Metabolic reprogramming by mutant GNAS creates an actionable dependency in intraductal papillary mucinous neoplasms of the pancreas.

Abstract

Oncogenic 'hotspot' mutations of <i>KRAS</i> and <i>GNAS</i> are two major driver alterations in intraductal papillary mucinous neoplasms (IPMNs), which are <i>bona fide</i> precursors to pancreatic ductal adenocarcinoma. We previously reported that pancreas-specific <i>Kras</i> ^G12D and <i>Gnas</i> ^R201C co-expression in p48^Cre; Kras^LSL-G12D; Rosa26^LSL-rtTA; Tg (TetO-Gnas^R201C) mice (<i>'Kras;Gnas</i>' mice) caused development of cystic lesions recapitulating IPMNs. We aim to unveil the consequences of mutant <i>Gnas</i> ^R201C expression on phenotype, transcriptomic profile and genomic dependencies. We performed multimodal transcriptional profiling (bulk RNA sequencing, single-cell RNA sequencing and spatial transcriptomics) in the <i>'Kras;Gnas</i>' autochthonous model and tumour-derived cell lines (<i>Kras;Gnas</i> cells), where <i>Gnas</i> ^R201C expression is inducible. A genome-wide CRISPR/<i>Cas</i>9 screen was conducted to identify potential vulnerabilities in <i>Kras^G12D;Gnas^R201C</i> co-expressing cells. Induction of <i>Gnas</i> ^R201C-and resulting G_(s)alpha signalling-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 <i>Gpi1</i> and <i>Slc2a1</i> in <i>Kras</i> ^G12D;<i>Gnas</i> ^R201C co-expressing cells. Real-time metabolic analyses in <i>Kras;Gnas</i> cells and autochthonous <i>Kras;Gnas</i> model confirmed enhanced glycolysis on <i>Gnas</i> ^R201C induction. Induction of <i>Gnas</i> ^R201C made <i>Kras</i> ^G12D expressing cells more dependent on glycolysis for their survival. Protein kinase A-dependent phosphorylation of the glycolytic intermediate enzyme 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 3 (PFKFB3) was a driver of increased glycolysis on <i>Gnas</i> ^R201C induction. Multiple orthogonal approaches demonstrate that <i>Kras</i> ^G12D and <i>Gnas</i> ^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.