PROJECT SUMMARY Pancreatic ductal adenocarcinoma (PDAC) is one of the most lethal human cancers, with a 5-year overall survival (OS) rate of 7% for metastatic disease and less than 20% for locally advanced disease. Benefit from current therapies including chemoradiation and surgery is often modest and transient. The significant challenge in the field is how to turn immunologically cold PDAC into hot tumors that respond to immune checkpoint blockade (ICB) therapy. We recently showed that irreversible electroporation (IRE), a tumor ablative technique currently used in the clinics, significantly sensitized PDAC to anti-PD-1 ICB, leading to long-term survival in ~40% mice in an aggressive orthotopic PDAC model. The remarkable anti-PDAC activity was attributed to efficient induction of immunogenic cell death and stromal perturbation in favor of tumor infiltration of CTLs. As part of an effort to define approaches to further enhance the efficacy of IRE + anti-PD-1 combination against PDAC, we uncovered novel immune suppressive mechanism through time-of-flight mass cytometry (CyTOF) immune profiling and single cell RNAseq of PDACs, which showed significant infiltration of CXCR2-expressing myeloid suppressive cells (MDSCs). Furthermore, we found that IRE collapsed glycolysis and oxidative phosphorylation (OxPhos) while upregulated glutaminase and glutamate, suggesting glutaminolysis as a compensatory mechanism to satisfy energy and biosynthesis needs of IRE-treated cells. These data, taken together with the known critical role of MDSCs and heightened glutamine metabolism in immune suppression, the findings by others that the anti-diabetic drugs metformin and phenformin fundamentally change the tumor metabolic program to sensitize tumors to ICB therapy, and our preliminary findings that both IRE and Re-Phen, a newly developed analogue of phenformin, downregulated the OxPhos pathway while displaying an opposite effect on glutamate production, lead us to hypothesize that attenuation of the immunosuppressive TME by depletion of MDSCs or suppression of glutaminolysis by Re-Phen potentiates IRE + ICB to further prolong overall survival and increase the rate of durable response. To test our hypothesis, we will pursue the following specific aims: 1) To identify immunosuppressive factors associated with long-term versus short-term response to IRE + ICB. We will use CyTOF immune profiling, scRNAseq, and cytokine array analyses to fully characterize the impact of IRE in the presence and absence of anti-PD-1 on the immunosuppressive TME. 2) To determine the extent to which therapies directed at MDSCs potentiate IRE + ICB. 3) To determine the extent to which disruption of the metabolic program by theranostic agent Re-Phen potentiates IRE + ICB. The findings from this project are expected to reveal previously undefined roles of MDSCs and deregulated metabolic programming in immune suppression in the context of combined IRE + ICB therapy. Success of this project will have exceptional impact because it will offer a potentially effective therapy for PDAC.

PROJECT NARRATIVE Novel treatments are urgently needed for pancreatic ductal adenocarcinoma (PDAC) because patients with advanced PDAC have a grave prognosis. The current project offers innovative solutions that overcome resistance to immunotherapy. This strategy, when successfully introduced into the clinic, will fulfill the unmet clinical need for improving the survival and quality of life of PDAC patients.