Project Summary The goal of this proposal is to dissect the mechanisms of self-DNA detection by the enzyme cyclic GMP-AMP synthase (cGAS), and to determine the relative contribution of its diverse signaling activities to inflammation in the microenvironment of implantable murine tumors. cGAS operates in virtually all cell types as a DNA sensory protein, which synthesizes the second messenger cyclic GMP-AMP (cGAMP) upon binding DNA. This second messenger stimulates interferon (IFN) and inflammatory activities via the downstream protein STING. Because cGAS detects the sugar-phosphate backbone of DNA, a major question relates to how this enzyme is regulated to ensure self-nonself discrimination. This question relates to much fundamental biology and the answer will impact the increasing number of clinical endeavors that target the cGAS-STING pathway. The cGAS-STING pathway is oft-discussed in the context of antitumor immunity, but the activities of this pathway that are beneficial (or not) to immunity remain unclear. For example, inflammatory activities induced by cGAS-STING in the tumor microenvironment (TME) have been reported to induce protective inflammatory and cytolytic CD8+ T cells. But cGAS-STING signaling events have also been reported to promote tumor growth and disease progression. A central idea that drives our work is that the ubiquitous presence of the cGAS-STING pathway in most cell types, along with its diverse signaling effectors (cGAMP, IFNs, cytokines), can create a complex TME prone to unpredictable outcomes (e.g. disease resolution or progression). In order to understand each activity of this pathway, new experimental tools are needed to disentangle its effector functions. Herein, we describe new synthetic biology-based genetic circuits that can dissect the effector functions of cGAS-STING, within cancer cells specifically. Notably, these systems led to the discovery of specifies-specific differences in the ability of primate and murine cGAS proteins to detect self-DNA. This finding raises questions of the suitability of mice and certain primates as accurate preclinical models for cGAS-STING function, and provide a mandate to define the mechanisms and consequences of cGAS-mediated self-DNA activities. The work in this proposal is based on the hypothesis that the cGAMP, IFNs and cytokines induced by the cGAS-STING pathway play differential roles in tissue inflammation and immunity, and that understanding the role of each of these activities, within specific cell types, requires a detailed characterization of the mechanisms of self (and nonself) DNA detection. To address this hypothesis, we propose to determine how distinct intra-tumoral cGAS activities influence protective T cell immunity (Aim 1). In Aim 2, we propose to determine mechanisms of self-DNA reactivity by human cGAS through comparative analysis of the human, mouse, chimpanzee and orangutan proteins, each of which display distinct means of self-DNA reactivity.