Mutation Based Resistance to Antiandrogens in Prostate Cancer
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Mutation Based Resistance to Antiandrogens in Prostate Cancer by Minna Delarae Balbas A Dissertation Presented to the Faculty of the Louis V. Gerstner, Jr. Graduate School of Biomedical Sciences, Memorial Sloan-Kettering Cancer Center in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy New York, NY May, 2013 Charles L. Sawyers, MD Date Dissertation Mentor Copyright by Minna Delarae Balbas 2013 Abstract Androgen receptor (AR) signaling plays a crucial role in the growth and progression of prostate cancer. Current therapies for prostate cancer rely on this dependence and aim to disrupt AR signaling through androgen deprivation and the use of AR antagonists (antiandrogens). While this is usually an effective treatment initially, most prostate tumors eventually regain the ability to grow. Reactivation of AR signaling has been implicated in resistance, often through overexpression or mutation of the androgen receptor. Several AR mutations have been described that broaden ligand specificity or convert AR antagonists into agonists of the mutant receptor. These mutations alter the conformation of antiandrogen-bound AR, such that the antiandrogen now activates the receptor. The second-generation antiandrogen enzalutamide (formerly MDV3100) recently received FDA approval for patients with castration resistant prostate cancer. Despite its success, the duration of patient response is often limited. To prospectively identify AR mutations that might confer resistance to enzalutamide, we developed and performed a reporter-based mutagenesis screen using an AR-regulated EGFP reporter and randomly mutagenized AR cDNA library. After several rounds of enzalutamide exposure and FACS- sorting, we enriched a population of cells that maintain EGFP expression in the presence of drug. Sequencing of the androgen receptor in these cells revealed a novel mutation (AR F876L) that converts enzalutamide into an agonist. Ectopic expression of AR F876L induces expression of AR target genes and rescues growth inhibition of enzalutamide treatment. This mutation also confers agonism to the antiandrogen ARN-509, which shares the same bisaryl-thiohydantoin core motif as enzalutamide. Molecular dynamics simulations on antiandrogen-AR complexes suggested a mechanism by which the F876L substitution alleviates antagonism, by repositioning the co- activator recruiting helix 12 of AR. This model then provided the rationale for a focused iii chemical screen which, based on existing antiandrogen scaffolds, identified three novel compounds that effectively antagonized AR F876L (and AR WT) to suppress the growth of prostate cancer cells resistant to enzalutamide. Employing recent advances in isolation of circulating tumor DNA and next generation sequencing, we have begun to address the question of whether this mutation occurs in patients who develop acquired resistance to enzalutamide or ARN-509 therapy. Preliminary results show that the AR F876L mutation emerged in a single patient, upon relapse with ARN-509 therapy. Further sequencing efforts are needed to determine the frequency of this mutation in a larger patient population. iv Acknowledgments I would like to thank my mentor, Dr. Charles Sawyers, for his support, guidance, and encouragement over the past several years. My experience in his lab has been life altering and I am extremely grateful to him for the opportunity to be a part of his lab. He has taught me how to be a good scientist, and to have confidence in my work. His passion for science is contagious. He has been supportive of my personal and professional decisions over the years. For all of this, I am deeply grateful. I would also like to thank the members of my thesis advisory committee, Dr. David Solit and Dr. Hans-Guido Wendel, and my first year mentor Dr. Ingo Mellinghoff, for their guidance and helpful suggestions. I am grateful to all of the members of the Sawyers lab; many of them have become close friends and are like a second family to me. In particular I would like to thank John Wongvipat, whose support I appreciate so much, and without whom the lab would fall apart. I am grateful to Karin Hepner, Kate Yen, Michael Burgess, and Brian Skaggs, who taught me so much and encouraged my curiosity during my early years in the lab. My deepest thanks go to Michael Evans, who has been a source of immeasurable support. Finally, I would like to thank my family for their love and encouragement, and for their patience during this journey. I want to thank everyone who helped establish our graduate program and make it a success, and all the GSK students and administrative staff, especially Iwona Abramek for her help in formatting my dissertation. v Table of Contents List of Figures............................................................................................................ vii List of Tables.............................................................................................................. ix List of Abbreviations....................................................................................................x Chapter 1 – Introduction .............................................................................................1 Androgens and the Androgen Receptor ............................................................................. 2 Androgen Receptor Domain Structure and Activation ........................................................ 4 Androgen Receptor Alterations........................................................................................... 6 Crystal Structures of Nuclear Receptors............................................................................. 7 Prostate Cancer ................................................................................................................ 11 Prostate Cancer Therapy.................................................................................................. 12 Mechanisms of Resistance to Targeted Therapy ............................................................. 17 Mechanisms of Resistance in CRPC ................................................................................ 18 Novel Antiandrogens: Enzalutamide and ARN-509 .......................................................... 22 Saturation Mutagenesis Screens ...................................................................................... 27 Chapter 2 – A reporter-based system to screen for AR activity................................28 Introduction ....................................................................................................................... 28 Materials and Methods...................................................................................................... 30 Results .............................................................................................................................. 35 Discussion......................................................................................................................... 48 Chapter 3 – Overcoming mutation-based resistance to antiandrogens with rational drug design ...............................................................................................................49 Abstract............................................................................................................................. 49 Introduction ....................................................................................................................... 49 Results .............................................................................................................................. 50 Discussion......................................................................................................................... 66 Materials & Methods ......................................................................................................... 69 Figure Supplements.......................................................................................................... 86 Chapter 4 – Future Directions.................................................................................107 Additional AR Mutations.................................................................................................. 107 Detection of AR F876L mutation in CRPC patient samples............................................ 110 Preliminary Patient Data ................................................................................................. 119 Conclusions .................................................................................................................... 120 Bibliography ............................................................................................................121 vi List of Figures Figure 1. Cartoon depicting androgen receptor signaling....................................................... 3 Figure 2. Cartoon depicting the domain organization of the androgen receptor..................... 4 Figure 3. Overlay of AR agonist crystal structures.................................................................. 8 Figure 4. The binding of agonists and antagonists promote different LBD conformations in the estrogen receptor (ER)............................................................................................ 10 Figure 5. Schematic of therapies targeting the AR signaling pathway.................................. 14 Figure 6. Typical response to hormonal therapy................................................................... 16 Figure 7. AR expression