Cellular features predicting susceptibility to ferroptosis: insights from cancer cell-line profiling Vasanthi Sridhar Viswanathan Submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy in the Graduate School of Arts and Sciences COLUMBIA UNIVERSITY 2015 © 2015 Vasanthi Sridhar Viswanathan All rights reserved ABSTRACT Cellular features predicting susceptibility to ferroptosis: insights from cancer cell-line profiling Vasanthi Sridhar Viswanathan Ferroptosis is a novel non-apoptotic, oxidative form of regulated cell death that can be triggered by diverse small-molecule ferroptosis inducers (FINs) and genetic perturbations. Current lack of insights into the cellular contexts governing sensitivity to ferroptosis has hindered both translation of FINs as anti-cancer agents for specific indications and the discovery of physiological contexts where ferroptosis may function as a form of programmed cell death. This dissertation describes the identification of cellular features predicting susceptibility to ferroptosis from data generated through a large-scale profiling experiment that screened four FINs against a panel of 860 omically-characterized cancer cell lines (Cancer Therapeutics Response Portal Version 2; CTRPv2 at http://www.broadinstitute.org/ctrp/). Using correlative approaches incorporating transcriptomic, metabolomic, proteomic, and gene-dependency feature types, I uncover both pan-lineage and lineage-specific features mediating cell-line response to FINs. The first key finding from these analyses implicates high expression of sulfur and selenium metabolic pathways in conferring resistance to FINs across lineages. In contrast, the transsulfuration pathway, which enables de novo cysteine synthesis, appears to plays a role in ferroptosis resistance in a subset of lineages. The second key finding from these studies identifies cancer cells in a high mesenchymal state as being uniquely primed to undergo ferroptosis. This susceptibility stems from a specific dependency of high mesenchymal-state cancer cells on the lipid hydroperoxide quenching mechanisms inhibited by FINs and is conserved across cancer cell lines of mesenchymal origin, epithelial-derived cancer cell lines that have undergone an epithelial-to-mesenchymal-transition, and patient-derived cancer cells exhibiting mesenchymal state-mediated resistance to anti-cancer therapies. The work presented herein formalizes frameworks for studying small molecule inducers of cell death through cell-line profiling. The results advance current mechanistic understanding of the cellular circuitry underlying ferroptosis sensitivity and lay the foundation for a novel therapeutic approach using ferroptosis inducers to target high mesenchymal-state cancer cells. TABLE OF CONTENTS LIST OF FIGURES AND TABLES ......................................................................................... iv LIST OF ABBREVIATIONS ....................................................................................................vii ACKNOWLEDGEMENTS ................................................................................................... .viii DEDICATION ......................................................................................................................... .x CHAPTER I. INTRODUCTION ........................................................................................ ..1 I. Cell death .......................................................................................................................... ..1 i. Classification of cell death .................................................................................... ..1 ii. Major forms of cell death ...................................................................................... ..3 iii. Ferroptosis............................................................................................................. ..7 II. Study of cell death ............................................................................................................17 i. Small molecules as probes of cell death ...............................................................17 ii. Cell-line profiling as a systematic method for studying cell death inducers ........19 III. Cancer and cell death ........................................................................................................23 i. Dysregulation of apoptosis ...................................................................................23 ii. Chemoresistance ...................................................................................................24 iii. The transformed mesenchymal cell state ..............................................................25 CHAPTER II. Omic predictors of cancer-cell response to ferroptosis inducers ............28 I. Introduction .......................................................................................................................28 II. Results ...............................................................................................................................30 i. Performance of ferroptosis inducers within CTRPv2 ...........................................30 ii. Lineage effects ......................................................................................................32 iii. Omic correlates of cell-line response to ferroptosis inducers ...............................36 III. Discussion ........................................................................................................................44 i i. Epithelial versus mesenchymal origin .................................................................44 ii. Molecular modulators of ferroptosis ....................................................................47 iii. Role of PI3K-AKT-mTOR pathway ....................................................................52 IV. Materials and methods .....................................................................................................53 V. Acknowledgements ..........................................................................................................55 CHAPTER III. Transformed mesenchymal state induces sensitivity to ferroptosis inducers ........................................................................................................... ..56 I. Abstract ............................................................................................................................57 II. Body of text......................................................................................................................58 III. Figures..............................................................................................................................66 IV. Methods............................................................................................................................83 V. Acknowledgements ..........................................................................................................91 CHAPTER IV. Transsulfuration mediates cancer cell-line dependence on cystine import .................................................................................................................94 I. Abstract ............................................................................................................................95 II. Body of text......................................................................................................................95 III. Figures............................................................................................................................ 104 IV. Methods.......................................................................................................................... 115 V. Acknowledgements ........................................................................................................ 119 CHAPTER V. Perspectives and future directions .......................................................... 121 I. Summary ........................................................................................................................ 121 II. Mesenchymal cell state and GPX4 ................................................................................ 122 III. Therapeutic implications ..................................................................................................123 ii IV. Ferroptosis as programmed cell death .............................................................................124 V. Electrophilic ROS inducers and cell-line profiling ..........................................................126 References .............................................................................................................................128 Appendix I. Extended omic correlation data ........................................................................146 Appendix II. Mesenchymal score correlation for compounds in CTRPv2 ..........................190 Appendix III. Gene-expression data for MCF-7 ER-Snail-1 ...............................................204 iii LIST OF FIGURES Figure 1.1. Structures of FINs..................................................................................................10 Figure 1.2. Ferroptosis pathway...............................................................................................11 Figure 1.3. Lipid peroxidation cycle ........................................................................................14 Figure 2.1. FINs share similar patterns of activity