Many cancer cells display enhanced glycolysis and suppressed mitochondrial metabolism. This phenomenon, known as the Warburg effect, is critical for tumor development. However, how cancer cells coordinate glucose metabolism through glycolysis and the mitochondrial tricarboxylic acid (TCA) cycle is largely unknown. We demonstrate here that phosphoglycerate kinase 1 (PGK1), the first ATP-producing enzyme in glycolysis, is reversibly and dynamically modified with O-linked N-acetylglucosamine (O-GlcNAc) at threonine 255 (T255). O-GlcNAcylation activates PGK1 activity to enhance lactate production, and simultaneously induces PGK1 translocation into mitochondria. Inside mitochondria, PGK1 acts as a kinase to inhibit pyruvate dehydrogenase (PDH) complex to reduce oxidative phosphorylation. Blocking T255 O-GlcNAcylation of PGK1 decreases colon cancer cell proliferation, suppresses glycolysis, enhances the TCA cycle, and inhibits tumor growth in xenograft models. Furthermore, PGK1 O-GlcNAcylation levels are elevated in human colon cancers. This study highlights O-GlcNAcylation as an important signal for coordinating glycolysis and the TCA cycle to promote tumorigenesis.

The pentose phosphate pathway (PPP) plays a critical role in macromolecule biosynthesis and maintaining cellular redox homoeostasis in rapidly proliferating cells. Upregulation of the PPP has been shown in several types of cancer. However, how the PPP is regulated to confer a selective growth advantage on cancer cells is not well understood. Here we show that glucose-6-phosphate dehydrogenase (G6PD), the rate-limiting enzyme of the PPP, is dynamically modified with an O-linked β-N-acetylglucosamine sugar in response to hypoxia. Glycosylation activates G6PD activity and increases glucose flux through the PPP, thereby providing precursors for nucleotide and lipid biosynthesis, and reducing equivalents for antioxidant defense. Blocking glycosylation of G6PD reduces cancer cell proliferation in vitro and impairs tumor growth in vivo. Importantly, G6PD glycosylation is increased in human lung cancers. Our findings reveal a mechanistic understanding of how O-glycosylation directly regulates the PPP to confer a selective growth advantage to tumours.

Programmed-death ligand 1 (PD-L1) and its receptor programmed cell death 1 (PD-1) mediate T cell–dependent immunity against tumors. The abundance of cell surface PD-L1 is a key determinant of the efficacy of immune checkpoint blockade therapy targeting PD-L1. However, the regulation of cell surface PD-L1 is still poorly understood. Here, we show that lysosomal degradation of PD-L1 is regulated by O -linked N -acetylglucosamine ( O -GlcNAc) during the intracellular trafficking pathway. O -GlcNAc modifies the hepatocyte growth factor-regulated tyrosine kinase substrate (HGS), a key component of the endosomal sorting machinery, and subsequently inhibits its interaction with intracellular PD-L1, leading to impaired lysosomal degradation of PD-L1. O -GlcNAc inhibition activates T cell–mediated antitumor immunity in vitro and in immune-competent mice in a manner dependent on HGS glycosylation. Combination of O -GlcNAc inhibition with PD-L1 antibody synergistically promotes antitumor immune response. We also designed a competitive peptide inhibitor of HGS glycosylation that decreases PD-L1 expression and enhances T cell–mediated immunity against tumor cells. Collectively, our study reveals a link between O -GlcNAc and tumor immune evasion, and suggests strategies for improving PD-L1-mediated immune checkpoint blockade therapy.

O-linked N-acetylglucosamine ( O-GlcNAc) is a dynamic post-translational modification occurring on myriad proteins in the cell nucleus, cytoplasm, and mitochondria. The donor sugar for O-GlcNAcylation...