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1 Targeting the Mitochondrial Dna Polymerase Gamma in Acute Myeloid Leukemia

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TARGETING THE MITOCHONDRIAL DNA POLYMERASE GAMMA IN ACUTE MYELOID LEUKEMIA By Sanduni U. Liyanage A thesis submitted in conformity with the requirements for the degree of Doctor of Philosophy Department of Medical Biophysics University of Toronto © Copyright by Sanduni U. Liyanage, 2017 1 TARGETING THE MITOCHONDRIAL DNA POLYMERASE GAMMA IN ACUTE MYELOID LEUKEMIA Sanduni U. Liyanage Doctor of Philosophy Department of Medical Biophysics University of Toronto 2017 ABSTRACT Part I Mitochondrial DNA (mtDNA) biosynthesis requires replication factors and adequate nucleotide pools from the mitochondria and cytoplasm. We performed gene expression profiling analysis of 542 human AML samples and identified 55% with upregulated mtDNA biosynthesis pathway expression compared to normal hematopoietic cells. Genes that support mitochondrial nucleotide pools, including mitochondrial nucleotide transporters and a subset of cytoplasmic nucleoside kinases, were also increased in AML compared to normal hematopoietic samples. Knockdown of cytoplasmic nucleoside kinases reduced mtDNA levels in AML cells, demonstrating their contribution in maintaining mtDNA. To assess cytoplasmic nucleoside kinase pathway activity, we employed a nucleoside analog 2’3’-dideoxycytidine (ddC), which is phosphorylated to the activated anti-metabolite, 2’3’-dideoxycytidine triphosphate (ddCTP) by cytoplasmic nucleoside kinases. ddC is a selective inhibitor of the mitochondrial DNA polymerase, POLG. ddC was preferentially activated in AML cells compared to normal hematopoietic progenitor cells. ddC treatment inhibited mtDNA replication, oxidative phosphorylation, and induced cytotoxicity in a panel of AML cell lines. Furthermore, ddC preferentially inhibited mtDNA replication in a subset of primary human leukemia cells and selectively targeted leukemia cells while sparing normal progenitors cells. In animal models of human AML, treatment with ddC decreased mtDNA, electron transport chain proteins, and induced tumor regression without toxicity. ddC also targeted leukemic stem cells in secondary AML xenotransplantation assays. Thus, AML cells have ii increased cytidine nucleoside kinase activity that regulates mtDNA biogenesis and can be leveraged to selectively target oxidative phosphorylation in AML. Part II Human mitochondrial DNA is replicated by the mitochondrial DNA polymerase gamma. Using proximity dependent biotin labelling (BioID), we characterized the POLG interactome and identified new interaction partners involved in mtDNA maintenance, transcription, translation and protein quality control. We also identified interaction with the nuclear AAA+ ATPase Ruvbl2, suggesting mitochondrial localization for this protein. Ruvbl2 was detected in mitochondria- enriched fractions in leukemic cells. Additionally, transgenic overexpression of Ruvbl2 from an alternative translation initiation site resulted in mitochondrial co-localization. Overall, POLG interactome mapping identifies novel proteins which support mitochondrial biogenesis and a potential novel mitochondrial isoform of Ruvbl2. iii Acknowledgements Throughout the course of my studies, I have been extremely fortunate to be surrounded by supportive and caring colleagues, family and friends. Despite the twists and turns during this journey, knowing that I have a supportive environment has been a great strength and I am forever grateful for the relationships and memories created these past few years. To my supervisor, Dr. Aaron Schimmer, thank you for providing me with the opportunity to grow and develop as a scientist through your constant support and mentorship. Your kind, enthusiastic and positive nature has made it a pleasure to have worked with you. I hope to continue these meetings and collaborate on projects in the future. The completion of this project would not have been possible without the excellent work of many colleagues, in particular, Rose. I truly admire the dedication, hard work and care you placed into every project you have participated it. I am grateful for your friendship, laughs, conversations and consistent support provided during my time at the Schimmer lab. To Marcela, thank you for all your advice, encouragement, support and friendship during this time. You both have served as great role models to me for your passion, commitment and caring attitude towards the people you have worked with. To the other staff in the Schimmer lab, Neil and XiaoMing, thank you for your technical assistance with this project. It’s a blessing to have worked with a great team, who can be consistently relied upon on for support and help. To the post-doctoral fellows in the Schimmer Lab, Danny J., Danny B. and Ian, thank you for serving as my mentors. In particular, thank you to Danny J. for your time and companionship. Our discussions both intellectual and personal, have been valuable to my growth on a personal and career level. To the other members of the Schimmer Lab, Lianne, Yan, Thirushi, Dilshad, Ayesh, Geethu, Dana, Samir, Iulia and others; thank you for your friendship, encouragement and support. iv I will cherish the many laughs, coffee breaks and conversations in between experiments, and otherwise. Each of you have made my time at the Schimmer lab a memorable experience. To my committee members, Rebecca Laposa and David Hedley, thank you for providing a positive and supportive framework to progress my project in a productive manner. Thank you in particular to Rebecca for your enthusiasm, understanding and mentorship during my graduate studies. The opportunity to collaborate with members of other programs on research projects has been a valuable training experience. Additionally, I extend my thanks to our collaborators at the Donnelly Centre and the Metabolomics Facility at McGill University, your technical expertise has been essential to the progress of this study. Lastly, but most importantly, I am grateful to my parents and my brother. The unconditional love and support provided by my parents have been the foundation for any success I have achieved. Thank you for all your sacrifices and efforts to ensure that I received the best education possible. Moving forward, I hope to use my scientific training to serve all beings to the best of my ability. v TABLE OF CONTENTS ABSTRACT………………………………………………………………………………………………..ii ACKNOWLEDGEMENTS…………………………………………..………………......……………….iv TABLE OF CONTENTS……….…………………………………...……………………………………vi LIST OF FIGURES…………....…………………………………..….…………………………………..x LIST OF ABBREVIATIONS…...……………………………………………………………….….…xii 1: INTRODUCTION ................................................................................................................................................... 1 1.1. ACUTE MYELOID LEUKEMIA ....................................................................................................................... 1 1.1.1 CLASSIFICATION ................................................................................................................................................ 4 1.1.2. STEM CELL CHARACTERISTICS OF AML ........................................................................................................... 5 1.1.2. MECHANISMS ................................................................................................................................................... 6 1.1.3. CURRENT TREATMENT STRATEGIES FOR AML ................................................................................................. 9 1.1.4. EMERGING THERAPIES FOR TREATMENT OF AML ............................................................................................ 9 1.2 MITOCHONDRIA .............................................................................................................................................. 11 1.2.1. STRUCTURE AND FUNCTION ........................................................................................................................... 11 1.2.2. MITOCHONDRIAL DNA ORGANIZATION ......................................................................................................... 14 1.2.3. MITOCHONDRIAL BIOGENESIS REGULATION ................................................................................................... 15 1.2.4. OXIDATIVE PHOSPHORYLATION ..................................................................................................................... 16 1.2.5. QUALITY CONTROL OF MITOCHONDRIA .......................................................................................................... 19 1.2.6. MITOCHONDRIAL REGULATION OF CELL DEATH ............................................................................................. 20 1.3. MITOCHONDRIAL DNA ................................................................................................................................. 23 1.3.1. STRUCTURE AND FUNCTION ........................................................................................................................... 23 1.3.2. MITOCHONDRIAL DNA REPLICATION ............................................................................................................ 26 1.3.3. MITOCHONDRIAL TRANSCRIPTION AND TRANSLATION ................................................................................... 27 vi 1.3.4. REGULATION OF MITOCHONDRIAL DNA CONTENT ........................................................................................ 29 1.3.5. MITOCHONDRIAL NUCLEOTIDE POOL MAINTENANCE ....................................................................................
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