PROJECT SUMMARY This application is being submitted in response to the Notice of Special Interest (NOSI) identified as NOT-CA- 23-045. Pancreatic ductal adenocarcinoma (PDAC) tumors exhibit regions of low oxygen tension (hypoxia) due to characteristically poor vascularization. The Lazzara lab recently showed that hypoxia drives an especially durable epithelial-mesenchymal transition (EMT) in transformed PDAC ductal cells that can last for weeks, compared to a persistence timescale of just days when EMT is driven by growth factors. The mechanism leading to durable EMT involves a positive feedback between histone methylation and MAP kinase activation that is initiated by loss of histone demethylase activity in low-oxygen settings. Because metabolic rewiring accompanies EMT in some cancer contexts, we propose the novel hypothesis that hypoxia promotes especially durable metabolic changes in PDAC cells that persist long after the hypoxic driving force is relieved. These durable changes to the metabolic state may promote disease progression by reconfiguring cellular energetic needs and by increasing the abundance of immune-suppressive metabolites. We will explore this novel hypothesis using multiple models of hypoxia-mediated PDAC cell EMT and metabolomic profiling. The work will be undertaken collaboratively by two principal investigator members of the NCI Cancer Systems Biology Consortium who are located at different academic institutions. In Phase I of the proposed work, we will use three different model systems to create samples of PDAC cell lines driven to undergo EMT in response to hypoxia or growth factors. The model systems include established human PDAC lines, cell lines derived from patient-derived xenografts, and cell lines derived from spontaneous PDAC tumors generated in a lineage- traced autochthonous mouse model. At several time points after removal of the EMT driving force (hypoxia or growth factors), cells will be flow-sorted into mesenchymal and epithelial populations, and frozen cell samples will be generated. In Phase 2, samples will be analyzed by liquid chromatography-mass spectrometry metabolomics to probe for changes in metabolomic programs and accumulation of hypoxia-associated immunosuppressive metabolites. Dimensionality reduction techniques will be applied to identify metabolite and metabolic pathway changes that maximally explain variance among the samples. In Phase 3, metabolic pathways nominated by the analysis of variance among samples will be tested for their ability to control EMT persistence. These investigations will uncover the degree to which durable hypoxia-driven EMT is accompanied by durable metabolic changes and how such changes may alter the phenotypes of mesenchymal PDAC ductal cells. Understanding these issues will ultimately reveal new druggable vulnerabilities that can be exploited for treating PDAC.

PROJECT NARRATIVE The Lazzara lab recently showed that hypoxia drives an especially durable epithelial-mesenchymal transition (EMT) in pancreas cancer that can last for weeks, compared to a relatively short-lived EMT driven by growth factors. Because cellular metabolic adaptations accompany EMT, and because hypoxia drives broad metabolic rewiring in pancreas cancer cells, we hypothesize that hypoxia-driven EMT in pancreas cancer could be accompanied by durable metabolic rewiring effects that promote disease progression. In this project, we will test this hypothesis through a collaboration between two labs associated with the Cancer Systems Biology Consortium that have expertise in pancreas cancer EMT (Lazzara) and metabolomic profiling (Graham).