Abstract A109: Single-cell epigenomic analysis reveals an important role of the receptor kinase Ror2 in the erosion of cellular identity during pancreatic carcinogenesis
Authors: Benitz Simone , Nasser Malak , Steep Alexander , Preall Jonathan , Mahajan Ujjwal , Loveless Ian , Davis Erick , Wen Hui-Ju , Long Daniel , Louw Michaela , Zwernik Samuel , Rempinski Donald , Moore Jacee , Salas-Escabillas Daniel , Metzler Thomas , Huang Ling , Steele Nina , Regel Ivonne , Bednar Filip , Crawford Howard 
Published: January 2024

Abstract
Introduction The major driver for pancreatic ductal adenocarcinoma (PDAC) is oncogenic KRAS. However, adult acinar cells, a probable origin of PDAC, are largely refractory to KrasG12D-mediated oncogenic transformation. With the concomitant loss of transcription factors that regulate acinar cell differentiation, such as Pdx1 (Pancreatic and Duodenal Homeobox 1), acinar cells undergo a rapid cell identity switch, known as acinar-to-ductal metaplasia (ADM). How loss of cell identity cooperates with oncogenic Kras to induce pancreatic transformation is largely unclear. Methods To elucidate mechanisms responsible for the accelerated cellular reprogramming in KrasG12D;Pdx1f/f animals, single-cell ATAC-seq (Assay for Transposase-Accessible Chromatin using sequencing) from frozen pancreatic bulk tissue was performed. Chromatin accessibility states were captured at early stages of carcinogenesis and correlated to RNA-seq data. Differentially regulated genes were validated by multiplex RNAscope and immunohistochemistry staining and functionally studied in pancreatic cancer cell lines. Results Single-cell ATAC-seq proved a powerful tool for defining cell-type identity, cellular reprogramming and target genes in early metaplastic transformation of pancreatic tissue. Notably, acinar cells of KrasG12D;Pdx1f/f animals as well as a proportion of metaplastic lesions in both, KrasG12D and KrasG12D;Pdx1f/f mice, showed elevated accessibility and expression of the Ror2 (Receptor Tyrosine Kinase Like Orphan Receptor 2) gene. As a receptor tyrosine kinase, Ror2 controls noncanonical Wnt signaling and other essential signaling pathways, such as PI3K/AKT or Ras-MAPK. Genetic ablation of Ror2 in a mouse model of pancreatic neoplasia resulted in a shift in ADM cell identity, enriching ADM lesions with a senescent duct cell phenotype. In PDAC, ROR2 expression correlates with the more aggressive basal-like subtype. Overexpression of ROR2 in pancreatic cancer cell lines with a classical differentiation induced epithelial-to-mesenchymal transition, characterized by the downregulation of multiple epithelial markers and upregulation of mesenchymal genes. Knockout of ROR2 in pancreatic cancer cells significantly decreased cell proliferation. Conclusions Our in-depth sequencing data revealed that expression of KrasG12D with the concomitant loss of Pdx1 leads to vast alterations of acinar cell identity. We identified the receptor kinase Ror2 as a regulator of pancreatic cancer initiation and driver of pancreatic cancer cell aggressiveness.
Citation Format: Simone Benitz, Malak Nasser, Alexander Steep, Jonathan Preall, Ujjwal Mahajan, Ian Loveless, Erick Davis, Hui-Ju Wen, Daniel Long, Michaela Louw, Samuel Zwernik, Donald Rempinski, Jacee Moore, Daniel Salas-Escabillas, Thomas Metzler, Ling Huang, Nina Steele, Ivonne Regel, Filip Bednar, Howard Crawford. Single-cell epigenomic analysis reveals an important role of the receptor kinase Ror2 in the erosion of cellular identity during pancreatic carcinogenesis [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 A109.