Abstract With an overall survival rate of about 8% after five years, ductal adenocarcinoma of the pancreas (PDAC) is one of the cancers with the worst prognosis (Cancer Research UK and American Cancer Society). A hallmark of PDAC is the immense contribution of desmoplastic stroma to the total tumour mass. Typically, epithelial PDAC cells represent only a minority of cells within the tumour (as low as 10%). The excessive stromal presence represents a major technical challenge when aiming to interrogate the specific molecular signals contributed by the neoplastic epithelial cell compartment from data generated using bulk tumour samples. To address this, we FACS-purified epithelial cells from PDAC and, as controls, healthy human pancreas and performed genome-wide transcriptome and DNA methylome analyses. Clustering based on DNA methylation revealed two distinct groups of PDAC with different methylation levels at genomic regions encoding repeat elements. Methylationlow tumours showed higher expression of endogenous retroviral (ERV) transcripts and a strong engagement of the dsRNA sensing machinery. This results in the cell intrinsic activation of an interferon response signature (IFNsign), leading to the reprogramming of stromal cells towards a pro-tumourigenic microenvironment and poor patient outcome. Methylationlow/IFNsignhigh and Methylationhigh/IFNsignlow PDAC cells harboured distinct lineage traits specific for normal ductal or acinar pancreatic epithelial cells at the methylation and transcriptional level. Moreover, ductal-cell-derived KrasG12D/Trp53-/- mutant mouse PDACs showed higher expression of IFNsign compared to tumors initiated by the same genetic drivers in acinar cells. Collectively, our data point to two distinct origins and etiology of human PDACs, with the aggressive Methylationhigh/IFNsignlow tumour subtype potentially targetable by agents blocking cell intrinsic IFN-signaling. Citation Format: Elisa Espinet, Andreas Trumpp. Viral Mimicry in Pancreatic Cancer [abstract]. In: Abstracts: AACR Special Virtual Conference on Epigenetics and Metabolism; October 15-16, 2020; 2020 Oct 15-16. Philadelphia (PA): AACR; Cancer Res 2020;80(23 Suppl):Abstract nr IA05.