Epigenomic alterations define lethal CIMP-positive ependymomas of infancy

Abstract

Ependymomas are common childhood brain tumours that occur throughout the nervous system, but are most common in the paediatric hindbrain. Current standard therapy comprises surgery and radiation, but not cytotoxic chemotherapy as it does not further increase survival. Whole-genome and whole-exome sequencing of 47 hindbrain ependymomas reveals an extremely low mutation rate, and zero significant recurrent somatic single nucleotide variants. Although devoid of recurrent single nucleotide variants and focal copy number aberrations, poor-prognosis hindbrain ependymomas exhibit a CpG island methylator phenotype. Transcriptional silencing driven by CpG methylation converges exclusively on targets of the Polycomb repressive complex 2 which represses expression of differentiation genes through trimethylation of H3K27. CpG island methylator phenotype-positive hindbrain ependymomas are responsive to clinical drugs that target either DNA or H3K27 methylation both in vitro and in vivo. We conclude that epigenetic modifiers are the first rational therapeutic candidates for this deadly malignancy, which is epigenetically deregulated but genetically bland.

Conflict of interest statement

The authors declare no competing financial interests.

Figures

Figure 1. Somatic SNVs are rare in the posterior fossa ependymoma genome

a, Summary of clinical and genomic details of PF ependymomas stratified according to group A and group B ependymoma (Wilcoxon rank-sum test). CNV, copy number variation; NA, not available; NS, not significant; WES, whole-exome sequencing; WGS, whole-genome sequencing. b, Bar graphs summarizing the numbers and frequencies of SNVs detected by whole-genome and whole-exome sequencing of PF ependymomas. c, Comparison of numbers of significant and recurrently mutated genes, and mutation rates, in several whole-genome and whole-exome sequencing studies of adult and paediatric cancers (false discovery rate (FDR) <0.1).

Figure 2. DNA-methylation profiles suggest that group A ependymomas demonstrate a CpG island methylator phenotype

a, Unsupervised hierarchical clustering of 79 ependymoma DNA-methylation profiles. SP, spinal cord; ST, supratentorial. b, Heatmap of 48 PF ependymoma DNA-methylation profiles. Group A and group B clinical differences were assessed using a two-sided Fisher’s exact test. c, Volcano plot comparing the number of significant methylated CpG sites between group A and group B (P < 0.05, Wilcoxon rank-sum test, FDR-corrected). d, e, Differences in the number of methylated genes (d) and methylated and silenced genes (e) in group A versus group B (P < 0.0001, binomial distribution test).

Figure 3. Group A (CIMP+) and group B (CIMP−) ependymomas are distinguished by CpG-hypermethylated and H3K27-trimethylated genes related to PRC2 occupancy in ES cells

a, b, CpG-methylated pathways in group A (CIMP+) and group B (CIMP−) ependymomas in a discovery (a) and validation (b) cohort. c, Differential H3K27me3 binding sites distinguishing group A and group B (P < 0.01 (MACSv2.0), P < 0.05 (R:DiffBind)). d, e, Venn diagrams comparing group A and group B H3K27me3 genes with ES cell PRC2 genes (d) and group A H3K27me3 and DNA-hypermethylated genes with ES cell H3K27me3 genes (e). f, Group B H3K27me3 and DNA-hypermethylated genes with ES cell H3K27me3 genes (binomial distribution test).

Figure 4. Whole-genome bisulphite sequencing validates a CpG island methylator phenotype in group A ependymoma

a, b, Heatmap of DNA methylation at CpG islands (a) and repetitive regions (b) in group A (CIMP+) versus group B (CIMP−). NB, normal brain. c, d, Proportion of hypermethylated versus hypomethylated regions at CpG islands (c) and repetitive elements (d) in group A and group B (P <2.2 × 10−16, binomial distribution test). LINE, long interspersed nuclear elements; LTR, long terminal repeats; SINE, short interspersed nuclear elements. e, f, Top 10 pathways upregulated (e) and differentially expressed (f) upon DAC treatment of E517-PF2 and E520-PF1 (P < 0.0001, binomial distribution test). g, Survival analysis of E478-ST2, E479-ST1, E517-PF2 and E520-PF1 cells treated for 7 days with DAC (P =0.05, two-sided t-test, error bars, s.e.m.; technical (n =6) over biological (n =2)). h, Limiting dilution assay of zero passage group A cells treated for 2 weeks with DAC (P =4.18 × 10−10, chi-squared test). DMSO, dimethylsulphoxide.

Figure 5. Modulation of H3K27 methylation has anti-neoplastic effects against group A ependymoma

a, Survival of E479-ST1, E478-ST2, E520-PF1 and E517-PF2 cells treated for 7 days with DZNep (P < 0.0001, two-sided t-test, error bars =s.e.m., biological (n =3)). b, EZH2, H3K27me3, H3K4me3 and cleaved-PARP protein expression in E520-PF1 and E479-ST1 cells treated for 7 days with DZNep (500 nM). c, E520-PF1 flank tumour volumes following treatment with DZNep (P =0.0087, Wilcoxon-test, error bars =s.d.). d, Survival of E520-PF1 PF tumour-bearing mice treated with DZNep (P =0.033, log-rank test). e, f, Top 10 pathways upregulated (e) and differentially expressed (f) upon treatment of E517 and E520 with GSK343. g, Cell proliferation of E520-PF1 and E479-ST1 cells treated for 11 days with GSK343 (active inhibitor) or GSK669 (inactive inhibitor) (P =0.0022, two-sided t-test, error bars =s.e.m., technical (n =9) over biological (n =3)). h, Limiting dilution assay of passage zero group A cells treated for 2 weeks with GSK343 (P =2.83× 10−5, chi-square-test). HCP, high-CpG-density promoter.