Abstract 350: VHL loss-driven accumulation of GCN5L1 drives clear cell Renal Cell Carcinoma by regulating acetylation of mitochondrial proteins and metabolic reprogramming.

Abstract VHL loss-associated clear cell Renal Cell Carcinoma (ccRCC) is the most common renal malignancy. VHL is a component of an E3 ubiquitin ligase that targets hypoxia inducible factor (HIF) transcription factors to proteasomal degradation under normoxic conditions. VHL loss-driven stabilization of HIF1 and HIF2 results in numerous metabolic changes favoring tumorigenesis and cancer cell survival. Although the VHL-HIF pathway is well characterized, the role of HIF independent VHL targets in ccRCC is less clear and carries immense therapeutic interest. Using patient-derived tumor cell lines, we show that loss of VHL in ccRCC leads to an accumulation of GCN5L1, which mediates lysine hyperacetylation of mitochondrial proteins, thereby attenuating the activity of several enzymes in the β-oxidation pathway. Specifically, we show that HADHA, LCAD, and SCAD enzymes show hypoacetylation and increased activity upon downregulation of GCN5L1 protein. Downregulation of GCN5L1 protein levels results in an increase in lipid metabolism and limits cytoplasmic lipid accumulation. Mechanistically, VHL interacts with and regulates GCN5L1 in a HIF-independent manner and targets GCN5L1 to ubiquitination mediated proteasomal degradation. We also show that GCN5L1 attenuates CPT1A protein levels, a rate limiting enzyme in the β oxidation pathway. In vitro assays show that loss of GCN5L1 expression selectively inhibited cell proliferation, cell invasion and anchorage independent cell growth in ccRCC cell lines but not in RCC subtypes with intact VHL. Interestingly, using etomoxir, an irreversible CPT1a/ β-oxidation inhibitor, we show that the growth repression following GCN5L1 loss is dependent on increased CPT1A expression and activity, directly linking GCN5L1 activity to lipid metabolism and ccRCC growth. Mouse xenograft studies demonstrated diminished tumor-forming ability of ccRCC cells upon loss of GCN5L1 expression compared to wild-type cells. Analysis of TCGA data revealed worse overall survival in ccRCC patients with high GCN5L1 expression, further supporting our observations from in vitro and in vivo studies. The importance of lipid metabolism in ccRCC is only beginning to be appreciated and may offer unique therapeutic targets. Lipid accumulation confers a growth and survival advantage in ccRCC and may help protect tumor cells against oxidative and endoplasmic reticulum stress. Our results identify a novel oncogenic pathway in which unregulated GCN5L1 expression drives mitochondrial lysine hyperacetylation and limits activity of β oxidation pathway enzymes, leading to increased lipid accumulation in ccRCC. Therefore, GCN5L1 could be investigated further as a possible therapeutic target in ccRCC. Citation Format: Bradley R. Webster, Kumarkrishna Raychaudhuri, Christopher Ricketts, W. Marston Linehan, Ramaprasad Srinivasan. VHL loss-driven accumulation of GCN5L1 drives clear cell Renal Cell Carcinoma by regulating acetylation of mitochondrial proteins and metabolic reprogramming 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 350.