PROJECT SUMMARY Metastatic prostate cancer (mPCa) is incurable and responsible for the majority of PCa associated mortality. Therefore, there is a critical need to identify interceptive therapeutic strategies to provide durable responses in patients. Androgen deprivation therapy (ADT) is the primary line of treatment for mPCa. ADT initially extends survival but is not curative as the patient’s tumor acquires castration resistance (mCRPC). A majority of mCRPC remain dependent on the function of the androgen receptor (AR). However, increasing reliance on more potent AR antagonists, such as enzalutamide, has led to the emergence, in a subset of cases (approximately 20%), of resistance mechanisms independent of AR activity (CRPC-AI). CRPC-AI adapt to ADT via lineage plasticity, adopting a phenotype no longer reliant on AR expression and signaling. These tumors may display neuroendocrine features, a stem or basal cell-like phenotype, altered kinase signaling, and characteristic epigenetic alterations. Recently, we and others have identified and validated new therapeutic targets and drivers of CRPC-AI, including loss of Retinoblastoma-1 (RB) and TP53, and induction of specific epigenetic/reprogramming factors such as (Enhancer of Zeste Homolog 2) EZH2 and SOX2. Additionally, our work validated the importance of loss of RB1 as the primary driver of transcriptional changes correlating with lineage plasticity, increased neuroendocrine features, and decreased sensitivity to ADT. Moreover, we demonstrated that this lineage reprogramming was largely dependent upon EZH2, as inhibition of EZH2 enabled lineage reversal and re-sensitized RB-deficient prostate cancer to ADT. Importantly, recent data from patients with mCRPC identified RB genetic aberrations as the strongest predictor of poor outcome. These data implicate RB as a dominant molecular mechanism driving lethal prostate cancer. Currently there is no therapeutic option able to provide durable response in patients with RB loss-of-function (LOF). Therefore, there is a critical need to delineate downstream effectors of RB LOF so that therapeutic targets can be identified and validated in clinical trials. We hypothesize that RB loss significantly alters EZH2 binding partners and enables formation of novel complexes, resulting in altered methylation patterns of EZH2 targets and contributing to the dependence on EZH2 function that we have previously observed. These altered complexes may provide exclusive vulnerabilities towards inhibiting lineage plasticity driven by RB loss. Specific to this application, we will characterize the changes which occur in EZH2-containing complexes and methyltransferase targets in the absence of RB and validate their functional relevance in tumor development and linage plasticity (Aim 1). We will further perform a functional genomic screen which will identify synthetic lethal targets which will cooperate with EZH2 inhibition in treatment of RB-deficient CRPC-AI and will validat...