Clear cell renal cell carcinoma (ccRCC) is the most frequent histological kidney cancer subtype, accounting for more than 75% of all kidney cancer cases. This malignant tumor has a high mortality rate in the advanced stage, particularly when metastases occur. ccRCC is a prototypical tumor characterized by histological appearance of cells filled with abundant lipids and glycogen. Due to alterations and reprogramming in multiple metabolic pathways, ccRCC is always classified as a metabolic disease. Identification of metabolic vulnerabilities in ccRCC is crucial for the development of new therapeutic targets and strategies. As a ferroptosis-susceptible tumor, the molecular and metabolic basis for ccRCC that underlies the dynamic regulation of ferroptosis sensitivity is poorly understood.

The study employed genome-wide CRISPR-Cas9 screening to identify essential genes in clear cell renal cell carcinoma (ccRCC), revealing TYMP as a key upregulated factor. Functional assays, including genetic knockout and pharmacological inhibition using TAS-102, demonstrated that TYMP is critical for ccRCC cell survival through the induction of ferroptosis. Mechanistic studies showed that TYMP inhibition leads to dThd and dUrd accumulation, mitochondrial dysfunction, and p53-mediated repression of SLC7A11/GPX4, triggering ferroptosis. Additionally, mitochondrial DNA release activated the cGAS/ZBP1-STING pathway, promoting type I interferon response and enhancing CD8+ T cell infiltration. In vivo experiments confirmed the synergy between TYMP inhibition and anti-PD-1 immunotherapy in suppressing tumor growth.

In this study, by performing metabolism-focused CRISPR-csa9 screens in renal cancer cells and in combination with sample analysis, we identified TYMP-mediated thymidine catabolism as an anti-ferroptotic process that is necessary for renal cancer cell survival and ICB resistance. Knockdown of TYMP or pharmacological inhibition induces dThd accumulation and selectively impairs mitochondria homeostasis, finally triggering ferroptosis and innate immune response. Mechanistically, TYMP inhibition causes excessive ROS production and p53-mediated ferroptosis via transcriptional repression of xCT/GPX4. Additionally, mitochondrial damage promotes cytosolic release of mtDNA to activate cGAS/ZBP1-STING-IFN-Is signaling, further intensifying T-cell mediated anti-tumor response. FDA-approved TYMP-associated inhibitor TAS-102 synergizes with anti-PD-1 therapy in suppressing ccRCC tumor growth, thereby identifying a potential combinational therapeutic strategy for ICB-resistant ccRCC patients.

Our results demonstrate that TYMP-mediated thymidine catabolism is a previously unidentified mechanism of ferroptosis suppression and indispensable for renal cancer cell survival. Targeting TYMP with FDA-approved TAS-102 induces ccRCC ferroptosis and enhances the sensitivity to anti-PD-1 treatment.