Role of Tafazzin in Hematopoiesis and Leukemogenesis by Ayesh Seneviratne A thesis submitted in conformity with the requirements for the degree of Doctor of Philosophy Institute of Medical Science University of Toronto © Copyright by Ayesh Seneviratne 2020 Role of Tafazzin in Hematopoiesis and Leukemogenesis Ayesh Seneviratne Doctor of Philosophy Institute of Medical Science University of Toronto 2020 Abstract Tafazzin (TAZ) is a mitochondrial transacylase that remodels the mitochondrial cardiolipin into its mature form. Through a CRISPR screen, we identified TAZ as necessary for the growth and viability of acute myeloid leukemia (AML) cells. Genetic inhibition of TAZ reduced stemness and increased differentiation of AML cells both in vitro and in vivo. In contrast, knockdown of TAZ did not impair normal hematopoiesis under basal conditions. Mechanistically, inhibition of TAZ decreased levels of cardiolipin but also altered global levels of intracellular phospholipids, including phosphatidylserine, which controlled AML stemness and differentiation by modulating toll-like receptor (TLR) signaling (Seneviratne et al., 2019). ii Acknowledgments Firstly, I would like to thank Dr. Aaron Schimmer for his guidance and support during my PhD studies. I really enjoyed our early morning meetings where he provided much needed perspective to navigate the road blocks of my project, whilst continuing to push me. It was a privilege to be mentored by such an excellent clinician scientist. I hope to continue to build on the skills I learned in Dr. Schimmer’s lab as I progress on my path to become a clinician scientist. Working in the Schimmer lab was a wonderful learning environment. I would like to especially thank Dr. Mingjing Xu who helped with critical experiments for the Cell Stem Cell paper that came out of this thesis. Her work ethic and love for science was very inspirational. I also want to thank Dr. Wei Xu, Dr. Danny Jeyaraju, Rose Hurren, Dr. Marcela Gronda, Mr. Neil MacLean, and Mrs. Xiaoming Wang for assisting me with experiments. I really appreciate your tireless effort to ensure that I have the resources to do my work, as well as your advice and help during the troubleshooting process. I also want to acknowledge our collaborators Dr. Juan J. Aristizabal, Dr. Ken Stark, Dr. Val Fajardo, and Dr. Paul Leblanc for your commitment to this project. Your expertise in lipid biology really pushed this work forward. Thanks to my advisory committee, Dr. Steven Chan, and Dr. Catherine O’Brien for reading all of my progress reports, and providing me with insightful comments, which helped to carry my project forward. I am very grateful for Dr. Auro Viswabandya, and Dr. Frank Michelis for letting me attend morning rounds, as well as bone marrow transplant clinics. I am also very appreciative of the late Dr. Hans Messner for giving me the opportunity to get involved in clinical research. The exposure to this extremely complicated medical specialty has inspired me to seriously pursue a carrier as a bone marrow transplant physician. I very grateful for the Canadian Institute of Health and Research and the MD/PhD program for their funding, without which this work would not be possible. I would like to especially thank my family, my father Dr. Charitha Seneviratne, my mother Mrs. Dilrukshi Seneviratne, and my sister Miss. Nayantara Seneviratne for their continuous guidance, emotional support, and encouragement during these four very testing years of graduate school. iii Your countless trips to Toronto with food to fill my fridge, and words of encouragement to fuel my spirit really kept me going. All my achievements reflect your commitment to my education. I also want to thank my new family Mr. Ravi Senanayake, Dr. Dhammika Senanayake, Mr. Jaliya Jayawardena, Mrs. Ramila Jayawardena, Master. Kanishka Jayawardena, Master. Kevaan Jayawardena, and Mr. Thimila Senanayake for their words of encouragement and support. Finally, I want to thank my wife Mrs. Uthumi Senanayake for her your support. I really appreciate the time you took to listen to my countless theories regarding my project, even though you were not familiar with the content, some of the pressing questions you asked pushed me to look at my project differently. I also want to thank you for taking the time-off from work to attend all of my talks. I appreciated your attendance not only for the sentiment, but also because it motivated me practice more, so I ended up giving a better presentation. Your tireless effort to make sure that I take time to: reflect on my achievements, travel, spend time with family and friends, and eat well, made the arduous task of completing a Ph.D. more pleasurable. I really do mean it when I say that this thesis is as much yours as it is mine. iv Contribution Statement The work in this thesis was published in Cell Stem Cell (Seneviratne et al., 2019). The majority of work presented in this thesis was done by Ayesh Seneviratne. Technical assistance was contributed as follows: Dr. Minjing Xu helped with investigating the role of phosphatidylserine decarboxylase in AML. She assisted with all experiments pertaining to this aspect of the project. Veronique Voisin performed all the bioinformatic analysis, under the supervision of Dr. Gary D. Bader. Rose Hurren provided technical assistance for qRT-PCR, under the supervision of Dr. Aaron Schimmer Mass spectrometric experiments were performed by. Dr. Juan J. Aristizabal, under the supervision of Dr. Ken Stark. Densitometric analysis of phospholipids were performed by. Dr. Val Fajardo, under the supervision of Dr. Paul Leblanc. Neil Maclean (Princess Margaret Cancer Centre, University of Toronto, Ontario, Canada) provided technical assistance with production of lentivirus for genetic knockdown experiments, under the supervision of Dr. Aaron Schimmer Rose Hurren and Xiaoming Wang (Princess Margaret Cancer Centre, University of Toronto, Ontario, Canada) performed and analyzed data for in vivo experiments, under the supervision of Dr. Aaron Schimmer v Table of Contents Acknowledgments .......................................................................................................................... iii Contribution Statement ....................................................................................................................v Table of Contents ........................................................................................................................... vi List of Tables ............................................................................................................................... xiii List of Figures .............................................................................................................................. xiv List of Abbreviations .................................................................................................................. xvii Chapter 1 ..........................................................................................................................................1 Literature Review ........................................................................................................................1 1.1 Hematopoiesis ......................................................................................................................1 1.1.1 Discovery of the Hematopoietic Hierarchy .............................................................1 1.1.2 Current Models of Hematopoiesis ...........................................................................2 1.1.3 Emergency Hematopoiesis .......................................................................................6 1.2 Leukemia ..............................................................................................................................6 1.3 Acute Myeloid Leukemia ....................................................................................................8 1.3.1 AML Pathogenesis ...................................................................................................8 1.3.2 Leukemia Stem Cells .............................................................................................10 1.3.3 Classification of AML ...........................................................................................12 1.3.4 Treatment Modalities of AML ...............................................................................17 1.3.4.1 Cytotoxic Therapies .................................................................................17 1.3.4.2 Hematopoietic Cell Transplant ................................................................18 1.3.4.3 All-trans retinoic Acid and Arsenic Trioxide (ATRA-ATO) ..................27 1.4 Energy Metabolism ............................................................................................................28 1.4.1 Catabolism .............................................................................................................28 1.4.2 Anabolism ..............................................................................................................31 vi 1.4.2.1 Nucleoside Synthesis ...............................................................................32 1.4.2.2 Lipid Synthesis ........................................................................................33 1.4.2.3 Amino Acid Synthesis .............................................................................34 1.5 Cancer Metabolism ............................................................................................................34