Therapies directed against the androgen receptor (AR) signaling axis represent the backbone of treatment for patients with metastatic prostate cancer, and death from prostate cancer most frequently occurs following the development of resistance to first- or second-line of AR antagonists that includes Enzalutamide and abiraterone. We have uncovered that in androgen- deficient environment or antagonist (e.g. enzalutamide)-rich condition, AR recruits a non- receptor tyrosine kinase, ACK1 (also known as TNK2), which deposits novel pY88-H4 epigenetic marks in the AR locus, facilitating AR and its splice variant, AR-V7 transcription. Significantly, immunohistochemical staining revealed that not only AR expression is upregulated as disease progress to CRPC stage, but also exhibited ACK1 upregulation. However, how ACK1 is up-regulated in CRPCs is not fully clear. In our quest to understand mechanistic details for CRPC recurrence, we observed that enzalutamide-resistant CRPCs exhibit AR acetylation at previously unknown site, Lys609 (ac609-AR). Further, ac609-AR bound to intron 1 of ACK1 gene (at ARBA1 site), upregulating its transcription in androgen-independent manner. These data reveal a previously unknown ACK1/acK609-AR/ACK1 feed-forward signaling loop that promotes CRPC recurrence. These data provided impetus to pursue development of ACK1 inhibitor, (R)-9bMS, which not only mitigated AR/AR-V7 and subsequently ACK1 mRNA expression, but also overcame enzalutamide resistance. Taken together these data suggests that (R)-9bMS with optimal pharmaceutical properties could emerge to be the `third generation' of inhibitors for CRPC treatment. The overall objective of this proposal is to examine the role of ACK1/acK609-AR/ACK1 signaling loop in CRPC recurrence and to perform drug development, biomarker, and preclinical therapeutic studies necessary to credential (R)-9bMS and its potent derivative SG4-176 as a treatment approach, with the long-term goal of ultimately advancing this therapy to the clinic. Specifically we will: Aim 1: Examine the ACK1/acK609-AR/ACK1 signaling loop as a mediator of CRPC progression and a therapeutic target. Aim 2: Investigate ACK1 & ac609-AR status as a biomarker of resistance to enzalutamide and abiraterone. Aim 3: Establish efficacy of (R)-9bMS & SG4-176 in vivo and perform toxicological studies.