Epigenomic mechanisms used by KrasG12D to regulate inflammatory gene clusters in epithelial pancreatic cancer cells, which are critical for reprogramming the tumor microenvironment.

Kras is one of the most frequently mutated genes in pancreatic cancer, with the G12D variant acting as a cancer driver mutation. Our previous work has integrated and defined gene networks induced by KrasG12D expression, particularly the expression of inflammatory gene clusters. Moreover, we observed that KrasG12D expression increases the overall protein levels of several histone marks, including H3K4me3, H3K27ac, H3K27me3, H3K9me3 and integrated data analysis correlated gene activation and silencing with the alteration of histone marks. Here we further consider KrasG12D-driven changes in gene expression and hypothesize that G9a-H3K9me2 may play an important role in the epigenomic regulation of this process. To investigate epigenomic mechanisms underlying KrasG12D-induced gene expression networks, CUT&RUN technology was used to profile H3K4me3 (promoter), H3K9ac (promoter, enhancer), H3K27ac (enhancer, super-enhancer), and H3K9me2 (silencer) histone marks at regulatory elements. We used a genetically engineered mouse model (GEMM)-derived inducible KrasG12D cell line and compared the epigenetic changes after 24hr of oncogene induction. Data was analyzed by using Sparse Enrichment Analysis for CUT&RUN (SEACR). At the genomic level, we found most H3K4me3 peaks (~90%) were close to a transcription start site (TSS), and the number of H3K4me3 peaks were reduced ~9x after KrasG12D activation. However, for the gene silencing mark, H3K9me2, >80% peaks were located at intronic or inter-genetic regions, and the number of peaks were increased >5x with KrasG12D. For H3K9ac and H3K27ac histone marks, we observed more peaks at intronic or inter-genetic regions than at the TSS, indicating the exchange of histone marks at enhancer, super-enhancer and silenced regions may play an important role in KrasG12D-induced gene expression changes. We next focused on histone modifications at regulatory elements of inflammatory gene clusters, such as IL11, CXCL9-CXCL10-CXCL11, and IL4-IL13 gene clusters. We further correlated epigenetic modifications with our gene expression data and found that the H3K4me3, H3K9ac, and H3K27ac, and H3K9me2 histone mark pathways are involved in the regulation of these inflammatory genes. Notably, these pathways are both, critical for pancreatic cancer development and potentially druggable, and therefore, these results bear both mechanistic and biomedical relevance to pancreatic carcinogenesis.