Combinational Analysis of Metabolomic and O-GlcNAcylation Omics Reveals the HBP Metabolic Regulation of Chemoresistance via GFPT1/NR3C1 O-GlcNAcylation/GPX4 Axis

Dysregulation of Ferroptosis is linked to chemoresistance, and reprogramming of glucose metabolism is involved in this progress. However, the underlying mechanisms remain obscure. Herein, using metabolic profiling, we find that hexosamine biosynthetic pathway (HBP) metabolism and the byproduct, UDP-GlcNAc, are substantially up-regulated in chemoresistant tumor tissues and cells. UDP-GlcNAc-derived O-GlcNAcylation levels increase with the decreased Ferroptosis and chemosensitivity in Cancer cells. Knockout of the rate-limiting enzyme GFPT1 in HBP metabolism inhibits O-GlcNAcylation, induces Ferroptosis, and mitigates chemoresistance of orthotopic bladder Cancer in Gfpt1^-/- mice. The global O-GlcNAcylation omics mapped the O-GlcNAcylated sites and proteins in resistant and nonresistant tumor cells, showing that NR3C1 is highly O-GlcNAcylated at Thr²⁹⁹ in response to chemotherapy. The chromatin immunoprecipitation Sequencing delineates that NR3C1 O-GlcNAcylation at Thr²⁹⁹ prominently enhances transcriptional activity of GPX4 by facilitating the binding of NR3C1 on GPX4 promoter, inhibiting Ferroptosis. Higher O-GlcNAcylation of NR3C1 improves protein stability and reduces proteasome-dependent degradation by suppressing ubiquitination. Inhibition of NR3C1 O-GlcNAcylation via Thr²⁹⁹ mutant or knockout of NR3C1 facilitates Ferroptosis and improves chemosensitivity of resistant Cancer cells in vitro and in vivo. In addition, we propose a novel predicting model for chemoresistance based on the GFPT1 and NR3C1 levels in pre-chemotherapy biopsy tissues through a training set and a validation set. These findings exemplify how metabolic and epigenetic reprogramming regulates Ferroptosis via the GFPT1/NR3C1/GPX4 axis, and implicate NR3C1 O-GlcNAcylation as a potential target for reversing chemoresistance.