Project Summary Pancreatic ductal adenocarcinoma (PDAC) is currently the third leading cause of cancer-related death in the United States. The five-year survival rate of less than nine percent is attributed mainly to a difficulty in early detection and a lack of effective treatments for PDAC. Novel immunotherapies such as immune checkpoint blockade which have revolutionized treatment of other cancers have failed to achieve efficacy in PDAC. This is thought to be due to the immunosuppressive microenvironment of PDAC which limits the effector T cell infiltration and activation necessary for effective immunotherapy. Understanding how to increase T cell activation and infiltration despite an immunosuppressive microenvironment is essential to increasing the efficacy of immunotherapies in PDAC. Using a genetically engineered mouse model (“KPCY”) of pancreatic cancer, our lab has demonstrated that clones derived from primary KPCY tumors can be divided into “T-cell-inflamed” or “non-T-cell-inflamed” phenotypes. While T-cell-inflamed tumors are responsive to combination immunotherapy, non-T-cell-inflamed tumors are resistant. A CRISPR screen of non-T-cell-inflamed cells found that DNMT1, a DNA methyltransferase involved in maintaining methylation marks through DNA replication, is important for tumor growth in PDAC. Knockout of DNMT1 in a non-T-cell-inflamed tumor line was found to significantly increase T cell infiltration and decrease tumor growth in vivo. T cell depletion, however, was found to rescue the wild type phenotype. Based on recent studies of the non-specific DNMT inhibitor azacytidine, loss of DNMT1 is hypothesized to de-repress endogenous retroviruses in the genome, leading to dsRNA induction which activates a “viral mimicry” immune mechanism. The goals of this proposal are: (1) to determine how loss of DNMT1 promotes T-cell dependent anti-tumor immunity, and (2) to assess the therapeutic potential of targeting DNMT1 in conjunction with immunotherapy. To examine the mechanism of tumor cell intrinsic DNMT1 in inhibition of T-cell dependent suppression of tumor growth, I will first assess the changes in DNA methylation at endogenous retroviruses and genes related to the viral mimicry pathway provoked by loss of DNMT1. I will then determine if loss of DNMT1 leads to activation of the viral mimicry pathway, and whether inhibition of this pathway rescues the wild type phenotype (Aim 1). To determine the translational potential of DNMT1 as a target, I will first identify anti-tumor changes in T cells and tumor cells provoked by loss of DNMT1. I will trial immunotherapy in mice with DNMT1 knockout tumors to determine changes in sensitivity to immunotherapy (Aim 2). Ultimately, the insights gained from this study will provide us with a better understanding of the role of DNMT1 in regulating the PDAC immune microenvironment and its potential as a therapeutic target.