KRAS 和 GNAS 的致癌“热点”突变是导管内乳头状粘液性肿瘤 (IPMN) 的两个主要驱动因素改变，而 IPMN 是胰腺导管腺癌的真正前体。我们之前报道过胰腺特异性 Kras G12D 和 Gnas R201C 在 p48Cre 中共表达；克拉斯LSL-G12D； Rosa26LSL-rtTA； Tg (TetO-GnasR201C) 小鼠（'Kras;Gnas' 小鼠）引起了重现 IPMN 的囊性病变的发展。我们的目标是揭示突变 Gnas R201C 表达对表型、转录组谱和基因组依赖性的影响。我们进行了多模式转录分析（批量） “Kras;Gnas”本土模型和肿瘤衍生细胞系（Kras;Gnas 细胞）中的 RNA 测序、单细胞 RNA 测序和空间转录组学），其中 Gnas R201C 表达是可诱导的。进行全基因组 CRISPR/Cas9 筛选，以确定 KrasG12D;GnasR201C 共表达细胞中的潜在漏洞。Gnas R201C 的诱导以及由此产生的 G(s)alpha 信号传导导致胃（幽门型）基因特征的出现）胰腺肿瘤上皮细胞的化生。 CRISPR 筛选鉴定了 Kras G12D;Gnas R201C 共表达细胞中糖酵解相关基因 Gpi1 和 Slc2a1 的合成必要性。 Kras;Gnas 细胞和本地 Kras;Gnas 模型的实时代谢分析证实 Gnas R201C 诱导糖酵解增强。 Gnas R201C 的诱导使 Kras G12D 表达细胞的生存更加依赖于糖酵解。糖酵解中间酶 6-磷酸果糖-2-激酶/果糖-2,6-二磷酸酶 3 (PFKFB3) 的蛋白激酶 A 依赖性磷酸化是 Gnas R201C 诱导糖酵解增加的驱动因素。多种正交方法证明 Kras G12D 和 Gnas R201C 共表达导致 IPMN 发病过程中胃幽门化生和糖酵解依赖性的基因特征。观察到的代谢重编程可能为 IPMN 的治疗和拦截提供潜在目标。© 作者（或其雇主）2024。禁止商业重复使用。请参阅权利和权限。英国医学杂志出版。

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 p48Cre; KrasLSL-G12D; Rosa26LSL-rtTA; Tg (TetO-GnasR201C) mice ('Kras;Gnas' mice) caused development of cystic lesions recapitulating IPMNs.We aim to unveil the consequences of mutant Gnas 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 'Kras;Gnas' autochthonous model and tumour-derived cell lines (Kras;Gnas cells), where Gnas R201C expression is inducible. A genome-wide CRISPR/Cas9 screen was conducted to identify potential vulnerabilities in KrasG12D;GnasR201C co-expressing cells.Induction of Gnas 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 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 on 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 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 3 (PFKFB3) was a driver of increased glycolysis on Gnas R201C induction.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.© Author(s) (or their employer(s)) 2024. No commercial re-use. See rights and permissions. Published by BMJ.