A RIPK2 activity signature in prostate cancer: Modulation by RIPK2 inhibition and clinical association

• This study defines an 8-gene RIPK2 activity signature in prostate cancer. • The signature dynamically responds to RIPK2 inhibition in vitro and in vivo . • The signature shows stronger associations with metastasis and survival than RIPK2 mRNA. • RIPK2-dependent transcriptional regulation involves c-Myc and KDM5A. Receptor-interacting protein kinase 2 (RIPK2) has emerged as a promising therapeutic target in multiple malignancies, including prostate cancer (PC). However, the lack of reliable biomarkers to assess RIPK2 activity limits patient selection and pharmacodynamic evaluation in anti-RIPK2 therapeutic strategies. To address this need, we performed RNA sequencing of three PC cell lines (22Rv1, DU145, and PC3) with CRISPR/Cas9-mediated RIPK2 knockout using two independent guide RNAs. This analysis identified 13 candidate RIPK2-regulated genes, eight of which were validated by reverse transcription quantitative PCR and incorporated into a RIPK2 activity signature. Pharmacological inhibition of RIPK2 using two structurally distinct inhibitors significantly reduced RIPK2 signature scores in five independent PC cell lines in a dose- and/or time-dependent manner. Consistently, RIPK2 inhibition progressively suppressed signature scores in vivo , supporting its utility as a pharmacodynamic readout of RIPK2 signaling output. Elevated RIPK2 signature scores were associated with metastatic disease and adverse clinical outcomes and demonstrated stronger clinical associations than RIPK2 mRNA expression alone. Mechanistic analyses identified c-Myc and KDM5A as candidate mediators of RIPK2-dependent regulation of the signature genes. Together, these findings define a RIPK2-regulated gene signature that provides a framework for patient stratification and pharmacodynamic assessment in future RIPK2-targeted clinical studies.