Abstract PR003: Mutant GNAS-mediated epithelial IL-33 upregulation promotes IPMN initiation and progression

Abstract

Intraductal Papillary Mucinous Neoplasms (IPMNs) are bona fide cystic precursors to pancreatic ductal adenocarcinomas (PDACs). We previously reported that pancreas-specific Kras;Gnas mutation develops IPMN which progress to PDAC (p48-Cre KrasLSL-G12D Rosa26LSL-rtTA TetO-GnasR201C + doxycycline; KG mice). Whereas this underscores the significance of mutant Gnas as a driver of IPMNs, little is known about the roles of its downstream effectors. Here, we revealed that mutant Gnas upregulates IL-33 via PKA/CREB signaling with mutant Kras background. IL-33 is a unique dual function cytokine that can act in two ways as a chromatin-associated nuclear factor or a secreted cytokine. This study aimed to clarify the significance of IL-33 in IPMN pathogenesis. First, we generated pancreas-specific IL-33-deficient KG mice (p48Cre KrasLSL-G12D Rosa26LSL-rtTA TetO-GnasR201C IL-33fl/fl + doxycycline; KGI mice). Compared to KG mice, KGI mice showed reduced cyst formation and normal acinar retention, indicating reduced tissue remodeling for IPMN initiation, leading to the prolonged survival. scRNA-seq revealed an impaired epithelial metaplastic trajectory. pySCENIC regulon analysis showed reduced activity of NKX6-2, which we previously identified as a master regulatory transcription factor of gastric metaplasia. Integrated scRNA/ATAC-seq revealed chromatin-associated repression of NKX6-2 in IL-33-deficient epithelial cells. Restoration of NKX6-2 upregulated gastric metaplasia markers in cell lines generated from KGI mice. In human, spatial transcriptomics showed positive correlation of NKX6-2 and IL-33 in IPMN epithelia. Collectively, loss of epithelial IL-33 suppressed early IPMN formation via NKX6-2 downregulation. To address the roles of IL-33 in cancer cells, we knocked out IL-33 in two IPMN-derived PDAC cell lines derived from KG mice (KGI cells). Loss of IL-33 suppressed orthotopic tumor growth and induced well-differentiated histology, together with the repression of EMT phenotypes. Integrated analyses of bulk RNA/ATAC-seq identified coordinated repression of Ror2 in KGI cells, which encodes a receptor tyrosine kinase involved in noncanonical WNT signaling. Restoration of ROR2 reverted the above phenotypes of KGI cells, indicating its contribution to the reduced aggressiveness. Finally, we investigated the alteration of tumor microenvironment in KGI mice. scRNA-seq showed that ST2, a receptor of secreted IL-33, was predominantly expressed in ILC2s, mast cells, and Tregs within the tumor microenvironment. Among them, Treg infiltration was significantly reduced in KGI mice compared to KG mice. KGI cells also showed decreased Treg infiltration in orthotopic tumors. Epithelial IL-33 thus promotes Treg accumulation, potentially creating an immunosuppressive microenvironment. In conclusion, IL-33 is a pleiotropic mediator of Kras;Gnas-driven IPMN tumorigenesis that affects initial metaplasia, tumor progression, and immune microenvironment. IL-33 represents a novel therapeutic target for IPMNs, for which no specific therapeutics are currently available.