PROJECT SUMMARY The goal of cancer immunotherapy is to awaken the body’s anti-tumoral immune response to fight against all cancers in all patients. Our lab recently identified a key mechanism by which cancer cells are detected by our innate immune system. We now know that cancer cells, with intrinsic chromosomal instability, constantly secret the soluble small molecule 2’3’-cyclic-GMP-AMP (cGAMP). Acting as an immunotransmitter, extracellular cGAMP is taken up by surrounding tissues and immune cells to activate its receptor STING in a paracrine fashion, resulting in downstream anti-cancer immune responses. The importance of extracellular cGAMP is demonstrated by our finding that it is required for the curative effect of ionizing radiation in a murine model of breast cancer. Furthermore, we identified the extracellular enzyme ENPP1 as the only detectable hydrolase of extracellular cGAMP that blocks its signaling pathway. Therefore, we hypothesize that ENPP1 is an innate immune checkpoint that could be targeted to expand the reach of cancer immunotherapy. Although ENPP1 is cGAMP’s only hydrolase, it hydrolyzes extracellular ATP at comparable potency. Therefore, genetic and pharmacological tools that are based on complete ablation of ENPP1 activity cannot be used to distinguish the physiological role between extracellular cGAMP and ATP. Here, we propose selective genetic and chemical biology approaches: we will characterize genetic tools that selectively abolish ENPP1’s hydrolase activity toward extracellular cGAMP but not ATP (dENPP1cGAMP); in parallel we will develop substrate- specific ENPP1 inhibitors as tool compounds and potential immunotherapeutics. In Aim 1, we will fully characterize the kinetics, selectivity, and mechanisms of action of mutant dENPP1cGAMP, as well as the pathophysiology of dEnpp1cGAMP mice. In Aim 2, we will evaluate multiple tumor models in the dEnpp1cGAMP mouse strain to determine the physiological roles of extracellular cGAMP in controlling tumor growth and synergism with checkpoint blockers. In Aim 3, we will first characterize mechanism of substrate selectivity of our lead cGAMP-selective ENPP1 inhibitor and then use this inhibitor to harness the anti-tumoral effects of extracellular cGAMP. This proposal combines careful biochemical analyses with mouse genetics and tool compound development to address the role of extracellular cGAMP in cancer, with the goal of improving cancer immunotherapy.