Investigation of KRAS Dependency Bypass and Functional Characterization of All Possible KRAS Missense Variants The Harvard community has made this article openly available. Please share how this access benefits you. Your story matters Citable link http://nrs.harvard.edu/urn-3:HUL.InstRepos:40050098 Terms of Use This article was downloaded from Harvard University’s DASH repository, and is made available under the terms and conditions applicable to Other Posted Material, as set forth at http:// nrs.harvard.edu/urn-3:HUL.InstRepos:dash.current.terms-of- use#LAA Investigation of KRAS Dependency Bypass and Functional Characterization of All Possible KRAS Missense Variants A dissertation presented by Seav Huong Ly to The Division of Medical Sciences in partial fulfillment of the requirements for the degree of Doctor of Philosophy in the subject of Biological and Biomedical Sciences Harvard University Cambridge, Massachusetts April 2018 © 2018 Seav Huong Ly All rights reserved. Dissertation Advisor: William C. Hahn Seav Huong Ly Investigation of KRAS Dependency Bypass and Functional Characterization of All Possible KRAS Missense Variants Abstract The importance of oncogenic KRAS in human cancers have prompted intense efforts to target KRAS and its effectors. To anticipate the development of resistance to these strategies, we previously performed a genome-scale expression screen to identify genes that bypass KRAS oncogenic dependency. Here we test thirty-seven genes that scored over five standard deviations and find that overexpression of LIM homeobox 9 (LHX9), a transcription factor involved in embryonic development, robustly rescues the suppression of KRAS in vitro and xenograft models. Furthermore, LHX9 substantially decreases cell sensitivity to KRASG12C and MEK1/2 inhibitors in KRAS-dependent cells. Based on RNA-seq and ChIP-seq analysis, we observe that LHX9 promotes both KRAS-associated and KRAS-unassociated expression profiles. Importantly, we show that LHX9-mediated upregulation of STAT3 and YAP1 expression is a major contributor to the rescue of KRAS suppression. Together these observations identify LHX9 as a transcription factor that regulates pathways that permit proliferation of KRAS-dependent cells following KRAS suppression. KRAS mutation is the major genetic alteration responsible for its oncogenicity, Although the majority of KRAS mutations occur at codon 12, 13, and 61, mutations that alter other positions in KRAS are found frequently in human cancers, and these variants of unknown significance (VUS) complicate the interpretation of clinical cancer profiling. Here we performed saturation mutagenesis of KRAS by creating a library of all possible alleles of KRAS. We characterized the transforming ability of each variant by measuring its ability to confer anchorage-independent growth to an immortalized human cell line. The strength of the observed transformation iii phenotypes correlates strongly with the frequency of observed mutations in human cancers. In addition, we identified alleles that are rarely mutated but exhibit a strong transformation phenotype and other alleles that do not appear to exhibit transforming functions. These observations provide strong evidence that oncogenic KRAS alleles are selected during tumorigenesis and establish a comprehensive resource to infer the significance of KRAS alleles identified by molecular analysis of human tumors. iv Table of Contents Abstract Table of Contents Acknowledgements CHAPTER ONE Introduction 1.1 Overview……………………………………………………………………………………………...2 1.2 RAS biology………………………………………………………………………………………….4 1.2.1 RAS function regulation…………………………………………………………………..5 1.2.2 RAS downstream effectors…….……………………………….………………………..7 1.2.3 RAS regulates cell proliferation….………………………………………………………9 1.3 RAS and cancers………………………………………………………………………………….10 1.3.1 RAS pathway dysregulation…………………………………………………………….11 1.3.2 RAS dependency………………………………………………………………………...13 1.3.3 RAS mutations and cancers……………………………………………………………17 CHAPTER TWO Investigation of KRAS dependency bypass 2.1 Introduction………………………………………………………………………………………….23 2.2 Results……………………………………………………………………………………………….25 2.3 Discussion…………………………………………………………………………………………...45 2.4 Materials and Methods……………………………………………………………………………..47 v CHAPTER THREE Functional characterization of all possible KRAS missense variants 3.1 Introduction………………………………………………………………………………………….53 3.2 Results……………………………………………………………………………………………….56 3.3 Discussion…………………………………………………………………………………………...77 3.4 Materials and Methods……………………………………………………………………………..82 CHAPTER FOUR Conclusion…………………………………………………………………………………………...…87 APPENDIX 92 REFERENCES 96 vi Acknowledgements I am very fortunate and privileged to have so much support from the Hahn lab, school, friends, and family. First and foremost, I am grateful for my mentor Bill Hahn. He gave me opportunities to learn, explore, and pursue my scientific interests. Along the way, he gave many needed scientific and life advice; “The long way is the short way” and “Do things because they are the right thing to do and not because they are what we can do” are some of the most helpful advice he gave. Additionally, he shows by example what it takes to be a great scientist, mentor, and leader. He has paved the way and established a great environment made of first-class financial/technical resources and outstanding and nice people, all of which allows me to do good science, become a good scientist, team member, and person. His mentorship and support allow me to accomplish my scientific and personal goals. Members of Hahn laboratory have been extremely supportive both scientifically and personally. I am especially grateful for Chao Dai, Nina Ilic, Justin Hwang, Ji Li, Andrew Hong and Andrew Giacomelli and previous members of Hahn lab Nicole Spardy, Joyce T. O’Connell, Elsa Krall, Belinda Wang, Eejung Kim, and Andrew Aguirre. They have been extraordinarily generous with their time, concern, knowledge, skill, and insights. They teach me benchwork techniques from pipetting and western blot tricks to procedures for RNA-seq library preparation and working with mice. They show me shortcuts and tricks in using Excel and big data analysis. They share their perspectives on the what and how to do good science and be a good scientist. They give encouragement and troubleshooting advice when experiments do not go well and cautions when my excitement goes overboard. I have also enjoyed our lab lunch discussion on topics ranging from science, politics, and life and I am thankful for their personal stories and advices that they kindly share. I would like to thank my collaborators who have contributed their time, scientific expertise and insights to our projects. Many people at the Genomic Perturbation Platform at the Broad vii Institute are instrumental to our projects. Special thanks go to Xiaoping Yang and Robert Lintner who are at the core of saturation mutagenesis library construction and sequencing. I am thankful for my Dissertation Advisory Committee – Dr. David Kwiatkowski, Dr. Kevin Haigis, Dr. Karen Cichowski, Dr. Peter Hammerman for their dedication, feedbacks, advice, and concerns. Additionally, I would like to thank my Dissertation Defense Committee – Dr. Kevin Haigis, Dr. Bruce Zetter, Dr. Larry Feig, Dr. Rameen Beroukhim for their time, efforts, and interests. Advisors Connie Cepko and Thomas Michel and friends from the Leder Human Biology and Translational Medicine (LHBTM) program and the Biological and Biomedical Science (BBS) program have been another source of support throughout my graduate studies. I want to thank my friends: Sovan Srun, Sotheary Sor, Rasi Yi, Mengheng Touch, Heng Sok, Chhay Ou Hak, and many others who have always been here for me despite the physical distance among us. I would like to especially thank my high school biology teacher Kim Eang Chea, a dedicated teacher who first instilled my appreciation and passion for biology. I am grateful for my aunt Nary Ly, a genocide survivor, a scientist, the first Cambodian woman marathon runner, and the first to represent Cambodia at the Olympic games. She was the first in our extended family to pursue higher education and also the first to get a PhD despite all the adversity and trauma that she had experienced. In doing so, she paved the way for me to higher education and to here. Her strength and resilience inspire me to never give up and her determination and ambition move me to do better and aim higher. Last but not least, I am eternally grateful to my family for this journey and everything in life. I am grateful for my mum who did not have the opportunity to even finish primary school and embraces my science pursuit, my dad whose dream to become a physician got deferred not because he did not work hard enough or was not smart enough or unkind but just because he was born at the wrong time and place, my brother Lucky who was born younger and so had to give up his chance at good education, and all their sacrifice so that I can be here pursuing science and my dream. viii To dad, mum, Lucky, and aunt Nary ជូនជំេះ េកពុក អក យ បនបស ក់ឃី និងមីងរ ix CHAPTER ONE Introduction 1.1 Overview RAS is a superfamily of small GTPase that is evolutionary conserved from budding yeast (Dhar et al. 1984; Powers et al. 1984) to human (Cox and Der 2010) (Figure 1.1). In human, RAS family is composed of four isoforms – HRAS, NRAS, KRAS-4A, and KRAS-4B. RAS plays many important biological roles and thus its function is regulated at multiple levels including post-translational modification, activation,