KRAS is the most frequently mutated gene in human cancers. Unfortunately, KRAS inhibitors have been elusive and attempts to target downstream signaling have had limited benefit. KRAS mutations are particularly common in two especially deadly cancers: non-small cell lung cancer (NSCLC) and pancreatic ductal adenocarcinoma (PDA). The basis for this multiple investigator R01 is our recent identification of a distinct molecular program in a genetically defined subset of KRAS mutant tumors?specifically those with concurrent KRAS/LKB1 mutations? that points to a novel therapeutic vulnerability. The LKB1/STK11 tumor suppressor encodes a serine-threonine kinase that integrates nutrient availability, cell metabolism, and cell growth. We recently discovered that KRAS activation and LKB1 loss synergize to drive tumorigenesis associated with pronounced rewiring of metabolism that is coupled to changes in epigenetic regulation. In particular, we find that oncogenic cooperation between these mutations is fueled by pronounced induction of the serine-glycine- one carbon network coupled to generation of the methyl donor S-adenosylmethionine. In concert, DNA methyltransferases (DNMTs) are upregulated, leading to increased DNA methylation, with particular enrichment at retrotransposon elements, which are consequently silenced. Accordingly, KRAS-LKB1 mutant NSCLC and PDA cell lines, xenografts, and GEMMs are highly sensitive to DNMT inhibitors (DNMTi), compared to equivalent cancers lacking LKB1 mutations. Importantly, we show that this sensitivity is associated with pronounced activation of retrotransposons and induction of the interferon (IFN)-mediated cytotoxic response. IFN signaling also alters immune surveillance, both positively by increasing antigen presentation, and negatively by upregulating PD-L1. Thus, combined immune checkpoint inhibition and DNMTi represents a novel translational strategy for this vulnerable group of patients. Our proposal seeks to dissect the differences in immune microenvironment and molecular circuitry of genetically defined NSCLC and PDA subsets and to investigate the efficacy and mechanisms of response to combination therapy. These studies will utilize both human tumors and GEMMs to further define the role of discrete genetic alterations in conferring this therapeutic vulnerability. Importantly, our data has also led to the development of an investigator-initiated Phase I study of a DNMTi and an immune checkpoint inhibitor in KRAS/LKB1 NSCLC with expansion planned for PDA. Assessment of on-study specimens will be integrated with other analyses described here to further extend the clinical translation of this therapeutic approach for patients whose options are otherwise limited.