Abstract 2229: RIPK2 and CDK2 form a targetable positive feedback loop in prostate cancer cells.

Abstract Background: Prostate cancer (PC) metastasis is the leading cause of PC-related mortality, yet effective therapies targeting this lethal process remain lacking. We previously identified RIPK2 as a promising drug target in PC metastasis, functioning in part through a noncanonical RIPK2/MKK7/c-Myc phosphorylation pathway (Nat. Commun., 2022). However, additional downstream effectors and upstream regulators of RIPK2 remain poorly characterized. This study aims to address this knowledge gap, which represents a major barrier to developing highly effective and low-toxicity combination therapies. Methods: Genetic manipulation was performed using CRISPR/Cas9 knockout, siRNA knockdown, and overexpression of wild-type or mutant genes. The metastatic potential of PC cells was assessed by cell proliferation, Matrigel invasion, clonogenicity, and soft-agar assays. RIPK2 activation of CDK2 was evaluated by western blotting and in vitro kinase assays. RIPK2-CDK2 interaction was examined using co-immunoprecipitation, GST pulldown, proximity-ligation assay, fluorescence colocalization, and fluorescence resonance energy transfer. Gene transcription and expression were measured by RT-qPCR. Protein stability was determined using cycloheximide chase and ubiquitination assays. Synergistic effects of RIPK2 and CDK2 inhibitors are assessed with a 6x6 matrix. At least two independent cell lines were used for each cell assay. Results: RIPK2 enhanced the metastatic potential of PC 22Rv1 and PC3 cells primarily through its kinase activity, cytoplasmic localization, and induction of c-Myc expression. RIPK2 bound directly to CDK2 and activated it, leading to phosphorylation of c-Myc at S62. Notably, active CDK2 increased both transcription and protein stability of RIPK2, establishing a positive feedback loop. Co-inhibition of RIPK2 and CDK2 synergistically reduced c-Myc protein levels and suppressed PC cell invasion and colony formation. Conclusion: RIPK2 and CDK2 form a multi-layered positive feedback loop that sustains c-Myc expression and drives PC metastatic progression. Low-dose co-inhibition of these two kinases using clinically evaluated inhibitors represents a promising therapeutic strategy to overcome c-Myc-dependent PC metastasis with high efficacy and minimal toxicity. Citation Format: Ahmed Elgehama, Jaceline Pires Sanches Sanches, Lili Guerra, Wei Yang. RIPK2 and CDK2 form a targetable positive feedback loop in prostate cancer cells abstract. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 2229.