Abstract Constitutive NRF2 activation is prevalent in human cancers and drives increased cystine uptake via SLC7A11-mediated xCT antiporter activity, exceeding cysteine demands for conventional pathways including glutathione and protein synthesis. The metabolic fates and functional consequences of this excess cysteine remain incompletely understood. To identify potentially unknown cysteine fates, we developed RMA tracing, an untargeted isotope tracing/mass spectrometry approach using equimolar mixtures of labeled 13C6,15N2 and unlabeled cystine to identify cysteine metabolic fates based on characteristic isotopologue peak pairs. Our LC-MS tracing identified 29 cysteine fates, including 20 previously unknown metabolites enriched in NRF2-activated cells and tumors. Many derived from reactions between cysteine thiols and glucose-derived sugar metabolites, forming irreversible thioether conjugates with sugar phosphates and reversible hemithioacetal/thiazolidine products with carbonyl compounds. We were able to identify these novel fates in greater abundance in NRF2-activated cultured cells, mouse tumors, and human tumor samples. We then asked if there was a functional phenotype associated with excess intracellular cysteine. We grew cells in media with increased cystine and observed a dose-dependent proliferation impairment rescued by SLC7A11 inhibition with erastin. We note this proliferation defect was independent of glutamate depletion or NADPH consumption. Additionally, inhibiting glutathione synthesis with buthionine sulfoximine intensified cysteine accumulation and proliferation defects by preventing enzymatic cysteine consumption. Alternate cysteine delivery methods (beta-mercaptoethanol or N-acetylcysteine supplementation) similarly increased intracellular cysteine and conjugate accumulation, and impaired cell growth in an SLC7A11-independent manner. We define “excess cysteine stress” as both accumulation of novel cysteine-derived metabolites and a proliferation defect in high cystine conditions. Mechanistically, we attribute NRF2 activation with this cancer-associated metabolic vulnerability to excess cysteine through constitutive SLC7A11 expression. These findings delineate novel cysteine conjugates, validate their physiological relevance across models, and identify excess cysteine stress as a distinct metabolic vulnerability in NRF2-activated cancers that may inform future therapeutic strategies. Citation Format: Jennifer A. Brain, Anna-Lena B. Vigil, Kristian Davidsen, Ayaha Itokawa, Abby C. Jurasin, Hannah J. Kerbyson, Maximilian Kobiesa, Madeleine L. Hart, Sang Jun Yoon, Gina M. DeNicola, Lucas B. Sullivan. Constitutive NRF2 activation drives excess cysteine stress 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 537.
Brain et al. (Fri,) studied this question.