Oncolytic Viruses Enhance NK Cell Activity Through AP-1 and IRF Signaling

Background: Natural killer (NK) cells play a crucial role in cancer immunosurveillance. They infiltrate the tumor microenvironment and kill tumor cells by integrating activating signals from stress-induced ligands on cancer cells and inhibitory signals, mediated by the major histocompatibility immune complex (HLA), that preserve self-tolerance. However, their effectiveness against solid tumors is limited by tumor-driven immune-suppression. Oncolytic viruses (OV) can potentially overcome this barrier by selectively infecting tumor cells and stimulating antitumor immunity. We thus combined the oncolytic adenovirus Delta24-RGD with NK cells to target aggressive solid tumors, aiming to define the mechanisms underlying virus-induced NK cell hyperactivation and supporting clinical translation of this approach. Methods: We conducted in vitro and in vivo studies to evaluate the cytotoxicity of ex vivo-expanded cord blood-derived NK cells against Delta24-RGD-infected pancreatic ductal adenocarcinoma and glioblastoma cells. In vitro, OV-infected tumor cells were co-cultured with NK cells, followed by CyTOF phenotyping and cytotoxicity evaluation using xCelligence and Incucyte. Long-term cytotoxic capacity was assessed by first co-culturing NK cells with OV-infected or non-infected tumor cells, isolating NK cells and then testing their serial killing against fresh tumor targets. In vivo efficacy was tested using patient-derived xenograft glioblastoma mouse models. Mechanistic workup included ATAC-seq, bulk RNA-seq, CUT&RUN, and CRISPR-Cas9 knockout of JUNB and FOS. Results: NK cells exhibited synergistic cytotoxicity against OV-infected PDAC and glioblastoma cells and acquired an enhanced activation phenotype with increased expression of DNAM-1, NKG2D, CD94, NKp30, CD25, CD69, ICOS, T-bet, TRAIL, and CD107a. OV-infected tumor cells upregulated stress ligands including B7-H6, MICA/B, and ULBP1 and downregulated HLA-I. In vivo, the combination therapy significantly reduced tumor growth and extended survival in glioblastoma models. NK hyperactivation required direct contact with OV-infected tumor cells, likely driven by virus-mediated modulation of NK-ligand expression on tumor cells. Robustly activated NK cells maintained enhanced cytotoxicity through repeated tumor rechallenges, indicating durable functional reprogramming. Mechanistic studies revealed AP-1 activation followed by type I interferon signaling. Disruption of JUNB and FOS, as well as chemical AP-1 inhibition reduced this effect. CUT&RUN confirmed AP-1 dependent transcriptional programs supporting cytotoxicity. Conclusion: Oncolytic viruses synergistically enhance sustained NK cell antitumor activity by reprogramming AP-1 driven transcriptional responses. This combination strategy provides a mechanistic foundation for advancing NK cell-based therapies against solid tumors. Citation Format: Hila Shaim, Cheryl Jiang, Mayra Shanley, Hind Rafei, Jiajinlong Kang, Yu-Sung Hsu, Huihui Fan, Patrick Zhang, Rafet Basar, May Daher, Qingnan Liang, Donghai Xiong, Joy Gumin, Viswakalyan Kotapali, Corry Jones, April Gilbert, Luis Muniz-Feliciano, Gary M. Deyter, Pinaki Banerjee, Madison Moore, Ye Ethan Li, Dexing Fang, Sunil Acharya, Inci Biederstädt, Hong Jiang, Nadima Uprety, Rejeena Shrestha, Byron Jia, Alexander Biederstadt, Paul Daniel, Maliha Munir, Mecit Kaplan, Mayela Mendt, Oluwatosin Banjo, Vakul Mohanty, Jinzhuang Dou, Xianli Jiang, Elizabeth Shpall, Ken Chen, Abhinav Jain, Frederick F. Lang, Candelaria Gomez-Manzano, Juan Fueyo, Katayoun Rezvani. Oncolytic virus infected tumors drive AP-1 and IRF signaling in NK cells to sustain anti tumor activity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 5194.