Project Summary Cancer immunotherapy leverages a patient’s immune system to recognize and destroy tumor cells. However, a major challenge to current therapeutics is the immunosuppressive nature of the tumor microenvironment that limits the potency of antitumor immune responses. A new approach for overcoming immunosuppression is the delivery of natural signals that stimulate innate immunity and transform tumors into an immunoactivated state to promote the infiltration and cross-priming of cytotoxic T-cells. In humans, the cGAS-STING innate immune pathway controls a potent antitumor response by detecting tumor-derived cytosolic DNA and inducing pro- inflammatory signaling. Mechanistically, DNA sensing activates the enzyme cGAS (cyclic GMP-AMP synthase) to synthesize the nucleotide second messenger 2′3′-cGAMP which then induces immune signaling through the cyclic dinucleotide receptor STING (STimulator of INterferon Genes). Thus, controlling cGAS-STING activation is an exciting new strategy for stimulating antitumor immunity. The clinical promise of natural and synthetic STING agonists underscores the importance of discovering and defining the function of novel nucleotide second messenger signals that can expand the immunotherapy toolset. For the F99 phase of this proposal, I will describe my discovery that cGAS is part of a large family of diverse immune sensors named “cGAS-like receptors” (cGLRs). The remarkable diversity of uncharacterized cGLRs in humans and animals supports that many new nucleotide signals in innate immunity remain to be discovered. My research identified cGLR1 as a dsRNA sensor in Drosophila that controls an antiviral immune response through the novel nucleotide signal 3′2′-cGAMP, demonstrating that the cGLR enzyme family can sense ligands beyond dsDNA and signal through distinct nucleotide second messengers. My current research investigates how a new Drosophila cGLR responds to a unique molecular pattern and signals through an uncharacterized nucleotide messenger. Ultimately, my research builds a mechanistic framework to define new cGLR signaling pathways in animals and humans and understand the role of diverse nucleotide second messengers in immunity. For the K00 phase of this proposal, I will leverage our expanded knowledge of nucleotide second messenger signaling to discover new molecules that stimulate antitumor immunity. Using a bacterial screening platform to deliver diverse cGAS-like enzymes to tumors I will identify chemically unique nucleotide signals that activate human innate immunity. As part of this new discovery pipeline, I will use in vivo tumor models and a mechanistic dissection of signaling in human cells to define new immune pathways and develop novel immunotherapy strategies. My research will harness the chemical diversity naturally generated by cGAS-like enzymes to expand our immunological toolset for treating cancers. In sum, my proposal will define new animal immune signaling pathways and discover n...