Abstract A065: Mutant GNAS drives cAMP/PDE/AMP–pAMPK signaling and therapeutic synergy with combined CDK4/6 and RAS inhibition in mucinous pancreatic neoplasia

Abstract

Intraductal Papillary Mucinous Neoplasm (IPMN) are pre-malignant lesions of the pancreas with frequent co-mutations in KRAS and GNAS oncogenes. We recently reported pancreatic neoplasia from both primary and metastatic disease with KRAS/GNAS mutations are sensitive to single agent treatment with the FDA approved cell-cycle CDK4/6 inhibitor, palbociclib, in ex-vivo patient derived 3D organoids, slices, and in a human patient in-vivo. While clinical benefit was observed, the mechanisms underpinning sensitivity and resistance remain unknown. Here we sought to understand why tumors with GNAS mutations uniquely depend on cellular CDK4/6 activity for proliferation. To understand mechanistic coupling of mutant GNAS to functional regulators of the cell cycle, we analyzed bulk RNAseq of doxycycline inducible GNAS mutant PDAC cell lines to identify pathways upregulated during mutant GNAS activation. We identified upregulated signaling in PDE enzymes which convert cAMP to 5’AMP, a major activator of pAMPK, as well as genes encoding for AMPK (PRKAA1 and PRKAA2). In analyses from human and murine PDAC tumors derived from IPMN pre-cursor lesions we identified that pAMPK protein expression was 2-fold higher than compared to GNAS-wild type PDAC tumors. To identify the role of AMPK signaling in GNAS mutant tumors, we established CRISPR knockouts of AMPK genetic subunits (PRKAA1 and PRKAA2) in a GNAS dox-inducible system and performed bulk RNAseq in control and AMPK-(KO) tumor cells to understand how AMPK signaling regulates gene expression in the context of mutant GNAS activation. Gene ontology analysis revealed AMPK contributes to suppression of the GNAS-cAMP-PKA axis, as well as EGFR signaling. Functional studies using pharmacological activation of AMPK using MK8722 in GNAS mutant 3D organoids had a reduction in cAMP levels during AMPK activation, as well as decreased size of organoids, which was not observed in AMPK-KO cells. Furthermore, AMPK-KO inhibited of transcriptional programs involving cell cycle repression machinery including p53, CDKN1A and CDKN2A pathways. Lastly, given that CDK4/6i is not curative for patients, we performed combination studies using CDK4/6i and RASi (Palbociclib and Daraxonrasib) in human organoids and living ex-vivo slices from IPMN and PDAC samples and identified synergy in GNAS mutant tumors compared to GNAS-wt tumors. These preliminary findings establish a unique role of AMPK as a suppressor of cAMP/PKA/CREB signaling in GNAS mutant tumors and a potential biomarker of response for combination CDK4/6i and RASi therapy in KRAS/GNAS mutant PDAC.