PROJECT SUMMARY/ABSTRACT Speckle-type POZ protein (SPOP), a substrate adaptor of the cullin3 (Cul3)-RING ubiquitin ligase (CRL3), demonstrates tumor- and context-specific oncogenic or tumor suppressor functions in various human tumors. The potential role of SPOP in prostate cancer (PCa) gained recognition through the results of multiple genomic studies during recent years that identified heterozygous missense mutations in the SPOP substrate-binding cleft (MATH domain) in up to 15% of PCas. However, the functional significance of PCa-associated SPOP mutants in PCa progression and their impact on current PCa treatment options are largely unknown. Interestingly, SPOP has been shown to regulate genomic stability through modulation of DNA double-strand break repair in primary PCa, and specific SPOP mutations can potentiate the cytotoxic effects of PARP inhibition. We have identified SPOP-binding consensus (SBC) motifs in multiple protein components of the cGAS-STING pathway (cGAS, STING, TBK1), and members of B7 family immune checkpoint proteins (PD-L1, CD276, VISTA). We have also shown that expression of SPOP mutation (F102C) can stabilize STING, TBK1, and PD-L1 and increase STING phosphorylation (p-S366/S365) in human and mouse PCa cells. We hypothesize that the presence of PCa- associated SPOP mutations leads to activation of cGAS-STING signaling and induction of B7 family immune checkpoint proteins including PD-L1, by reducing SPOP substrate ubiquitination. We further hypothesize that the presence of PCa-associated SPOP mutations represents a therapeutic vulnerability that can be exploited by PARP inhibition combined with immune checkpoint blockade targeting of these B7 family proteins (such as anti– PD-L1) to maximize direct intrinsic DNA damage–induced cytotoxicity and cGAS-STING–mediated T-cell anti- tumor response. We will use biochemical methods, genetic approaches [including SPOP wild-type and SPOP mutants (F133V and F102C) doxycycline-inducible expression human and mouse PCa cell lines], and novel syngeneic mouse PCa models to test our hypothesis. Importantly, state-of-the-art proteomics and single cell RNA-seq analyses will be used to determine the molecular pathways and tumor microenvironment immune cell treatment responses altered by SPOP-F133V and SPOP-F102C mutations, to identify novel, relevant SPOP targets (markers) in our PCa models. Overall, the results of these studies will establish PCa-associated SPOP mutations as critical regulators of PARP inhibitor and anti–PD-L1 combination therapies, and define a subset of PCas that respond to this therapeutic approach. More broadly, these studies may lead to the development and clinical testing of specific SPOP mutations as predictive markers for DNA damage response–targeting and immune checkpoint blockade combination therapies for PCa.