Project Summary/Abstract Prostate cancer arises as an androgen driven disease, and systemic therapies that target the androgen receptor (AR) are used to treat patients at all stages of the disease. In recent years, with the earlier and more potent targeting of the AR with newer drugs, AR-independent prostate cancer has emerged. We have found that this is associated with lineage plasticity in which upon selective therapeutic pressure, tumors evade AR-therapy through loss of luminal prostate identity (including AR) and the acquisition of alternative lineage programs including neuronal/neuroendocrine, stem-like, and developmental pathways. In extreme cases, tumors may completely transition from an AR- positive prostate adenocarcinoma (PADC) toward an AR-negative small cell/neuroendocrine carcinoma (NEPC). This phenotypic change is associated with clinical and molecular features similar to small cell lung cancer, manifest by rapid progression and lethal disease. We have integrated patient and preclinical data to identify and molecularly characterize genes and pathways that drive lineage plasticity including the combined loss of TP53/RB1, suppression of the Notch signaling pathway, and up-regulation of lineage-determining transcription factors (LDTFs) including ASCL1 and INSM1. We hypothesize that loss of Notch signaling activates LDTFs, which act coordinately with super-enhancers and chromatin regulators to drive lineage plasticity, loss of AR signaling dependence, and NEPC progression. To test this hypothesis, we will investigate the role of NOTCH- INSM1 signaling in regulating LDTFs to drive NEPC progression and treatment resistance (Aim 1); extensively characterize the super-enhancer landscape and transcriptional reprogramming that governs lineage plasticity (Aim 2); and elucidate the transcriptional network of LDTFs that promote tumor evolution from an AR-driven state towards non-AR driven disease (Aim 3). This proposal will not only enhance our understanding of tumor evolution and cell identity, but will also identify new therapeutic approaches to target lineage plasticity. These are critical steps towards improving the early detection, treatment, and mortality of prostate cancer patients developing treatment resistance. Results may also have relevance in other cancer types that develop lineage plasticity to evade effective targeted therapies, such as lung cancer, melanoma, and breast cancer.