PROJECT SUMMARY/ ABSTRACT Changes in cellular glycosylation support cancer growth and progression, but the biosynthetic pathways that supply the required nucleotide-sugar precursors are resource intensive. The cell therefore utilizes salvage pathways to recycle free sugars following glycan degradation or extracellular uptake, offsetting the need for their de novo synthesis. Little is known about the extent to which cancer cells rely on sugar salvage pathways or how these pathways are regulated. Recently, the salvage kinase responsible for phosphorylating N- acetylglucosamine (GlcNAc) for reuse by the hexosamine biosynthetic pathway, N-acetyl-D-glucosamine kinase (NAGK), was found to support tumor growth, providing early evidence that cancer cells may indeed rely heavily on salvage. Preliminary data presented here show that NAGK is deprotonated at two cysteines in response to nutrient limitation, a condition frequently encountered in the tumor microenvironment. Cysteine deprotonation can alter protein activity and promote subsequent oxidation. This deprotonation is observed not just on NAGK, but on proteins throughout the cell in what may be a previously unrecognized adaptation to stress. Cysteines, especially in the deprotonated thiolate state, serves as ready sites of covalent inhibition by small molecule electrophiles. Thus, the wide-spread increase in thiolates in response to nutrient limitation may represent a class of proteins that are more responsive to covalent inhibition within stressed tumor cells than when in their protonated thiol state in healthy, perfused tissue. The aims detailed in this proposal will first characterize the effect of cysteine deprotonation on NAGK activity and its role in tumor growth and will then focus on identifying the global changes in cysteine protonation status in response to nutrient limitation as well as the environmental factors that promote this deprotonation. This work will characterize, both mechanistically and broadly, a novel stress response that may promote tumor progression despite nutrient limitation but that may also render the tumor more vulnerable to therapeutic intervention with small molecule electrophiles.

PROJECT NARRATIVE Tumor cells encounter many types of stress, such as nutrient scarcity, throughout disease progression that can promote adaptation and resilience but may also yield tumor-specific therapeutic vulnerabilities. The research proposed here describes a novel response to nutrient stress. Studying this stress response mechanism and understanding how it is employed by the cancer cell to support disease progression may thus facilitate the development of cancer therapeutics aimed at exploiting this response.