RIPK2/MKK7/c-Myc Signaling as a Therapeutic Target in Prostate Cancer Metastasis ABSTRACT: Despite progress in prostate cancer (PC) therapeutics, distant metastasis remains a major cause of morbidity and mortality of PC, imposing significant social and economic burdens. Three recent phase III clinical trials (i.e., SPARTAN, PROSPER, and ARAMIS) unequivocally demonstrated that preventing or delaying PC metastasis provides strong clinical benefits, prolonging the median overall survival by 10-14 months. To keep up the momentum and further improve the metastasis-free survival (a strong surrogate of overall survival) of PC patients, there is an urgent unmet need to identify novel druggable targets in PC metastasis and delineate their mechanisms of action (MoAs). Through an integrated analysis of three clinical omics databases, we identified receptor-interacting protein kinase 2 (RIPK2) as a top druggable target candidate for PC metastasis. RIPK2 is amplified/gained in 65% of lethal metastatic castration-resistant PC and its mRNA overexpression is associated with disease progression and poor prognosis. RIPK2 knockout (RIPK2-KO) or treatment with a potent RIPK2 inhibitor (e.g., the FDA-approved ponatinib) significantly suppressed PC cell invasion and colony formation but not proliferation in vitro and reduced the metastasis of 22Rv1 cells by up to 92% in vivo. Mechanistically, distinct from the canonical NOD/RIPK2/NF-κB pathway, RIPK2 strongly regulates the stability and activity of c-Myc (a driver of PC metastasis), largely by binding and activating MKK7, which we identified as a novel direct c-Myc- S62 kinase. This noncanonical RIPK2/MKK7/c-Myc signaling pathway can be potently inactivated by RIPK2-KO or ponatinib and thus is a very promising drug target in PC metastasis. Here, our overall goal is to provide a strong scientific rationale for a clinical trial by testing a central hypothesis: RIPK2/MKK7/c-Myc signaling is associated with PC progression, metastasis, and poor prognosis in patients and is critical for RIPK2-dependent PC metastasis, and its inhibition is the primary (albeit not the only) MoA of ponatinib in suppressing PC metastasis. The PI has assembled an outstanding multi-disciplinary team to pursue three distinct but interrelated specific aims: 1) assess the clinical correlations and refine the molecular basis of RIPK2/MKK7/c-Myc signaling, 2) determine whether RIPK2-KO inhibits PC metastasis primarily by inactivating the MKK7/c-Myc signaling pathway, and 3) determine whether ponatinib impairs PC metastasis primarily by inactivating RIPK2/MKK7/c- Myc signaling and define RIPK2-independent MoAs of ponatinib in suppressing PC metastasis. If successful, the proposed studies will expose a novel Achilles’ heel for PC metastasis and reveal the major MoAs of RIPK2- KO and ponatinib in suppressing PC metastasis. They will provide valuable preclinical data to guide the development of a clinical trial repurposing ponatinib to substantially impro...