PROJECT SUMMARY Prostate cancer (PCa) is the most diagnosed cancer among American men and many patients with primary PCa will eventually develop metastatic disease. Metastatic disease is often resistant to the current “standard-of-care” treatment, androgen receptor (AR) targeted therapies, such as enzalutamide. Resistance to AR targeted therapies largely limits the clinical outcome of patients with advanced PCa. Therefore, there is an unmet need for revealing the molecular mechanisms of resistance and identifying novel vulnerabilities to overcome it. Through a comprehensive in vivo library screen, we have identified Ubiquitin Conjugating Enzyme E2 J1 (UBE2J1), which is lost in 8-15% of patients with PCa, as one of the top candidate modifiers of response to AR targeted therapy. Through CRISPR-mediated knock-out, I demonstrated that UBE2J1 depletion in LNCaP/AR PCa cells confers resistance to AR targeted therapies. Furthermore, UBE2J1 depletion also confers resistance to Androgen Deprivation Therapy (ADT). Mechanistically, I have observed significantly increased expression of AR and AR target genes in cells with UBE2J1 depletion, suggesting the loss of UBE2J1 may slow down the ubiquitin mediated degradation of AR. This hypothesis is further supported by the observation of a significant increase of AR binding in AR regulated enhancer sites upon UBE2J1 depletion. Strikingly, restoring AR degradation in UBE2J1 deleted cells with two innovative AR PROTACs rescues the sensitivity to ADT. Furthermore, as UBE2J1-loss promotes ER associated degradation pathway (ERAD), it might rescue cell death caused by a ER stress inducing microenvironment including therapy treatment. I have observed a significant increase of key ER-stress regulator, GRP78. Collectively, my preliminary data suggests the loss of UBE2J1 may confer AR targeted therapy resistance through impaired AR degradation and promoting ERAD and rescuing tumor cell apoptosis caused by AR targeted therapy. I propose two specific aims to examine this hypothesis and elucidate the function and molecular mechanism of UBE2J1-loss conferred resistance to AR therapy. For the first aim, I will comprehensively elucidate the consequence of UBE2J1 depletion both in vitro and in vivo, using other PCa cell lines and SCID mouse xenograft models. For the second aim, I will reveal the mechanism by which UBE2J1 causes AR-targeted therapy resistance. I will explore whether UBE2J1, as an E2 ubiquitin conjugating enzyme, targets AR for degradation and regulates AR signaling. On the other hand, I will examine ERAD and ER stress in cells with UBE2J1 depletion and determine whether rescuing cell death is the mechanism of resistance to AR targeted therapy. The successful completion of this study will contribute novel insights into the mechanism of resistance to AR targeted therapies, advancing our understanding of the connection between AR degradation and ERAD to therapy resistance. It may also lead to the identification of ne...