Genetic manipulation of OGT enhances NK cell-mediated cytotoxicity in tumor immunity

Natural killer (NK) cells are essential effectors in immune surveillance and cancer immunotherapy, but their function is often compromised by metabolic stress and environmental factors within the tumor microenvironment (TME). O-GlcNAcylation, a post-translational modification, regulates immune responses, yet its impact on NK cell function and therapeutic potential in immune cell-based therapies remains underexplored. This study investigates the effects of O-GlcNAcylation on NK cell-mediated cytotoxicity and its potential as a therapeutic target to enhance tumor immunity. We investigated the impact of O-GlcNAcylation on NK cell cytotoxicity, focusing on its regulation under cytokine stimulation and pharmacological modulation. Mass spectrometry identified O-GlcNAc-modified proteins involved in NK cell cytotoxicity. NK92 cells were genetically engineered to delete the O-GlcNAc transferase (OGT) intronic splicing silencer (ISS) to ensure stable O-GlcNAcylation. The effects were evaluated under adverse TME conditions and in vivo tumor models. Gene expression analysis was performed to uncover the molecular networks underlying the observed effects. Cytokine stimulation and the O-GlcNAcase (OGA) inhibitor Thiamet G increased O-GlcNAc levels, enhancing NK cell cytotoxicity. Proteomic analysis identified key O-GlcNAc-modified proteins, including NK cell regulators and LRPPRC, which modulate NK function. Genetically engineered NK92 cells lacking the OGT-ISS region exhibited stable O-GlcNAcylation, preserving potent cytotoxicity under tumor-mimicking conditions and superior tumor-killing activity in vivo. Whole-transcriptome analysis of OGT-ISS-deleted NK cells revealed downregulation of TGF-β signaling and upregulation of Type I interferon signaling, as well as genes involved in cell adhesion and mobility, suggesting enhanced target recognition and cytotoxic function of NK cells. Stabilization and enhancement of O-GlcNAcylation improve the target-killing capacity of NK cells while overcoming suppressive factors in the TME. These findings highlight advanced strategies, including genetic engineering of O-GlcNAc pathways, as potent approaches to augment NK-based immunotherapies against cancer.