PROJECT SUMMARY/ABSTRACT Tumor suppressor gene speckle-type POZ protein (SPOP), a substrate adaptor of cullin3-RING ubiquitin ligase, demonstrates heterozygous missense mutations in up to 15% of prostate cancers, yet the functional significance of these SPOP mutations is largely unknown. We identified SPOP-binding consensus motifs in multiple proteins of the canonical cGAS-STING and non-canonical STING-NF-κB pathways and demonstrated that human and mouse STING protein is a bona fide SPOP target. Analysis of SPOP mutant (SPOPmut) and SPOP wild-type (SPOPwt) castration-resistant prostate cancer (CRPC) clinical data sets revealed a 29-gene “SPOPmut gene signature”, which reflected STING-NF-κB signaling activity and suggested a role for the recruitment of tumor microenvironment (TME)–facilitated tumor cell growth and survival in SPOPmut CRPC. Further evaluation of the “SPOPmut gene signature” in primary, untreated prostate cancer from TCGA revealed a subset of SPOP mutant tumors, as well as a subset of CHD1 mutant (CHD1mut) tumors, that are enriched for the signature. Importantly, co-mutations in SPOP and CHD1 (chromatin remodeling factor) are well documented. In stably transduced human and mouse SPOPmut (SPOPF102C and SPOPF133V)–expressing CRPC models, we demonstrated upregulation of non-canonical STING-NF-κB-IL-6 pathway proteins, STAT3, and HMG proteins involved in promoting secretory pathway activities. We also showed that PARP inhibitor (PARPi) treatment of SPOPmut CRPC cells induces DNA damage, activates canonical cGAS-STING-TBK-IFN-ß signaling and suppresses non- canonical STING-NF-κB-IL-6/STAT3, in part through inhibitory phosphorylation (S754-STAT3), leading to growth suppression and apoptotic signaling. Olaparib (OLA) increased IFN-β secretion and reduced viability to a greater extent in SPOPmut prostate cancer cells than in control cells in coculture with macrophages versus monoculture. In addition, activation of cGAS-STING and induction of IFN-ß in macrophages were demonstrated only in OLA- treated coculture models, and neutralizing antibody experiments showed that paracrine regulation of OLA- mediated growth suppression involved IFN-β induction and IL-6 suppression of SPOPmut prostate cancer cells. We hypothesize that SPOPmut, CHD1 deletion (CHD1del) and SPOP + CHD1 co-mutations in prostate cancers promote PARPi or PARPi + anti-IL-6 therapeutic efficacy through enhanced synthetic lethality driven by increased, unrepaired DNA damage, which leads to a shift in the balance toward canonical cGAS-STING-IFN-ß signaling and suppression of IL-6/STAT3. We propose to analyze the underlying mechanisms of the SPOPmut and CHD1del prostate cancer phenotype and define these genetically driven TME alterations and their protein effectors through macrophage reprogramming using state-of-the-art proteomics (Aim 1); analyze the efficacy of PARPi and anti-IL-6 treatment in SPOPmut (F133V), CHD1del, and SPOPmut;CHD1del RM-1-BM syngeneic CRPC models (Aim 2); an...