Project Summary Immune checkpoint blockade (ICB) targeting the PD-1/PD-L1 pathway has induced dramatic and durable clinical responses in melanoma and other cancers. Despite the success of ICB, effective treatment strategies to overcome resistance to cancer immunotherapy are lacking. TANK-binding kinase 1 (TBK1) is a versatile innate immune protein kinase nominated as a candidate immune evasion gene in a number of pooled genetic screens. Using genetic and pharmacologic tools across multiple experimental model systems, we have confirmed a role for TANK-binding kinase 1 (TBK1) as an immune evasion gene. Targeting TBK1 enhances response to ICB by lowering the cytotoxicity threshold to effector cytokines (TNFa/IFNg) secreted by immune cells. Tumor cells lacking TBK1 are primed to undergo RIPK1-dependent cell death in response to TNFa/IFNg. Beyond its effect on cancer cells, targeting TBK1 in immune cells appears to augment this effect as TBK1 inhibition +/- PD-1 blockade not only promoted accumulation of effector/progenitor exhausted CD8 T cells and M1-like macrophages, but was sufficient to enhance production of inflammatory cytokines (e.g., IFNg, TNFa) from these cells. Taken together, our results demonstrate that targeting TBK1 is a novel and effective strategy to overcome resistance to cancer immunotherapy and raise important questions about the function of TBK1 in cancer cells versus immune cells in tumor immunity. In this proposal, we aim to confirm and extend these initial observations and resolve the cell type specific roles of TBK1 in tumor immunity. In Aim 1, we will determine the cancer cell-specific roles and regulation of TBK1 in resistance to cancer immunotherapy, by defining the downstream substrates of TBK1 in the setting of ICB resistance and defining upstream the mechanism(s) of TBK1 regulation promoting immune evasion. In Aim 2, we define the T cell-specific role of TBK1 in anti-tumor immunity by examining the effect of conditional deletion of TBK1 in CD8+ T cells. We will use CD8+ T cell specific TBK1 conditional knockout mouse models to define the effect of TBK1 deletion in T cells on the efficacy of ICB and profile of tumor-infiltrating immune cells, and CD8+ T cell effector function/dysfunction. In Aim 3, we will dissect the role of TBK1 in regulating intratumoral myeloid cells. Using myeloid-specific TBK1 conditional knockout mouse models, we will define the effect of myeloid-specific TBK1 deletion on the efficacy of ICB and the landscape of tumor infiltrating immune cells, as well as the effector function of macrophages and other myeloid cell populations.