Project Summary/Abstract Castration-resistant prostate cancer is associated with substantial clinical, pathologic, and molecular heterogeneity; most tumors remain driven by androgen receptor (AR) signaling, which has clinical implications for patient selection for AR-directed therapies. However, histologic and clinical resistance phenotypes can also emerge after prolonged AR pathway inhibition, in which the tumors become less dependent on AR signaling (referred to as ‘androgen indifferent’). These highly aggressive and lethal tumors, termed treatment-emergent neuroendocrine prostate cancer (t-NEPC), are clonally derived from adenocarcinoma through lineage plasticity or transdifferentiation. There is an urgent need for novel targets and therapies for t-NEPC. t-NEPC cells carry recurrent genetic and epigenetic alterations as an adaptive response, thus suggesting that key molecular pathways and drivers controlling cell fate may be used as targets for therapeutic intervention. N6- methyladenosine (m6A) is an abundant internal RNA modification in eukaryote messenger RNAs. Despite its functional importance in different types of cancer, their specific role in prostate cancer progression and therapy resistance still remains elusive. Our integrative analysis of phosphoproteome, epitranscriptome, transcriptome, and ribosome profiling using in vitro and in vivo models identified m6A as exciting new epigenetic mark underlying prostate cancer lineage transition and therapeutic resistance. We therefore hypothesize that m6A drives lineage plasticity and is dynamically regulated by antiandrogen in prostate cancer, and that targeting m6A can reverse the lineage transformation, thereby restoring sensitivity to antiandrogen therapy in t-NEPC. We will test our central hypothesis by pursuing the following specific aims: (1) Determine the functional significance of m6A RNA epigenetics for therapeutic resistance in prostate cancer; (2) Elucidate the molecular mechanisms of m6A function in prostate cancer lineage plasticity and antiandrogen resistance; and (3) Establish the therapeutic potential of inhibitors tageting m6A for treatment of t-NEPC. The outcomes of this project are expected to open new avenues for t-NEPC therapeutics in linking m6A RNA epigenetics to lineage plasticity-mediated therapy resistance, and should have a profound impact on our approach to tackle the greatest challenges facing patients with treatment-emergent maligancies.