Inhibiting the Hexosamine Biosynthetic Pathway Lowers O-GlcNAcylation Levels and Sensitizes Cancer to Environmental Stress

Abstract

The amounts of the intracellular glycosylation, O-GlcNAc modification, are increased in essentially all tumors when compared to healthy tissue, and lowering O-GlcNAcylation levels results in reduced tumorigenesis and increased cancer cell death. Therefore, the pharmacological reduction of O-GlcNAc may represent a therapeutic vulnerability. The most direct approach to this goal is the inhibition of O-GlcNAc transferase (OGT), the enzyme that directly adds the modification to proteins. However, despite some recent success, this enzyme has proven difficult to inhibit. An alternative strategy involves starving OGT of its sugar substrate UDP-GlcNAc by targeting enzymes of the hexosamine biosynthetic pathway (HBP). Here, we explore the potential of the rate-determining enzyme of this pathway, glutamine fructose-6-phosphate amidotransferase (GFAT). We first show that CRISPR-mediated knockout of GFAT results in inhibition of cancer cell growth in vitro and a xenograft model that correlates with O-GlcNAcylation levels. We then demonstrate that pharmacological inhibition of GFAT sensitizes a small panel of cancer cells to undergo apoptosis in response to diamide-induced oxidative stress. Finally, we find that GFAT expression and O-GlcNAc levels are increased in a spontaneous mouse model of liver cancer. Together these experiments support the further development of inhibitors of the HBP as an indirect approach to lowering O-GlcNAcylation levels in cancer.

Hyaluronic Acid Fuels Pancreatic Cancer Growth

“…The HBP is activated in a Kras-dependent manner in PDA (5), and it is similarly elevated in numerous cancers to provide a diverse set of functions, including the regulation of proliferation, survival, angiogenesis, and metastasis (9). As such, we and others have proposed that the HBP may provide a selective vulnerability for cancer therapy, with GFAT1 as an attractive therapeutic target (5,10,40,41). Indeed, several pan glutamine-deamidase inhibitors (e.g.…”

“…The resulting CRISPR/Cas9 plasmid was transformed into chemically competent Stbl3 cells, miniprepped for plasmid DNA, and sequence-verified. sgRNA oligonucleotide pairs for GFAT1 (10) and NAGK are as follows: GFAT1 (sg1 Fwd 5′-CACCgCTTCAGAGACTGGAGTACAG-3′; sg1 Rev 5′-AAACCTGTACTCCAGTCTCTGAAGc-3′) and NAGK (sg1 Fwd 5'-CACCgTAGGGGAGGCACACGATCCG; sg1 Rev 5'-AAACCGGATCGTGTGCCTCCCCTAc-3'; sg2 Fwd 5'-CACCgGCCTAGGGCCTATCTCTGAG-3'; sg2 Rev 5'-AAACCTCAGAGATAGGCCCTAGGCc-3'). Human PDA were transiently transfected using Lipofectamine 3000 according to the manufacturer's instructions.…”

“…Because the HBP integrates nutrients from several major macromolecular classes to produce UDP-GlcNAc, predictably it also acts a nutrient sensing mechanism for available energy within a cell (7). Indeed, numerous studies across cancer subtypes have demonstrated how HBP activity is enhanced to support tumor survival and growth (8)(9)(10)(11) and even immune evasion through alteration of extracellular glycosylation content (12).…”

Molecular Interrogation to Crack the Case of O‐GlcNAc

“…However, O‐GlcNAc regulation can go beyond aiming at OGT or OGA. For instance, CRISPR‐Cas9 genome editing and small‐molecule inhibitors have been coupled to knockout glutamine fructose‐6‐phosphate amidotransferase (an essential enzyme in the HBP) and block the production of UDP‐GlcNAc . This approach resulted in low cellular O‐GlcNAc levels and suppressed cancer‐cell proliferation.…”

O-GlcNAcylation in Chronic Lymphocytic Leukemia and Other Blood Cancers

“…ER stress in CLL cells is partly related to signaling through the BCR ( 199 , 200 ) and may increase HBP activity through a IRE1/XBP1s/GFAT axis that can protect cells from death ( 15 ). Inhibition of this O-GlcNAcylation-mediated regulatory loop helps sensitize cancer cells to stress ( 201 ). Taken together, these observations are consistent with a view that O-GlcNAcylation may sustain cancer cells and that blocking OGT and the hexosamine pathway offers a novel approach to treatment for many cancers including CLL.…”