Project Summary/Abstract Despite the progress in the treatment of advanced melanoma brought by the approval and use of immune checkpoint inhibitors (ICIs), most patients are resistant to therapy initially or respond but then relapse. A critical pathway that controls responses to ICIs is regulated by interferon-gamma (IFNγ). While IFNγ signaling increases expression of immunostimulatory genes (IStGs) required for response to ICIs, it also induces the expression of immunosuppressive genes (ISpGs) that support tumor immune escape and promote resistance to ICIs. This dual and opposing role of IFNγ signaling supports the need to identify an approach to inhibit expression of ISpGs, without limiting the expression of IStGs to improve the response rates to ICI-based therapies. We have identified a new transcriptional regulatory function for the Unc-51-like kinase 1 (ULK1) downstream of the IFNγ receptor, independent of its autophagy-related function. We have shown that, in malignant melanoma cells, inhibition of ULK1 represses IFNγ-induced transcription of ISpGs, without affecting the expression of IStGs, both in vitro and in vivo. Importantly, we have found that ULK1 interacts with IRF1 in the nuclear compartment of melanoma cells and show that inhibition of ULK1 kinase activity reduces the binding of IRF1 to ISpG’s promoter region, but not to IStGs. Remarkably, high levels of ULK1 correlate with poor survival and with an immunosuppressive tumor microenvironment (TME) in melanoma patients. Moreover, in preliminary studies we show that drug-targeted inhibition of ULK1 with anti-PD-1 (αPD-1) therapy induces significant increases in the percentages of IFNγ and perforin producing CD8+ T and natural killer (NK) cells and reduction in the frequency of Tregs compared to αPD- 1 treatment alone in co-culture assays using B16 melanoma cells and pmel-1 splenocytes. The goal of this project is to determine if blocking ULK1 will limit IFNγ-induced immunosuppressive effects and promote an immunostimulatory TME, overcoming resistance to ICIs in melanoma. Aim 1 will determine the mechanism by which ULK1 mediates the immunosuppressive function of IFNγ in melanoma cells. It includes generation of specific CRISPR knockout (KO) cells, and studies to determine the role of IFNγ-induced ULK1-mediated phosphorylation of IRF1 in melanoma cells. Aim 2 will determine the direct role of ULK1 in immune cell function against melanoma and in response to ICIs in vivo using conditional Ulk1 KO mice. Additionally, we will use CRISPR/Cas9 gene editing to specifically target ULK1 in primary human CD8+ T cells and study its role in regulation of signaling cascades, gene transcription, and CD8+ T cell activity against melanoma. Aim 3 will determine the role of ULK1 in ICI-resistant melanoma models. We will determine the effects of genetic or pharmacological inhibition of ULK1 in response to ICIs in vivo. Additionally, we will correlate the expression of ULK1 and IRF1 proteins in melanoma pa...