Abstract Immune checkpoint inhibitors (ICIs) have transformed the treatment landscape for metastatic melanoma, yet more than half of patients do not respond to these therapies, and about 40% of responders relapse. Responses to immunotherapies are tightly controlled by interferon gamma (IFNγ) signaling. While acute IFNγ signaling promotes antigen presentation and cytotoxic immune activation, chronic IFNγ signaling induces immunosuppressive gene expression and immune escape. IRF1 is a key transcription factor activated downstream of IFNγ regulating expression of both immunostimulatory and immunosuppressive IFNγ-stimulated genes (ISGs). Therefore, precisely defining the mechanisms that control IRF1’s selective transcriptional function may uncover signaling events that could serve as biomarkers of resistance to ICIs, as well as targets to improve responses. Prior studies from our lab have shown that inhibition of ULK1 kinase activity decreases the binding of IRF1 to the promoter region of immunosuppressive IFNγ-stimulated genes, inhibiting their expression, without affecting the expression of IFNγ-induced immunostimulatory genes. However, the precise mechanism by which ULK1 regulates IRF1 transcriptional function is unknown. Using a kinase-library scoring method, we identified five putative ULK1 phosphorylation sites within IRF1 and showed that ULK1 directly phosphorylates these sites with differing efficiencies in kinase assays. To evaluate the functional relevance of these phosphorylation events, we performed rescue assays using IRF1 knockout melanoma cells and defined the ULK1-dependent phosphorylation sites on IRF1 that drive expression of immunosuppressive ISGs. Together, these findings identify IRF1 as a direct ULK1 substrate and characterize the precise mechanism by which IFNg-ULK1-IRF1 signaling axis may promote resistance to immunotherapies. These results highlight ULK1-dependent phosphorylation of IRF1 as a selective mechanism to induce expression of immunosuppressive ISGs and as a potential therapeutically actionable target to precisely modulate IFNγ signaling and enhance sensitivity to ICIs. Citation Format: Nikita Mehta, Sofia Geroyska, Eric J. Berns, Milan Mrksich, Leonidas C. Platanias, Diana Saleiro. Uncovering interferon gamma signaling programs that drive resistance to cancer immunotherapy abstract. In: Proceedings of the AACR Immuno-Oncology Conference (AACR IO): Discovery and Innovation in Cancer Immunology: Revolutionizing Treatment through Immunotherapy; 2026 Feb 18-21; Los Angeles, CA. Philadelphia (PA): AACR; Cancer Immunol Res 2026;14(2 Suppl):Abstract nr C029.
Mehta et al. (Wed,) studied this question.