Fig. 2: Cysteine reacts with glucose-derived metabolites to generate metabolites enriched in NRF2-activated cells and tumours.
From: Excess cysteine drives conjugate formation and impairs proliferation of NRF2-activated cancer cells
a, Table describing ten unknown CYS fates with sugar-like added masses, each annotated by ‘C,’ the predicted neutral molecular weight integer, and the retention time. Each includes values for the average abundance enrichment across NRF2on cell lines compared with NRF2off cell lines (NRF2on enrichment), the base metabolite, the implied added mass (by subtracting the monoisotopic exact mass of the base metabolite), and the resulting predicted elemental composition of the added mass. b, Fractional isotopologue enrichment of glycolytic intermediates glucose-6-phosphate (glucose-6P), DHAP, phosphoenolpyruvate (PEP), pyruvate and unknown CYS fates as measured by LC–MS metabolomics after culturing SSP25 cells in [U-13C] glucose containing medium for the indicated times. For each analyte the dominant isotopologue is shown, which corresponds to all carbons for glycolytic intermediates or the number of carbons in the predicted added elemental composition for unknown CYS fates. Mean value for n = 3 per time point is shown. Full isotopologue distributions can be found in Extended Data Fig. 3e,f. c, Relative abundance of known and unknown CYS fates measured by LC–MS metabolomics from SSP25 cells extracted in standard conditions or in extraction solvent containing the thiol-reactive conjugating agent N-ethylmaleimide (NEM). n = 3 replicate wells per condition. GSH, C209_7.0, C175_4.0, C175_4.4, C193_5.1, C191_10.4, C469_10.1: P < 0.0001, GSF P = 0.5401, C253_8.8 P = 0.7037, C193_7.7 P = 0.9959, C193_7.3 P = 0.5822 and C283_9.5 P = 0.6516. d, LC–MS measurements of C253_8.8, C193_7.7, C193_7.3, C283_9.5 or C191_10.4 after purified chemical standards of glucose metabolic fates (substrates) were incubated with or without cysteine for 1 h at 37 °C. n = 2 separate reactions. e, Schematic of two proposed groups of identified CYS fates, deriving from reactions between CYS and sugar phosphates to generate stable sugar–CYS thioether conjugates or CYS and carbonyl-containing molecules to generate reversible hemithioacetal and thiazolidine-4-carboxylic acid products. f, Schematic depicting the 13C6-CYS2 4-h infusion in autochthonous murine LUAD tumours without (WT) or with activation of a genetically encoded mutant of NRF2 (NRF2D29H) and subsequent LC–MS metabolomics. g, Mass isotopologue fraction of labelled species (M + 1-M + 3) of sugar–CYS conjugates extracted from LUAD murine tumours as depicted in f. 3GC: WT, n = 16 tumours, NRF2D29H, n = 10 tumours, M + 3: P = 0.004. 1DC: WT, n = 11 tumours, NRF2D29H, n = 10 tumours, M + 3: P = 0.0017. h, Relative ion count (total of all isotopologues) for sugar–CYS conjugates measured by LC–MS metabolomics. Relative abundance was calculated to the average of the WT group for each metabolite. n = 10, 1DC: P = 0.0356. i, Schematic depicting the collection and LC–MS metabolomics of primary human SqCLC samples without (WT) or with annotated KEAP1 or NRF2 mutations (Mut). j, Relative ion count for sugar–CYS conjugates in primary SqCLC tumours measured by LC–MS metabolomics. 3GC: WT, n = 47 tumours, Mut, n = 29 tumours, P < 0.0001. 1DC: WT, n = 44 tumours, Mut, n = 29 tumours, P = 0.0004. Error bars show s.e.m. (c,d,g,h,j). Statistical significance was assessed using two-way analysis of variance (ANOVA) with Sidak’s correction for multiple comparisons (c,g) or two-tailed unpaired Student’s t-test (h,j). For g, highlighted comparison is for M + 3 species, whereas comparisons of M + 1 and M + 2 were not significant. NS, not significant, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. Panels f and i created in BioRender; Brain, J. https://biorender.com/wtofmpq (2026).
