IDENTIFICATION AND VALIDATION OF GERMLINE GENETIC VARIANTS THAT ASSOCIATE WITH SORAFENIB CLINICAL OUTCOMES AND CYTOTOXICITY Daniel James Crona A dissertation submitted to the faculty of the University of North Carolina at Chapel Hill in partial fulfillment of the requirements for the degree of Doctor of Philosophy in the Eshelman School of Pharmacy (Pharmacotherapy and Experimental Therapeutics). Chapel Hill 2015 Approved by: Federico Innocenti William Kim Karen Mohlke John Valgus Timothy Wiltshire © 2015 Daniel James Crona ALL RIGHTS RESERVED ii ABSTRACT Daniel James Crona: Identification and validation of germline variations that associate with overall survival in metastatic renal cell carcinoma patients treated with sorafenib (Under the direction of Federico Innocenti ) Sorafenib is a potent inhibitor of multiple oncogenic, stromal and angiogenic receptor tyrosine kinases. Germline variants in VEGF-pathway genes and in sorafenib pharmacology genes might associate with prognosis and/or sorafenib efficacy in metastatic renal cell carcinoma (mRCC patients). A total of 295 mRCC patients from the phase III TARGET trial were genotyped using candidate germline variants from 56 candidate genes implicated in angiogenesis, sorafenib pharmacology and/or RCC prognosis/pathogenesis. Seven variants that significantly associated with overall survival (OS) in mRCC patients treated with sorafenib, and an additional two variants associated with OS in a combined analysis of both treatment arms. Statistical associations between genetic variants and outcomes in cancer studies should be supported with molecular mechanistic evidence of variant function to aid in biomarker validation. Variants identified in Aim 1 that significantly associated with OS were analyzed using in silico bioinformatic tools to prioritize in vitro validation assays. Cell viability assays validated one non-synonymous variant in FLT-4, and dual reporter gene luciferase assays validated two intronic VEGFA variants in three different cell lines. Novel pathways and targets of sorafenib activity remain to be identified. Primary mouse embryonic fibroblasts (MEFs) from 32 inbred strains were profiled for sorafenib iii cytotoxicity utilizing high content imaging and simultaneous evaluation of cell health parameters (cell viability, membrane permeability, mitochondrial membrane potential, and cytochrome C release). One quantitative locus (QTL) on chromosome 9, which reached genome-wide significance and significantly associated with cytochrome C release, was identified. A total of nine genes, expressed in MEF cells at mRNA level, were present in this QTL. A second QTL associated with cell viability was also identified. A total of 13 candidate genes, expressed in MEF cells at mRNA level, were present in this QTL. In the future, functional validation of candidate genes under these two identified QTLs, using knockdown and overexpression approaches, will be conducted in MEF and human cell lines. iv I would like to dedicate this work to my one and only true love: Dr. Lana Crona. Thank you for sticking by me through all of this, and for being my rock. I love you, my dear Nannybelle. Without you, I am nothing. v ACKNOWLEDGEMENTS Federico Innocenti, you were precisely what I needed in a mentor. You taught me self-reliance, critical thinking skills, and self-confidence. You helped me see the big picture of why I am here and why I want to continue as a clinical pharmacologist and pharmacogeneticist in oncology: to make impactful changes and advancements that better the lives of our patients. I truly value your hands-off mentoring approach and that you did not micro-manage my every move. I appreciate that, from the beginning, you treated me like you would a post-doctoral researcher and gave me the latitude to pursue new ideas and test new hypotheses. As a result, you engendered within me a sense of independence. I hope I met your high expectations for a first graduate student. Amy Etheridge, you have certainly been a great teacher and mentor to me as well. Thank you for helping me design and develop my assays, for making sure supplies were always available, for babysitting my cells, for providing me with sage career advice, for talking about biking and running, and for listening to all of my thoughts and concerns on so many different subjects. I literally could not have done this without you. My friend and colleague from “down under,” Dylan Glubb, I only have one thing to say to you: shot bro. I could not have asked for a better colleague, and role model when you moved here from Chicago. I appreciate all of your advice on pharmacogenetics, genetics, and molecular biology. Moreover, thank you for being a really great friend. And Eric Seiser, thank you so much for being a great resource since you joined the lab. I am so glad I can count on you for your expertise in bioinformatics, and biostatistics. I am so excited for you to get to start your life in Washington DC. I would also like to thank vi one of our collaborators from University of Chicago, Andrew Skol. You are an amazing statistician, and were integral to the analysis of the data and my overall learning in the early stages of my dissertation. I cannot thank you enough. I also would like to thank Carol Peña from Bayer for providing me access to the TARGET patient samples, as well as to thank Habibul Ahsan at the University of Chicago and Jason Luo from UNC for their assistance with the genotyping portion of my research. Tim Wiltshire, I sincerely could not have asked for a better Chair of my Dissertation Committee. You are so kind, intelligent, easy to talk with, and truly personify the words “mentor” and “teacher.” I would not be at UNC if not for you because, as you know, you literally convinced me to attend UNC while I was waiting in the lift line while snowboarding in Colorado. You were my first lab PI here, and I am so thankful that I will soon get the opportunity to be your collaborator and colleague. A million times thank you. I cannot say enough good things about two more members of the Wiltshire lab: Oscar Suzuki and Amber Frick. Oscar, you are simply amazing. I wish I knew half as much about genetics, bioinformatics, and molecular biology (you are also one heck of a swimmer). And Amber, you are no less amazing. You are so smart, so kind, are an incredible teacher, and wonderful at explaining difficult concepts (and you are also on heck of a baker). I am also very grateful to Rusty Thomas, Bethany Parks, Joe Trask, and all of the members of the Hamner Institute for collaborating with me on the MEF study. And, thank you very much to Tammy Havener for helping guide me on cell viability assays, and molecular biology. I learned so much from each and every one of you. Thank you for being integral to my learning. John Valgus, of all my mentors and Dissertation Committee members, I relate best to you because we are both pharmacists. I value every single one of our discussions on GU vii oncology topics, your advice on my different HOPS presentations, you advice on my dissertation, your explanations regarding supportive care topics, as well as how you’ve advised me extensively on career and on life. You have truly made this training experience unique and special for me. You are an oncology pharmacist of the highest quality, an insightful pharmacogenetics researcher, and have incredible perspective. You are everything I want to be as a clinician, as a clinical researcher and as a person. Thank you so very much for taking a chance on me. Billy Kim, I wanted to thank you for being such a great part of my Dissertation Committee and for being a mentor to me in clinic. I will always remember the first time you introduced me to one of your patients as “Dr. Crona.” That was such a validating moment for me. Thank you for challenging me to think deeply in my Dissertation Committee meetings, and for all of our discussions about science while in clinic. Thank you so much for your generosity. Giving me the Caki-1 cells REALLY helped me, and all of your lab members were so kind and giving of their time. You are clearly one of the most intelligent people I have ever met, and I really look forward to working together in the future, both in clinic and hopefully through lab collaborations. Karen Mohlke, I sincerely appreciate all of your contributions to my research as part of my Dissertation Committee. You are simply amazing when it comes to your knowledge of human genetics. I hope someday that I understand even a fraction of what you know. You and your lab members, most notably Tamara Roman, have been essential to the directions that I took in designing, implementing and analyzing my validation experiments. I also appreciate how you challenged me on difficult genetics and statistics issues during my Dissertation Committee meetings. You have definitely helped me grow as a researcher. I viii hope that you will continue to mentor me in the future. I value you and your expertise so very much. As part of my education, I have been lucky enough to engage in a longitudinal clinic experience. I have essentially served as the clinical pharmacist specialist in the outpatient GU oncology clinic of over four years. During that time, I have gotten to meet some really extraordinary individuals who have truly shaped me as a clinician and researcher. Matt Milowsky, I cannot adequately express just how much I gain from you as a mentor. You are a brilliant clinician, and an even better clinical researcher. Thank you for providing me with opportunities to collaborate with researchers at Memorial Sloan Kettering, and thank you for all you do for me (which I am sure is more than I know).