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Genetic and Chemical Genetic Approaches to Identifying Liabilities in Malignant Brain Cancer

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Genetic and Chemical Genetic Approaches to Identifying Liabilities in Malignant Brain Cancer Genetic and Chemical Genetic Approaches to Identifying Liabilities in Malignant Brain Cancer by David Tieu A thesis submitted in conformity with the requirements for the degree of Master of Science Department of Molecular Genetics University of Toronto © Copyright by David Tieu 2019 Genetic and Chemical Genetic Approaches to Identifying Liabilities in Malignant Brain Cancer David Tieu Master of Science Department of Molecular Genetics University of Toronto 2019 Abstract Glioblastoma (GBM) remains the most common primary malignant brain tumor in adults. Patients face a dismal prognosis despite efforts to identify novel therapeutic targets. Following conventional therapy, patients relapse with treatment-refractory GBM, which remains largely unexplored. This thesis explores the functional genetic landscape of recurrent GBM (rGBM) using unbiased CRISPR-Cas9 knockout screening. Negative selection screens in two patient- derived rGBM cell models provided evidence for the essential role of EGLN1, PTP4A2 and TIMELESS in treatment-refractory GBM. Pursuing the idea of identifying targets in rGBM, I explored modulators of BMI1, a master-regulator of brain cancer tumorigenesis. I carried out a chemical genetic screen to identify perturbations that sensitize cells to the effects of PTC-028, a small molecule thought to inhibit BMI1. I validated the loss of WNK1 gene as the strongest positive chemical-genetic interaction with PTC-028. In summary, this thesis describes new rGBM genetic vulnerabilities and sheds light on the mode of action of PTC-028. ii Acknowledgments I would like to express my gratitude to my Principal Investigator Dr. Jason Moffat of the Faculty of Medicine at the University of Toronto, who supported me throughout the course of my degree and provided me with excellent opportunities to conduct my research. I am thankful for his aspiring guidance, invaluably constructive criticism and friendly advice through my research and thesis writing. In addition, I must also thank my committee members Drs. David Kaplan and Scott Gray-Owen and my advisor Dr. Sheila Singh at McMaster University, who have challenged me and provided me with their insights into my projects. Without their regular support, I would not have experienced the same success. I would like to especially thank my collaborator Chirayu Chokshi from the Singh lab at McMaster University for his consistent support and scientific discussions to forward the project. We powered together through long days and nights to complete screens and analyses. I would like to acknowledge my colleagues in the Moffat lab for their thought-provoking discussions and willingness to provide me with protocols and ideas. Every one of you has provided me with immense support. I am particularly grateful for my family, who have supported me in my pursuit of higher education and allowed me the privilege to follow my dreams. I would also like to thank my partner Lisa Shao, who has been with me on this journey since the very beginning and providing me with unfailing support and continuous encouragement. Finally, I am thankful for my friends, who were of great support in providing a distraction to refresh my mind outside of my research. iii Table of Contents Acknowledgments ................................................................................................................. iii Table of Contents ................................................................................................................... iv List of Tables ......................................................................................................................... vii List of Figures ....................................................................................................................... viii List of Appendices ................................................................................................................... x List of Abbreviations .............................................................................................................. xi Chapter 1 ................................................................................................................................ 1 Introduction ............................................................................................................................ 1 Chapter One: Introduction ............................................................................................... 2 1.1 Introduction to Glioblastoma .............................................................................................. 2 1.1.1 Glioblastoma (GBM): Burden of the disease ......................................................................................... 2 1.1.2 Current treatments, biomarkers and actionable targets ...................................................................... 3 1.1.3 Major pathways involved in the pathogenesis of primary and secondary GBM .................................. 5 1.1.4 Brain tumor initiating cells (BTICs) ........................................................................................................ 6 1.1.5 PTC-028 as a therapeutic molecule targeting the BTIC population ...................................................... 7 1.1.6 Differences between untreated primary and treatment refractory recurrent GBM ............................ 9 1.2 Introduction to genome-wide CRISPR-Cas9 functional screens ........................................... 10 1.2.1 Genome-wide CRISPR-Cas9 functional screens in mammalian cells .................................................. 10 1.2.2 Fitness and genetic liabilities: Core vs. context-dependent essential genes ...................................... 11 1.2.3 Chemogenomic profiling using genome-wide CRISPR-Cas9 screens: Identifying sensitizers and suppressors of small molecules ........................................................................................................................ 13 1.3 Thesis rationale ................................................................................................................. 14 1.3.1 Goals and objectives ........................................................................................................................... 14 Chapter 2 .............................................................................................................................. 16 Functional genetic screen in rGBM ........................................................................................ 16 Chapter Two: Exploring the genetic landscape of rGBM ................................................. 17 2.1 Introduction ...................................................................................................................... 17 iv 2.2 Materials and Methods ..................................................................................................... 17 2.2.1 Cell culture .......................................................................................................................................... 17 2.2.2 Primary cell line characterizations ...................................................................................................... 18 2.2.3 Pooled genome-wide CRISPR-Cas9 screens using the TKOv3 library .................................................. 19 2.2.4 Cloning individual sgRNA into lentiCRISPRv2 vector ........................................................................... 19 2.2.5 Lentivirus production .......................................................................................................................... 20 2.2.6 Screenability and editing efficiency assessment ................................................................................. 20 2.2.7 Multiplicity of infection estimation ..................................................................................................... 21 2.2.8 Pooled genome-wide CRISPR dropout screens in GBM cells .............................................................. 21 2.2.9 Bayesian Analysis of Gene EssentiaLity ............................................................................................... 22 2.2.10 Gene Set Enrichment Analysis ........................................................................................................ 22 2.2.11 Gene knockouts using lentiCRISPRv2 lentiviral vectors ................................................................. 23 2.2.12 PrestoBlue assay ............................................................................................................................ 23 2.2.13 Sphere formation assay (in vitro limiting dilution assay) ............................................................... 23 2.3 Results .............................................................................................................................. 24 2.3.1 Editing efficiency of CRISPR-Cas9 in GBM cell lines ............................................................................ 24 2.3.2 Estimating relative lentiviral infection rates for BT241 and BT972 ..................................................... 25 2.3.3 CRISPR-Cas9 sequencing quality control ............................................................................................. 26 2.3.4 BAGEL analysis .................................................................................................................................... 27 2.3.5 Gene set enrichment analysis (GSEA) ................................................................................................
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