THE SMALL GTPASE RAB35 IS A NOVEL ONCOGENIC REGULATOR OF PI3K/AKT SIGNALING A Dissertation Presented to the Faculty of the Weill Cornell Graduate School of Medical Sciences in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy by Douglas B. Wheeler May 2015 © 2015 Douglas Berg Wheeler THE SMALL GTPASE RAB35 IS A NOVEL ONCOGENIC REGULATOR OF PI3K/AKT SIGNALING Douglas Berg Wheeler, Ph.D. Cornell University 2015 The phosphatidylinositol 4,5-bisphosphate 3’-OH kinase (PI3K) is a lipid kinase that regulates cell survival, proliferation and metabolism in response to external growth factors. PI3K signaling regulates a diverse set of effectors via the phosphoinositide dependent kinase 1 (PDK1), AKT/PKB, and the mechanistic target of rapamycin complexes 1 and 2 (mTORC2). The components of this pathway are frequently mutated in human cancers, which give rise to oncogenic signals that drive tumorigenesis. As such, the proteins involved in PI3K/AKT signaling are attractive targets for targeted therapies. It is thought that most tumors upregulate PI3K/AKT signaling in some way. However, a large portion of tumors do not have any identifiable genetic lesions in the genes that code for proteins that regulate this pathway. Thus, we reasoned that there are likely to be many proteins that regulate PI3K/AKT signaling that are currently unappreciated. To identify novel regulators of the PI3K axis, we undertook an arrayed loss-of-function RNAi screen using a library of shRNA reagents that targeted all known human kinases and GTPases to identify proteins whose depletion altered AKT phosphorylation. Further, we triaged genes from this screen using oncogenomic databases to identify screen hit genes that were mutated in human tumor samples or cell lines. We reasoned that this approach could identify novel components of PI3K/AKT signaling that also play a role in tumorigenesis. This screen identified the small GTPase RAB35 as a novel positive regulator of PI3K/AKT signaling. Depletion of RAB35 in a variety of human and murine cell lines suppressed phosphorylation of PI3K-dependent proteins like AKT, FOXO1/3A and NDRG1. Moreover, stable expression of a GTPase-deficient, constitutively active allele of RAB35—but not wildtype RAB35—potently activated AKT signaling in the absence of growth factors. Further, we identified two RAB35 mutations in human tumor genome sequence databases that activated PI3K signaling and transformed NIH-3T3 cells in vitro in a PI3K-dependent manner. Finally, we find that RAB35 is likely regulating PI3K signaling by trafficking the platelet derived growth factor receptor (PDGFR) to a RAB7-positive compartment in the cell where the receptor actively signals PI3K. Thus, RAB35 is a novel oncogenic regulator of PI3K/AKT signaling. BIOGRAPHICAL SKETCH Douglas Berg Wheeler was born in 1981 in Utica, NY to Nancy Berg and Albert Charles Wheeler. Doug attended Poland Central School in Poland, NY for elementary, middle and high school. During high school, he participated annually in the Poland Central School and Utica College Science Fairs, which gave him an early taste for both biology and failure. After high school, Doug attended Hamilton College in Clinton, NY from 1999 to 2001, where he worked in the endocrinology lab of Dr. David A. Gapp. After two years at Hamilton, he transferred to Cornell University in Ithaca, NY in the fall of 2001. During his time at Cornell, he taught undergraduate Biochemistry (BIOBM 330) and worked in the lab of yeast geneticist Dr. Susan A. Henry. He graduated from Cornell in May, 2003 with a Bachelors of Science in Biology. After his time at Cornell, Doug moved to Cambridge, MA and began working as a research technician in the lab of Dr. David M. Sabatini at the Whitehead Institute for Biomedical Research at the Massachusetts Institute of Technology. While at the Whitehead, he developed a tool for rapid, high- throughput loss-of-function RNA interference screens in Drosophila cells [3- 4]. This technology—where RNA reagents are printed onto glass slides and cells seeded on these spots could be assayed for loss of gene function— spurred Doug’s interest in high-throughput technology and the examination of gene function. Further, his time in the Sabatini lab sparked in Doug a desire to study the genes that regulate survival and growth in cancer cells. iii After two years at the Whitehead, Doug moved to New York City in July of 2005 to begin his time at the Tri-Institutional MD/PhD program. During the two preclinical years of medical school, Doug decided that he wanted to start his graduate work with an RNAi screen to discover new components the PI3K/AKT signaling pathway. In July of 2007 he began his graduate work in the lab of Dr. Charles L. Sawyers at Memorial Sloan- Kettering Cancer Center. With the cooperation of Dr. Sawyers and Dr. Olaf S. Andersen, Doug was fortunate enough to concoct a thesis that allowed him to be mentored jointly by Dr. Sawyers and his previous mentor, Dr. Sabatini. Doug spent the first two and a half years of his graduate training primarily working at the Whitehead and Broad Institutes. While there, he performed the RNAi screens that nucleate this thesis. After finishing these screens, Doug returned to New York City in the beginning of 2010 to continue his thesis work full time in the Sawyers laboratory. After six years in the lab, Doug will ultimately defend his Ph.D. in December of 2013, after which he will begrudgingly put down his pipettes and return to medical school. He is looking forward to spending the rest of his days studying cancer. iv This dissertation is dedicated to my parents, Nancy Berg Wheeler and Albert Charles Wheeler. Without the two of them I would not have had the inquisitiveness or stubbornness that science demands. They have been very patient. v ACKNOWLEDGEMENTS I would first like to thank the director of the Tri-Institutional MD/PhD program, Dr. Olaf Sparre Andersen. Without Dr. Andersen’s willingness to allow me to pursue my interests, I would not have been able to design and carry out my thesis work the way I wanted to. In the middle of my second year of medical school, I approached Dr. Andersen with the idea for my thesis project—which involved me moving to Boston for an indeterminate amount of time. Rather than turn down my request, Dr. Andersen allowed me to return to the Whitehead Institute without any hesitation. I thank Dr. Andersen for his flexibility in allowing me to pursue my scientific interests. I would also like to thank my two thesis advisors: Dr. David M. Sabatini and Dr. Charles L. Sawyers. I have known David since 2003, when I joined his lab as a technician. David has always encouraged my pursuit of science, and his passion and excitement for biology are contagious. Moreover, his sense of humor taught me that science can be fun, even when the results are not. I also thank my MSKCC mentor, Dr. Charles Sawyers, for being willing to take me on as a graduate student when I approached him to ask if I could be in his lab, but from a distance. In the years that Dr. Sawyers has been my thesis advisor, I have come to admire his sense of humor, his patience, and his dedication to helping cancer patients with translational science and medicine. Further, I would like to extend my thanks to my thesis committee members for their patience, scientific insights, and flexible schedules: Dr. vi Scott C. Blanchard, Dr. Robert Darnell, Dr. Timothy E. McGraw, Dr. Neal Rosen and Dr. Agata Smogorzewska. Each member of my committee has been generous in sharing their time and wisdom with me. They stand as examples of busy faculty who are nevertheless willing to selflessly extend themselves to trainees. I am in their debt and will seek to emulate their generosity throughout my career. I would also like to acknowledge the many people who have encouraged my pursuit of science at various stages of my life. During middle and high school, I was privileged to have teachers like William Newman and Edward Rosenburgh, my biology and earth science teachers, respectively. Both Mr. Newman and Mr. Rosenburgh encouraged my science fair projects all throughout my time at Poland Central School—despite the fact that not one of my projects ever succeeded. They helped me to learn very early on that science is not about proving one’s hypothesis right, but rather about asking interesting questions. Without their involvement in the PCS Science Fair, it is unlikely that I would have chosen the training path that I have. I was very fortunate during my undergraduate years to have a number of teachers who encouraged me to take risks and try new things. In particular, my advisor from Hamilton College—Dr. David A. Gapp—was instrumental in my early years at the bench. Another of my mentors at Hamilton—my organic chemistry professor Dr. Ian Rosenstein—helped to stoke my passion for biochemistry at an early age. While at Cornell, I was lucky enough to teach undergraduate biochemistry under the guidance of vii James E. Blankenship and Dr. Helen T. Nivison. Their enthusiasm for teaching left an indelible mark on me. My time at the bench at Cornell was overseen by Dr. Stephen Jesch in the lab of Dr. Susan A. Henry. I thank both Steve and Susan for their commitment to teaching and their patience for my marginal skills at yeast genetics. I have been lucky to have been a part of two wonderful labs during my graduate training, and was blessed with many friends from these two groups. From the Sabatini laboratory I would like to thank Dr.