PROJECT SUMMARY Pancreatic ductal adenocarcinoma (PDA) has the worst five-year survival rate of any major cancer. The lethality of PDA is largely due to lack of effective treatment options. A major obstacle in PDA treatment is conferred by the tumor microenvironment, composed mainly of dense fibroinflammatory stromal response. The stroma is populated mainly by immune cells and cancer-associated fibroblasts (CAF). CAFs deposit extensive extracellular matrix components, including a high degree of hyaluronic acid (HA). HA is a ubiquitous, hydrophilic carbohydrate polymer. The high HA levels in PDA tumors retain water, leading to supraphysiological interstitial pressure. The high pressure collapses the vasculature, limiting drug penetrance, and oxygen and nutrient availability. To sustain viability in the austere microenvironment, cancer cells undergo metabolic reprogramming via mutant Kras, the signature transforming oncogene in PDA. Mutant Kras enhances flux through the hexosamine biosynthesis pathway (HBP) by regulating the rate-limiting enzyme glutamine-fructose 6-phosphate transamidase (GFAT). The HBP is a highly conserved pathway that integrates glucose and glutamine metabolism. In addition, the HBP is the only way to synthesize glycosylation substrate de novo. Therefore, the HBP represents an attractive therapeutic target in PDA. In contrast to this necessary role, my preliminary data show that the HBP is not an effective therapeutic target in vivo. Rather, I found that cancer cells are able to utilize HA, a carbohydrate polymer, to fuel the HBP and support growth independent of its activity. My data illustrate that one of the ways cancer cells achieve this is by utilizing N-acetyl-glucosamine (GlcNAc) from HA via GlcNAc salvage pathway. The data implicate HA as a novel nutrient source for cancer cells. They also point to a novel metabolic reprogramming that cancer cells undergo to survive and proliferate in the nutrient-poor tumor microenvironment. The working hypothesis of this proposal is that CAFs support PDA metabolism via the release of GlcNAc as HA. This will be examined in two parts. First, mechanistic studies involving genetic and pharmacological inhibition of HA synthesis by CAF and HA uptake by cancer cells will be performed; functional consequences of targeting these features will be defined in vitro and in vivo (Specific Aim 1). Second, mechanistic studies involving genetic inhibition of HA catabolism and GlcNAc salvage pathway will be performed; similarly, the functional consequences of targeting these pathways will be defined in vitro and in vivo (Specific Aim 2). The in vitro studies will be performed on a patient-derived CAF and human PDA cell lines. Further, I will demonstrate the role for this pathway using orthotopic co-injection in vivo studies with genetically-modified CAF and PDA cells . This proposal aims to functionally and mechanistically define a novel metabolic rewiring, which can identify a new therapeutic target(s...