PROJECT SUMMARY/ABSTRACT Castration-resistant prostate cancer (CRPC) emerges following androgen deprivation therapy (ADT), where variable degrees of dependence on the androgen receptor (AR) are observed, and features of neuroendocrine (NE) carcinoma often arise. We have identified the developmental transcription factor, ONECUT2 (OC2/HNF6β), as a master regulator of AR networks in metastatic (m)CRPC. OC2 governs a lethal differentiation program, drives metastasis, and interacts with the AR at several levels, including as a multiprotein complex and a transcriptional regulator of AR target genes. Our studies indicate that OC2 appears to override AR-dependent mechanisms in a subset of mCRPC, and activates an NE differentiation program within the context of adenocarcinoma. To inhibit OC2, we developed a novel class of small molecules that bind OC2 directly and suppress growth and metastasis of AR/AR-V7-positive mCRPC xenografts. Additionally, we have developed profiling and immunohistochemistry (IHC) methods to identify OC2 activity in clinical specimens, laying a foundation for an OC2-targeted treatment approach in select patients. This project will test the hypothesis that OC2 is an actionable target in a subset of aggressive prostate cancer where OC2 is active. The Specific Aims are: Aim 1. Study the mechanism by which OC2 activity promotes aggressive behavior of CRPC. Determine whether OC2 can compensate for AR in CRPC. Determine whether OC2 upregulation can confer independence from AR. Determine whether OC2 interactors in AR-dependent and AR-independent transcription complexes are distinct. Determine whether OC2 is required for NE differentiation and growth of NE-CRPC. Aim 2. Develop and optimize OC2 inhibitors for use in patients with early mCRPC. Synthesize and test derivatives of the OC2 targeting compound CSRM617. Perform in silico and high-throughput screening for structurally unrelated OC2 inhibitors. Test the safety and efficacy of OC2 inhibitors in pre-clinical models. Aim 3. Identify the clinical scenarios where OC2-driven tumors emerge. Refine multiplex IHC detection of OC2/AR expression in OC2- active tumors. Determine the correlation between OC2 activity and IHC detection of OC2 and AR in independent sets of clinical samples. Evaluate OC2 activity along the clinical spectrum of prostate cancer progression, including sequential pre/post-ADT tumor specimens, metastases and xenograft models. Measure OC2/AR activity in diagnostic prostate needle biopsies (PNBX) of untreated men with high-grade prostate cancer, and determine the impact of OC2 activity on clinical outcome in univariate and multivariate analyses. These experiments will help clarify the role of alternative drivers of progression and ADT resistance that emerge to cause lethal disease. They will also advance, toward a phase I clinical trial, a therapeutic approach against a novel master regulator that we estimate operates in 1/3 or more of all patients with CRPC tumors.