Unveiling the Role of UBE2J1 as the E2 Ubiquitin Conjugating Enzyme in Androgen Receptor Degradation

PROJECT SUMMARY Despite the significant clinical success of AR-targeted therapies in the management of prostate cancer (PCa), resistance to these treatments remains a significant challenge. This resistance often manifests as persistent or even elevated levels of AR and AR signaling. Emerging evidence indicates that the dysregulation of the

ubiquitination-based protein degradation process is pivotal in the accumulation of oncogenic proteins like AR, contributing to therapeutic resistance. While the role of various E3 ligases in the degradation of AR and the tumorigenesis of PCa has been extensively studied, there is a crucial yet unaddressed gap in our understanding

regarding the specific E2 enzyme responsible for AR degradation. Our research identifies UBE2J1 as the authentic E2 ubiquitin conjugating enzyme accountable for AR ubiquitination in PCa. Preliminary results reveal that the frequent loss of UBE2J1 in 10-15% of PCa patients leads to dysregulated AR ubiquitination and

degradation, contributing to its accumulation and resistance to AR-targeted therapy. By utilizing ubiquitination- based AR degraders, we have successfully reinstated AR degradation and impeded the growth of therapy- resistant PCa tumors. In light of these compelling findings, we propose three aims to validate the central

hypothesis that UBE2J1 serves as the E2 ubiquitin conjugating enzyme in regulating AR degradation and resistance to AR-targeted therapies. The overarching goal of this study is to unravel the molecular functions of UBE2J1 in governing AR ubiquitination, degradation, and various facets of PCa tumorigenesis and therapy

resistance. In Aim 1, we will undertake a comprehensive analysis of the role of UBE2J1 in regulating AR functions across multiple stages of PCa, employing both in vitro and in vivo models. We will utilize single-cell RNA sequencing and spatial transcriptomics to assess the effect of UBE2J1-loss on tumor heterogeneity and

resistance at single-cell resolution. In Aim 2, we will comprehensively dissect the molecular mechanisms through which UBE2J1 regulates AR degradation. We will identify the critical domains of UBE2J1, pinpoint the specific interaction and ubiquitination sites on AR, and reveal the E3 ligase responsible for UBE2J1-mediated AR

degradation. We will also explore alterations in the AR cistrome and transcriptome landscape in the context of UBE2J1-loss. In Aim 3, we will initially evaluate the in vitro and in vivo effectiveness of restoring AR degradation using PROTAC-based AR degraders AC67 and AC176 in various UBE2J1-KO PCa models. We will

subsequently assess the predictive value of UBE2J1 expression as a biomarker for responses to both AR antagonists and AR degraders. Completion of this project will not only substantially refine our comprehension of the molecular mechanisms governing AR degradation and therapy resistance in advanced PCa but pave the

way for the development of an impactful predictive biomarker, as well as a new therapeutic approach to combat this lethal complication of modern targeted therapies, thereby improving the clinical outcomes for patients.