Abstract B061: Identification of cell identity pathways that cooperate with oncogenic Kras in pancreatic cancer initiation by single-cell epigenomic analysis
Authors: Benitz Simone , Loveless Ian , Nasser Malak , Wen Hui-Ju , Long Daniel , Davis Erick , Moore Jacee , Straub Tobias , Regel Ivonne , Bednar Filip , Crawford Howard 
Published: November 2022

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 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 (two and ten weeks after CreERT-induced recombination) and compared to those of KrasG12D and CreERT control animals. Changes in the epigenome were 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 very powerful and robust tool for defining cell-type identity, cellular reprogramming and target genes in early metaplastic transformation of pancreatic tissue. By analyzing tissue of KrasG12D;Pdx1f/f mice at different time points, we captured the complete metaplastic conversion of acinar cells into ADM and different subsets of precancerous lesions. Despite initially appearing phenotypically normal, the chromatin profiles of Pdx1 knockout acinar cells showed a striking similarity to ADM cells. Interestingly, acinar and ADM cells of KrasG12D;Pdx1f/f animals as well as ADM lesions of KrasG12D mice showed elevated accessibility and expression of the Ror2 gene. As a receptor protein tyrosine kinase, Ror2 regulates essential signaling pathways, such as Ras-MAPK signaling. Immunostaining of pancreatic cancer tissues from KrasG12D;p53mut (KPC) mice and human PDAC specimens also revealed Ror2 expression in a subset of neoplasms and cancer cells. Knockdown of Ror2 in pancreatic cancer cell lines with a basal-like subtype significantly decreased cell proliferation and expression of cell cycle regulators, while ectopic overexpression in PDAC lines with a classical differentiation induced a dramatic change in cell identity and massively increased cell proliferation. Conclusions: Our in-depth sequencing data revealed that expression of KrasG12D with the concomitant loss of Pdx1 lead to vast alterations of acinar cell identity and significantly accelerated transformation. We identified de-regulation of the receptor kinase Ror2, which cooperates with KrasG12D to induce and promote pancreatic carcinogenesis and proliferation.

Citation Format: Simone Benitz, Ian Loveless, Malak Nasser, Hui-Ju Wen, Daniel Long, Erick Davis, Jacee Moore, Tobias Straub, Ivonne Regel, Filip Bednar, Howard Crawford. Identification of cell identity pathways that cooperate with oncogenic Kras in pancreatic cancer initiation by single-cell epigenomic analysis [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer; 2022 Sep 13-16; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2022;82(22 Suppl):Abstract nr B061.