Abstract 5751: 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 , Loveless Ian , Nasser Malak , Wen Hui-Ju , Long Daniel , Davis Erick , Moore Jacee , Regel Ivonne , Bednar Filip , Crawford Howard 
Published: April 2023

Background The major genetic 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 in mouse models. 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). Consequently, KrasG12D;Pdx1f/f (Pdx1 knockout) mice present with massively accelerated tumor formation. How loss of cell identity cooperates with oncogenic Kras to induce pancreatic transformation is largely unclear.

Methods To elucidate mechanisms responsible for the cellular reprogramming in KrasG12D;Pdx1f/f animals, single-cell ATAC-seq from pancreatic bulk tissue was performed. Chromatin accessibility states were captured at early stages of carcinogenesis and correlated to RNA-seq data. Expression of differentially regulated genes was validated by RNAscope and immunohistochemistry staining. The role of identified target genes was studied in pancreatic cancer cell lines.

Results Single-cell ATAC-seq proved as a powerful tool for defining cell-type identity, cellular reprogramming and target genes in early metaplastic transformation of pancreatic tissue. While sole expression of oncogenic Kras lead to reduced accessibility of acinar differentiation genes in acinar cells, these changes were much more prominent in KrasG12D;Pdx1f/f mice, promoting metaplastic conversion. 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 gene. As a receptor protein tyrosine kinase, Ror2 controls essential signaling pathways, such as Ras-MAPK signaling. By analyzing Ror2 knockout mice, we found that the receptor kinase regulates the identity of metaplastic epithelia. Immunostaining of pancreatic cancer tissues from KrasG12D;p53mut (KPC) mice and human PDAC specimens also revealed ROR2 expression in a subset of cancer cells. Knockdown of ROR2 in pancreatic cancer cell lines significantly decreased cell proliferation, while overexpression induced a profound increase in proliferation and in epithelial-to-mesenchymal transition based on the downregulation of multiple epithelial markers and an upregulation of an array of mesenchymal genes.

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 and significantly accelerated transformation. We identified induced expression of the receptor kinase Ror2, which regulates pancreatic cancer initiation and drives pancreatic cancer cell aggressiveness.

Citation Format: Simone Benitz, Ian Loveless, Malak Nasser, Hui-Ju Wen, Daniel Long, Erick Davis, Jacee Moore, 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 American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5751.