PROJECT SUMMARY/ABSTRACT Pancreatic ductal carcinoma (PDAC) is the most common form of pancreatic cancer and is highly lethal and resistant to therapy. There is a need to explore new, effective, strategies to treat PDAC, given that only ~10% of the patients survive beyond five years. PDAC overutilize extracellular nutrients to sustain their growth. This nutrient dependency, coupled with a low blood supply, limits nutrient availability in the PDAC microenvironment. To achieve therapy and improve patient survival outcome, it is important to understand how PDAC survive in the nutrient-limited condition and the tumor-intrinsic or microenvironmental factors that sustain their survival. In this proposal, we show that PDAC cells rely on cysteine at a far greater extent than other amino acids. Metabolomics profiling revealed that the PDAC cells almost exclusively use cysteine to sustain intracellular glutathione (GSH). While some PDAC cells rapidly generate GSH when starved of cysteine, others maintain their GSH pool when starved of both cysteine and arginine, indicating the use of various mechanisms to sustain GSH and survival in PDAC cells. In addition, we found that under the same cysteine starvation, macrophages produce GSH, which is an important discovery given the high abundance of macrophages in PDAC microenvironment, their arginine catabolic function, and that the macrophage-derived GSH could sustain PDAC. In multiple gene expression datasets of patient tumors, we observed that PDAC express a high level of GSH pathway genes. Based on these data, we hypothesize that GSH is a core nutrient required for PDAC growth, is potentially sustained by tumor-associated macrophages, and that disrupting GSH utilization could improve therapy in PDAC. The aims of this study are 1). to determine the molecular mechanisms driving the dependency of PDAC on GSH – including the epigenetic regulation of GSH pathway, and 2). to determine the role of tumor-associated macrophages as a source and modulator of GSH in PDAC. The overarching goal is to explore whether blocking GSH utilization alone or alongside macrophage activities could be a way to improve PDAC therapy. Aim 1 will be pursued at the K99 phase, while most of Aim 2 will be pursued at the R00 phase. Methods will include gene interference (e.g., CRISPR/Cas9, shRNA, siRNA), pharmacological inhibitors of GSH pathways (including the pentose phosphate pathway), cell culture assays, metabolomics (including stable isotope tracing), dietary mouse models, bioinformatics, promoter analysis/epigenetic methods, RNA sequencing (single cell and bulk), immunohistochemistry, flow cytometry and mass cytometry. The project will receive input from a 5-person mentorship team that have expertise in tumor immunology, metabolism, bioinformatics, and epigenetics. The expected results could a) offer new insights on disrupting GSH pathway to suppress PDAC growth, b) reveal new microenvironmental mechanisms that enable tumor adaptation in nutrient-limited state, and c) reveal new opportunities to overcome resistance to chemotherapy or immunotherapy in PDAC.

PROJECT NARRATIVE Pancreatic cancer is a difficult-to-treat cancer associated with several undesirable features, including uncontrolled nutrient consumption, high invasiveness, therapeutic resistance, dense fibroinflammatory microenvironment, and low patient survival rate. In this proposal, we will focus on glutathione (GSH), which pancreatic cancer cells use to survive. Our goal is to determine how pancreatic cancer cells acquire and use GSH, assess the role of tumor-associated macrophages as a GSH source, and determine how preventing pancreatic cancer from using GSH could help to improve therapeutic outcome.