Genetic and Chemical Modifiers of EGFR Dependence in Non- Small Cell Lung Cancer The Harvard community has made this article openly available. Please share how this access benefits you. Your story matters Citation Sharifnia, Tanaz. 2014. Genetic and Chemical Modifiers of EGFR Dependence in Non-Small Cell Lung Cancer. Doctoral dissertation, Harvard University. Citable link http://nrs.harvard.edu/urn-3:HUL.InstRepos:11745725 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 Genetic and Chemical Modifiers of EGFR Dependence in Non-Small Cell Lung Cancer A dissertation presented by Tanaz Sharifnia 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 December 2013 © 2013 Tanaz Sharifnia All rights reserved. Dissertation Advisor: Professor Matthew Meyerson Tanaz Sharifnia Genetic and Chemical Modifiers of EGFR Dependence in Non-Small Cell Lung Cancer ABSTRACT The term ‘oncogene addiction’ has been used to describe the phenomenon whereby tumor cells exhibit singular reliance on an oncogene or oncogenic pathway for their survival, despite the accumulation of multiple genetic lesions. In non-small cell lung cancer (NSCLC), this principle is perhaps best exemplified with the finding that epidermal growth factor receptor (EGFR) mutations predict response to EGFR-targeted therapies and thus represent a dependency in the subset of tumors harboring these alterations. Yet while EGFR-mutant tumors often respond dramatically to EGFR inhibition, nearly 30% of cases are refractory to therapy at the outset, and all responsive patients ultimately develop resistance to therapy. A deeper understanding of the genetic underpinnings of EGFR dependence, and of the mechanisms by which EGFR-mutant cells can overcome addiction to EGFR, may improve clinical outcomes. In this work, we have applied systematic functional screening approaches to identify modifiers of EGFR dependence in NSCLC. Recent advances in large-scale functional screening libraries have made it possible to query a wide-range of genetic or chemical perturbations for their ability to modulate specific cellular phenotypes. Using the model of EGFR-mutant, erlotinib- sensitive NSCLC cells, we have performed systematic open reading frame (ORF)- and shRNA- based screens to identify genetic perturbations that can complement loss of EGFR in an EGFR- dependent context. Additionally, by integrating screening findings with an unbiased, gene- expression-based approach, we have identified chemical compounds that can modulate the degree to which cells rely on EGFR. Our findings indicate broad potential for EGFR substitution iii in the setting of EGFR dependence, with compensatory mechanisms commonly conferring EGFR-independent activation of the phosphoinositide 3-kinase (PI3K)-AKT and MEK-ERK signaling pathways. These data support the idea that the EGFR-addicted state can be redundantly driven by diverse genetic inputs that commonly converge on shared downstream signaling nodes. iv TABLE OF CONTENTS Preface .......................................................................................................................................... i Abstract .................................................................................................................................. iii Table of contents ................................................................................................................... v Acknowledgements ............................................................................................................... vii Introduction: EGFR Dependence in Non-Small Cell Lung Cancer ......................................... 1 Summary ............................................................................................................................... 2 Lung cancer classification and treatment paradigms ............................................................ 2 Overview ...................................................................................................................... 2 Histological classification of lung cancer ...................................................................... 3 Conventional treatments for non-small cell lung cancer .............................................. 4 Oncogene addiction and targeted therapies ................................................................ 5 Molecular classification of lung cancer ........................................................................ 5 EGFR oncogene addiction in non-small cell lung cancer ..................................................... 8 The ErbB family of receptor tyrosine kinases .............................................................. 8 EGFR signaling and oncogene addiction ..................................................................... 8 Resistance to EGFR-targeted therapies ............................................................................. 10 Primary resistance ..................................................................................................... 10 Acquired resistance ................................................................................................... 11 Secondary mutations in EGFR ....................................................................... 11 Bypass mechanisms ....................................................................................... 13 Histological transformation .............................................................................. 15 Context for the current work and thesis summary .............................................................. 15 References .......................................................................................................................... 17 Chapter 1: Diverse Kinase Genes Can Induce EGFR-Inhibitor Resistance ......................... 27 Abstract ............................................................................................................................... 28 Introduction ......................................................................................................................... 28 Results ................................................................................................................................ 30 Discussion ........................................................................................................................... 46 Materials and Methods ........................................................................................................ 49 References .......................................................................................................................... 53 Chapter 2: A Gene-Expression-Based Approach to Identify Chemical Modifiers of EGFR Dependence ............................................................................................................................... 57 Abstract ............................................................................................................................... 58 Introduction ......................................................................................................................... 58 Results ................................................................................................................................ 60 Discussion ........................................................................................................................... 74 Materials and Methods ........................................................................................................ 75 References .......................................................................................................................... 79 Chapter 3: A Genome-Scale shRNA Screen for Mediators of EGFR-Inhibitor Response .. 84 Abstract ............................................................................................................................... 85 Introduction ......................................................................................................................... 85 Results ................................................................................................................................ 86 v Discussion ......................................................................................................................... 100 Materials and Methods ...................................................................................................... 100 References ........................................................................................................................ 105 Conclusions ............................................................................................................................ 108 Appendix .................................................................................................................................. 114 vi ACKNOWLEDGEMENTS This work would not have been possible without the strong support and key contributions of many people over the last several years. First, I would like to acknowledge and thank my advisor, Matthew Meyerson, for his sage mentorship, his open and boundless approach to science, and his enthusiastic and unwavering