Androgen deprivation therapy (ADT) is effective in treating metastatic prostate adenocarcinoma (PADC), but all patients inevitably relapse with castrate resistant prostate cancer (CRPC). Most CRPCs remain dependent on androgen receptor (AR) signaling, but a significant fraction lack AR expression, become AR signaling independent, and aberrantly express neuroendocrine lineage markers (NEPC). The incidence of NEPC variants among CRPC has increased as more patients benefit from improved ADTs like enzalutamide and abiraterone acetate. This suggests increasingly stringent AR signaling blockade is driving development of NEPC. This is an important clinical problem because NEPC is aggressive and lethal; development of effective therapies is hampered by limited understanding of relevant molecular mechanisms. NEPC clearly arises from ARpos CRPC as they share clonal origin in patients that harbor both. We have determined that genetic inactivation of the RB1/TRP53 tumor suppressor genes cooperate to facilitate transformation of ARpos PADC to NEPC through derepression of epigenetic reprogramming factors. Inhibiting these reprogramming factors reverses NEPC transformation and restores ADT sensitivity, demonstrating that epigenetic changes are involved. We hypothesize that a change in NOTCH-ASCL1 signaling triggers the epigenetic reprogramming underlying NEPC transformation. This hypothesis has clinical ramifications as the pathway could conceivably be manipulated therapeutically to delay or reverse NEPC transformation, extending the duration of beneficial ADT clinical responses in some patients. We propose three specific aims using novel prostate cancer mouse models and unique human clinical specimens to test this hypothesis, characterize how PADC cells transform into NEPC cells, and explore novel therapeutic approaches for the treatment of this lethal form of prostate cancer. We will: 1) Test if NOTCH signaling is sufficient to maintain an androgen dependent PADC phenotype; 2) Characterize how prostate cancer cells transition from PADC to NEPC; 3) Determine whether epigenetic modulating drugs reverse NEPC transformation and ADT resistance via NOTCH-ASCL1 signaling. The long term goal of this project is to improve prostate cancer therapy by advancing mechanistic understanding of lineage plasticity as a mechanism of acquired therapeutic resistance.