Abstract Clear cell renal cell carcinoma (ccRCC) harbors metabolic vulnerabilities beyond canonical HIF signaling, yet non-genetic dependencies remain largely undefined. Here, using patient-derived regulatory networks (SJARACNe→NetBID2) coupled with in vitro and in vivo functional genomics, we identify never-in-mitosis A–related kinase 5 (NEK5) as a hidden driver selectively required for VHL-deficient ccRCC. NEK5 activity—but not mRNA expression—is elevated in VHL-mutant tumors. Genetic ablation of NEK5 suppresses anchorage-independent growth, delays orthotopic tumor formation, and reduces metastasis, while sgRNA-resistant NEK5 fully restores tumorigenicity. Mechanistically, NEK5 is a mitochondrial protein that binds the translocator protein TSPO to control respiration, membrane potential, and redox homeostasis. NEK5 loss increases oxygen consumption yet decreases ΔΨm and ATP/ADP ratios, producing uncoupled oxidative phosphorylation and excessive mitochondrial ROS; ROS scavenging restores growth. A kinase-dead NEK5 mutant rescues proliferation and ATP-competitive inhibition is ineffective, demonstrating a largely kinase-independent mechanism. Epistasis positions TSPO downstream of NEK5, and VHL loss strengthens NEK5–TSPO association, linking this axis to the VHL pathway. Guided by these mechanistic insights, we developed Cereblon-based PROTACs that degrade NEK5, recapitulate the phenotypes of genetic loss, and selectively impair VHL-deficient ccRCC growth. These findings unveil NEK5 as an activity-defined, mitochondria-centered vulnerability in ccRCC and demonstrate how network-inference frameworks can expose non-enzymatic targets amenable to protein degradation therapies. Citation Format: Cheng Zhang, Zhen Xie, Shanshan Bradford. Yu, Jamie Jarusiewicz, Nina Gildor, Gisele Nishiguchi, Kaiwen Yu, Qingfei Pan, Junmin Peng, Jiyang Yu, Qing Zhang. Activity-based network analysis identifies NEK5 as a PROTAC-targetable vulnerability in ccRCC abstract. In: Proceedings of the AACR Special Conference in Cancer Research: Innovations in Kidney Cancer Research: From Molecular Insights to Therapeutic Breakthroughs; 2026 Mar 13-16; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2026;86 (5Suppl₂): Abstract nr PR018.
Zhang et al. (Fri,) studied this question.