Abstract 1947 Investigating chemical reactions associated with newly discovered cysteine conjugates

Cancer cells exhibit altered metabolism compared to healthy cells.Constitutive activation of the transcription factor NRF2 drives tumorigenesis and alters metabolism, including increasing cystine uptake, but comprehensive understanding of the intracellular fates of this cysteine dimer remains unknown.We find NRF2 activation upregulates SLC7A11, a cystine/ glutamate antiporter, explaining the increase in cystine uptake and driving up intracellular cysteine levels.We recently developed an untargeted isotope tracing/mass spectrometry approach for the unbiased identification of cysteine metabolic fates, we reveal that NRF2 drives accumulation of known and previously uncharacterized cysteine-derived metabolites in NRF2-activated cancer cells.Calculating exact masses and using isotope tracing, we find a subset of unknown cysteine fates arise from reactions between the thiol group of cysteine and endogenous sugar phosphate metabolites in cells and tumors.To confirm their identities, we perform in vitro syntheses of newly identified cysteine conjugates and measure them with mass spectrometry.Unexpectedly, we also observe that these reactions generated colored pellets and reduced cysteine conjugates levels when sugar phosphates were in excess, indicating that sugar phosphates react not only with the thiol group of cysteine, as predicted, but also through other chemical reactions.Based on the formation of color pellets and the reactivity on amine and thiols groups of cysteine with sugar phosphates, we discover that sugar phosphates react with cysteine's amine group via the Maillard reaction, a browning process observed in cooking.Additionally, our analysis of cysteine derivatives, such as N-acetyl cysteine, revealed that both free amine and thiol groups are required for sugar phosphate reactivity.These findings highlight the power of our tracing approach to identify new and unexpected metabolites in cells.To further understand these newly identified metabolites, future studies will investigate the potential enzymes that are involved in the reaction, the cellular consequences of accumulating these metabolites, and potential downstream pathways.