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221 Background: Androgen targeted therapy remains the mainstay for advanced prostate cancer (PC). Second-generation androgen receptor (AR) antagonist, enzalutamide (ENZ), re-targets persistent AR activity in castrate-resistant (CR) PC tumors, and is approved for CRPC. Despite initial clinical activity, acquired resistance to ENZ arises rapidly and most patients succumb to PC. Mechanisms underlying resistance to ENZ are incompletely understood. Prior work has established Stat5 as a potent inducer of PC growth. Here, we investigated the significance of Jak2-Stat5 signaling in ENZ-resistant growth of PC. Methods: Levels of Jak2 and Stat5 activation in PC cells, tumors and patient samples were evaluated by immunohistochemistry, 3D tumor explant cultures and western blotting. Jak2 and Stat5 were inhibited by lentiviral (expression of) shRNA or pharmacologically. Levels of mRNA were assessed by QPCR and gene expression profiling. Results: ENZ induced a robust increase in Stat5 activation in PC cells in vitro, in xenograft tumors in vivo and in patient-derived PCs during ENZ treatment. Mechanistically, ENZ activation of Stat5 involves a positive feed-forward mechanism where ENZ-liganded AR induces rapid and sustained Jak2 phosphorylation in PC cells through a process involving Jak2-specific phosphatases. This results in a formation of a positive feed-forward loop in PC where activated Stat5 induces Jak2 mRNA and protein levels in PC. We showed that active Stat5 increased viability of PC cells during ENZ treatment and, at the same time, inhibition of Stat5 as a second-line treatment induced excessive death of PC cells surviving ENZ treatment. Importantly, pharmacological Stat5 blockade inhibited CR growth of PC xenograft tumors after ENZ resistance developed. Conclusions: Collectively, this work introduces a novel concept for a pivotal role of Jak2-Stat5 signaling in mediating resistance of PC to ENZ. Pharmacological Jak2-Stat5 inhibition may provide efficacious therapy in advanced PC in combination with ENZ or after ENZ fails.

110 Background: Effective drug development does not always occur successfully in prostate cancer despite discovery of pathways and targeted agents due to lack of pre-clinical models that effectively mimic prostate cancer in humans. We optimized a novel Ex vivo organ-culture model system using clinical human prostate cancers to test novel therapeutic agents targeting the Jak2/Stat5a/b pathway in prostate cancer; this pre-clinical model may be useful to test novel drugs in prostate cancer for target validation and efficacy. Methods: Organ explant cultures of prostate cancers from 16 patients undergoing radical prostatectomy were performed within 2 hours of surgery, 1-2 mm fresh tissue explants were cultured in a medium containing different doses of pharmacologic inhibitor of Stat5a/b (IST5-002) or a small molecule Jak2 inhibitor with appropriate controls and treated with the drug daily for 7 days. Explants were fixed and analyzed for viability by hematoxylin-eosin staining and “in situ DNA end-labeling” assays, Stat5 activation was analyzed by immunohistochemistry for phosphorylated Stat5a/b. Results: Morphology of prostate cancer was well maintained and dose-dependent tumor responsesoccurred with the Stat5a/b and Jak2 inhibitors. The minimum effective dose of each drug was 25 micromoles and considerable effects on morphology and apoptosis were seen at 50 micromoles. Conclusions: Inhibition of the /Stat5a/b pathway showed promising preclinical activity in prostate cancer as demonstrated by our 3D-ex vivo explant organ culture model system using clinical human prostate cancers, corroborating the data from prostate cancer cell lines and xenograft prostate tumors. This model system may also serve as an effective pre-clinical tool to explore new targets and analyze the effects of different targeted therapies in prostate cancer. Finally, since this uses Ex vivo prostate cancer explants from individual prostate cancer patients, once could test different targeted agents in parallel and based on the efficacy of individual drugs, select patients who would best respond to a particular drug, thus providing an effective personalized medicine approach.

17 Background: There are no effective treatments for metastatic or castration resistant prostate cancer. We have shown that transcription factor Stat5a/b is constitutively active in high-grade prostate cancer, but not in normal human prostate epithelium. Stat5a/b is active in 95% of clinical castration resistant prostate cancers, and the expression of active Stat5a/b in primary prostate cancer predicts early disease recurrence. Stat5a/b is critical for the viability of prostate cancer cells in vitro and for growth of prostate xenograft tumors in nude mice. Stat5a/b synergizes with androgen receptor (AR) and Stat5a/b promotes metastatic behavior of human prostate cancer cells in vitro and in vivo. Here, we hypothesize that Stat5a/b is a molecular target for rational drug design for prostate cancer. Methods: We identified a small- molecule inhibitor of Stat5a/b dimerization by structure-based virtual screen from a database of 30 million chemical structures. The efficacy of the Stat5a/b inhibitor was determined by reporter gene assays, dimerization by co-immunoprecipitations, nuclear translocation by cytochemistry and binding to DNA by EMSA. Cell viability was analyzed by MTT assay. Results: The novel Stat5a/b inhibitor IST5-002 inhibited transcriptional activity of Stat5a/b at IC50 of 1.5 μ M for Stat5a and 3.5 μ M for Stat5b, but not of Stat3 in prostate cancer cells. IST5-002 inhibited dimerization, nuclear translocation, and binding of Stat5a/b to the Stat5 DNA consensus sequence. Furthermore, IST5-002 inhibited expression of Stat5a/b target gene cyclin D1, and induced massive apoptosis of DU145, CWR22Rv1 and LNCaP human prostate cancer cells. IST5-002 blocked prostate cancer xenograft tumor growth in nude mice and induced death in clinical prostate cancers ex vivo in 3D organ cultures. Conclusions: We have identified a small molecule Stat5a/b inhibitor IST5-002 for therapy development for prostate cancer. Future work will focus on chemical modifications of IST5-002 to achieve IC50 below 1 μ M and oral administration. No significant financial relationships to disclose.