Fei, G. Q.; Marando, V. M.; Olszewski, K. L.; Gibbs, B. E.; Hall, A. M. J.; Jost, M.; Walker, S.

O-GlcNAc transferase (OGT) is an essential mammalian glycosyltransferase that installs O-GlcNAc onto thousands of cytosolic and nuclear proteins. Dysregulated glycosylation by OGT is associated with disease states, including cancer and neurodegeneration. However, cellular consequences of acute loss of O-GlcNAc remain poorly understood. Here, we employed a cell-permeable small molecule inhibitor of OGT, OSMI-4, coupled with transcriptomic and lipidomic analyses to characterize the effects of O-GlcNAc depletion in human cell lines. Loss of O-GlcNAc led to changes in expression of lipid metabolism genes and remodeling of the lipidome, with unsaturated phosphatidylcholines (PCs), saturated lysophosphatidylcholines (LPCs) and unsaturated storage lipids accumulating. We also observed increased lipid droplets. Our results suggest that polyunsaturated fatty acids (PUFAs) accumulate in the membrane under low O-GlcNAc conditions and that cells buffer this accumulation by removing PUFAs from membrane lipids and sequestering them in lipid droplets. We found that treatment with OSMI-4 sensitizes cells to ferroptosis inducers, likely because accumulated cell membrane PUFAs undergo peroxidation. Taken together, our results highlight lipid remodeling as an important process that mediates the downstream effects of loss of O-GlcNAc. It may be possible to exploit the synergy between OGT inhibitors and ferroptosis inducers for therapeutic benefit.