Abstract PR-18: RNF43 Loss Induces an IRE1-dependent Metabolic Reprogramming in Pancreatic Cystic Neoplasms
Authors: Sagara Akiko , Lv Xiangdong , Chen Brandon , Makino Yuki , So Shui Ping , Woermann Sonja M , Lyssiotis Costas A , Shah Yatrik M , Ghosh Bidyut , Chen Xi , Fahrmann Johannes F , Maitra Anirban 
Published: September 2024

Abstract
Background: RNF43 is frequently mutated in intraductal papillary mucinous neoplasms (IPMNs), which are cystic precursor lesions of pancreatic ductal adenocarcinoma (PDAC). RNF43 encodes for an E3 ubiquitin ligase and was initially identified as a negative regulator of Wnt signaling in colorectal cancer. Clinical trials targeting ligand dependent Wnt signaling in RNF43-mutated cancers have failed to demonstrate any efficacy, suggesting other mechanisms may be involved. Methods: To elucidate the role of RNF43 in an organ specific context, we generated a genetically engineered mouse model with pancreas-specific loss of Rnf43 and co-expression of mutant Kras (referred to as Kras;Rnf43 mice). The Kras;Rnf43 mice develop pancreatic cystic lesions resembling human IPMNs, a subset of which progressive to invasive cancers. We generated derivative cell lines from the IPMN-associated cancers, referred to as Kras;Rnf43 lines. Single-cell RNA sequencing (scRNA-seq) was performed to compare murine PDAC arising in Kras;Rnf43 mice with that of the Kras;p53 (“KPC”) mice. To determine if observed perturbations were dependent on Rnf43, we also created isogenic Kras;Rnf43 cell lines with re-expression of full-length Rnf43 (Kras;Rnf43-RNF43OE). Results: Kras;Rnf43 tumors showed upregulation of several mitochondrial transcripts (Mt-Atp8, Atp5o.1, and Atp6v0c), as well as mitochondrial biogenesis markers (PPARGC1A and NFE2L2), compared to KPC mice. Concurrently, the oxidative phosphorylation (OXPHOS) pathway was enriched on proteomics analyses of Kras;Rnf43 cells, relative to isogenic cell lines with RNF43 restitution. Seahorse metabolic flux analyzer revealed enhanced mitochondrial OXPHOS activity in Kras;Rnf43 cell lines, along with increased mitophagy. Kras;Rnf43 cells harbored gene signatures indicative of endoplasmic reticulum (ER) stress compared to KPC lines. Endoplasmic reticulum-mitochondria contact sites (ERMCS) regulate ER stress and mitochondrial dynamics. Notably, Kras;Rnf43 cells had significantly increased ERMCS compared to KPC cells, and these were attenuated upon RNF43 re-expression. We further identified the ER stress sensor inositol-requiring protein 1 (IRE1) was markedly elevated in Kras;Rnf43 cells compared to KPC cells, and decreased upon RNF43 re-expression. Furthermore, IRE1 localized at the mitochondrial-associated membrane (MAM) and enhanced ERMCS formation via a non-canonical scaffolding function. Deletion of IRE1 by CRISPR/Cas9 editing in Kras;Rnf43 cells inhibited OXPHOS and significantly impaired cell viability. Conclusion: Our data suggests that bi-allelic loss of RNF43 caused increased mitochondrial function and OXPHOS dependency. This is largely attributed to upregulated level of IRE1, which localizes at MAM to promote ERMCS formation and OXPHOS in Kras;Rnf43 cells. IRE1-dependent reprogramming of mitochondrial bioenergetics serves as a cell survival mechanism. Therefore, targeting OXPHOS or the noncanonical function of IRE1 could be a potential strategy to inhibit the progression of RNF43-mutated IPMNs to PDAC.
Citation Format: Akiko Sagara, Xiangdong Lv, Brandon Chen, Yuki Makino, Shui Ping So, Sonja M Woermann, Costas A Lyssiotis, Yatrik M Shah, Bidyut Ghosh, Xi Chen, Johannes F Fahrmann, Anirban Maitra. RNF43 Loss Induces an IRE1-dependent Metabolic Reprogramming in Pancreatic Cystic Neoplasms [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Pancreatic Cancer Research; 2024 Sep 15-18; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(17 Suppl_2):Abstract nr PR-18.