ABSTRACT 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 Kras G12D and Gnas R201C co-expression in p48 Cre ; Kras LSL-G12D ; Rosa26 LSL-rtTA ; Tg (TetO- Gnas R201C ) mice (“ Kras;Gnas ” mice) caused development of cystic lesions recapitulating IPMNs. Here, we aim to unveil the consequences of mutant Gnas R201C 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 Gnas R201C expression is inducible. A genome-wide CRISPR/ Cas 9 screen was conducted to identify potential vulnerabilities in Kras G12D ;Gnas R201C co-expressing cells. Results Induction of Gnas R201C – 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 Kras G12D ; Gnas R201C co-expressing cells. Real-time metabolic analyses in Kras;Gnas cells and autochthonous Kras;Gnas model confirmed enhanced glycolysis upon Gnas R201C induction. Induction of Gnas R201C made Kras G12D 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 Gnas R201C induction. Conclusion Multiple orthogonal approaches demonstrate that Kras G12D and Gnas R201C 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. SUMMARY What is already known on this topic Activating “hotspot” mutations of KRAS and GNAS are found in a majority of Intraductal Papillary Mucinous Neoplasms (IPMNs). Expression of mutant KRAS and GNAS drives development of IPMN-like cystic lesions in the murine pancreas that eventually progress to pancreatic ductal adenocarcinoma (PDAC). What this study adds Mutant GNAS and the resulting aberrant G (s) alpha signaling drives a transcriptional signature of gastric (pyloric type) metaplasia in IPMNs with mucin production. Aberrant G (s) alpha signaling enhances glycolysis via protein kinase A-dependent phosphorylation of the glycolytic enzyme PFKFB3. Enhanced glycolysis in KRAS;GNAS -mutated IPMN cells is validated via multiple orthogonal approaches in vitro and in vivo and represents an actionable metabolic vulnerability. How this study might affect research, practice or policy The present study provides mechanistic insight into how aberrant G (s) alpha signaling alters the biology of Kras -mutant pancreatic epithelial neoplasia through metaplastic and metabolic reprogramming. Targeting glycolysis in IPMNs may represent both a therapeutic avenue as well as an opportunity for intercepting progression to invasive cancer.

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