemo- and radioresistance. This information is invaluable in developing DNA repair inhibitors, the latest effort in creating more targeted anticancer treatments that cause less toxicity to normal cells. DNA repair inhibitors are essential in the application of synthetic lethal combinations of drugs and genetic deficiencies. This chapter traces the history of DNA repair, explains its transition from academia to translational medicine, and notes important firsts in the field of DNA repair inhibition.

History of DNA Repair

Ever since Swiss physician Friedrich Miescher isolated something he called nuclein from cell nuclei in 1868, scientists have strived to unravel the secrets of DNA. Knowledge of how genetic material is stored, copied, maintained, and used gave rise to the field of molecular biology – which opened new vistas for modulating biological processes. ¹

In the 1930s, genes were presumed to be (1) made of proteins and (2) intrinsically stable, with mutations being rare events. ² Key discoveries such as Oswald Avery’s 1944 work with bacteria revised that thinking. Avery showed that disease could be transferred to a harmless strain of bacteria, then passed on to the next generation of that strain, giving the world its first glimpse of the fact that DNA is subject to alterations. Other scientists’ work gradually revealed the structure of DNA, which was a key step in starting to learn how its structure could be changed. Notably, in 1949, biochemist Erwin Chargoff discovered that DNA contains equal amounts of adenine and thymine (30% each in humans) and also equal amounts of guanine and cytosine (20% each in humans). This paved the way for Watson and Crick’s 1953 visualization of the double helix structure of DNA. ¹

By then, 20 years of studies regarding radiation’s mutagenic effects on DNA had transpired²– but the elaborate genome maintenance systems that control DNA damage were yet to be elucidated. The concept of DNA repair did not become a fixture in the lexicon of molecular and cellular biology until the 1960s. ² That same decade, the base excision repair pathway (BER) was discovered. ² Although more repair pathways were discovered over the next decade (Table 1.1), ² their importance in maintaining the integrity of the human genome and preventing malignant transformation was largely ignored in a clinical sense. By and large, the study of DNA repair pathways languished in the realm of academia.

However, the concept of irreparably damaging DNA to kill cancer cells pharmacologically, in addition to radiation, took center stage as a number of chemotherapeutics moved through clinical trials into everyday use. Although the mode of action for many anticancer agents is to cause DNA damage, it is ironic that early development of chemotherapeutics did not take into consideration how the study of DNA repair pathways could help determine what treatments might be most efficacious. Instead, the first paradigm for treating cancer followed an infectious disease approach. ³ Akin to matching a drug to a bug, scientists attempted to find a specific chemotherapeutic that would eradicate a particular type of cancer. A parallel path of concerted research effort was expended in finding an individual gene that would correspond to a particular type of cancer, with the goal of either restoring lost genetic functionality or silencing a tumorigenic gene function. ⁴

With very few exceptions, neither approach met real-world expectations. Scientists observed indirectly, then learned empirically, that (1) cancers are more heterogeneous than bacterial targets, (2) genes govern a plethora of functions that collectively contribute to carcinogenic transformation, and (3) cells have numerous built-in mechanisms of DNA damage repair that thwart chemotherapeutic efficacy. In attempts to overcome intrinsic or acquired treatment resistance, many combinations of treatments have been and are being tried.

As scientists looked for unique features of cancers to target for treatment, the biggest treatment conundrum reared its head: how to kill cancer cells without causing similar damage to normal cells. Developing more targeted chemotherapeutics and better delivery methods has remained paramount as new treatments go from bench to bedside. Only in recent years have scientists started to tap into their knowledge of DNA repair pathways as a means for solving this ominous problem. The study of rare genetic diseases paved the way for this new paradigm.

The critical role of DNA repair in preventing cancer in humans first came to light in studying individuals with xeroderma pigmentosum (XP), a rare recessive genetic disorder characterized by the inability to repair DNA damage caused by ultraviolet light. This deficiency leads to premature aging and multiple forms of skin cancer. Investigation of XP’s underlying causes revealed a mutation in an enzyme in the nucleotide excision repair pathway (NER) of people with XP; this defect reduces or eliminates one or both sub-pathways of NER activity. ⁵. and ⁶. Since then, scientists have uncovered a handful of other hereditary conditions including Fanconi anemia and certain cancers (non-polyposis colon cancer, familial breast and ovarian cancers) that are constitutively deficient in a particular DNA repair pathway. ². and ⁷.

Studies on aging have provided additional understanding of DNA repair processes. DNA damage does not always lead to mutagenesis. The body can eliminate cells with low-level DNA damage; this protects the body from cancer but at the expense of accelerating aging. An extreme example of this is Cockayne’s syndrome, which causes severe progeroid syndromes. ⁵ Mutations in the genes that encode two proteins in a NER sub-pathway called transcription coupled repair (TCR) cause global premature cell death. And, although premature aging is a hallmark of this disease, no person with Cockayne’s syndrome has ever been documented as developing cancer. ⁵ This underscores the relationship between DNA damage, cancer, and aging – and sheds light on the arsenal of options that cells have for preserving genome integrity.

In addition, DNA damage can initiate cancer, but cells may also induce DNA injury to protect against cancer. This is seen in the loss of protective telomeric repeats at chromosome ends. Precancerous cells have critically short telomeres that behave like DSBs in that they awaken the DNA damage-response system, triggering cell-cycle arrest and death. This shows what extent the body will go to in order to protect genome integrity, and it highlights one of the maladaptive processes that almost all tumors exhibit. Approximately 90% of all cancers possess reactivated telomerase to overcome this natural barrier to growth. ⁵

Thus, DNA damage can elicit one of four cellular responses: repair, senescence, death, or mutation. The decisions that lead to one response or another are governed by multiple repair pathways and various replication apparatuses including checkpoints, signal-transduction and effector systems, all of which influence transcription, recombination, chromatin remodeling and differentiation. The fact that DNA’s integrity normally remains intact is remarkable when one considers that

• DNA is the only biologic molecule that relies solely on its survival and the integrity of its information by repairing existing molecules instead of synthesizing new ones.

• DNA accumulates damage from both endogenous and exogenous sources over its lifetime.

• Most cells contain only one copy of its information.

• DNA undergoes approximately 10 ⁴ spontaneous base losses and single-strand breaks (SSBs) per day per cell. ⁵

DNA’s stability is assaulted from three sides. ⁵. and ⁸. Hydrolysis and other spontaneous intracellular reactions can create abasic sites and cause deamination. Cellular metabolism generates reactive oxygen and nitrogen species that can cause SSBs as well as numerous oxidative base and sugar products. In addition, lipid peroxidation products, carbonyl species, endogenous alkylating agents, and estrogen and cholesterol metabolites cause other types of DNA damage. Exogenous physical and chemical agents, naturally occurring and synthetically made (such as chemotherapeutics), cause many types of DNA damage. ⁵

Given the complexity of DNA damage response and repair, it is a virtual certainty that part of its machinery goes awry in cancers. Similar to when bad data is resaved to a hard drive, mutagenic DNA information becomes more garbled, fragmented, and transformed with subsequent saves. Recent studies provide evidence that defective DNA damage repair is present in virtually all tumors, ⁵ but scientists are just starting to exploit that clinically to its greatest advantage.

MGMT Inhibition: First Foray into DNA Repair Inhibition

The first effort to tap into the clinical importance of understanding DNA repair processes with respect to cancers came to light when the first nitrosourea was introduced in the early 1970s as a treatment for glioblastomas and other malignant gliomas. ⁹

Nitrosoureas are highly lipid-soluble, enabling them to cross the blood–brain barrier to reach gliomas. As a polyfunctional alkylating agent, nitrosourea achieves its therapeutic effect by alkylating DNA at various positions of guanine – particularly N ⁷, and, to a lesser extent, O ⁶ and O ⁴. ¹⁰ These adducts subsequently cause single- or double-strand damage (the latter by crosslinking). ¹¹ Early studies, such as one described in a 1975 article explaining the mechanisms of action of BCNU (1,3-bis [2-chloroethyl]-1-nitrosourea) and related nitrosoureas, trumpeted this drug class’s highly selective anti-tumor activity. ¹² However, scientists soon learned that something could reverse the DNA damage that those alkylating agents inflicted on tumors. That something was O ⁶-methylguanine-DNA methyltransferase (MGMT), a DNA repair protein that removes alkyl groups in a single-step suicide reaction. ¹³ Thus, the end of the 1970s heralded the first attempts to study DNA repair pathways in depth for their potential clinical influence. ²

MGMT presented investigators with a unique opportunity in early studies of DNA repair because this protein has a restricted mechanism of direct action. ¹⁰ MGMT removes only alkyl groups at only the O ⁶ position of guanine, transferring them from the oxygen of the amino acid to MGMT in a stoichiometric reaction that subsequently causes ubiquitination and degradation of MGMT. This requires cells to continually manufacture more MGMT to help maintain DNA integrity. ¹³

More than 20 years of studying MGMT¹⁰ has made it the most widely studied gene that encodes a DNA repair protein. ¹³ Those studies have revealed many aspects of DNA repair in general, which continue to be translated into clinical applications today. MGMT is not critical for survival, but its pleiotropic effects make it an important linchpin in the overall scheme of DNA repair. As such, MGMT possesses many characteristics worthy of study: ¹³

• It protects normal cells from naturally occurring alkylating agents, contributing to genome stability – which also diminishes the effectiveness of alkylating chemotherapeutics. ¹⁰

• Loss of MGMT activity increases the risk of carcinogenesis. ¹⁰

• MGMT levels are altered in many cancers, ¹⁰. and ¹³. which can give us insight into malignant transformation and may provide a potential biomarker for early tumor detection. ¹⁰

• Low MGMT activity may indicate intrinsic drug sensitivity in certain cancers (although findings to date are conflicting). ¹⁴

• Effects beyond MGMT itself may affect tumor susceptibility, ⁹ which alludes to pathway cross-talk.

• MGMT interacts with other DNA repair pathways to help maintain the genome. ¹⁵

• MGMT activity is somewhat inducible (although this is transient). ¹⁰

MGMT was successfully inhibited more than 10 years ago, and that success shed light on how manipulation of a DNA repair pathway could be used clinically. This constituted an important first, as MGMT was the first entity studied for DNA repair inhibition. ¹⁵ Methylating the promoter of the gene that encodes MGMT inhibits the cells’ ability to make more MGMT, eliminating one avenue that cells have for repairing DNA damage caused by alkylating chemotherapeutics such as TMZ, BCNU, and ACNU.

Based on this knowledge, O ⁶-BG (O ⁶-benzylguanine) was identified in 1990 as a potent inhibitor of MGMT. This was a first as well because (1) it was the first anticancer drug developed as a chemosensitizer and (2) it was developed on the basis of a target effect rather than on a maximally tolerated dose. ¹⁰. and ¹⁶. Clinical trials are ongoing in this arena to determine to what extent that MGMT inhibition can increase treatment responsiveness when alkylating chemotherapeutics are administered. ¹⁰

Another potential application stemming from MGMT research is gene therapy for myeloprotection. Myeloablation is the most common toxicity that keeps anticancer treatments from reaching truly therapeutic levels. Because MGMT is a potent drug-resistance gene, its induced overexpression in bone marrow stem cells prior to chemotherapy can transduce drug resistance and protect against myelosuppression of healthy cells – thus overcoming dose-limiting toxicities while sparing healthy cells. ¹⁰

This represented another first: MGMT was the first molecule targeted as both an inhibitor and a bone marrow protectant.

Although an MGMT inhibitor was not the first DNA inhibitor to make it out of clinical trials, the ongoing study of MGMT continues to reveal more information about DNA repair pathways, their cross-talk and overlapping functions. For example, although MGMT repairs only one lesion that comprises a very small portion of all DNA methyl adducts, MGMT activity or lack of activity can have many different effects on tumor cells, including repair, clastogenicity, mutagenicity, or apoptosis – all based on interactions with other repair pathways (Table 1.2).

The study of MGMT inhibition is the earliest example of how a DNA inhibitor was initially considered for therapeutic use to sensitize tumors to chemotherapy in an attempt to overcome treatment resistance. However, inhibition of MGMT did not occur only in the tumor cells; it also sensitized normal cells to alkylating agents. In some cases, this resulted in an obligatory reduction in dose of alkylator therapy, which compromised the agent’s efficacy. Therefore, despite MGMT’s contribution, scientists again faced the question of how to selectively kill tumors while sparing normal cells. That question remains today.

Virtually all of today’s anticancer drugs, including the targeted ones, still target fundamental cellular processes (such as DNA replication) that transpire in both healthy and mutagenic cells. ¹⁰ But researchers are coming closer to an answer in the continuing quest of how to selectively kill cancer cells (Table 1.3).

If key processes in the continuum of cancer transformation and progression can be interrupted, then it should be possible to stop cancer in its tracks. The key processes that have manifested themselves to date are primarily deficiencies. As mutations progress, loss of heterozygosity occurs, also called genetic streamlining³ (Figure 1.1). With respect to DNA repair pathways, deficiencies in a particular pathway can lead to increased levels of other DNA repair proteins, either in the same pathway or a different one. Compensating for a deficiency is paramount to efficient DNA repair, and by extension, cancer survival. These altered levels of DNA repair proteins contribute to acquired or intrinsic cellular resistance to DNA-damaging agents. ¹⁷ Capitalizing on cancer-cell deficiencies to turn them against themselves is the conceptual framework for synthetic lethality – a new approach being pursued in the war against cancer.

Synthetic Lethality

Synthetic lethality is a situation in which a mutation in one of two genes separately still supports cellular viability, but the combination of the two mutations leads to cell death. If one of those genes is important to a cancer, then discovering the second entity that can create a synthetic lethal interaction should be both highly effective and highly selective in causing cancer cell death, because cells that contain a normal copy of either gene should not be affected. ³. and ⁸.

The idea of synthetic lethality is not new; Dobzhansky first described it in 1946. However, it took another half century before anyone suggested that the concept could be applied to cancer therapeutics, when Hartwell et al. posited this in 1997. ⁸ Thus, targeting a weakness of cancer, such as abnormal expression (deficiency, overactivity) or some other feature that promotes cancer growth, ³. and ⁵. could provide a solution to the conundrum of killing cancer cells while sparing normal cells.

The most likely opportunities to induce synthetic lethality are related to DNA damage response and repair. Although much remains to be learned regarding the sequential biochemical changes that transform normal cells into cancer cells, we do know that many cancer cells are defective in their ability to recognize and respond to DNA damage. Such defects can lead to point mutations, copy number changes, structural abnormalities such as translocations, and other mutagenic transformations. If those defects can be exploited – especially early in the process of carcinogenesis – then it would keep cancerous cells from acquiring a more virulent mutational phenotype (Figure 1.2). Furthermore, developing the right inhibitors to capitalize on such defects would create a lethal liability in cancer cells. Moreover, such agents should have higher therapeutic indices than current anticancer drugs because relatively low doses of inhibitors should affect cancer cells without causing collateral damage. ³

Real-world scenarios are more complicated than conceptualizations, and many questions regarding synthetic lethality have yet to be answered. At the top of that list are (1) how to find the targets that are most amenable to this type of attack, and (2) whether cancer-cell selectivity can be achieved by inhibiting proteins that are also important for cellular homeostasis. In addition, researchers have just started to identify the quantitative and qualitative biochemical differences in cancer cells versus normal cells. In the absence of uncovering clear-cut patterns of deficiencies or mutagenic changes such as those seen in certain familial cancers, today it is still difficult to discern overlapping but slightly different functionalities of paralogous proteins. ³

Despite these challenges, adopting the study of DNA repair inhibition as a tool in the arsenal of fighting cancer has become a worldwide priority. ¹⁸ Carcinogenic mutations to DNA repair genes and their regulators are important diagnostic and therapeutic targets – and most of them are still waiting to be discovered. ¹⁹ Methylation of the promoter of MGMT was the first therapeutic foray into inhibiting DNA repair, but it fell short of being a synthetic lethal agent. Today’s most exquisite clinical example of synthetic lethality is the story behind PARP (poly[ADP-ribose] polymerase) inhibitors.

PARP: The Archetypical Inhibitor

PARP was first described in 1963, ⁸ but it took 40 more years for it to enter clinical trials in the fight against cancer. Like MGMT, PARP is not required for survival, but it is important for maintaining genetic stability. ²⁰ There are 18 members in the PARP superfamily; ²¹ PARP1 is part of the enzymatic machinery of the BER pathway in humans. PARP1’s job is to sense single-strand breaks (SSBs), then bind to the damage site and undergo a conformational change to recruit scaffolding and repair proteins to the site. ¹⁸ In addition to its DNA repair duties in BER, PARP1 is involved in transcriptional regulation and, under certain circumstances, induction of cell death. ²¹

In 2003, the first PARP inhibitor entered clinical trials, ¹⁵. and ¹⁸. much like MGMT inhibitors did: as a chemosensitizer without regard to tumor selection for its DNA repair function. ⁸ Interestingly, a PARP inhibitor was first tested in clinical trials in combination with temozolomide (TMZ), a chemotherapeutic that was previously tested with an MGMT inhibitor. Clinical trials of non-selective combination therapy using a PARP inhibitor continued until 2005, when two seminal papers published in Nature provided proof of concept regarding the efficacy of using PARP inhibitors as single agents to treat BRCA-deficient cell lines. ²⁰ It was then that the concept of treating a weakness was born. ⁸

BRCA forms of breast cancer contain an inherited defect in the proteins encoded by the BRCA1 and BRCA2 genes. Normal cells would retain one of the alleles, but breast cancers that have lost both alleles have no ability to repair double-strand breaks (DSBs) using the homologous recombination (HR) repair pathway. BRCA cancer cells survive by repairing DSBs using the non-homologous end joining (NHEJ) pathway. However, in the absence of PARP, SSBs accumulate, subsequently leading to recombinogenic lesions or DSBs during replication, the collapse of replication forks, and cell death²⁰ (Figure 1.3).

Normal cells can live without PARP1. Even though SSBs would still accumulate, the HR pathway would repair them when they become DSBs during replication. Thus, a PARP deficiency by itself is not lethal; neither is a BRCA deficiency. But the combination of the two becomes lethal to cells deficient in both. This phenomenon, called BRCAness, is a powerful example of synthetic lethality.

Today almost a dozen third-generation PARP inhibitors are in clinical trials ¹⁵. and ¹⁸. (Table 1.4), and PARP’s clinical efficacy remains one of the most exciting recent developments in clinical oncology. ¹⁸. and ²². In addition, the idea of a PARP inhibitor as a combination drug has not been discarded. Clinical trials are ongoing to test PARP inhibitors in combination with chemotherapy for triple-negative breast cancers (another BRCAness phenotype), as that type of cancer historically has had few treatment options to draw from. ¹⁸

The characterization of BRCAness has several important wide-ranging clinical implications: ⁸

• Other types of cancer can exhibit this phenotype.

• In cells where one BRCA allele is still functional, it can be silenced by methylating its promoter or the gene F of Fanconi anemia, which is present in breast, ovarian, cervical head, and neck cancers, as well as squamous cell and non-small-cell lung carcinomas.

• Investigators must determine to what extent PARP must be inhibited to achieve maximal dose responsiveness.

• Investigators need to develop ways to identify tumors with BRCAness.

The plethora of clinical trials that are in progress today for various PARP inhibitors (see Table 1.4) attests to the potential widespread clinical applications for exploiting the synthetic lethality conferred by PARP inhibition. These applications are extending beyond the field of oncology, as seen by the last entry in Table 1.4. As noted in Chapter 4, there is also abundant evidence that PARP inhibitors can protect the body against a variety of insults: ischemia-reperfusion injury (e.g., after stroke or heart attack), as well as chronic and acute inflammation (e.g., caused by arthritis, asthma, septic shock, diabetes); hence, those trials are included in Table 1.4.

The hunt is underway to develop more biomarkers that can reliably identify other BRCA-like tumors to extend the benefit of PARP inhibitors. ¹⁸ In the quest to find additional drug-able synthetic lethal targets, EZH2 may be the next. EZH2 mediates proliferation in breast cancer cell lines, and studies in mammals show that it requires BRCA1 to do so. Although this relationship is not yet fully elucidated, theoretically EZH2 inhibition could be combined with PARP inhibition in a double synthetic lethal strategy to treat BRCA1-deficient breast cancers. ²²

In similar fashion, silencing of XRCC1 (X-ray repair cross-complementing group 1) has been demonstrated to sensitize cells to PARP inhibition. XRCC1 is a substrate of PARP, and it also has functionality in the NER pathway. ²³ Such possibilities of enhancing or inducing BRCA-ness to create synthetic lethalities are tantalizing.

DNA Damage Checkpoints and Their Inhibition

As DNA repair pathways began to be studied in more depth, the working definition of DNA repair expanded. DNA repair in its strictest sense means biological processes during which alterations in the chemistry of DNA are removed and the integrity of the genome is restored. ² Today’s broader definition now includes many biological responses to DNA damage, including arrest of DNA synthesis in the presence or absence of defined DNA damage and cellular decisions on when to induce apoptosis versus attempt repair. Although this textbook’s discussions of repair pathways operate on the narrower definition of DNA repair, it is worthy to mention the damage response side to DNA repair, as (1) its contributions help maintain genome integrity and (2) concerted efforts are under way to develop checkpoint inhibitors therapeutics as well. ⁵.²⁰. and ²⁴.

Cell replication is a highly regulated process by necessity; this helps guarantee accurate and complete transfer of genetic material. ²⁵ Key contributors to this regulation are DNA checkpoints. DNA checkpoints function much like a quality control manager, precisely monitoring DNA status throughout the cell cycle and determining whether to signal it to halt when DNA damage is sensed. DNA checkpoints allow cells to respond to critical situations such as exposure to genotoxic agents or to cope with DNA lesions that cannot be repaired immediately. In addition to controlling replication efficiency and accuracy, checkpoints may engage in any combination of strategies if any aspect of replication goes astray. They may:

• Prevent cell cycle progression

• Segregate damaged chromosomes

• Prevent generation of secondary lesions

• Modify transcription

• Potentiate repair actions and modulate levels of repair proteins

• Direct lesions to the most appropriate repair pathway. ⁷

Thus, DNA damage checkpoints are intrinsic to cell cycle integrity and DNA damage response. ⁷.²⁴. and ²⁵.

Briefly, checkpoint signaling comes from five sources: sensors, proximal and distal transducer kinases, mediators, and effectors. Sensors recognize structural abnormalities of damaged DNA or chromatin; proximal transducer kinases function like sensors but require activation from additional proteins. Mediators assess both the temporal and spatial progression of the DNA damage response. When activated, both the proximal and distal transducer kinases phosphorylate a plethora of effector molecules involved in DNA damage response. p53, known as the keeper of the genome, is the most prominent of those. The effectors signal CDK-cyclin complexes, which drive the consecutive phases of the cell cycle and can halt cell cycle progression and transcription. Various cyclins are expressed in different phases of the cell cycle, and cyclin levels rise or fall according to what phase is in progress ²⁴. and ²⁵. (Figure 1.4).

Checkpoints are activated at times of cell stress. If stress in the form of DNA damage is minimal, checkpoints may not be activated. Scientists are still discerning exactly how checkpoint signaling cascades are activated. However, there is evidence that the checkpoint factors do not directly recognize the lesions; DNA repair mechanisms do that, then they activate the apical checkpoint kinases. If necessary, the checkpoint signaling cascade initiates cell cycle arrest to allow adequate time to repair DNA damage or to induce apoptosis. Checkpoints also appear to play a role in determining the most efficient method of DNA repair to employ. For example, checkpoint-dependent phosphorylation of BRCA1 and Nej1 seems to affect whether the HR or NHEJ pathway is used to repair DSBs. ⁷

Although the two proximal transducer kinases, ATM and ATR, appear to have distinct divisions of labor – the former responding to DSBs, the latter responding to stalling of replication forks during replicative stress – there is overlap and coordinated cross-talk, which is still being elucidated. ²⁴ That is true with the distal transducer kinases as well. Chk1 regulates both the S and G2-M checkpoints via downstream effectors. But Chk2 can do the same, through p53 as well as other effectors that Chk1 influences²⁴ (Figure 1.5).

Cancers demonstrate numerous checkpoint abnormalities; this dysfunction is a hallmark of tumor progression and neoplastic transformation. For example, expression of p53, which has signaling and regulation roles at both cell cycle checkpoints, is decreased in many cancers. ²⁵ In contrast, overexpression of cyclins, the regulatory subunits of CDK-cyclin complexes, is common in cancers. ²⁵ In addition, chemotherapy and ionizing radiation activate cell cycle checkpoints. ²⁴ Further disruption of these DNA damage-response systems that are already dysfunctional in tumors could be exploited as a new route for creating selectivity in anticancer treatments and enhancing sensitivity to cytotoxic agents. ⁵. and ²⁴.

The first checkpoint inhibitor, caffeine, was studied in 1982 and was found to be a non-specific inhibitor of ATM and ATR. Enthusiasm for the idea of checkpoint inhibition resurged when the staurosporine analog UCN01 (7-hydroxystaurosprine) was tested. It proved to be a potent but non-selective inhibitor of Chk1; unfortunately, its toxicities presented problems that halted its further development. Since then, many other checkpoint inhibitors have been studied, and close to 30 (including CDK inhibitors) are in clinical trials today. ²⁴. and ²⁵.

Checkpoint inhibition follows one of two approaches, depending on the protein under discussion. One approach is to look at a dysfunctional protein and determine the best single target that could induce synthetic lethality, similar to how DNA repair inhibitors work. However, cyclin-dependent protein kinases (CDKs) are more multitasking than DNA repair inhibitors and even other checkpoint proteins. Because CDKs function in multiple cellular processes, the current train of thought is to create pan-selective CDK inhibitors. Targeting multiple pathways with them increases the chances of eliminating tumor cells with varying mitotic potential – before they become treatment-resistant. Thus, most currently available CDK inhibitors affect two or more kinases²⁵ (Table 1.5). An example of how this works can be found in utilizing a CDK inhibitor to treat chronic lymphocytic leukemia. The affected peripheral blood cells are almost always in a resting state; however, their precursors in the bone marrow are highly mitotic. This creates a very heterogeneous tumor target. If you simultaneously block cell cycle progression, transcription, facilitation of apoptosis, and reactivation of the p53 tumor suppressor, you have a better chance of effectively attacking all the tumor cells. ²⁵ As attractive as this sounds, such less-selective CDK inhibitors must be screened for toxicity in animal models, due to the importance of these enzymes in other cell types. Monospecific CDK inhibitors also have their place, but they seem to be better suited to conditions where primarily one kinase is deregulated, as in certain cardiovascular disorders. ²⁵

One common thread that is emerging in using inhibitors of DNA repair proteins and DNA checkpoints is that the inhibitors enhance the toxicities of the anticancer agents given with them, requiring downward dosage adjustments to the traditional chemotherapeutics. Myelosuppression remains the most common dose-limiting toxicity, even with these more targeted approaches. ¹⁵ This raises yet-unanswered questions:

• What dosing approach is optimal: combination, intermittent, sequential?

• What combinations of treatment approaches can yield true synthetic lethality (with less toxic results)?

These questions continue to remain uppermost in the minds of everyone working in translational research of DNA repair and damage response inhibitors.

Overview of DNA Repair Pathways

Every cell in the body employs an intricate system of repair, damage tolerance, and checkpoint pathways to counteract DNA damage and maintain genome stability. DNA damage induces more than 900 distinct phosphorylation events involving more than 700 proteins. ⁵ The manner in which the repairs are made is predicated on the type and extent of the damage done. Six major DNA repair pathways, each with distinct (but sometimes overlapping) modes of action, dispatch the damage caused by endogenous and exogenous DNA-damaging agents, including chemotherapy and radiotherapy. ⁶.¹⁷. and ²⁶. It is those DNA repair pathways, and how they are influencing emerging treatments for cancer, that is the focus of this book. Separate chapters are dedicated to each pathway, as well as select repair proteins and signaling targets within those pathways that are being culled for clinical development as therapeutics. This book ends with discussions of the role of DNA repair in cancer therapeutic toxicities and future directions for development of therapeutics.

A short description of each DNA repair pathway follows. (See also Figure 1.6).

Direct Repair (DR)

As mentioned previously, the DR pathway removes alkyl groups by a direct transfer to MGMT in a one-time reaction. It is notable because it repairs only one type of lesion, and the repair tool is not an enzyme. However, it may be the most efficient of all repair paths. The cell must be able to continually manufacture more MGMT to perform this function. If it does not, the lack of MGMT can influence the functions of other repair pathways. ¹³

Base Excision Repair (BER)

BER repairs subtle, non-bulky lesions produced by alkylation, oxidation or deamination of bases. A hallmark of BER is its varied glycosylases; they can be mono- or bi-functional; they act only on specific substrates, and their action is to cleave the lesion, leaving an abasic site that APE1 processes. Then APE1 recruits repair proteins to the site to complete the repair. The BER pathway consists of two sub-pathways, called short-patch and long-patch. The short-patch pathway is used more frequently; it repairs normal AP sites. The long-patch pathway preferentially repairs oxidized and reduced AP sites, replacing sequences of 2 to 8 nucleotides. PARP1 is a component of the long-patch pathway. ²⁶ Because BER activity produces cytotoxic intermediates, it is important for BER to complete its repairs before cell replication starts.

Nucleotide Excision Repair (NER)

NER repairs large adducts and bulky DNA lesions, such as those induced by crosslinking agents and base-damaging carcinogens. ²⁶ NER works on this helix-distorting damage when only one of the two DNA strands is affected. Its multistep cut-and-patch process involves more than 30 proteins. ⁵ Two sub-pathways comprise NER: global genome repair (GGR) and transcription coupled repair (TCR). The pathway chosen is predicated on the protein complexes that initiate the repair. ²⁶ GGR repairs damage throughout the genome, on both transcribed and non-transcribed strands of active genes. TCR preferentially repairs transcribed strands in active genes, ²⁷ removing distorting lesions that block elongating RNA polymerases. ⁵ NER’s capacity and the expression of genes related to this pathway can be modulated by oxidative stress. ⁵

Mismatch Repair (MMR)

MMR recognizes and repairs single-base mismatches and misaligned short nucleotide repeats, such as small insertion/deletion loops introduced during normal DNA replication ²⁶. and ²⁸. that escape the proofreading activity of DNA polymerases. Such errors occur with a frequency of about 1 in 10 ⁹–10 ¹⁰ base pairs per cell division. ⁷ Nucleotide mispairing can also occur after exposure to exogenous agents or endogenous reactive species that may cause base modifications. Varying recognition complexes are formed based on the type of mismatch to be repaired. Historically, the repair was thought to be completed specifically on the new strand; but the most recent evidence indicates that MMR repair can happen before, during, or after mitosis (see Chapter 9). ⁷ Loss of MMR leads to a mutator phenotype; this predisposes one to cancer and affects, among other processes, DNA damage response, signaling, and recombination. ⁷ That is why MMR inhibitors are not currently in clinical development. However, new tumor cell targeting approaches may overcome these challenges. ⁶

Homologous Recombination (HR)

HR is the repair pathway used to fix double-strand breaks that are detected during the S/G2 portion of the cell cycle. Because it uses a homologous template to reconstruct the damaged DNA strand, it is highly accurate in its repairs. ²⁸ Unrepaired SSBs can become DSBs when cell replication begins; this activates one or more DNA checkpoints. The checkpoint responses finely regulate DSB ends processing, dictating which DSBs should be repaired by HR versus NHEJ. This is a crucial stage in the recombination process. ⁷

Non-Homologous End Joining (NHEJ)

NHEJ rejoins the ends of double-strand breaks regardless of sequence homology. It works at the ends of broken DNA without using an identical copy of DNA as a template, which creates the possibility of losing or adding bases in the process. This occurs during the G1 phase of the cell cycle, before replication, which tends to make it prone to errors such as loss of nucleotides. ⁵. and ²⁸. Inactivation of CDK1 increases NHEJ events in the G2 phase of the cell cycle. ⁷ DNA strands that are not repaired completely by NHEJ are subject to repair by HR. ²⁹

DNA repair inhibitors for most pathways are either in preclinical or clinical trials. Many inhibitors affect more than one pathway; for example, PARP inhibitors affect both BER and double-strand break repair; APE1 inhibitors affect BER as well as transcription of other proteins; RAD51 inhibitors affect double-strand break repair; ATM and ATR inhibitors affect cell cycle checkpoints and double-strand break repair. The preclinical history, reasoning, challenges, and successes involved in discovering cancer biomarkers and developing inhibitors of each pathway – either as single agents or in rational combinations – are discussed in the following chapters.

Our ever-expanding knowledge of DNA repair and DNA damage response is paramount to the ongoing translational research that seeks to create new ways of fighting diseases. Much is yet to be learned about the regulation of DNA damage response and repair processes. The concept of relatively straight, strictly hierarchical DNA repair pathways that operate in a cascade sequence is being replaced with a model of robust networks of pathway crosstalk and interactions (Table 1.6). As researchers uncover more of those interactions, it paints a picture of how complex normal DNA repair processes function, how rescue signaling is provided from a number of proteins – and how carcinogenesis can modify signaling networks to compensate for mutagenic losses. ³⁰