CRISPRi Screens to Identify Combination Therapies for the Improved Treatment of Ovarian Cancer By Erika Daphne Handly B.S. Chemical Engineering Brigham Young University, 2014 Submitted to the Department of Biological Engineering in partial fulfillment of the requirements for the degree of Doctor of Philosophy in Biological Engineering at the MASSACHUSETTS INSTITUTE OF TECHNOLOGY February 2021 © 2020 Massachusetts Institute of Technology. All rights reserved. Signature of author………………………………………………………………………………… Erika Handly Department of Biological Engineering February 2021 Certified by………………………………………………………………………………………… Michael Yaffe Director MIT Center for Precision Cancer Medicine Department of Biological Engineering and Biology Thesis Supervisor Accepted by………………………………………………………………………………………... Katharina Ribbeck Professor of Biological Engineering Chair of Graduate Program, Department of Biological Engineering Thesis Committee members Michael T. Hemann, Ph.D. Associate Professor of Biology Massachusetts Institute of Technology Douglas A. Lauffenburger, Ph.D. (Chair) Ford Professor of Biological Engineering, Chemical Engineering, and Biology Massachusetts Institute of Technology Michael B. Yaffe, M.D., Ph.D. (Thesis Supervisor) David H. Koch Professor of Science Prof. of Biology and Biological Engineering Massachusetts Institute of Technology 2 CRISPRi Screens to Identify Combination Therapies for the Improved Treatment of Ovarian Cancer By Erika Daphne Handly B.S. Chemical Engineering Brigham Young University, 2014 Submitted to the Department of Biological Engineering in partial fulfillment of the requirements for the degree of Doctor of Philosophy in Biological Engineering ABSTRACT Ovarian cancer is the fifth leading cause of cancer death for women in the United States, with only modest improvements in patient survival in the past few decades. Standard-of-care consists of surgical debulking followed by a combination of platinum and taxane agents, but relapse and resistance frequently occur. To identify genes that confer sensitivity or resistance in tumor cells treated with platinum chemotherapeutics, I performed genome-wide screens combining cisplatin or oxaliplatin with CRISPRi pooled gene knockdowns. Screens were analyzed at 9-days to mimic patient care, and at 48-hours to isolate the short-term DNA damage response. Genes whose knockdown caused sensitivity to the platinum chemotherapeutics were identified through a multi-objective optimization approach to account for knockdown efficiencies and variances in sequencing depth. To filter the noise in the genome-wide screen and more confidently identify ‘hits,’ a smaller pooled CRISPRi screen of four hundred targets was designed, and a few ‘hits’ were validated. Interestingly, knockdown of FAAP24, a component of the FA core complex, was found to sensitize multiple ovarian cancer cells to platinum compounds, and thus may be a promising candidate for a combination treatment with oxaliplatin and cisplatin. Chapter 5 details an implementation of a combination therapy with cisplatin using peptide nanoparticles. Peptide nanoparticles are a promising therapeutic for the delivery of siRNA and allow for targeting of specific proteins that are difficult to inhibit with small molecular inhibitors; specifically, nanoplexes allowed for the targeting of the REV3 protein, the catalytic component of the translesion synthesis polymerase. Interfering with REV3 expression through siRNA has a synergistic effect with cisplatin treatment in both human and mouse models of lung cancer, indicating that REV3 is an excellent target to combine with cisplatin therapies. This REV3 knock- down sensitivity was also extended to human ovarian cancer cell lines, indicating the potential of the combination treatment for both lung and ovarian cancers. Thesis Supervisor: Michael B. Yaffe Title: David H. Koch Professor of Science; Prof. of Biology and Biological Engineering 3 Acknowledgments I would like to thank everyone who made the work for my thesis possible, specifically the Ovarian Cancer Research Foundation (OCRF), the Koch Institute of Integrative Cancer Research, the Biological Engineering Program at MIT, and the Center for Precision Cancer Medicine (CPCM) for providing the funding for this work. I would thank my advisor, Michael Yaffe, for his scientific insights, support both in science and in life, and dedication to the proper training of young scientists. He is an excellent hypothesis generator and has an ability to connect topic that most people would think are disparate fields due to his eclectic interests. My thesis committee, Douglas Lauffenburger and Michael Hemann, gave fantastic ideas and progressed the work on the project greatly. My lab-mates were instrumental for teaching me lab techniques, engaging in scientific discussion, and keeping me sane. Special acknowledgements for Yi Wen Kong, Christian Braun, Tatiana Netterfield and Jesse Patterson for directly helping me on my work. Thank you for also being kind and fun to work with. Also, special thanks to my bay mates Tatiana Netterfield and Sriram Ganapathy for talking about life and science, and making coming into work fun and positive. Brian Joughin and Pau Creixell were very patient helping me with any and all computation questions. Additionally, thank you to all the people who entertained me in tissue culture and kept me entertained during long tissue culture sessions; notable good talkers include Ian Cannell, Bert van de Kooij, Karl Merrick and Sanjeev Dhara. Leny Gocheva, the director of research at the Koch Institute, has always taken time to be supportive of me, provide words of encouragement, and is amazing at editing (with quick turnarounds!). Thank you to our lab support team – Susanne Swartwout, for keeping the lab functional and working with a chaotic group of scientists, and Tom Dietzel, for keeping Mike on track and being master of the schedule. I had some great mentors through undergraduate work as well that should be mentioned. William Pitt let me research in his lab as a freshman in undergraduate, and gave me one of my first experiences with biomedical research. Brian Woodfield also mentored me as an early researcher and was very fun, intelligent, and inspirational. Christian Metallo let me study under him for a summer internship with the Amgen program, and the skills I learned in that lab were the most relevant to my research today. I would not have been prepared for MIT without that internship and his guidance. Finally, I would like to thank Dr. Todo, Dr. Hiroshi, and Dr. Asahi, who let me study with them at Hokkaido University and who worked extremely hard to teach me in English when it was not their primary language (I speak Japanese, but my science Japanese vocabulary is limited). I was very lucky to have the opportunity to study with them. I come from a wonderful family who has been supportive of every venture I have chosen to pursue. My parents, Paul and Kyoko Handly, are a constant source of support and encouragement, and obviously played a crucial role in who I am today. I am very grateful for everything they have done, and continue to do. My siblings, Naomi and Jonah, are a source of inspiration and fun, and my older sister Naomi has been leading the way in academic studies since I was born. I have a great role model in her. My husband, Seth Vogel, has done a great job encouraging me to work hard and has been a source of comfort and commiseration when science does not work. With Seth, 4 I gained a whole other family as well, who have been supportive and encouraging to me as well. My grandpa, Tatsuo Izumi, believed in my scientific abilities very early on, and invested time in learning about the biomedical sciences and set me up with my first internship studying diabetes. He emphasized hard work and I miss his random phone calls in my life. My thanks extend to a whole community of friends, roommates, and classmates (not necessary in distinct categories) who have been inspirational and supportive of me. The Biological Engineering program has been a great community of classmates to study and grow with. Specific thanks to Ally Huang – for being a great roommate and friend, George Sun – for supporting me through my first few times trying to do computational work and being a hilarious friend, and Malvika Verma – for being my running buddy and keeping me physically and mentally healthy through graduate school. Corinne Saltzman was a great roommate through graduate school, and still motivates me to work hard and have fun. My undergraduate roommate, Heidi Carlston, is still one of my best friends and person I go to for random fun ideas and just generally life advice. My church community in general has always provided support and reassurance, and helped keep my faith strong through graduate school. Finally, I would like to acknowledge some key inspirations for going into cancer research. I have multiple family members who has have died of cancer, which has shaped my career trajectory and has motivated me to join the "fight" against cancer. My grandmother, Setsuko Izumi, died while I was young of pancreatic cancer and my aunt, Oko Izumi, died of breast cancer while I was in High School. Both cases were jarring and demonstrated the devastating effects that cancer can have on a family. While I was in college, my grandmother, Cristel Handly, died of high grade serous ovarian cancer, which is the same type of cancer I have the opportunity to study today. I am grateful I have had so many good people in my life who still continue