Pancreatic cancer remains almost incurable and has recently surpassed breast cancer as the third leading cause of death from malignant disease in the United States; an estimated 53,070 Americans will be diagnosed with pancreatic cancer and 41,780 will die from the disease this year (statistics from the Pancreatic Cancer Action Network). This project builds on two metabolic glycoengineering (MGE) approaches that our group has developed in previous funding periods for the parent R01 grant that reverse the disease-driving impact of two types of abnormal glycosylation broadly associated with cancer. One approach exploits our “high flux” ManNAc analogs that increase sialylation, which masks galectin binding sites on highly-branched N-glycans and thereby attenuates “galectin lattice” strength and reduces multiple aspects of cancer progression (our work focuses on EGFR but surveys a range of additional oncogenic surface markers). This approach will test the non-natural azide-modified form of ManNAc as a step towards developing theranostic treatment options where the sugar analog not only sensitizes drug resistant cancer cells to tyrosine kinase inhibitors (TKIs) but can also be used to image cancer using “click chemistry” probes. In a complementary approach, we will use an alternative strategy to reduce cancer-driving glycosylation by inhibiting metabolic flux through the hexosamine biosynthetic pathway (HBP), which prevents the initial formation of the galectin lattice as well as knocking down other oncogenic glycoforms (for example, the “O-GlcNAc” protein modification). This project will compare each of these approaches in cell lines that provide genetic diversity found in human patients (our expectation is that our “glyco” approach will transcend genetic diversity and be broadly applicable) and then demonstrate efficacy in rodent models of pancreatic cancer using patient-derived cells and xenografts available to our team through the Johns Hopkins Medical Institute’s Division of Gastrointestinal and Liver Pathology (Dr. Anne Le, the co- investigator on this project is a faculty member of this division and has several years of experience conducting research with cell- and xenograft models of pancreatic cancer).