Metabolic Reprogramming by Mutant GNAS Creates an Actionable Dependency in Intraductal Papillary Mucinous Neoplasms of the Pancreas

Objective: Oncogenic ″hotspot″ mutations of KRAS and GNAS are two major driver alterations in Intraductal Papillary Mucinous Neoplasms (IPMNs), which are bona fide precursors to pancreatic ductal adenocarcinoma. We previously reported that pancreas–specific KrasG12D and GnasR201C co–expression in p48Cre, KrasLSL–G12D, Rosa26LSL–rtTA, Tg (TetO–GnasR201C) mice (″Kras,Gnas″ mice) caused development of cystic lesions recapitulating IPMNs. Here, we aim to unveil the consequences of mutant GnasR201C expression on phenotype, transcriptomic profile, and genomic dependencies. Design: We performed multimodal transcriptional profiling (bulk RNA sequencing, single cell RNA sequencing, and spatial transcriptomics) in the ″Kras,Gnas″ autochthonous model and tumor–derived cell lines (Kras,Gnas cells), where GnasR201C expression is inducible. A genome–wide CRISPR/Cas9 screen was conducted to identify potential vulnerabilities in KrasG12D,GnasR201C co–expressing cells. Results: Induction of GnasR201C – and resulting G(s)alpha signaling – leads to the emergence of a gene signature of gastric (pyloric type) metaplasia in pancreatic neoplastic epithelial cells. CRISPR screening identified the synthetic essentiality of glycolysis–related genes Gpi1 and Slc2a1 in KrasG12D,GnasR201C co–expressing cells. Real–time metabolic analyses in Kras,Gnas cells and autochthonous Kras,Gnas model confirmed enhanced glycolysis upon GnasR201C induction. Induction of GnasR201C made KrasG12D expressing cells more dependent on glycolysis for their survival. Protein kinase A–dependent phosphorylation of the glycolytic intermediate enzyme PFKFB3 was a driver of increased glycolysis upon GnasR201C induction. Conclusion: Multiple orthogonal approaches demonstrate that KrasG12D and GnasR201C co–expression results in a gene signature of gastric pyloric metaplasia and glycolytic dependency during IPMN pathogenesis. The observed metabolic reprogramming may provide a potential target for therapeutics and interception of IPMNs. ### Competing Interest Statement A. M. is listed as an inventor on a patent that has been licensed by Johns Hopkins University to Thrive Earlier Detection. A.M. serves as a consultant for Tezcat Biosciences.