PROJECT SUMMARY The commensal bacteria that inhabit our microbiomes have a direct impact on human health. Recent studies have shown that bacteria in the gut can even alter patient responsiveness to immune checkpoint inhibitors, the latest generation of anticancer immunotherapy. The mechanisms of how bacteria influence the immune response to cancer are poorly understood; therefore, understanding bacterium-human interactions at the molecular level is critical to our understanding of the immune system, disease, and therapeutics. An important way that bacteria influence human cell signaling is through the production of small molecules that activate or inhibit human cellular receptors. We are specifically focused on how diverse bacteria manipulate the mammalian cGAS-STING pathway, a clinically relevant pathway that is critical for the immune response to cancer, as well as viruses and pathogenic bacteria. In humans, cGAS generates a cyclic dinucleotide second messenger that activates STING, which is the crucial step for initiating anticancer signaling. We recently made the unexpected finding that bacteria of the gut microbiome encode enzymes homologous to cGAS. Bacterial cGAS synthesizes cyclic dinucleotides that agonize STING, and cyclic dinucleotides released by bacteria can be taken up by mammalian cells. Cyclic dinucleotides thus provide a molecular basis for bacteria to manipulate the human cGAS-STING pathway and offer a shared molecular language for crosstalk between the microbiota and mammalian cells. Although bacterial and mammalian cGAS are highly similar, there are important differences in the heterogeneity of their cyclic dinucleotide products and studying these differences will decipher the crosstalk between these domains of life. Mammals synthesize one potent second messenger while bacterial cGAS-like enzymes are highly diverse. Different bacteria produce unique cyclic dinucleotide products that vary in their ability to agonize STING and additional human immune pathways. This proposal investigates the hypothesis that bacteria in the microbiome expressing different bacterial cGAS alleles can alter host STING signaling, the host immune response to cancer, and the effectiveness of cancer immunotherapy. First, we will investigate the role of specific bacteria expressing cGAS homologues in altering effectiveness of cancer immunotherapy. Next, we will interrogate known bacterial strains that alter patient responsiveness to cancer immunotherapy for production of cyclic dinucleotides. Finally, we will explore the human immune pathways, including STING, that are activated by the full range of diverse cyclic dinucleotides synthesized by bacteria. This proposal is focused on the mechanisms by which bacteria alter patient responsiveness to cancer immunotherapy; however, I expect results from these studies to be broadly relevant to our understanding how specific bacteria in the human microbiota influence human health and will advance the clinical utili...