Project Summary Pancreatic ductal adenocarcinoma (PDAC) is almost universally lethal and is projected to become the second-leading cause of cancer related deaths in the US by 2030. Conventional (genotoxic) chemotherapy approaches that make up the current standard of care are mostly ineffective and prolong survival of advanced PDAC patients by less than one year on average. Similarly, small molecule drugs targeting aberrantly activated oncogenic signaling pathways have shown disappointing clinical results and accordingly have failed to gain FDA approval for PDAC. An alternative strategy to these two approaches is to exploit metabolic dependencies that are unique to malignant cells by virtue of their deranged cellular metabolism. While there are well-characterized resistance mechanisms to genotoxic and targeted therapies, deprivation of certain nutrients critical for proliferation of malignant cells appears to be an insurmountable barrier for cancer progression. However, cells have redundant means of acquiring these critical nutrients, and so inhibition of a single metabolic enzyme is generally not sufficient to deny them to cancer cells. Thus, combinatorial blockade of multiple metabolic pathways could be required to impose deficiency of key metabolites on malignant cells. Pyrimidine nucleotides represent a class of metabolites that has been shown in numerous studies to be essential for PDAC and a host of other malignancies. Importantly, there are several clinical grade inhibitors of pyrimidine synthesis enzymes that have shown preclinical promise as anticancer agents. However, these drugs have uniformly failed to show efficacy in clinical trials in which they were used as monotherapy against various malignancies. One potential explanation for this is that there are two major pathways by which cells generate nucleotides, termed the de novo and salvage pathways, and these inhibitors block the key de novo pathway enzyme dihydroorotate dehydrogenase (DHODH), thus leaving the salvage pathway fully intact. We’ve characterized the response of various PDAC cell lines to the DHODH inhibitor brequinar (BQ). We found that some PDAC cell lines are highly resistant to BQ in cell viability assays compared to their more sensitive counterparts. We then screened some 350 known kinase inhibitor compounds to probe for any that could restore BQ sensitivity in our resistant PDAC cell lines, and this nominated the preclinical BTK inhibitor CNX-774 as the strongest hit. Follow-up studies have shown that combined BQ/CNX-774 treatment leads to profound cell viability loss and pyrimidine depletion, compared to either drug alone, in BQ-resistant PDAC cells. Furthermore, we have strong evidence that CNX-774 is acting in an off-target manner to inhibit pyrimidine salvage. Thus, the goal of this study is to uncover the mechanism by which CNX-774 is sensitizing PDAC cells to BQ and determine if this drug combination is efficacious in our preclinical PDAC mouse models. Our ...