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Modeling and Therapeutic Development for the Tuberous Sclerosis Related Neoplasm Lymphangioleiomyomatosis

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Modeling and Therapeutic Development for the Tuberous Sclerosis Related Neoplasm Lymphangioleiomyomatosis Modeling and therapeutic development for the tuberous sclerosis related neoplasm lymphangioleiomyomatosis Sean Phillip Delaney A thesis submitted to the Faculty of Graduate and Postdoctoral Studies in partial fulfillment of the requirements for the Doctorate of Philosophy degree in Cellular and Molecular Medicine Department of Cellular and Molecular Medicine Faculty of Medicine University of Ottawa © Sean Phillip Delaney, Ottawa, Canada, 2019 Abstract The multisystemic tumors characteristic of the monogenic neoplastic diseases, tuberous sclerosis complex (TSC) and lymphangioleiomyomatosis (LAM), share common signaling aberrations upon the loss of heterozygosity in either the TSC1 or TSC2 genes. However, their physical manifestations are vastly different and can generally be classified as being either neurological (TSC) or mesenchymal (TSC & LAM; referred to herein as LAM for simplicity) in origin. In this study, I present a comprehensive stem cell model of LAM utilizing multiple TSC2 knockout (TSC2-/-) pluripotent stem cell lines differentiated to the putative cell of origin for mesenchymal tumors, neural crest cells (NCCs). TSC2-/- NCCs faithfully recapitulate LAM phenotypes and temporal RNA-seq analysis of neural and neural crest differentiation was performed to model disease pathogenesis. Analysis revealed immediate activation of stress response signaling resulting in protein aggregation and lysosome and autophagosome accumulation upon neuralization in TSC2-/- cells. This resulted in acute and lasting effects specific to neural progenitor cells (NPCs), that are transient and ameliorated in NCCs. These lineage-specific effects resulted in selective sensitization of NPCs to cell death via proteasome inhibition, suggesting a potential therapeutic avenue for neurological TSC, but not LAM. Thus, a genome-wide CRISPR knockout screen was performed in TSC2-/- NCCs. Analysis of synthetic lethal genes reveals pathways previously targeted for LAM, but provides gene-level resolution to the vulnerable nodes within these pathways. Importantly, 18 novel gene targets were identified that display synthetic lethality to TSC2-/- cells with high specificity. 3 genes within this list were targetable using commercially available small molecule inhibitors, one of which, FGFR1, shows highly selective lethal targeting of TSC2-/- NCCs. Importantly, this model system, paired with the expansive resource of transcriptomic and synthetic lethal data, serves as a foundation for the development of next generation treatment strategies for LAM, and potentially the entire spectrum of TSC manifestations. ii Authorization Adaptations, Figures, Tables: Permission to adapt content from Delaney S.P., Julian L.M., Pietrobon A., Yockell- Lelièvre J., Doré C., Wang T.T., Doyon V.C., Raymond A., Patten D.A., Harper M.E., Sun H., Stanford W.L. (2019) Stem cell models identify lineage-specific catabolic signaling, neoplastic mechanisms and therapeutic vulnerabilities in tuberous sclerosis. bioRxiv doi: https://doi.org/10.1101/683359 is granted by bioRxiv under the Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International Public License. Permission to adapt content from Delaney S.P., Julian L.M., Stanford W.L (2014) The neural crest lineage as a driver of disease heterogeneity in Tuberous Sclerosis Complex and Lymphangioleiomyomatosis. Front Cell Dev Biol. 69. doi: 10.3389/fcell.2014.00069 is granted from Frontiers under the Creative Commons Attribution 4.0 International Public License. iii Acknowledgements The first and foremost person that I owe much gratitude is Catherine Lawrence. Catherine, leading with laughs in the face of what would otherwise be serious circumstances, your efforts and attitude are simply inspiring. It‘s contagious too! Research is not easy, and when it is tough, a good hearty laugh will bring you back around; though I do not think anyone can laugh quite as whole-heartedly as you. It has always been a pleasure sharing our work with you and having a good laugh together at Laugh Out LAM. All the work I performed during my studies, in the end, was always for you. I would also like to thank the Judith R. Raymond Scholarship fund for the substantial support that I have received during my studies. The financial support greatly alleviated much worry and stress that would have otherwise occupied my mind, allowing me to focus on my studies to help advance my research and, in turn, their mission. I also thank the University of Ottawa for the generous bursaries awarded during my studies. I wish to acknowledge and thank my thesis advisory committee, Dr. Jeff Dilworth, Dr. Bernard Thébaud, and Dr. Steffany Bennett, for their guidance throughout my studies. Your valuable comments, critiques, and suggestions honed the mission of my research project and helped keep me on course within an expansive project. Very importantly, I would like to thank the members of the Stanford Lab, both past and present. Not quite knowing exactly what I was getting into, I was fortunate to have landed in such a supportive, collaborative, and productive work environment. Working with such colleagues made any hardships endurable, victories that more enjoyable, and it is a pleasure to walk in through the door every day. I would like to especially thank Dr. Lisa Julian for her valuable advice and perspective while working on this project, especially as I was rounding the final bend of my studies. I would also like to iv express my deep gratitude to Carole Doré and Angela Raymond for making sure the lab world was always spinning, and Dr. Julien Yockell-Lelièvre for his technical expertise. The amount of help I have received from them, and other members of the lab, both in and outside of work surpasses anything that I could possibly have expected, and I will always be indebted to them. One person who will undoubtedly have lasting influence in my life is my advisor, Dr. William L. Stanford. Bill, I thank you for bringing me into your lab and providing me with a research project in which I could work with independence to develop my skills as a scientist. Your enthusiasm for pursuing the unknown and pushing the limits of our capabilities has challenged me throughout my PhD project, and I believe has made me a better researcher. You have always pushed for excellence and believed in my capabilities, and I have enjoyed trying my best to deliver. I thank you for your mentorship. Whenever I asked for help or advice, you would, without fail, take the time to properly consider and offer whatever council you could. Throughout all of the challenges I, along with my family, have faced over the years, your patience, understanding, and generosity have been most greatly appreciated. We thank you. I would also like to thank my father, for always instilling the value of education and his constant support throughout the many long years of university. I wouldn‘t be in this mess if it wasn't for you, and I‘m grateful for it. Finally, I would like to quite especially thank my wife, Trischa. I thank you for pushing me out of the plane (figuratively), over and over again in life. Your efforts to pull me back into the reality that is the world outside of work have always been appreciated. You give me perspective and remind me of what counts. You are always supportive, always my advocate, and always a comfort when it all seems to spiral. I thank you for your patience throughout these years. I dedicate this to you, and those two wonderful (not so) little knuckleheads of ours. v Table of Contents Abstract ........................................................................................................................... ii Authorization .................................................................................................................. iii Acknowledgements ........................................................................................................ iv List of Tables .................................................................................................................. ix List of Figures .................................................................................................................. x List of Abbreviations ...................................................................................................... xii Preface ......................................................................................................................... xvi Chapter 1: General Introduction ..................................................................................... 1 1.1: Tuberous sclerosis complex and lymphangioleiomyomatosis .............................. 2 1.1.1 The TSC Genes ............................................................................................. 5 1.1.2 The mTOR pathway and the TSC1/2 complex ..............................................10 1.1.3 Molecular characteristics of TSC and LAM tumors ........................................14 1.1.4 Current treatment options for TSC and LAM .................................................15 1.2 The Cells of Origin for TSC and LAM ...................................................................17 1.2.1 The Cell of Origin for Neurological TSC Manifestations.................................20 1.2.2 The neural crest as a putative cell of origin for mesenchymal TSC and LAM 21 1.2.3 The embryonic neural crest cell of origin theory ............................................25 1.2.4 The adult neural crest cell of origin theory .....................................................26
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