Prostate cancer (PCa) accounts for an estimated 26,120 deaths in 2016 representing the second-greatest cause of cancer death among men. Recent molecular characterization of PCa has revealed striking genomic heterogeneity and defined distinct molecular subclasses that may provide insight to the variable clinical course. Recurrent mutations in SPOP are the most common point mutations in prostate cancer, occurring in about 10% of patients across early and advanced disease. SPOP is a substrate receptor of the Cullin 3-based ubiquitin ligase, which recruits androgen receptor (AR), TRIM24 and SRC-3 and other key regulators for ubiquitination and degradation, thereby governing the threshold of AR transcription, DNA damage repair, and tumor suppression. SPOPmut defines a distinct molecular class of prostate cancer characterized by activation of AR signaling and impairment of DNA double strand break (DSB) repair with transcriptional response (gene set signature) similar to that of BRCA1 inactivation. We have interrogated the TCGA RNA-seq datasets on primary (untreated) prostate cancer to define a SPOPmut transcriptional signature of 213 differentially expressed genes, and validated in independent RNA-seq cohorts for significant enrichment of this gene set in multiple cohorts of SPOPmut cases. Surprisingly, we also detected primary prostate tumors without mutation of the SPOP gene, yet showing the same gene expression characteristics to the SPOP mutant subclass, indicating that other events can phenocopy SPOP mutations in these primary “SPOPmut-like” PCa. To further understand the function and regulation of SPOP in prostate tumorigenesis, we performed tandem affinity purification coupled with mass spectrometry analysis, and identified G3BP1 as a novel SPOP- binding protein. G3BP1 is not a substrate of SPOP, but acts as a potent inhibitor of the SPOP ubiquitin ligase. This first-in-kind SPOP inhibitor revealed previously unrecognized means of SPOP inactivation that is independent of PCa-associated SPOP gene mutations. Importantly, we detected abnormally high levels of G3BP1 in PCa either with or without SPOP mutations. We hypothesize that dysregulation of G3BP1 defines a new subclass of prostate cancer with SPOPmut-like molecular signature, pathophysiological characteristics, and sensitivity to AR- and PARP targeting therapeutics. This application is built upon the unique and complementary strengths and resources of a team of investigators in prostate cancer genomics and pathology (Rubin), molecular classification, clinical management and precision therapy of PCa patients (Barbieri), computational genomics (Sboner), and cullin-based ubiquitin ligases (Zhou). In this proposal, we will 1) determine the clinical value of G3BP1 dysregulation in PCa classification and potential therapeutic implications, 2) determine the biochemical mechanisms underlying SPOP inhibition by G3BP1 and the roles of this newly discovered G3BP1-SPOP ubiquitin signaling pathway in AR ...