Background: Enhanced glucose metabolism is a feature of most tumors, but downstream functional effects of aberrant glucose flux are difficult to mechanistically determine. Metabolic diseases including obesity and diabetes have a hyperglycemia component and are correlated with elevated pre-menopausal cancer risk for triple-negative breast cancer (TNBC). However, determining pathways for hyperglycemic disease-coupled cancer risk remains a major unmet need. One aspect of cellular sugar utilization is the addition of the glucose-derived protein modification O-GlcNAc (O-linked N-acetylglucosamine) via the single human enzyme that catalyzes this process, O-GlcNAc transferase (OGT). The data in this report implicate roles of OGT and O-GlcNAc within a pathway leading to cancer stem-like cell (CSC) expansion. CSCs are the minor fraction of tumor cells recognized as a source of tumors as well as fueling metastatic recurrence. The objective of this study was to identify a novel pathway for glucose-driven expansion of CSC as a potential molecular link between hyperglycemic conditions and CSC tumor risk factors. Methods: We used chemical biology tools to track how a metabolite of glucose, GlcNAc, became linked to the transcriptional regulatory protein tet-methylcytosine dioxygenase 1 (TET1) as an O-GlcNAc post-translational modification in three TNBC cell lines. Using biochemical approaches, genetic models, diet-induced obese animals, and chemical biology labeling, we evaluated the impact of hyperglycemia on CSC pathways driven by OGT in TNBC model systems. Results: We showed that OGT levels were higher in TNBC cell lines compared to non-tumor breast cells, matching patient data. Our data identified that hyperglycemia drove O-GlcNAcylation of the protein TET1 via OGT-catalyzed activity. Suppression of pathway proteins by inhibition, RNA silencing, and overexpression confirmed a mechanism for glucose-driven CSC expansion via TET1-O-GlcNAc. Furthermore, activation of the pathway led to higher levels of OGT production via feed-forward regulation in hyperglycemic conditions. We showed that diet-induced obesity led to elevated tumor OGT expression and O-GlcNAc levels in mice compared to lean littermates, suggesting relevance of this pathway in an animal model of the hyperglycemic TNBC microenvironment. Conclusions: Taken together, our data revealed a mechanism whereby hyperglycemic conditions activated a CSC pathway in TNBC models. This pathway can be potentially targeted to reduce hyperglycemia-driven breast cancer risk, for instance in metabolic diseases. Because pre-menopausal TNBC risk and mortality are correlated with metabolic diseases, our results could lead to new directions including OGT inhibition for mitigating hyperglycemia as a risk factor for TNBC tumorigenesis and progression.Keywords: Chemical biology; Epigenetics; Glycobiology; Hyperglycemia; Metabolic disease; O-GlcNAc transferase; Obesity; TET1; TNBC; Tumorigenesis.