Abstract 790: Identification of cell identity pathways that cooperate with oncogenic Kras in pancreatic cancer initiation by single-cell epigenomic analyses
Authors: Benitz Simone , Loveless Ian , Nasser Malak , Wen Hui-Ju , Long Daniel , Moore Jacee , Davis Erick , Straub Tobias , Regel Ivonne , Bednar Filip , Crawford Howard 
Published: June 2022

Abstract
Introduction: The major genetic driver for pancreatic ductal adenocarcinoma (PDAC) is oncogenic KRAS. However, adult acinar cells, the most probable origin of PDAC, are largely refractory to KrasG12D-mediated oncogenic transformation in conditional 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, the so-called acinar-to-ductal metaplasia (ADM). Consequently, 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 that accelerate the cellular reprogramming after loss of acinar differentiation, conditional Pdx1 knockout animals were analyzed. By performing single-cell ATAC-seq (Assay for Transposase-Accessible Chromatin using sequencing) from frozen bulk tissue, genome-wide chromatin accessibility states were captured at different stages of carcinogenesis. Changes in the epigenome were correlated to RNA-seq data. Moreover, expression of differentially regulated genes was validated by RNAscope and immunofluorescence staining.
Results: While sole expression of KrasG12D in adult acinar cells induces only a few transcriptional and epigenetic changes, loss of the acinar differentiation factor Pdx1 leads to a previously unidentified switch of acinar cell identity. Notably, the chromatin profiles of Pdx1 knockout acinar cells, despite appearing phenotypically normal, show a striking similarity to ADM cells. One of the most differentially regulated genes in acinar cells from wild type versus Pdx1 knockout mice is Ror2. As a mediator of the non-canonical Wnt signaling, Ror2 regulates essential signaling pathways, such as Ras-MAPK signaling. Pdx1 knockout acinar cells exhibit a high accessibility of the gene, while it is repressed in Pdx1 wild type cells. Moreover, gene expression is significantly increased in Pdx1 knockout mice. To test if Ror2 is a potential regulator of pancreatic carcinogenesis, immunostaining of pancreatic cancer tissues from KrasG12D;p53mut (KPC) mice and human PDAC specimens was performed. Staining revealed Ror2 expression in a subset of neoplasms, suggesting a distinct subtype of early lesions.
Conclusion: Our data reveal that the loss of the acinar differentiation factor Pdx1 leads to massive alterations in the transcriptome and epigenome of acinar cells, significantly accelerating Kras-dependent pancreatic carcinogenesis. In-depth sequencing and molecular analyses identified de-regulation of the receptor kinase Ror2, an important mediator of multiple intracellular signaling pathways, which we hypothesize cooperates with KrasG12D to induce and promote pancreatic carcinogenesis.
Citation Format: Simone Benitz, Ian Loveless, Malak Nasser, Hui-Ju Wen, Daniel Long, Jacee Moore, Erick Davis, 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 analyses [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 790.