Project Summary/Abstract Neuroendocrine prostate cancer (NEPC) is the most virulent form of the disease. Loss of the tumor suppressors TP53/RB1 is ubiquitous in NEPC, but molecular mechanisms that explain how loss of TP53/RB1 promotes NEPC development are largely unknown. Further, there are no effective therapies for NEPC. Thus, there is a clear need to clarify targetable mechanisms in NEPC so we may develop new treatments. Loss of epithelial differentiation is one critical hallmark of NEPC. We identified specific transcriptional repressors turned on by TP53/RB1 loss that block androgen receptor signaling and epithelial differentiation. Activation of a neuroendocrine differentiation/epithelial-mesenchymal transition (EMT) program is another hallmark of NEPC. We identified important activators of this program that are also turned on by TP53/RB1 loss. Importantly, blocking the repressor proteins with drugs approved for use in other cancers or blocking the activator proteins with a drug that is currently in clinical trials in prostate cancer reduces growth of NEPC tumors in vitro and in vivo. However, we find that combination therapy is particularly effective. This application is designed to clarify mechanisms by which this treatment combination blocks NEPC cell survival. We hypothesize that TP53/RB1 loss activates key factors that suppress epithelial differentiation while also activating those that promote NEPC differentiation; co-targeting this combined axis is more effective because it restores epithelial differentiation and blocks neuronal/EMT pathways, and—in doing so—blocks adaptive, single agent resistance mechanisms. Aim 1: Determine mechanisms by which loss of TP53/RB1 loss represses epithelial differentiation gene expression. Completion of this aim will provide a detailed understanding of how TP53/RB1 loss turns on key downstream repressors to block epithelial differentiation so we can prevent that from occurring. Aim 2: Determine mechanisms by which blocking key transcriptional repressors reactivates epithelial differentiation. Completion of this aim will determine how key transcriptional repressors block epithelial differentiation so we can identify the most suitable pharmacologic agents to suppress their function. Aim 3: Block NEPC lineage plasticity, tumor growth, and metastasis by targeting key transcriptional activators + repressors induced by TP53/RB1 loss in NEPC tumor models in mice. Completion of this aim will identify in vivo response biomarkers, measure safety, and provide the rationale to test this combination in future clinical trials focused on patients with NEPC. We expect that the studies proposed will mechanistically clarify the function of a network induced by TP53/RB1 loss that is critical for survival of NEPC tumors. These results may help translate a new combination treatment strategy to the clinic to block this network in men with NEPC in the near-term.