Summary The objective of this proposal is to determine the molecular mechanisms underlying luminal-to-neuroendocrine transdifferentiation (NET) of prostate cancer (PCa). NET underlines the critical progression of prostate adenocarcinoma (AdPC) to neuroendocrine PCa (NEPC), a lethal disease with no cure. Understanding the molecular mechanisms that drive NET may pave the way to discovering critical druggable steps and identifying biomarkers of NEPC progression. In our preliminary studies, we generated a model system where we push the prostate luminal epithelial cell LNCaP towards NE cell type over a period of 28 days via the overexpression (OE) of a single gene FOXA2, a chromatin-pioneering factor. Time-course RNA-seq analyses of FOXA2-OE cells revealed a gradual loss of epithelial gene expression and gain of NE transcriptional program. This transcriptomic shift is accompanied by substantial alterations in TF binding and chromatin remodeling at luminal and NE enhancers, accompanied by remarkable changes in DNA methylation and 3D chromatin architecture. Interestingly, FOXA2 induced the transcription of a neural TF called NKX2-1 on day 14 (D14) of OE, and the expression of NKX2-1 is required for the completion of FOXA2-driven NET. Further, NKX2-1 and FOXA2 bind to promoters and enhancers, respectively, and they interact with each other through chromatin looping to co-occupy active enhancers. Further, we found regional DNA demethylation around FOXA2-binding sites and a remarkable up-regulation of TET1, the key enzyme that catalyzes DNA demethylation, on D14 following FOXA2 OE. These preliminary data lead to our central hypothesis that enhancer-bound FOXA2 interacts with promoter-bound NKX2-1 to drive luminal-to-NE transdifferentiation, involving 3D chromatin reorganization and enhancer priming and activation, which requires regional DNA demethylation mediated by TET1. To test these hypotheses, Aim 1 will first determine whether the abilities of NKX2-1 to bind DNA and interact with FOXA2 are both required for its role in facilitating FOXA2-induced NET and then validate this pathway in additional NEPC models. Aim 2 will evaluate whether TET1 induction at D14 is required for FOXA2- driven NET through modulating regional DNA demethylation and NE enhancer priming. Lastly, Aim 3 will validate the genomic and epigenomic reprogramming in clinical samples by comparative analyses of the spatial transcriptome, whole-genome DNA methylation, and 3D chromatin structure in AdPC vs. NEPC samples. We will also examine the downstream genes regulated by a TET1 inhibitor and test its efficacy in suppressing NEPC growth in vitro and in vivo.