Targeting mLST8 in mTORC2-dependent cancers By Laura Chonghae Kim Dissertation Submitted to the Faculty of the Graduate School of Vanderbilt University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY in Cancer Biology June 30th, 2020 Nashville, Tennessee Approved: Albert B. Reynolds, Ph.D. (chair) Mark R. Boothby, M.D., Ph.D. Christine M. Lovly, M.D. Ph.D. William P. Tansey, Ph.D. Jin Chen, M.D., Ph.D. (adviser) DEDICATION This dissertation is dedicated to all cancer patients and their loved ones for their strength and courage in the fight against this disease. This is also dedicated to my family (Kalhee and Jenny Kim, Stephanie Kim) for their unwavering love and support. ii ACKNOWLEDGEMENTS Completion of this work would have been impossible without the support of many, and I would like to express my sincere gratitude here. First and foremost, I must thank my mentor, Jin Chen. Her support, mentorship, and high-fives have been invaluable in my development as a scientist, and I plan to continue seeking her guidance even when I am many years beyond graduate school. I would also like to acknowledge my committee members Al Reynolds, Bill Tansey, Mark Boothby, Rebecca Cook, and Christine Lovly, who continuously challenged me and provided scientific guidance. Much of this work stems from their insightful questions, and I am extremely appreciative of the time they put into thinking about my projects. I would also like to thank the past and present members of the Chen Lab: Shan Wang, Deanna Edwards, Wenqiang Song, Dana Brantley-Sieders, Katherine Hastings, Victoria Youngblood, Tammy Sobolik, Yoonha Hwang, Eileen Shiuan, Kalin Wilson, Verra Ngwa, Ashwin Inala, and Chris Rhee. I am tremendously grateful for their training, scientific brilliance, and most importantly their friendship throughout this journey. In particular, I would like to highlight Deanna, Shan, Wenqiang, and Eileen, who were always willing to lend an ear to my questions and musings for the last five years. I am going to miss our morning coffee runs, breakroom lunches, and even the long weekends in lab! I also need to thank those who supported me financially throughout my graduate career, specifically Dr. Jin Chen for supporting me through the Microenvironmental Influences in Cancer Training Grant and the National Cancer Institute for the Predoctoral Ruth L. Kirschstein National Research Service Award (F31). I would also like to thank all the members of the Program in Cancer Biology for day-to-day logistical support. iii Last, I must thank my friends and family. Meeting such wonderful people around Nashville and in graduate school at Vanderbilt has made this process infinitely easier. I am incredibly grateful for the wonderful memories and enduring friendship. I also feel immensely lucky to have friends from back home in North Carolina that always supported me from afar. From weekend trips to incessant messages, they all put in an incredible amount of energy into staying connected despite the distance. I cannot thank them enough for this effort. Finally, I must thank my parents, Kalhee and Jenny Kim, and sister, Stephanie, for their unconditional love and trust. Their support gave me the confidence to persevere against every hurdle, and I feel extraordinarily privileged to have them in my corner. iv TABLE OF CONTENTS DEDICATION.............................................................................................................................. ii ACKNOWLEDGEMENTS .......................................................................................................... iii LIST OF TABLES .................................................................................................................... viii LIST OF FIGURES .................................................................................................................... ix LIST OF ABBREVIATIONS ....................................................................................................... xi CHAPTERS I. Introduction .............................................................................................................. 1 Overview .............................................................................................................. 1 Lung Cancer ........................................................................................................ 2 Targeted Therapy ..................................................................................... 3 Immunotherapy ........................................................................................ 7 The mTOR Complexes ........................................................................................ 9 mTORC1 Signaling ................................................................................ 14 mTORC1 in Cancer ................................................................................ 16 mTORC2 signaling ................................................................................. 19 mTORC2 in Cancer ................................................................................ 21 mTOR Inhibitors for Cancer Therapy ...................................................... 21 mTOR in Vasculature ............................................................................. 26 mTOR in Tumor Immunity ...................................................................... 38 Summary and Thesis Projects ........................................................................... 30 II. Disruption of the scaffolding function of mLST8 selectively inhibits mTORC2 assembly and function and suppresses mTORC2-dependent tumor growth in vivo ......................................................................................................................... 33 Abstract ............................................................................................................. 33 Significance ....................................................................................................... 33 Introduction ........................................................................................................ 34 Materials and Methods ....................................................................................... 34 Cell Lines and cell culture ....................................................................... 34 Generation of the mTOR E2285A knock-in cell line by CRISPR-Cas9 technology .............................................................................................. 35 Xenograft Assay ..................................................................................... 35 Plasmids................................................................................................. 36 Lentivirus production and transduction ................................................... 36 Western Blot and Co-immunoprecipitation .............................................. 36 In Vitro Kinase Assay ............................................................................. 37 Results ............................................................................................................... 39 v mLST8 is required for mTORC2, but not mTORC1, integrity and function ............................................................................................................... 39 mLST8 mutations that uncouple binding to LBE domain of mTOR disrupt mTORC2 complex formation and signaling ............................................ 42 mLST8 serves as a molecular bridge between mTOR and SIN1 ............ 46 mLST8 point mutations that block mLST8-mTOR interactions impair mTORC2 signaling and tumor growth in vivo .......................................... 50 Discussion ......................................................................................................... 54 III. Rictor promotes NSCLC cell proliferation through formation and activation of mTORC2 at the expense of mTORC1 ................................................................... 56 Abstract ............................................................................................................. 56 Introduction ........................................................................................................ 57 Results ............................................................................................................... 58 RICTOR is amplified and overexpressed in non-small cell lung cancer .. 58 Alterations in Rictor expression lead to corresponding changes in mTORC2 and mTORC1 ......................................................................... 64 Rictor promotes proliferation of NSCLC cells .......................................... 67 Increased Rictor expression promotes NSCLC tumor growth in vivo ...... 68 Targeting mTORC2 for loss-of-function inhibits tumor growth of RICTOR- amplified NSCLC .................................................................................... 70 Discussion ......................................................................................................... 73 Materials and Methods ....................................................................................... 75 Cell lines and cell culture ........................................................................ 75 Plasmids and sgRNA Sequences ........................................................... 76 Lentivirus Production and Transduction .................................................. 76 Xenograft Assay ....................................................................................