Abstract
Introduction: Natural Killer (NK) cell therapy has shown strong potential for cancer treatment; however, NK cell efficacy is limited by their short lifespan and suppression within the tumor microenvironment. One factor contributing to this suppression may be the activation of the cyclic GMP-AMP synthase (cGAS) pathway. Since the discovery of cGAS as a DNA sensor, there has been renewed interest in DNA sensing mechanisms, although the role of DNA sensing-mediated innate immune responses on NK cells remains unclear. Poxin, a protein derived from DNA viruses, is known to degrade the cGAS signaling product cyclic GMP-AMP (cGAMP) and could potentially alleviate DNA sensing-related stress in NK cells. Objectives: This study aims to investigate whether Poxin expression can inhibit DNA sensing pathways in NK cells, thereby enhancing their cytotoxic function and anti-tumor activity. Methods: We generated NK-92 cells expressing a Poxin transgene and evaluated their response to DNA sensing activation. The cGAS-STING pathway was stimulated through either exogenous plasmid transfection or irradiation to induce endogenous double-stranded DNA (dsDNA). We assessed NK-92 and umbilical cord blood derived primary NK (pNK) cells activity through cytotoxicity assay and analysis of perforin and granzyme B expression levels, performed RNA-seq analysis to explore the mechanism and conducted functional assays to evaluate anti-tumor efficacy of NK-92 and chimeric antigen receptors (CAR)-NK-92 cells. Results: Poxin expression significantly inhibited the cGAS-STING pathway, reducing activation in response to both plasmid transfection and irradiation-induced dsDNA. RNA-seq analysis indicated increased levels of cytotoxic mediators, including perforin and granzyme B, in Poxin-expressing NK-92 cells. Furthermore, these transgenic NK-92/CAR-NK-92 cells exhibited enhanced anti-tumor activity compared to controls. Conclusion: Poxin effectively suppresses DNA sensing-mediated innate immunity in NK cells, enhancing their cytotoxicity and anti-tumor effectiveness.