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

Se-Chan Oh; Bong Chan Jeon; In-Hwan Jang; Mi-Ran Song; Heeyoun Hwang; Dasom An; Liu Yue; Yuri Jung; Yunhee Lee; Seona Jo; Sung-Kyun Park; Tae-Don Kim

doi:10.1016/j.jare.2025.08.062

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

J Adv Res. 2025 Sep 4:S2090-1232(25)00678-2. doi: 10.1016/j.jare.2025.08.062. Online ahead of print.

Authors

Se-Chan Oh¹, Bong Chan Jeon², In-Hwan Jang¹, Mi-Ran Song³, Heeyoun Hwang⁴, Dasom An⁴, Liu Yue³, Yuri Jung³, Yunhee Lee⁵, Seona Jo⁶, Sung-Kyun Park⁷, Tae-Don Kim⁸

Affiliations

¹ Center for Gene and Cell Therapy, Korea Research Institute of Bioscience and Biotechnology (KRIBB), Daejeon 34141, Republic of Korea.
² Infectious Disease Research Center, Korea Research Institute of Bioscience and Biotechnology (KRIBB), Daejeon 34141, Republic of Korea; Department of Functional Genomics, KRIBB School of Bioscience, Korea University of Science and Technology (UST), Daejeon 34113, Republic of Korea.
³ Infectious Disease Research Center, Korea Research Institute of Bioscience and Biotechnology (KRIBB), Daejeon 34141, Republic of Korea.
⁴ Digital OMICs Research Center, Korea Basic Science Institute, Cheongju 28119, Republic of Korea.
⁵ Immunotherapy Research Center, Korea Research Institute of Bioscience and Biotechnology (KRIBB), Daejeon 34141, Republic of Korea.
⁶ Center for Gene and Cell Therapy, Korea Research Institute of Bioscience and Biotechnology (KRIBB), Daejeon 34141, Republic of Korea; KRIBB School of Advanced Bioconvergence, University of Science and Technology (UST), Daejeon 34113, Republic of Korea.
⁷ Infectious Disease Research Center, Korea Research Institute of Bioscience and Biotechnology (KRIBB), Daejeon 34141, Republic of Korea; Department of Functional Genomics, KRIBB School of Bioscience, Korea University of Science and Technology (UST), Daejeon 34113, Republic of Korea. Electronic address: skpark@kribb.re.kr.
⁸ Center for Gene and Cell Therapy, Korea Research Institute of Bioscience and Biotechnology (KRIBB), Daejeon 34141, Republic of Korea; KRIBB School of Advanced Bioconvergence, University of Science and Technology (UST), Daejeon 34113, Republic of Korea; Department of Biopharmaceutical Convergence, School of Pharmacy, Sungkyunkwan University, Suwon 16419, Republic of Korea. Electronic address: tdkim@kribb.re.kr.

PMID: 40914422
DOI: 10.1016/j.jare.2025.08.062

Abstract

Introduction: 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.

Objectives: This study investigates the effects of O-GlcNAcylation on NK cell-mediated cytotoxicity and its potential as a therapeutic target to enhance tumor immunity.

Methods: 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.

Results: 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.

Conclusion: 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.

Keywords: Cancer immunotherapy; Cytotoxicity; NK cell; O-GlcNAcylation; OGT-ISS deletion.