Mechanism and therapeutic targeting of castration resistance in SPOP-mutated prostate cancer

PROJECT SUMMARY Prostate cancer (PCa) is the most commonly diagnosed malignancy and effective treatment of castration- resistant PCa (CRPC) remains the major challenge in clinic. SPOP is a substrate-binding adaptor of the CULLIN3-RBX1 E3 ubiquitin ligase complex and the SPOP gene is mutated in 10-15% of primary and non- castrate metastatic PCa. Notably, approximately 50% of SPOP-mutated PCa remain resistant to androgen receptor (AR) pathway inhibitors such as enzalutamide (Enza) and therefore there is an urgent need to elucidate the underlying mechanism of resistance and devise new treatment strategies. Our preliminary studies demonstrated that SPOP mutation (SPOPmut) co-occurred with aberrant activation of the MAPK pathway (MAPKalt) such as genomic alterations in the components of the RAS/RAF/MEK/ERK signaling axis and that two lesions cooperated to induce prostate oncogenesis and progression in mice. We found that SPOPmut/MAPKalt PCa cells are castration-resistant in culture and in mice. Mechanistically, we demonstrated that SPOP mutation induced stabilization of GLP and G9a proteins and augmented MAPKalt-induced expression of DNA methyltransferase DNMT1, thereby promoting DNA hypermethylation and epigenetic silencing of a group of tumor suppressor genes (TSGs), including AR-interacting proteins such as KDM6A. We confirmed hypermethylation in KDM6A gene locus and its downregulation in TCGA and SU2C PCa patient samples and in cultured PCa cells with high-level ERK1/2 phosphorylation (MAPKalt). We further showed that KDM6A interacts with AR and that SPOP mutation increases AR protein methylation in a manner dependent on KDM6A. We showed that co-treatment with MEK inhibitor re-sensitized SPOPmut/MAPKalt PCa cells to Enza. Based on these novel preliminary data, we hypothesize that SPOP mutation cooperates with aberrantly activated MAPK pathway to induce aberrant GLP/G9a protein stabilization and DNMT1 upregulation, which in turn promote DNA hypermethylation at the loci of TSGs such as KDM6A, increased AR protein methylation, AR signaling reprograming, and castration-resistant progression of PCa, thereby representing a viable target to overcome the antiandrogen resistance in SPOPmut/MAPKalt PCa. We will determine the molecular mechanism and extent to which KDM6A regulates AR protein demethylation and AR activities in SPOPmut/MAPKalt PCa cells (Aim 1), determine molecular mechanism underlying the deregulations of the GLP/G9a/DNMT1-KDM6A axis and their impact on castration-resistant growth of SPOPmut/MAPKalt PCa cells (Aim 2), and determine the mechanism, disease relevance and preclinical therapeutic targeting of functional cooperation between SPOP mutation and aberrant activation of the MAPK pathway in PCa (Aim 3). Findings from this innovative proposal will not only shed new light on our understanding of the tumor biology of SPOP mutations and molecular mechanisms of antiandrogen therapy resistance in SPOP mutated PCa, but also lead to identification of new therapeutic targets or strategies for effective treatment of PCa, thereby influencing the PCa field both scientifically and clinically.