Identification and Characterization of Modulators of Chemotherapeutic Response in Mouse Models of Cancer

Chemotherapeutic drug resistance is a major cause of cancer treatment failure. While much attention has been focused on the genetics of tumor development, less is known about the genetic determinants of therapeutic outcome. As a central focus of my graduate work, I have employed pool-based shRNA-mediated screening methodologies using the Ep-myc lymphoma model to identify genes that modulate the response to front-line chemotherapeutics. In addition to identifying known mediators of chemotherapeutic response, these screens also uncovered several novel genetic targets that influenced the cellular response to selected chemotherapies. Initial in vitro screening experiments identified topoisomerase 2A levels as critical determinants of doxorubicin response. Subsequent follow-up experiments including one demonstrating doxorubicin sensitization in the presence of topoisomerase 1 shRNAs revealed important insights into topoisomerase biology and the clinical heterogeneity associated with topoisomerase poisonbased therapies. In a related screen, I also found Nek4 levels to be hitherto unappreciated determinants of the cellular response to microtubule poisons. Importantly, I went on to demonstrate that this effect on microtubule poisons was attributable, in part, to Nek4 regulation of microtubule function. In addition to screening-based approaches, I also show that targeted shRNA-mediated knockdown strategies are useful for probing in vivo chemotherapeutic response. By suppressing translesion synthesis, initially in Bcell lymphoma and later in a newly developed transplantable model of murine lung adenocarcinoma, I show that tumors are not only sensitized to clinicallyrelevant chemotherapeutics, but are also partially protected from drug-induced mutagenesis and acquired resistance. Taken together, these results described in this thesis underscore the importance of both large-scale, unbiased gene discovery and target-based interrogation of genetic determinants of chemotherapeutic response. Thesis Supervisor: Michael Hemann Title: Latham Family Career Development Assistant Professor of Biology Biographical Note Jason Doles Contact Information: 61 Winter Street Apt #1 Cambridge, MA 02141 Phone: (608) 239-4216 Email: jasondoles@gmail.com Education: 2005-present: Ph.D. (Biology), Massachusetts Institute of Technology (MIT), Cambridge, MA, USA Thesis: Interrogating genetic determinants of chemotherapeutic response in mouse models of cancer 1999-2003: B.A. (Political Science, Biology) Brown University, Providence, RI, USA Research Experience: 2005-present: Graduate Student, MIT Ph.D. Advisor: Michael T. Hemann Selected projects: Evaluating the role of Nek4 in modulating a differential response to microtubule poisons. Sensitizing lung adenocarcinoma to cisplatin chemotherapy using Rev3 RNAi. 2003-2005: Associate Research Specialist, University of Wisconsin-Madison Pl: Wade Bushman Project: Evaluating the significance of hedgehog signaling during mouse prostate development Summer 2002: Research Assistant, Massachusetts General Hospital Pl: Robert Peterfreund Project: Assisting in modeling/experimentally determining efficacy of intravenous drug delivery adaptors. Teaching Experience: Fall 2006: Teaching Assistant, Introductory Biology (7.012), MIT Spring 2009: Teaching Assistant, Project Laboratory (7.16), MIT Funding Awards: Ludwig Center Graduate Student Fellowship (2009-2010) Scientific Publications: Doles J, Oliver T, Hsu G, Jacks T, Walker GC, Hemann MT. Rev3 suppression sensitizes drug-resistant lung tumors to chemotherapy. Submitted to PNAS. Xie K*, Doles J*, Hemann MT, Walker GW. Error-prone translesion synthesis mediates acquired chemoresistance. Submitted to PNAS. *Equal Contributors Doles J, Hemann MT. Nek4 status differentially alters sensitivity to distinct microtubule poisons (2010). Cancer Res. 70(3):1033-41. Burgess DJ, Doles J, Zender L, Xue W, Ma B, McCombie WR, Hannon GJ, Lowe SW, Hemann MT. Topoisomerase expression levels determine chemotherapy response (2008). Proc Natl Acad Sci U S A. 105(26):9053-8. Doles J, Cook C, Shi X, Valosky J, Lipinski R, Bushman W. Functional compensation in Hedgehog signaling during mouse prostate development (2006). Dev Biol. 295(1):13-25. Lipinski RJ, Gipp JJ, Zhang J, Doles JD, Bushman W. Unique and complimentary activities of the Gli transcription factors in Hedgehog signaling (2006). Exp Cell Res. 312(11):1925-38. Doles JD, Vezina CM, Lipinski RJ, Peterson RE, Bushman W. Growth, morphogenesis, and differentiation during mouse prostate development in situ, in renal grafts, and in vitro (2005). Prostate. 65(4):390-9. Lovich MA, Doles J, Peterfreund RA. The Impact of Carrier Flow Rate and Infusion Set Dead-Volume on the Dynamics of Intravenous Drug Delivery (2005). Anesth Analg. 100(4):1048-55.