Project Summary While checkpoint blockade has provided unprecedented clinical benefit, there are still many cancer patients who remain unresponsive to therapy. Given that the presence of tumor-infiltrating CD8+ T cells predicts clinical responses, there has been increased focus on what spontaneously generates this “inflamed” tumor microenvironment. In answering this question, current approaches have identified phagocytosed tumor DNA as the ligand that activates cGAS-STING pathway in BATF3-dependent DCs to elicit type I IFN signaling that ultimately matures DCs and effectively primes T cells. These approaches, however, depend primarily on tumor models that highly express infectious endogenous retroviruses (ERVs), which is absent in human cancers. Because there is the possibility that type I IFN and innate sensors are being erroneously induced, we propose to use ERV-free immunogenic YUMMER melanoma lines developed by Marcus Bosenberg to study the innate sensors involved in activating DCs. Based on preliminary data demonstrating that tumor rejection is STING- dependent but cGAS-independent and recent studies showing that chromosomal instability (CIN) in cancer can autonomously activate cGAS-STING-NFkB pathway, we hypothesize that CIN is activating tumor production of cGAMP which then transactivates STING in nearby BATF3-dependent DCs, leading to NFkB signaling and DC maturation. To directly test this, I will pursue the following aims. For my first aim, I will determine the host cell type in which STING expression is required for cancer immunosurveillance. To accomplish this, I will subcutaneously implant YUMMER cells in mice lacking various DC lineages and screen for any deficiencies in tumor rejection, tracking tumor volume and measuring DC activation and T cell infiltration by flow cytometry. For my second aim, I will identify the tumor-derived ligand that is responsible for activating host STING. To do this, I will induce CIN in YUMM cells (the non-immunogenic parental cell line of YUMMER lines) by overexpressing the dominant negative allele of microtubule depolymerizing kinesin (MCAK) to test whether CIN is sufficient to induce a host STING-dependent rejection. We will also stably knockout cGAS in YUMMER cells to assess whether spontaneous rejection can be abrogated in the absence of tumor cGAMP production. For my third aim, I will elucidate the downstream signaling pathway elicited by host STING. By conditionally knocking out NFkB pathway components in the identified DC lineage, we will verify whether NFkB is required for cancer immunosurveillance. Next, we will utilize RNA-Seq of sorted DCs to identify potential cytokines driving tumor rejection and verify that these identified cytokines are required for immunosurveillance by administration of depleting cytokines. I...