Lowered H3K27me3 and DNA hypomethylation define poorly prognostic pediatric posterior fossa ependymomas

Abstract

Childhood posterior fossa (PF) ependymomas cause substantial morbidity and mortality. These tumors lack recurrent genetic mutations, but a subset of these ependymomas exhibits CpG island (CpGi) hypermethylation [PF group A (PFA)], implicating epigenetic alterations in their pathogenesis. Further, histological grade does not reliably predict prognosis, highlighting the importance of developing more robust prognostic markers. We discovered global H3K27me3 reduction in a subset of these tumors (PF-ve ependymomas) analogous to H3K27M mutant gliomas. PF-ve tumors exhibited many clinical and biological similarities with PFA ependymomas. Genomic H3K27me3 distribution showed an inverse relationship with CpGi methylation, suggesting that CpGi hypermethylation drives low H3K27me3 in PF-ve ependymomas. Despite CpGi hypermethylation and global H3K27me3 reduction, these tumors showed DNA hypomethylation in the rest of the genome and exhibited increased H3K27me3 genomic enrichment at limited genomic loci similar to H3K27M mutant gliomas. Combined integrative analysis of PF-ve ependymomas with H3K27M gliomas uncovered common epigenetic deregulation of select factors that control radial glial biology, and PF radial glia in early human development exhibited reduced H3K27me3. Finally, H3K27me3 immunostaining served as a biomarker of poor prognosis and delineated radiologically invasive tumors, suggesting that reduced H3K27me3 may be a prognostic indicator in PF ependymomas.

Figure 1. H3K27me3 is globally reduced in a subset of pediatric PF ependymomas

A. Mass spectrometry of H3K27 and H3K4 modifications in pediatric PF (n=5) and ST (n=3, except for H3K27ac n=2) ependymomas. *p=0.016 B. Quantification of H3K27me3 and H3K27ac Western blots normalized to total histone H3 content in PF (n=6), ST (n=6), and SC (n=3) ependymomas. **p=0.002 for H3K27me3 and **p=0.005 for H3K27ac. C. Western blots for H3K27me3, H3K27ac, and total histone H3 quantified in B. D–I. Representative H3K27me3 IHC staining in pediatric PF (D–F) ependymomas; H3K27M mutant GBM (G); pediatric ST (H) and adult PF (I) ependymomas. Arrows indicate H3K27me3 positive endothelial and immune cells as internal control. Scale bars represent 200 μM. J. Quantification of H3K27me3 IHC in pediatric and adult ependymomas, and H3K27M and WT GBM (for n see table S7); ***p<0.0001. K–L. Representative composite immunofluorescence images for endothelial marker (K, CD31, red) or leukocyte common antigen (L, CD45, red); H3K27me3 (green); and DAPI (blue) in a PF−ve ependymoma. Arrowheads indicate H3K27me3 positive non-tumor cells. Scale bars represent 20 μM. Data are represented as mean ± SEM. Statistical significance in A, B and J was determined by two-sided, unpaired, two-tailed, Student’s t test. SC = spinal cord ependymoma, MP = myxopapillary spinal ependymoma, SE= subependymoma, and WT= H3 wild type glioma.

Figure 2. PF−ve tumors share clinical and biological features with PFA ependymomas

A–B. Age (A) and gender (B) distribution in ependymomas (6) immunostained with H3K27me3. *p=0.026. (for n see table S7; number of cases is indicated when n<3). C. Comparison of gene expression between PF−ve/PF+ve (n=11/4, Y-axis) and PFA/PFB ((6) n=18/19, X-axis) ependymomas. D. Principal component (PC) analysis of CpGi methylation in PF−ve (n=8), PF+ve (n=4), and ST (n=4) ependymomas. E. CpGi methylation per chromosome (bar plot and ideogram) indicating hypermethylated (pink) and hypomethylated (green) regions from PF−ve (n=8) and PF+ve (n=4) ependymomas. F. H3K27me3 immunohistochemistry from previously defined PFA (n=13) and PFB (n=3) pediatric ependymomas ***p<0.0001. Arrows indicate H3K27me3 positive endothelial and immune cells used as internal control. Scale bars represent 60 μM. Data are represented as means ± SEM. Statistical significance was determined in A and F by two-sided, unpaired, two-tailed Student’s t test; in B by two-tailed Chi square test, and in C by two-tailed Pearson’s Correlation.

Figure 3. PF−ve ependymomas exhibit global DNA hypomethylation that drives gene expression

A–B. Chromosomal methylation bar plot and ideogram indicating hypermethylated (pink) and hypomethylated (green) regions. C. Cumulative distribution frequency (CDF) plots of global DNA methylation in PF−ve, PF+ve, and ST ependymomas (arrows indicate direction of hypo or hypermethylation). ****Overall: p= 1.56e-93 for PF−ve vs PF+ve and negligible (reported as 0.00) for PF−ve vs ST. D. Percent methylation (overall, CpG islands, CpG shores, exons, introns, and promoters) in ependymomas. (p values provides in table S7). Data are represented as means ± SD. E. Differentially methylated regions (DMR) in CpG shores, CpG shelves, 5′ untranslated regions (UTR), 3′ UTR, introns, coding DNA sequences (CDS), gene bodies, and promoters. F. Integration of promoter DNA methylation (X-axis) and gene expression (Y-axis) for differentially expressed genes (n=213) where DMR at promoters were identified. For all graphs, PF−ve (n=8), PF+ve (n=4), and ST (n=4). For C and D, statistical significance was determined by Mann Whitney U/Wilcoxon test. Other p values are provided in table S7.

Figure 4. H3K27me3 distribution inversely correlates with CpGi methylation

A. Gene expression (Log2 FD, PF−ve/ST; n=5/3, ***p<0.0001 and PF−ve/PF+ve; n=11/4, ** p=0.0022) in H3K27me3 high versus H3K27me3 low regions. B. H3K27me3 distribution in the SIX1 locus. C. Comparison of BAMBI and SIX1 gene expression (Log2 FD) between PF−ve/ST (n=5/3), PF−ve/PF+ve (n=11/4), and PFA/PFB (n=18/19 (6)) ependymomas. D. Kaplan-Meier survival curves in PF ependymomas (6) with low (red, n=16) or high (blue, n=21) expression of SIX1/BAMBI mRNA. E. Fold difference (PF−ve/ST) of H3K27me3 enrichment for hyper- or hypo-methylated CpGi in PF−ve tumors. ***p<0.0001. F. The DMRT2A locus on chromosome 1 with CpGi hypomethylation and enriched H3K27me3. G. The LHX2 locus on chromosome 9 with CpGi hypermethylation and low H3K27me3. H. Correlational analyses between H3K27me3 distribution (FD, PF−ve/ST, Y-axis) and DNA methylation (mean methylation difference, X-axis). I. Functional grouping of all genes that are epigenetically deregulated. Red indicates pathways related directly to neuronal development and cell differentiation (altered promoter DNA methylation and/or marked by promoter H3K27me3) and show altered gene expression. Data in A and E are represented as means ± SEM. Statistical significance was determined by two-sided, unpaired, two-tailed Student’s t test; the log-rank test was used in D, and two-tailed Pearson’s Correlation in H.

Figure 5. PFA/PF−ve ependymomas show similarities to H3K27M gliomas in DNA methylation and H3K27me3 genome-wide distribution

A. Unsupervised clustering analyses of Illumina 450K array DNA methylation data from PFA (n=14) and PFB (n=11) ependymomas (7) and H3K27M (n=18), G34 (n=18), and IDH1 mutant (n=19) gliomas (20). All methylation sites were included in the analysis. B. Heat map of representative hyper and hypomethylated CpGi from A. C. Comparison of S100B and PCDH9 gene expression (Log2FD between PFA/PFB (n=18/19, left Y-axis, (6)), H3K27M/H3wt (n=12/21 (20), right Y-axis), and G34/H3wt (n=10/21 (20), right Y-axis). D. Kaplan-Meier curves in PF ependymomas with (6) high (n=25) or low expression (n=12) of S100B/PCDH9 mRNA. E. Heat map displaying H3K27me3 peak intensities in PF−ve (n=3), ST (n=3), and neuronal progenitor cells (NPC) expressing H3.3K27M (K27M) or H3.3 wild type (WT) (22). Unguided hierarchical clustering was used for clustering samples based on H3K27me3 enrichment. **p = 0.0075. F. H3K27me3 distribution in the EMX2 locus in PF−ve and ST ependymomas and H3K27M-expressing and H3WT NPC. G. Numbers of specific H3K27me3 peaks common to PFA (n=6) or PFB (n=4) ependymomas (7) and H3K27M or WT NPC (22). ***p< 0.0001. H. Comparison of EMX2 and HOXA7 gene expression (Log2FD) between PFA/PFB (brown, left Y-axis, n=18/19, (6)), H3K27M/H3WT (orange (20), right Y-axis, n=12/21), and G34/H3wt (green, right Y-axis (20), n=10/21). I. Kaplan-Meier survival curves in PF ependymomas (6) with high (n=27) or low expression (n=10) of EMX2/HOXA7 mRNA. J. Overlap of differentially DNA or H3K27 methylated genes in PFA and H3K27M tumors with those in human radial glial gene expression data sets (23). Statistical significance in D and I was determined by the log-rank test. In E and G, the significance of differences in peak numbers was evaluated by two-tailed Chi square test.

Figure 6. Radial glia in early cerebellar development show reduced H3K27me3

A–B. Representative confocal immunofluorescent images of human cerebellar tissues stained with antibodies against GFAP (green), H3K27me3 (red), and DAPI (blue) when the EGL is present before birth (A) or when EGL migration is complete (B). Arrows indicate H3K27me3 negative (A) or positive (B) nuclei in GFAP-positive radial glia. Arrowheads indicate radial glial processes. Dotted circles indicate granular cells. Solid lines indicate the pial surface, and the dotted lines indicate the extent of the EGL. Scale bars represent 20 μM. C–D. Higher power images of radial glia. Large arrowheads indicate H3K27me3 negative (C) or positive (D) nuclei in GFAP-positive radial glia. Small arrowheads indicate radial glial processes. Dotted circles indicate granular cells. Scale bars represent 8 μM. E–F. Representative confocal immunofluorescent images from the human 4^th periventricular region. Scale bars represent 20 μM. Arrows indicate H3K27me3-positive nuclei in GFAP-positive radial glia. G. The percentage of GFAP-positive/H3K27me3-negative or GFAP/H3K27me3-positive radial glia before and after development (n=3, each). ***p<0.0001 H. Amount of H3K27me3 in each cell (H3K27me3 pixel units) quantified using NIH imageJ from various cell types in the PF before and after development. ***p<0.0001, for n, see table S7. I. Expression (Log2 FD) of factors that differentiate PFA from PFB ependymomas in PFA/PFB (n=18/19 (6)) and radial glia from P6/P30 mice (n=5 each (29)). ND = not detected. Data in G and H are represented as means ± SEM; statistical significance was determined in G using two-tailed Chi square test and two-sided, unpaired, two-tailed Student’s t test in H. NS = not significant. RG= radial glia, EGL= external granular layer, ML = molecular layer, PC = Purkinje cell, IGL = internal granular layer, GL= granular layer, 4V= 4th ventricle.

Figure 7. Decreased H3K27me3 is a poor prognostic biomarker in pediatric PF ependymomas

A–B. Representative axial and coronal T2 weighted MRI images from PF−ve (A) and PF+ve (B) ependymomas (white arrows). C Percentage of cases with or without invasion in PF−ve (n=20) versus PF+ve (n=4). ***p < 0.0001 D Tumor invasion and infiltration into surrounding brain tissue was compared between PF+ve (n=4) and PF−ve (n=28) by detecting neurofilaments in axons within tumor tissues (arrowheads) using immunohistochemistry (representative images from PF−ve and PF+ tumors and quantification). Scale bars represent 60 μM. ***p=0.0017. E Extent of resection (GTR=gross total resection/no detectable tumor on post-surgical MRI, STR=subtotal resection/portion of tumor is detectable on post-surgical MRI, and NTR=near total resection/detection of minimal tumor on post-surgical MRI) in pediatric PF−ve (n=30) and PF+ve (n=4) ependymomas. **p=0.0047. F–G. Kaplan-Meier survival curves from pediatric (F) or combined pediatric and adult (G) cohorts of PF−ve (n=50 in both F and G) and PF+ve (blue, n=11 in F and n=15 in G). H. Cox proportional regression analyses for overall survival in PF−ve (n=50) and PF+ve (n=11) ependymomas. Exp(B) is a measure of the hazard ratio. I. H3K27me3 in matched primary and recurrent PF−ve and ST ependymomas (n=3 for each). Representative images and quantification are shown. Scale bars represent 200 μM. Data are represented as means ± SEM in D and I. Statistical significance in C and E was determined by two-tailed Chi square test, in D and I by unpaired, two-sided, two-tailed t test, and in F and G by the log-rank test.