Work

Pdtm-02. Next-generation Sequencing of Diffuse Intrinsic Pontine Glioma Cells Reveals Altered Epigenetic Regulation and Distinct Tumor Subgroups

Year: 2017

Type: article

Abstract: Diffuse intrinsic pontine glioma (DIPG) is an infiltrative neoplasm with the highest mortality rate of all pediatric solid tumors. Recurrent somatic mutations of histone H3 isoforms result in methionine for lysine substitution (H3K27M) in 80% of DIPGs, thought to contribute to tumorigenesis via altered chromatin structure and function. To characterize the effects of the H3K27M mutation on gene expression, we analyzed the transcriptomes of rare DIPG primary cell lines with three distinct bioinformatics approaches. H3K27M DIPG (n=7), wild-type pediatric high-grade glioma (HGG, n=3), neural stem cells (n=1) and astrocytes (n=1) were analyzed (RNASeq, 50bp single end, Illumina NexSeq 500) in triplicate (36 runs). Reads were trimmed from 3’ to a final base quality score >30 (Trimmomatic), aligned to the human genome (UCSC hg19, Tophat), then normalized and quantified for 1) specific gene locus (GRanges), 2) exons of a specific gene locus (HTSeq), and 3) alternative splicing isoforms (Rsem). Differential expression was quantified (edgeR), with functional analyses for biological relevance of data sets (TopGO, Ingenuity Pathways Analysis). Epigenetic regulation of transcription, including increased EZH2 function (FC>5, p<0.05), SMARC4A inactivation (z-score >2), and Polymerase II activity at proximal promoter and enhancer regions (p>0.01), were identified in H3K27M mutant lines. Neurogenesis and neuronal differentiation were downregulated in H3K27M lines, consistent with morphologic changes observed in DIPG cell lines after EZH2 and BRD inhibition. Unsupervised clustering of GRanges and HTSeq demonstrated two DIPG subgroups, distinct from wild type and control lines. Rsem data demonstrates similar clustering of DIPGs, with less overlap of HGGs. We present novel transcriptome analysis of rare DIPG cell lines, indicating specific genes and signaling pathways that may promote tumor formation in the presence of H3K27M mutation. These data will be integrated with ChIP-Seq to correlate potential epigenetic mechanisms of aberrant transcriptional regulation secondary to H3K27M mutation.

Source: Neuro-Oncology

DOI: 10.1093/neuonc/nox168.767