CRISPR Activation Screening of Circulating Tumor Cells Identifies Enhancers of Blood-Based Metastasis The Harvard community has made this article openly available. Please share how this access benefits you. Your story matters Citation Ebright, Richard Yon. 2020. CRISPR Activation Screening of Circulating Tumor Cells Identifies Enhancers of Blood-Based Metastasis. Doctoral dissertation, Harvard University, Graduate School of Arts & Sciences. Citable link https://nrs.harvard.edu/URN-3:HUL.INSTREPOS:37365157 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 CRISPR activation screening of circulating tumor cells identifies enhancers of blood-based metastasis A dissertation presented by Richard Yon Ebright 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 September 2019 © 2019 Richard Yon Ebright All rights reserved. Dissertation Advisors: Daniel A. Haber & Shyamala Maheswaran Richard Yon Ebright CRISPR activation screening of circulating tumor cells identifies enhancers of blood-based metastasis Abstract Over ninety percent of cancer mortality is attributable to metastasis, most commonly due to the blood-borne dissemination of cancer cells from a primary tumor to secondary tissues. However, the vast majority of these cancer cells in the circulation, known as circulating tumor cells (CTCs), never go on to form clinically relevant metastases, instead dying or senescing in the circulation or at distant sites. As such, identification of factors that promote the metastatic ability of CTCs will shed light upon CTC biology, as well as suggest novel targets for therapeutic intervention to prevent or slow metastatic growth. We conducted in vivo genome-wide CRISPR activation screens to identify genes capable of accelerating distal metastasis by breast cancer patient-derived circulating tumor cells following direct intravascular inoculation in mice. From these screens, we identified and conducted follow-up studies on two classes of hits: ribosomal proteins, primarily RPL15, and transcriptional regulators, primarily ID3. Expression of RPL15, a component of the large ribosome subunit, is sufficient to increase metastatic growth in multiple organs. RPL15 overexpression selectively increases translation of cell cycle regulators and other ribosomal proteins. Unsupervised analysis of single-cell RNA sequencing of freshly isolated CTCs from breast cancer iii patients identifies a subset with strong ribosomal and protein translation signatures, correlated with increased proliferative markers, CTC clusters and poor clinical outcome. Thus, ribosome protein expression identifies an aggressive subset of CTCs, whose therapeutic targeting may suppress metastatic progression. Expression of ID3, an inhibitor of bHLH transcription factors, is also sufficient to increase metastatic growth in multiple organs. siRNA screening of bHLH transcription factors inhibited by ID3 identifies knockdown of ARNTL2, a transcription factor involved in circadian clock signaling, as able to increase CTC growth. Clinically, ID3 and ARNTL2 expression are correlated with worse and better outcome, respectively, in triple negative breast cancer, matching our in vitro and in vivo results. Thus, ID3 and ARNTL2 represent novel genes involved in breast cancer metastasis, and dysregulation of circadian clock signaling is implicated in progression through the metastatic cascade. iv Table of Contents 1. Title Page………………………………………………………..…………………..……………i 2. Copyright…………………………………………………………………………………....……ii 3. Abstract…………………………………………………………………….…………………….iii 4. Table of Contents……………………………………………………………………..…………v 5. Acknowledgements…………………………………………………………………..…………vi 6. Main Text 1. Introduction………………………………………………………………………1 2. Genome-wide in vivo CRISPR activation screening in CTCs identifies enhancers of metastasis………………………………...………………….…19 3. Deregulation of ribosomal protein expression and translation promotes breast cancer metastasis……………………………………………………...37 4. Deregulation of ID3 and circadian clock signaling promotes breast cancer metastasis……………………………...…………………..………...………...78 5. Conclusion…………………………………………………………...............107 v Acknowledgements Throughout the past three years of my PhD, I’ve received an incredible amount of support, guidance, and mentorship from my thesis advisers, Drs. Daniel Haber and Shyamala Maheswaran. They’ve unconditionally supported my development as a physician- scientist every step of the way, and I’m extraordinarily thankful and grateful for having had the opportunity to work in their laboratory. I look forward to continuing to learn from Daniel and Shyamala in the years to come as I continue through med school and into a career in academic medicine. Along with Daniel and Shyamala, my dissertation committee has provided invaluable guidance and support; the combined advice of Drs. Raul Mostoslavsky, Lee Zou, and Mario Suvá helped me learn to focus my efforts on the pressing questions for each of the projects I’ve worked on, and their recommendations helped elevate the quality of the projects as well. I am thankful to the hugely collaborative MGH Cancer Center environment, where collaborators such as Drs. David Ting, Shobha Vasudevan, Willi Haas, Aditya Bardia, and Mehmet Toner come together to work at the intersection of cancer biology, medicine, and bioinformatics, and whose generosity with time and resources has allowed our projects to answer questions in cancer biology more deeply and fully. Celia Harris, our administrative wizard, has always helped me at every turn to get all these people in the same room, and to keep us well fed, too. Similarly, the Harvard-MIT MD/PhD Program and Harvard Biological and Biomedical Sciences PhD Program have served as vi tremendous sources of friends and mentors, with special thanks to Dr. Loren Walensky and Amy Cohen for always being in my corner. I owe every member of the laboratory my deep gratitude, but I want to especially thank Drs. Doug Micalizzi, Marcus Zachariah, Hongshan Guo, Taronish Dubash, Nicole Vincent-Jordan, and Matteo Ligorio for their guidance and support in molecular biology and mouse experiments, and Drs. Ben Wittner, Linda Nieman, Vishal Thapar, and newly- minted-Dr. Mark Kalinich for continually making up for my lack of computational skills. Doug deserves an additional shout-out for our very close collaboration on the ribosome project, and Mark deserves an additional shout-out for keeping me sane (or going insane with me) during the last few years as the lab’s other grad student. It has also been a privilege to work with many of the technicians as well, including Kira Niederhoffer, Devon Wiley, Ben Wesley, Ben Nicholson, Rushil Desai, Ryan O’Keefe, Joseph LiCausi, Brian Chirn, and Andy Mai, and I look forward to watching their own careers continue to grow. In particular, it has been a privilege and blessing of the last two years to work closely with and mentor Kira, whose technical expertise kept our projects afloat and whose inquisitive nature made working together a joy. Frequently, I was the one learning from her, not the other way around. Finally, the support of my family and friends has, of course, been crucial to surviving and thriving throughout my PhD. I cannot thank them enough for understanding that I was too busy with lab to spend time together, and then dragging me out of lab anyway for international trips, music festivals, and everything in between. vii Chapter 1 1 Introduction: Metastasis, the process of tumorigenic spread from a primary tumor to secondary sites, is responsible for the vast majority of cancer deaths, as metastatic disease is incurable in the vast majority of patients. Cancer cells progressing through the metastatic cascade which have entered into the bloodstream, known as circulating tumor cells (CTCs), represent an intermediate stage of metastasis and face cellular challenges unique to the vasculature, including shear and oxidative stresses and the lack of growth factors or cytokines. As such, the vast majority of CTCs die in the circulation. Those that survive and successfully exit the bloodstream need to be competent to proliferate at distant sites, but the vast majority of such disseminated tumor cells never form metastatic lesions. The low frequency of cancer cells being capable of forming metastases raises the question of the identity of genes and pathways that promote disseminated cells to successfully establish metastatic tumors. Identification of such metastasis-promoting genes and pathways or signaling nodes will shed light on biological pathways coopted by cancer and will identify targets for pharmacological intervention aimed at limiting metastatic spread. To that end, this thesis describes a genome-wide search for genes capable of enhancing the metastatic potential of CTCs and highlights two targets identified through that search. Chapter 1 reviews our current knowledge of the metastatic cascade, with a particular focus on the role of CTCs. Given the key role of bloodborne dissemination in the spread 2 of cancer, our group has focused on CTC biology for the last fifteen years, developing tools to isolate, analyze, and culture patient-derived CTCs. Chapter 1 also describes these