Preclinical Evaluation of Synergistic Drug Combinations in Acute Myeloid Leukemia by Lianne Emily Rotin A thesis submitted in conformity with the requirements for the degree of Doctor of Philosophy Institute of Medical Science University of Toronto © Copyright by Lianne E. Rotin (2016) Preclinical Evaluation of Synergistic Drug Combinations in Acute Myeloid Leukemia Lianne E. Rotin Doctor of Philosophy Institute of Medical Science University of Toronto 2016 Abstract The FDA-approved Bruton’s tyrosine kinase (BTK) inhibitor ibrutinib has significantly improved patient outcomes in B-cell malignancies, where BTK signaling is implicated in disease progression. Documented expression of constitutively active BTK in AML cells has generated interest in evaluating the potential therapeutic role of ibrutinib in the treatment of AML. We investigated the role of ibrutinib in AML by screening this drug against libraries of approved drugs in AML cell lines, identifying the poly(ADP-ribose) glycohydrolase inhibitor ethacridine lactate as a profoundly synergistic hit. This drug combination was preferentially cytotoxic to patient-derived AML cells over normal controls, and synergistic cell death was preceded by reactive oxygen species (ROS) production. Ibrutinib similarly synergized with current first-line AML chemotherapy agent daunorubicin. Interestingly, neither ethacridine, nor daunorubicin’s synergy with ibrutinib appeared to be BTK-dependent. Further study with the epidermal ii growth factor receptor (EGFR) inhibitor erlotinib—which also has preclinical anti- AML activity—revealed equally profound EGFR-independent synergy with ethacridine, with an increase in ROS that paralleled that induced by the ibrutinib- ethacridine combination. We determined that erlotinib-mediated potentiation of ethacridine accumulation was responsible for this combination’s synergistic cytotoxicity, and hypothesize that ibrutinib and ethacridine likely synergize via the same mechanism. In summary, we have identified a novel BTK-independent role for ibrutinib in AML, and for the first time, report a potential role for PARG inhibition as a combination candidate for AML therapy. iii Acknowledgements I extend my deepest gratitude to my supervisor, Dr. Aaron Schimmer, for his guidance and encouragement throughout my PhD studies. Aaron is a wonderful mentor and a role model for the clinician-scientist I hope to one day become. I would also like to thank my program advisory committee members, Drs. Mark Minden and Meredith Irwin, for their helpful advice and constructive feedback during and in between committee meetings. Furthermore, I would like to thank Dr. Minden for providing me with the opportunity to attend his weekly leukemia clinic for the better part of two years; observing these patient visits gave me a better understanding of this disease and its impact on patients and their families, as well as a sincere appreciation for the need for new ways to tackle it. Working in the Schimmer Lab has been a truly incredible experience; it has been a privilege to train alongside a team of such bright, creative, and enthusiastic scientists. I would especially like to thank Marcela Gronda, Rose Hurren, and Neil MacLean: you have taught me countless lab techniques, assisted with many important experiments, and you have helped me become a better researcher. Finally, this acknowledgements section would be incomplete without mention of my wonderful support team outside of the lab. I’m grateful to my parents, Daniela and Robbie, for instilling in me the importance of education, hard work, and putting your heart into everything you do. I am also thankful for their continued and unwavering support throughout this long educational journey. Lastly, I would like to thank my fiancé Zach, who has patiently sat through every single one of my practice talks, asked some impressively pertinent questions, and who has always had something positive and encouraging to say. Thank you! iv Contributions This thesis consists of 3 data chapters. Chapter 3 was published in the journal Oncotarget (Rotin et al., 2016b) and Chapter 4 was published in the journal Leukemia and Lymphoma (Rotin et al., 2016a). Chapter 5 has yet to be published. The author performed all experiments and analyses outlined in the thesis, except as indicated below: Mr. Neil MacLean – performed combination high-throughput drug screens against ibrutinib and ethacridine, provided assistance with combination drug screens against erlotinib, analyzed screen data, and prepared lentiviral stocks Ms. Marcela Gronda – performed immunoblots (BTK, phospho-BTK, BMX, RLK, TEC, ITK, EGFR), olaparib assays, reactive oxygen species measurements depicted in figures 4-10 and 4-11, and provided assistance with the PARG inhibitor assay Ms. Rose Hurren – conducted in vivo combination ibrutinib-ethacridine studies, radiolabeled daunorubicin uptake studies, and Z-VAD-FMK experiments Ms. XiaoMing Wang – carried out in vivo combination ibrutinib-ethacridine studies Dr. Ahmed Aman – mass spectrometry analysis of ethacridine accumulation in AML cell lines v Dr. Feng-Hsu Lin – designed software program for excess-over-Bliss analysis of drug screen data Dr. Alessandro Datti – guidance in planning high-throughput drug screening procedures vi Table of Contents ACKNOWLEDGEMENTS……….………………………………..……………….IV CONTRIBUTIONS………….………………………………………..…….............V TABLE OF CONTENTS……………………………………………..……………VII LIST OF TABLES………………………………………………………………….XI LIST OF FIGURES………………………………………………….…….……….XII LIST OF ABBREVIATIONS………………………………………………………XIV Table of Contents Preface ................................................................................................................. 1 Chapter 1: Literature Review ............................................................................. 2 1.1 Acute Myeloid Leukemia .............................................................................. 3 1.1.1 Normal Hematopoiesis ...................................................................................... 3 1.1.2 Acute Myeloid Leukemia ................................................................................... 5 1.1.2.1 AML Pathogenesis ........................................................................................ 5 1.1.2.2 Epidemiology of AML .................................................................................... 6 1.1.2.3 AML Classification and Prognostication ........................................................ 6 1.1.2.4 AML Management ......................................................................................... 7 1.2 Tyrosine Kinase Inhibitor Therapy in AML ............................................... 10 1.2.1 Targeted Cancer Therapies ............................................................................. 10 1.2.1.1 Tyrosine Kinase Inhibitors ........................................................................... 10 1.2.2 Oncogenic Tyrosine Kinases in AML ............................................................. 11 1.2.2.1 FMS-Related Tyrosine Kinase 3 ................................................................. 11 1.3 Ibrutinib ........................................................................................................ 14 1.3.1 Bruton’s tyrosine kinase: background & role in signal transduction from the B-cell receptor .................................................................................................... 14 1.3.1.1 BTK domains ............................................................................................... 14 1.3.1.2 BTK expression ........................................................................................... 15 1.3.1.3 BTK: Role in B-cell Maturation .................................................................... 15 1.3.1.4 BTK Signaling in B-Cells ............................................................................. 15 1.3.2 A Rationale for Targeting BTK in B-cell Malignancies ................................. 16 1.3.2.1 Chronic Lymphocytic Leukemia .................................................................. 17 1.3.2.2 Mantle-Cell Lymphoma ............................................................................... 17 1.3.2.3 Waldenström Macroglobulinemia ................................................................ 17 1.3.3 Development of Ibrutinib as a Selective and Irreversible BTK Inhibitor with In Vivo Activity .......................................................................................................... 18 1.3.4 Preclinical and clinical activities of ibrutinib in B-cell cancers ................... 19 1.3.4.1 Chronic Lymphocytic Leukemia .................................................................. 19 vii 1.3.4.2 Mantle Cell Lymphoma ............................................................................... 20 1.3.4.3 Waldenström Macroglobulinemia ................................................................ 21 1.3.5 B-cell independent BTK signaling: myeloid-lineage cells ........................... 22 1.3.5.1 Mast Cells ................................................................................................... 23 1.3.5.2 Macrophages .............................................................................................. 24 1.3.5.3 Erythroid Cells ............................................................................................. 25 1.3.5.4 Platelets ...................................................................................................... 26