PROJECT SUMMARY Pancreatic ductal adenocarcinoma (PDAC) is one of the most aggressive forms of cancer, known for its profound immune suppression and resistance to most therapies. One strategy to overcome these barriers is to leverage the effects of standard-of-care chemotherapy to increase extracellular ATP and promote anti-tumor inflammation. However, PDACs express high levels of ectonucleotidases ENPP1, CD39, and CD73 that quickly convert extracellular ATP released by dying tumor cells to immunosuppressive adenosine. In a recent Phase IA/1B clinical trial at UCLA for patients with Borderline Resectable PDAC (NCT03970252), neoadjuvant FOLFIRINOX chemotherapy and PD-1 inhibition produced changes of a more permissive anti-tumor immune microenvironment (TME) and was associated with excellent overall survival. However, compensatory adenosine signaling increased in post-treatment tumors, potentially hindering anti-tumor immunity. To strategically address this problem, a follow-up Phase 1/2 trial (NCT05688215) was initiated at UCLA in collaboration with Arcus Biosciences that introduces a small molecule inhibitor of CD73 to neoadjuvant FOLFIRINOX and PD-1 inhibition to limit adenosine in the PDAC TME. This proposal aims to extensively evaluate pre- and post-treatment patient specimens from this trial to define the impact of CD73 inhibition on tumor adenosine metabolism, signaling and -mediated immunosuppression (Aim 1). Predictors of response and resistance to CD73 inhibition will be identified. In Aim 2, autochthonous tumor models, implantable models of metastasis and human tumor explants will be used to define the roles of ENPP1 and CD39 as regulators of ATP breakdown and whether they cooperate with CD73 to generate adenosine. In addition to ATP, the ectonucleotidase ENPP1 degrades the natural STING ligand cGAMP, also released by tumor cells after DNA damaging chemotherapy, to further reduce anti-tumor inflammation. Therefore, the proposed studies in Aim 3 will build on novel preliminary data showing that adenosine directly inhibits STING activation in myeloid cells and will explore whether ENPP1 inhibition cooperates with CD73 inhibitor-mediated adenosine depletion to enhance STING signaling in the TME. These experiments will draw on a newly developed anti-human ENPP1 antibody and ENPP1 humanized mouse model. A transdisciplinary team with expertise in PDAC biology, tumor immunology, clinical trials, bioinformatics, biostatistics and mass spectrometry with a strong track record of working together has been assembled to complete the proposed studies. This project not only will provide a comprehensive understanding of ATP and adenosine metabolism in the PDAC TME but also identify the immunologic consequences of these changes. Overall, it has the potential to advance treatment outcomes for patients with this challenging cancer type by effectively reversing adenosine-mediated immunosuppression to unleash the full potency of chemo-immunotherapy.