Abstract B058: Mutant GNAS drives glycolytic dependency in intraductal papillary mucinous neoplasms of the pancreas

Abstract

Abstract Oncogenic “hotspot” mutations of KRAS and GNAS are two major driver alterations in Intraductal Papillary Mucinous Neoplasms (IPMNs), which are bona fide precursor lesions of pancreatic ductal adenocarcinoma (PDAC). We previously reported that pancreas-specific KrasG12D and GnasR201C expression caused IPMN development, with subsequent progression to PDAC in p48-Cre; LSL-KrasG12D; Rosa26R-LSL-rtTA-TetO-GnasR201C mice (referred to as “Kras;Gnas” mice). This study aimed to clarify the functional roles of mutant GNAS, thereby investigating therapeutic dependencies that exist in the context of mutant KRAS and GNAS co-expression in IPMN. We established cell lines derived from IPMN-associated PDAC in Kras;Gnas mice (referred to as “Kras;Gnas” cells), in which KrasG12D is constitutively active whereas GnasR201C expression is inducible by the addition of doxycycline. RNA-sequencing of Kras;Gnas cells identified the enrichment of glycolysis-related gene signatures upon GnasR201C induction. This was also confirmed on single cell RNA sequencing data of IPMN-associated PDAC in Kras;Gnas mice, when compared with tumors arising in mice expressing mutant KrasG12D alone. Genome-wide CRISPR/Cas9 screening identified multiple glycolysis-related genes as essential genes in the setting of KrasG12D and GnasR201C co-expression in Kras;Gnas cells. Further, Seahorse metabolic flux analyzer showed the enhancement of glycolysis by GnasR201C induction in Kras;Gnas cells. In vivo, 13C-hyperpolarized MRI (HP-MRI) revealed the enhancement of glycolytic flux (pyruvate to lactate conversion) in Kras;Gnas mice with concurrent GnasR201C expression, compared to mice with KrasG12D alone. The GNASR201C-expressing Kras;Gnas cells were also more susceptible to glycolysis inhibition either by glucose deprivation or upon exposure to inhibitors of glycolysis, when compared to cells with only KrasG12D expression. Knockout of Gpi1 or Slc2a1, two synthetic lethal glycolysis associated genes identified on CRISPR/Cas9 screening, significantly suppressed cell growth in GNASR201C-expressing Kras;Gnas cells both in vitro and in vivo. Of note, Gpi1 deletion increased compensatory ATP production from mitochondrial oxidative phosphorylation (OXPHOS) and autophagy in GNASR201C-expressing Kras;Gnas cells and as a result, these cells were more susceptible to both inhibitors of OXPHOS and of autophagy. We identified two candidate effector pathways downstream of GNASR201C activation - PKA/PFKFB3 and EPAC/DRP1 – which were responsible for the increased glycolytic activity observed via multiple orthogonal approaches in Kras;Gnas cells. The expression of phospho-PFKFB3, Gpi1, and Glut1 proteins were elevated in human IPMN lesions with GNAS mutations. In conclusion, the co-occurrence of hot spot GNAS mutations enhances glycolysis in IPMNs arising in the context of KRAS mutations. Targeting glycolysis might be a potential avenue for treatment and cancer interception in IPMN lesions with co-mutations of KRAS and GNAS. Citation Format: Yuki Makino, Kimal Rajapakshe, Nathaniel Yee, Megan Siemann, Jimin Min, Benson Chellakkan Selvanesan, Fredrik Thege, Akiko Sagara, Prasanta Dutta, Pratip Bhattacharya, Merve Dede, Traver Hart, Anirban Maitra. Mutant GNAS drives glycolytic dependency in intraductal papillary mucinous neoplasms of the pancreas [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Pancreatic Cancer; 2023 Sep 27-30; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(2 Suppl):Abstract nr B058.