Abstract C049: Loss of RNF43 creates an OXPHOS dependency in early pancreatic neoplasia

Abstract

Abstract Pancreatic ductal adenocarcinoma (PDAC) has a dismal 5-year survival of 12%. RNF43 is frequently mutated in pancreatic cystic neoplasms, including intraductal papillary mucinous neoplasms (IPMNs), which are precursor lesions of PDAC. RNF43 encodes for an E3 ubiquitin ligase and was initially identified as a negative Wnt regulator. To elucidate the role of RNF43-mutated neoplasms in an organ specific context, we have generated a genetically engineered mouse model which has pancreas specific loss of RNF43 (Hosein, Gastroenterology 2022). In this study we examine some of the metabolic dependencies created by Rnf43 loss on the background of mutant Kras, and how the Kras;Rnf43 model differs from the commonly used Kras;p53 model of PDAC. We have generated cell lines harboring mutant Kras and bi-allelic loss of Rnf43 from the resulting lesions in these mice (henceforth referred to as Kras;Rnf43 lines).Isogenic Kras;Rnf43 cell lines with re-expression of full-length Rnf43 (Kras;Rnf43-RNF43OE) were generated to confirm if observed phenotypes were Rnf43 dependent. Single-cell RNA sequencing (scRNA-seq) was performed for comparison between murine PDAC arising in Kras;Rnf43 versus Kras;p53 mice. We further compared metabolic parameters in Kras;Rnf43 and Kras;Rnf43-RNF43OE cells using the Seahorse platform. Mitochondrial ultrastructure by transmission electron microscopy (TEM) was analyzed in Kras;Rnf43 compared to Kras;p53 cells. To visualize endoplasmic reticulum (ER)-mitochondria contact sites (ERMCS), a split green fluorescent protein-based contact site sensor (SPLICS) was transfected and analyzed. Compared to Kras;p53 PDAC, Kras;Rnf43 tumors demonstrated upregulated mitochondrial transcripts and gene signatures of ER stress. Further, the ER stress response protein IRE1 was markedly elevated in Kras;Rnf43 cells versus Kras;p53 cells. The Kras;Rnf43 lines had enhanced mitochondrial oxidative phosphorylation (OXPHOS) activity and demonstrated evidence of enhanced mitochondrial quality control. ERMCS is a dynamic contact region between ER and mitochondria, which regulates biological processes including calcium homeostasis, ER stress and mitochondrial dynamics. The Kras;Rnf43 cells had significantly increased ERMCS compared to Kras;p53 cells. Finally, using IACS-010759, a small molecule inhibitor of OXPHOS, we demonstrate that Kras;Rnf43 cells had both increased in vitro sensitivity as well as in vivo susceptibility to tumor growth inhibition in orthotopic models, which translated into increased overall survival of mice. In conclusion, we provide evidence showing that bi-allelic loss of RNF43 is associated with increased ER stress and enhanced IRE1 dependent stress response. ER stress is mitigated via increased mitochondrial OXPHOS and ERMCS. Importantly, our results suggest that this creates a metabolic synthetic essentiality in the context of RNF43 loss, providing an avenue to suppress the progression of RNF43-mutated IPMNs, and potentially other solid cancers by targeting mitochondrial OXPHOS. Citation Format: Akiko Sagara, Brandon Chen, Nathaniel G. Yee, Megan J. Siemann, Costas A. Lyssiotis, Yatrik M. Shah, Sonja M. Woermann, Anirban Maitra. Loss of RNF43 creates an OXPHOS dependency in early pancreatic neoplasia [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 C049.