Justin M. Drake, Ph.D. University of California, Los Angeles RESEARCH STATEMENT Pre-clinical and Translational Approaches to Assess Activated Signaling Pathways in Prostate Cancer The goal of my research program will be to generate more effective, rational, and personalized treatment options for advanced prostate cancer patients. I will do this through the mechanistic understanding of the signaling pathways and therapeutic resistance mechanisms underlying advanced prostate cancer. My lab will combine my experience using in vitro (Drake et al., 2010a; Drake et al., 2009) and in vivo (Drake et al., 2005; Drake et al., 2012) models of mouse and human prostate cancer (graduate and postdoctoral studies) with phosphoproteomic (Drake et al., 2013; Drake et al., 2012) and other mass spectrometry-based ‘omics’ technologies (postdoctoral studies). My research laboratory will openly collaborate with clinical, basic, and computational scientists to facilitate productive science towards my overall research program goals. The use of mass spectrometry for proteomics will be conducted through the collaboration with other labs and core facilities. Finally, my expertise is in prostate cancer and my studies will be initiated using prostate cancer models, but these approaches would be applicable to numerous other cancer types and model systems increasing the collaboratory potential of my research. OVERVIEW Prostate cancer is an androgen regulated disease that is the second-leading cause of cancer death for men in the United States. The standard of care for aggressive prostate cancer patients is androgen deprivation therapy (Fig. 1). Unfortunately, these tumors inevitably recur resulting in castration resistant prostate cancer (CRPC) concomitant with metastatic disease. The development of new, potent anti-androgen agents have provided increased survival benefit in CRPC patients, although the development of resistance to these therapies is common, often leading to the development of a highly lethal variant termed small cell neuroendocrine carcinoma (SCNC). These clinical findings have prompted the intense investigation into other signaling mechanisms beyond the androgen receptor (AR) that are driving the progression of metastatic CRPC in patients who fail these antiandrogen therapies. My work and others suggest that enhanced tyrosine kinase signaling develops as a result of acquired resistance to anti-androgens in CRPC patients and should be evaluated further as viable therapeutic targets (Fig. 1) (Drake et al., 2013; Drake et al., 2012). The identification of genetically altered tyrosine kinases has transformed the way we treat cancers. Typically, these genetic lesions render the kinase constitutively active resulting in hyperactive pathways that lead to continued cancer growth and survival. Hence, the development of targeted therapies designed to block the kinase’s activity have resulted in significant clinical benefits. The history of genetic alterations in prostate cancer, however, has revealed a paucity of activating mutations in kinase genes (Grasso et al., 2012) although kinase activity is elevated (Drake et al., 2012). Accordingly, there are examples where non-mutated, overactive kinases can drive disease such as BTK tyrosine kinase in hematopoietic malignancies (Rawlings et al., 1996) or SRC tyrosine kinase in prostate and colon cancer (Cai et al., 2010; Cartwright et al., 1990). Recently, the approval of a BTK specific inhibitor for chronic lymphocytic leukemia provides evidence that non-mutated hyperactive pathways can be therapeutically targeted with clinical success (Byrd et al., 2013). PREVIOUS AND CURRENT RESEARCH During my Ph.D. training in the laboratory of Dr. Michael Henry at the University of Iowa, I investigated the cellular and molecular mechanisms of metastatic prostate cancer. To understand the metastatic process in more detail, I initially developed and characterized an in vivo xenograft model of metastatic disease by monitoring prostate cancer cell dissemination via intracardiac injection coupled with bioluminescence imaging (BLI) (Drake et al., 2005). I was able to show that dissemination of prostate cancer cells resembled metastatic tissue tropism that is Justin M. Drake, Ph.D. University of California, Los Angeles observed clinically. Using this model, I investigated the efficacy of an endothelin receptor antagonist, atrasentan, on prostate cancer cell metastasis (Drake et al., 2010b). Antagonism with atrasentan resulted in striking differences in metastatic prostate cancer cell growth as tumor cells that seeded bone were growth repressed while cells that seeded soft tissues (such as the liver) grew unimpeded. I also developed a new in vitro model of metastasis, transendothelial migration, and found that the epithelial-to-mesenchymal (EMT) transcription factor, ZEB1, regulated the transendothelial migration and in vivo metastatic potential of prostate cancer cells (Drake et al., 2009). I was able to expand upon these findings and demonstrate that ZEB1 regulated the expression of key basement membrane attachment proteins known as laminins and integrins, thereby promoting metastatic disease (Drake et al., 2010a). As a Postdoctoral Fellow in the laboratory of Dr. Owen Witte at the University of California, Los Angeles (UCLA), I have focused on uncovering non-mutated kinases and kinase pathway targets in metastatic CRPC using unbiased phosphopeptide enrichment strategies coupled with quantitative mass spectrometry in collaboration with Dr. Thomas Graeber at UCLA. My recent work has revealed that tyrosine phosphorylation is heightened in a panel of advanced non-tyrosine kinase-driven mouse tumors with SRC, JAK2, and EGFR tyrosine kinase activity (Drake et al., 2012). A follow up to this work confirmed that human CRPC tissues also display robust tyrosine phosphorylation when compared to organ confined, treatment naïve prostate tissues (Drake et al., 2012). In collaboration with Dr. Kenneth Pienta and the University of Michigan I acquired rare lethal human metastatic CRPC tissues via a rapid autopsy program to identify the activated tyrosine kinases. I observed that these kinase activation patterns were highly conserved in multiple anatomically distinct metastatic lesions from the same patient, but differed between patients and that co-targeting the SRC and MAPK pathways may be clinically beneficial (Drake et al., 2013). These findings support clonally derived metastatic disease and that matching activation patterns in an individual to a specific inhibitor or inhibitor combinations would be fruitful clinically. Currently, I am assessing phosphoserine and phosphothreonine kinase activities in these clinical tissues to identify more candidate kinases and kinase pathway targets for diagnostic and functional evaluation. In collaboration with Dr. Josh Stuart at the University of California, Santa Cruz, we are developing computational approaches to delineate the predominant kinase networks within this dataset for subsequent functional analysis. FUTURE RESEARCH Project 1: Evaluation of the signaling pathways that distinguish adenocarcinoma and small cell neuroendocrine carcinoma (SCNC) using pre-clinical models of prostate cancer. Background: Prostate SCNC is very rare in untreated adenocarcinomas. However, frequency of SCNC increases to greater than 20% after resistance to anti-androgens with low survival rates. The identification of candidate kinases driving SCNC may lead to new diagnostic and predictive biomarkers that will classify which patients are at a greater risk of developing this highly lethal variant prior to selective therapy. This project is an extension of my currently funded Department of Defense Exploration-Hypothesis Development Award. Approach: I will use phosphoproteomic enrichment and quantitative mass spectrometry to evaluate the differential kinase signaling pathways between the neuroendocrinebased transgenic adenocarcinoma of the mouse prostate (TRAMP) mouse model (Greenberg et al., 1995) compared to transgenic adenocarcinoma mouse models such as PTEN-/- (Wang et al., 2003) and MYC (Ellwood-Yen et al., 2003) (Fig. 2A). Candidate kinases activated in the TRAMP model, but not in the adenocarcinoma models, would then be evaluated for their capability to induce SCNC using mouse or human tissue recombination cancer models alone or in combination with other oncogenes or anti-androgens (such as abiraterone acetate or enzalutamide) which drives the phenotype clinically (Fig. 2B). Further, I will evaluate clinical SCNC tissues for the candidate kinase’s activity and diagnostic biomarker potential. Project 2: Evaluation of therapeutic resistance in advanced prostate cancer. Background: I plan to evaluate therapeutic resistance mechanisms in aggressive primary mouse and human tissue recombination cancer models via 3 ways: (1) Define the resistance mechanisms that arise upon administration of anti-androgens (primary resistance and data already collected from CRPC patients, Drake et al., 2013), (2) Pharmacologically Justin M. Drake, Ph.D. University of California, Los Angeles target the primary resistance mechanisms in combination with anti-androgens, and (3) Evaluate new resistance mechanisms to these combination therapies (secondary resistance). Once the mechanisms of primary and secondary resistance to specific combinations of pharmacologic agents are understood, clinicians will then be able to better predict which inhibitors to use early in the tumor life cycle. This project is the focus for my submitted K22 Transition Career Development Award. Approach: Several genetic mouse models exist for prostate cancer but I will focus initially on the PTEN-/- or MYC mouse models as my source for prostate epithelial cells. Initially, the mouse prostate recombination cancer model will be utilized to infect prostate epithelial cells from the genetic mouse models (i.e. PTEN-/-) with lentivirus expressing oncogenes such as the androgen receptor (AR) to develop aggressive prostate cancer (Drake et al., 2012; Xin et al., 2006) (Fig. 3A). Aggressive prostate tumors will be re-implanted into castrated mice at which point kinase activity will be assessed (Fig. 3B). Since I have previously shown that tyrosine kinase activity is robust in aggressive mouse prostate tumors expressing oncogenes relevant to metastatic CRPC and have utilized phosphoproteomics to characterize the activated kinases in these tumors, I can begin to perturb the tumors with kinase inhibitors in combination with other agents (e.g. anti-androgens or PI3K inhibitors). During perturbation, I would plan to monitor tumor response using in vivo imaging such as positron emission tomography (PET) or BLI as well as evaluate the acute biochemical resistance mechanisms to these therapies via phosphoproteomic and metabolomic enrichment methods coupled to quantitative mass spectrometry (Fig. 3C). Project 3: Development of a diagnostic and predictive biomarker platform for CRPC. Background: Due to the increasing resistance to anti-androgen therapies in metastatic prostate cancer, the development of biomarkers that can either predict disease progression or stratify cancer patients for personalized therapy are urgently needed. I plan to use targeted selected ion monitoring (SIM) mass spectrometry to evaluate druggable kinase targets as diagnostic or predictive biomarkers for personalized therapy in prostate cancer. Targeted SIM mass spectrometry will provide a viable alternative to traditional immunohistochemistry by increasing the sensitivity and eliminating the need for an application specific antibody toward the kinase of interest (Fig. 4A, B). In addition, this approach has the capability to evaluate dozens of kinase activation states simultaneously from one tissue source. This project is the focus for my submitted Department of Defense Idea Development Award. Approach: To develop the most clinically relevant and actionable phosphopeptide list for targeted SIM mass spectrometry, I plan to identify and list the FDA-approved kinase inhibitors indicated for cancer treatment. Once identified, I can then construct phosphopeptides flanking the activation site of each kinase of interest. Initially, test runs will be performed using established prostate cancer cell line-derived xenograft tumors and then subject them to phosphoproteomic enrichment techniques coupled to targeted mass spectrometry identification (Fig. 4C-F). The ability to biopsy organ confined, treatment naïve prostate tumors or metastatic CRPC lesions for activated kinases would be the ultimate goal of this project. The capability to evaluate kinase activities in real time using this approach could aid in diagnostic, prognostic, predictive, or pharmacologic biomarker detection and help stratify CRPC patients for personalized, inhibitor combinations. Justin M. Drake, Ph.D. University of California, Los Angeles REFERENCES Byrd, J. C., Furman, R. R., Coutre, S. E., Flinn, I. W., Burger, J. A., Blum, K. A., Grant, B., Sharman, J. P., Coleman, M., Wierda, W. G., et al. (2013). Targeting BTK with ibrutinib in relapsed chronic lymphocytic leukemia. N Engl J Med 369, 32-42. Cai, H., Babic, I., Wei, X., Huang, J., and Witte, O. N. (2010). Invasive prostate carcinoma driven by c-Src and androgen receptor synergy. Cancer Res. Cartwright, C. A., Meisler, A. I., and Eckhart, W. (1990). Activation of the pp60c-src protein kinase is an early event in colonic carcinogenesis. Proc Natl Acad Sci U S A 87, 558-562. Drake, J. M., Barnes, J. M., Madsen, J. M., Domann, F. E., Stipp, C. S., and Henry, M. D. (2010a). ZEB1 coordinately regulates laminin-332 and {beta}4 integrin expression altering the invasive phenotype of prostate cancer cells. J Biol Chem 285, 33940-33948. Drake, J. M., Danke, J. R., and Henry, M. D. (2010b). Bone-specific growth inhibition of prostate cancer metastasis by atrasentan. Cancer Biol Ther 9. Drake, J. M., Gabriel, C. L., and Henry, M. D. (2005). Assessing tumor growth and distribution in a model of prostate cancer metastasis using bioluminescence imaging. Clin Exp Metastasis 22, 674-684. Drake, J. M., Graham, N. A., Lee, J. K., Stoyanova, T., Faltermeier, C. M., Sud, S., Titz, B., Huang, J., Pienta, K. J., Graeber, T. G., et al. (2013). Metastatic castration-resistant prostate cancer reveals intrapatient similarity and interpatient heterogeneity of therapeutic kinase targets. Proc Natl Acad Sci U S A 110, E4762-4769. Drake, J. M., Graham, N. A., Stoyanova, T., Sedghi, A., Goldstein, A. S., Cai, H., Smith, D. A., Zhang, H., Komisopoulou, E., Huang, J., et al. (2012). Oncogene-specific activation of tyrosine kinase networks during prostate cancer progression. Proc Natl Acad Sci U S A 109, 1643-1648. Drake, J. M., Strohbehn, G., Bair, T. B., Moreland, J. G., and Henry, M. D. (2009). ZEB1 Enhances Transendothelial Migration and Represses the Epithelial Phenotype of Prostate Cancer Cells. Mol Biol Cell. Ellwood-Yen, K., Graeber, T. G., Wongvipat, J., Iruela-Arispe, M. L., Zhang, J., Matusik, R., Thomas, G. V., and Sawyers, C. L. (2003). Myc-driven murine prostate cancer shares molecular features with human prostate tumors. Cancer Cell 4, 223-238. Grasso, C. S., Wu, Y. M., Robinson, D. R., Cao, X., Dhanasekaran, S. M., Khan, A. P., Quist, M. J., Jing, X., Lonigro, R. J., Brenner, J. C., et al. (2012). The mutational landscape of lethal castration-resistant prostate cancer. Nature 487, 239-243. Greenberg, N. M., DeMayo, F., Finegold, M. J., Medina, D., Tilley, W. D., Aspinall, J. O., Cunha, G. R., Donjacour, A. A., Matusik, R. J., and Rosen, J. M. (1995). Prostate cancer in a transgenic mouse. Proc Natl Acad Sci U S A 92, 3439-3443. Rawlings, D. J., Scharenberg, A. M., Park, H., Wahl, M. I., Lin, S., Kato, R. M., Fluckiger, A. C., Witte, O. N., and Kinet, J. P. (1996). Activation of BTK by a phosphorylation mechanism initiated by SRC family kinases. Science 271, 822-825. Wang, S., Gao, J., Lei, Q., Rozengurt, N., Pritchard, C., Jiao, J., Thomas, G. V., Li, G., Roy-Burman, P., Nelson, P. S., et al. (2003). Prostate-specific deletion of the murine Pten tumor suppressor gene leads to metastatic prostate cancer. Cancer Cell 4, 209-221. Xin, L., Teitell, M. A., Lawson, D. A., Kwon, A., Mellinghoff, I. K., and Witte, O. N. (2006). Progression of prostate cancer by synergy of AKT with genotropic and nongenotropic actions of the androgen receptor. Proc Natl Acad Sci U S A 103, 7789-7794. Justin M. Drake, Ph.D. University of California, Los Angeles TEACHING STATEMENT Great teachers create the foundation for a prosperous community. Throughout my student life, I have been very fortunate to be exposed to excellent teachers in all disciplines and that has directly influenced my career path and impacted how I teach and will teach in the future. My primary goal as a teacher will be to get the students excited about the course material. To do this, I must first demonstrate my own enthusiasm for the topic as well as teach the material in a stimulating and logical manner. From my experiences, I find students become more engaged if the topic you are discussing can be connected to their everyday experiences and can demonstrate value that links the knowledge gained to their career goals such as getting into professional school or being successfully employed. As a teacher, it will be my duty to convey that consistently. At the University of Iowa I was afforded 2 excellent teaching opportunities: (1) Teaching assistant for graduate level human physiology and (2) Ambassador for the University of Iowa Health Sciences Program. The human physiology course taught dental and graduate students, and my responsibilities included leading weekly research paper discussions with graduate students to develop scientific comprehension, weekly review sessions on topics pertaining to human physiology, and writing exam questions. As a result of my instruction, I received the 2008 Dr. Byron A. Schottelius Teaching Award, which is presented annually to a graduate student who shows exceptional promise as a teacher in the physiological sciences. Further, as an ambassador for the University of Iowa in 2008, I traveled to high schools to educate junior high and high school students on the day to day activities of being a scientist, the truths and stereotypes of scientists, and the wide array of career paths that a Ph.D. can take. I took pride in both of these opportunities as I realized that my instruction, demeanor, and advice may positively influence the students I was teaching. I have also had the opportunity to mentor several undergraduate and graduate students during my postdoctoral studies at the University of California, Los Angeles in the laboratory of Dr. Owen Witte. As a mentor, my responsibilities were to train my students to become better scientists, through the development of critical thinking skills and knowledge of experimental design, and also to help them achieve their career goals. Excellent mentors realize that each student is unique and can adjust their teaching/mentoring style accordingly. Students can reach their full potential only if they are put into an environment that is best suited for that particular student. As I have a background in athletics, I find that academic teachers and coaches are very similar. In either case, the teacher/coach must be able to recognize the strengths and weaknesses of each individual to ultimately position that individual into a position of success. My scientific background is broad, and this is reflected in my teaching interests. Further, my research program in cancer biology will directly influence my enthusiasm about teaching courses related to Biochemistry, Cell and Molecular Biology, Physiology, and Cancer Biology to undergraduate, graduate, and medical students. As my life has been directly impacted by good teachers, I am committed to the education of young scientists.
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