O-GlcNAc transferase (OGT) functions as a nutrient sensor, utilizing a substrate whose synthesis incorporates major metabolites such as glucose, glutamine, and acetyl-CoA. O-GlcNAcylation has a well-characterized role in regulating central glucose metabolic pathways, however, a potential role in regulating acetate metabolism has not yet been explored. Additionally, this post-translational modification has been shown to be elevated by increasing OGT levels in a variety of cancers and reduced O-GlcNAcylation can block cancer growth. Glioblastomas (GBMs) preferentially generate acetyl-CoA from acetate as a fuel source to promote biosynthesis and tumor growth. Here, we identify a novel mechanism whereby OGT regulates acetate-dependent acetyl-CoA production through regulation of phosphorylation of acetyl-CoA synthetase 2 (ACSS2) by cyclin dependent kinase 5 (CDK5). OGT is required and sufficient for GBM cell growth and regulates acetate conversion to acetyl-CoA and lipids. Elevating O-GlcNAcylation in GBM cells increases phosphorylation of ACSS2 on Ser-267 in a CDK5-dependent manner. Importantly, we show that Ser-267 phosphorylation of ACSS2 regulates its protein stability, by reducing polyubiquitination and proteasomal degradation. ACSS2 Ser-267 is critical for OGT-mediated GBM growth and regulation of acetate metabolism as overexpression of ACSS2 Ser-267 phospho-mimetic rescues lipid droplets and growth in vitro and in vivo. Thus, O-GlcNAcylation regulates cancer acetate metabolism via regulation of CDK5-mediated ACSS2 phosphorylation.

OGT plays an essential role in embryonic development, as OGT null mice fail to be viable; however, the effects of whole body inhibition of OGT and O-GlcNAcylation in adult mice are unknown. Here, we show that inducible global knockout of OGT (iOGTKO) in adult mice was lethal due to development of a severe loss of gastrointestinal (GI) intestinal homeostasis and development of inflammatory bowel disease (IBD)-like phenotype. Conditional loss of OGT led to weight loss, reduced food consumption, and detection of fecal blood. Histopathological analysis showed severe defects in GI homeostasis, including widespread cryptic abscesses, GI neutrophil infiltration, and disruption of the mucus layer. OGT-depleted GI tissue and isolated epithelial organoids contained reduced MUC2 and elevated apoptosis suggesting a defect in mucin-secreting goblet cells potentially associated with elevated TNF-alpha levels. Furthermore, OGT deletion disrupted the epithelial architecture of enteroids, which was reversible via anti-TNF neutralizing antibody treatment. Treatment of iOGTKO mice with antibiotics ameliorated the GI-associated symptoms including fecal blood, weight loss, and enhancing overall survival, implicating microbial dysbiosis as a contributor to the GI-associated phenotype. Importantly, we show that patients with IBD have reduced total O-GlcNAcylation compared to healthy patients. These results identify O-GlcNAcylation as a regulator of intestinal homeostasis in adult mice and potential intervention point for treating inflammatory bowel disease.