Abstract B010: VHL inhibits autophagy and tumor development by modulating ATG14 hydroxylation in a PHD1-dependent manner

Introduction & Objectives The Von Hippel–Lindau (VHL) protein, frequently mutated in clear-cell renal cell carcinoma (ccRCC), is primarily recognized for its regulatory role over hypoxia-inducible factors (HIFs) and its ability to manage oxidative stress. However, less is known about its potential tumor-suppressive functions that are independent of the HIF pathway. This study focuses on its role in modulating autophagy in response to nutrient deprivation. Materials & Methods To investigate the HIF-independent role of VHL in ccRCC, we analyzed VHL-RCC samples and studied the molecular interactions between VHL and autophagy regulators under nutrient-deprived conditions. We identified a post-translational modification of ATG14, hydroxylation at Proline 54, facilitated by PHD1. The study then used mutant forms of ATG14(P54A) that cannot be hydroxylated to assess the importance of this modification for VHL's inhibition of autophagy. Results Our findings revealed that VHL plays a critical role in suppressing autophagy triggered by nutrient stress. In ccRCC samples lacking functional VHL, autophagic activity was significantly elevated, which was linked to worse patient outcomes, suggesting a correlation between increased autophagy and tumor progression. Mechanistically, we discovered that VHL binds to ATG14, but only after it undergoes hydroxylation at Pro54, a modification mediated by PHD1. This hydroxylation is crucial for preventing ATG14 from forming autophagy-initiating complexes with VPS34 and ATG14L. Mutations in ATG14 that prevent its hydroxylation (P54A) abolished VHL’s ability to inhibit autophagy, and tumors expressing this mutant form of ATG14showed reduced sensitivity to VHL's tumor-suppressing effects. Finally, the combination treatment in mouse models with VHL-deficient tumors, targeting both autophagy and HIF2α, led to a significant reduction in tumor growth. Conclusions This study uncovers a previously unknown HIF-independent mechanism by which VHL suppresses tumor growth through the regulation of autophagy. Our findings offer new insights into the role of VHL in ccRCC and suggest that therapies combining autophagy inhibitors and HIF2α inhibitors may offer a more effective treatment strategy for patients with VHL-deficient tumors. Citation Format: Chuandong Wang, Kan Gong. VHL inhibits autophagy and tumor development by modulating ATG14 hydroxylation in a PHD1-dependent manner [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(5_Suppl_2):Abstract nr B010.