R. Kerketta1, A. Mathison3, A. Zeighami3, J. Abrudan3, W. Demos3, M. Zimmermann2,4, G. Lomberk1, R. Urrutia1,3 1Medical College Of Wisconsin,Division Of Research, Department Of Surgery,Milwaukee, WI, USA 2Medical College Of Wisconsin,Bioinformatics Research And Development Laboratory, Genomic Sciences & Precision Medicine Center,Milwaukee, WI, USA 3Medical College Of Wisconsin,Genomic Sciences And Precision Medicine Center,Milwaukee, WI, USA 4Medical College Of Wisconsin,Clinical And Translational Sciences Institute,Milwaukee, WI, USA
Introduction: Pancreatic ductal adenocarcinoma (PDAC) develops through accumulation of genetic alterations, with the KRAS oncogene being the earliest genetic mutation found, which drives the progression of preneoplastic pancreatic intraepithelial neoplasia (PanIN) lesions into carcinoma. Direct targeting of the KRAS gene has been clinically unsuccessful and the downstream impact that the constitutive activation of KRAS has on chromatin remains unknown. Thus, in order to identify chromatin events downstream of oncogenic KRAS which can be clinically targeted, we investigated the earliest changes at the transcriptomic and epigenomic levels that occur following activation of this oncogene.
Methods: Our in vitro pancreatic cell model was derived from a genetically engineered mouse, carrying a doxycycline-inducible KRASG12Dtransgene. At different time points following doxycycline treatment, western blot was used to evaluate levels of oncogenic KRAS. Subsequently, mRNA, cross-linked chromatin and DNA were isolated for next generation sequencing (NGS). These NGS technologies included RNA sequencing (RNA-seq) for gene expression, chromatin immunoprecipitation sequencing (ChIP-seq) of a series of histone marks to assess active and silenced chromatin, and reduced representation bisulfite sequencing (RRBS) for DNA methylation. We used advanced bioinformatics tools to process, integrate and analyze changes in the gene pathways and epigenetic landscape.
Results:Induction of oncogenic KRAS was confirmed by western blot using a G12D specific antibody. RNA-seq data indicated that following KRAS induction, genes involved in the regulation of epithelial to mesenchymal transition (EMT) and metabolic pathways were downregulated, while genes involved in KRAS signaling and cellular proliferation were upregulated. ChIP-seq revealed an increase in the deposition of histone marks associated with enhancers/super-enhancers (H3K27ac and H3K4me1), activated promoters (H3K4me3), and regions silenced by polycomb (H3K27me3). Integration of RNA-seq and ChIP-seq data demonstrated that up- or down-regulated genes also had corresponding alterations of the H3K27ac and H3K4me3 activating histone marks near their promoters. DNA methylation levels of several CpG islands were also altered following KRAS induction.
Conclusion:Based on our results, exposure to oncogenic KRAS induced pancreatic cells to acquire a more epithelial-like phenotype with increased proliferation, which coincides with changes in the transcriptome and epigenome. RRBS indicated that KRAS induction resulted in differentially methylated regions across the genome. Through the analysis of histone marks, we observed a marked increase in active enhancers and super-enhancers, as measured by H3K27ac and H3K4me1 peaks, implicating the role of histone acetyltransferases as downstream epigenetic modulators of the KRAS signaling pathway. Thus, these enzymes may serve as potential drug therapy targets for mitigating the progression of PDAC.