51Antioxidant dependencies in fumarate hydratase–deficient kidney cancer

Abstract Background Fumarate hydratase (FH)-deficient renal cell carcinoma (RCC), associated with hereditary leiomyomatosis and RCC (HLRCC), is a highly aggressive kidney cancer with limited treatment options. FH loss leads to accumulation of the oncometabolite fumarate, which drives tumor progression not only by inhibiting key enzymes but also by covalently modifying cysteine residues on proteins (succination). Fumarate accumulation leads to persistent oxidative stress, disrupting redox homeostasis and contributing to DNA damage, metabolic reprogramming, and immune evasion. Yet how FH-deficient tumors survive this oxidative pressure—and whether these adaptations reveal druggable vulnerabilities—remains unclear. To address this, we previously functionally characterized FH variants of uncertain significance, generating a panel of RCC cell lines with graded FH activity and fumarate accumulation. This system enables precise dissection of fumarate-driven metabolic rewiring and the identification of therapeutic targets. Methods We employed an integrated approach combining transcriptomic profiling of FH-deficient RCC tumors, metabolomics, isotope tracing, and CRISPR-based functional genomics. Using our FH-VUS cell line panel to model increasing intracellular fumarate, we examined antioxidant pathway activation, glutathione (GSH) metabolism, and redox balance. Targeted CRISPR dropout screens were used to identify metabolic dependencies, with a focus on nutrient transporters. Liquid chromatography-mass spectrometry (LC-MS) and stable isotope tracing were used to quantify antioxidant metabolite levels and map substrate utilization. Results Both FH-deficient tumors and high-fumarate cell lines showed robust upregulation of NRF2 target genes, particularly those involved in GSH and thioredoxin metabolism—two key antioxidant systems. CRISPR-mediated knockout of NRF2 resulted in rapid cell death in FH-deficient cells, confirming its essential role in counteracting fumarate-induced oxidative stress. Although intracellular GSH levels were markedly elevated in high-fumarate cells, the GSH: GSSG ratio remained unchanged, suggesting that these cells maintain redox balance through enhanced antioxidant capacity rather than by altering the redox potential itself. Stable isotope tracing revealed that most of the cysteine in FH-deficient cells is diverted to GSH synthesis. Re-expression of wild-type FH reversed this effect, reducing cysteine incorporation into GSH. A focused CRISPR screen identified SLC7A11 as a selective dependency in FH-deficient cells. Finally, cystine deprivation triggered irreversible cell death, indicating that these tumors are metabolically addicted to cystine import to sustain their antioxidant defenses. Conclusions Our findings define a central redox adaptation in FH-deficient RCC: sustained activation of NRF2-dependent antioxidant programs and a reliance on cystine import to fuel glutathione synthesis. This cystine addiction creates a metabolic vulnerability that may be therapeutically exploited using SLC7A11 inhibitors or cystine-lowering strategies. By leveraging a tunable FH-VUS cell line model, we directly linked fumarate accumulation to redox rewiring and nutrient dependency. These insights uncover actionable metabolic liabilities in FH-deficient RCC and provide a framework for targeting redox adaptations in malignancies with high oxidative stress.