Aberrant patterns of H3K4 and H3K27 histone lysine methylation occur across subgroups in medulloblastoma

Abstract

Recent sequencing efforts have described the mutational landscape of the pediatric brain tumor medulloblastoma. Although MLL2 is among the most frequent somatic single nucleotide variants (SNV), the clinical and biological significance of these mutations remains uncharacterized. Through targeted re-sequencing, we identified mutations of MLL2 in 8 % (14/175) of MBs, the majority of which were loss of function. Notably, we also report mutations affecting the MLL2-binding partner KDM6A, in 4 % (7/175) of tumors. While MLL2 mutations were independent of age, gender, histological subtype, M-stage or molecular subgroup, KDM6A mutations were most commonly identified in Group 4 MBs, and were mutually exclusive with MLL2 mutations. Immunohistochemical staining for H3K4me3 and H3K27me3, the chromatin effectors of MLL2 and KDM6A activity, respectively, demonstrated alterations of the histone code in 24 % (53/220) of MBs across all subgroups. Correlating these MLL2- and KDM6A-driven histone marks with prognosis, we identified populations of MB with improved (K4+/K27-) and dismal (K4-/K27-) outcomes, observed primarily within Group 3 and 4 MBs. Group 3 and 4 MBs demonstrate somatic copy number aberrations, and transcriptional profiles that converge on modifiers of H3K27-methylation (EZH2, KDM6A, KDM6B), leading to silencing of PRC2-target genes. As PRC2-mediated aberrant methylation of H3K27 has recently been targeted for therapy in other diseases, it represents an actionable target for a substantial percentage of medulloblastoma patients with aggressive forms of the disease.

Fig. 1

MLL2 mutations in medulloblastoma are independent of molecular subgroup and lack prognostic utility. a Incidence of MLL2 mutations across a discovery (n = 175) (left) and validation cohort (n = 398) (right). b Percent distribution of MLL2 homozygous versus heterozygous mutations based on allele frequency, identifies a single homozygous mutation (1/53) across the combined cohorts. c Analysis of the functional consequences of MLL2 mutations highlights a significant fraction of discovery (45 %) and validation (67–80 %) single nucleotide variants that disrupt the MLL2 coding sequence through frameshift and nonsense mutations ablating enzymatic methyltransferase activity. d Subgroup-specific examination of MLL2 mutations describes the extensive variability across both sequencing cohorts suggesting MLL2 mutations occur independent of molecular subgroup. e Schematic representation of the MLL2 mutational landscape in medulloblastoma indicates single nucleotide variants scattered throughout the coding sequence and the absence of a mutational hotspot. A single recurrent mutation (R5501*) was identified across 53 MLL2 mutations. f MLL2 immunohistochemical (IHC) analysis provides no prognostic utility based on presence/absence of MLL2 protein expression for WNT and SHH subgroups (left), whereas loss of MLL2 protein expression was significantly (P < 4.63E–2) associated with survival for Group 3 and Group 4 (i.e., non-WNT, non-SHH) tumors (right)

Fig. 2

Identification of focal deletions of KDM6A, and mutations that are enriched in Group 4 tumors. a Analysis of medulloblastoma copy number aberrations reveals one (0.5 %, 1/203) tumor with a homozygous deletion spanning KDM6A. b Subgroup-specific distribution of X-chromosome loss across 190 primary medulloblastomas studied by Affymetrix 500 K SNP arrays demonstrates a significant (P = 4.00E−3) enrichment of this cytogenetic event in Group 4 medulloblastomas (22 %, 8/36). c Targeted exon-capture and sequencing identifies a medulloblastoma (0.6 %, 1/175, 1M2) with a KDM6A intragenic deletion spanning exons 5 through 13. d Frequency and percent of KDM6A mutations across a discovery cohort of 175 primary tumors (left) and a validation cohort of 398 tumors (right) reveals mutations in 4–8 % of tumors. e Allele frequency inference of KDM6A mutation zygosity highlights the abundance of homozygous mutations across both cohorts. f Percent distribution of KDM6A mutations which disrupt the coding sequence (frameshift or nonsense mutations) versus non-disruptive nucleotide changes (missense and in-frame INDELs) accents the frequent disruptive nature of KDM6A mutations across the discovery and validation cohorts. g Subgroup-association of KDM6A mutations demonstrates predominant targeting of Group 4 medulloblastomas. h Mutational landscape of KDM6A across 573 tumors (combined discovery and validation cohort) calls attention to the 3′ localized, damaging and Group 4-enriched nature of KDM6A single nucleotide variants

Fig. 3

H3K4me3 and H3K27me3 staining reveals deregulation of the histone code in a significant fraction of medulloblastomas. a Analysis of MLL2 and KDM6A mutations demonstrates the mutually exclusivity (P = 8.60E–6) and mutational inactivation of chromatin-modifier genes in 12 % (21/175) of the discover cohort. The validation cohort recapitulated these findings (P = 4.85E–10); however, a single tumor was identified with both MLL2 and KDM6A mutations. Importantly, the MLL2 mutation in this case was a missense variant with no known deleterious effect to protein functionality. b H3K4me3 (K4) and H3K27me3 (K27) immunostaining results identifies populations of medulloblastomas with modifications from the expected (K4+/K27+) staining patterns in 23.5 % (53/222) of tumors analyzed, suggesting deregulation of the histone code is a common occurrence in medulloblastoma (left). The molecular distribution of medulloblastomas with specific histone marks (right). c Survival analysis of medulloblastomas with normal (K4+/K27+) or aberrant (K4+/K27−; K4−/K27+; K4−/K27−) histone code demonstrates distinct clinical differences with improved outcome associated with K4+/K27− and dismal outcome associated with dual negativity (K4−/K27). d Subgroup-specific analysis of chromatin marks demonstrates the clinical significance associated with H3K4me3 and H3K27me3 staining patterns are driven by Group 3 and Group 4 medulloblastomas

Fig. 4

Identification of a genomic and transcriptional H3K27me3-enrichment phenotype (K27+) in Group 4 medulloblastomas. a Cytogenetic distribution of H3K27me methyltransferases (EZH2, chr7q36.1) and demethylases (KDM6A, chrXp11.3; KDM6B chr17p13.1) (left) and a subgroup-specific analysis of cytogenetic aberrations resulting in the accumulation of H3K27me3 identifies a significant (P = 2.87E−2) enrichment in Group 3 and Group 4 medulloblastomas versus WNT and SHH tumors (right). b Over-expression of H3K27-methyltransferases and down-regulation of H3K27-demethylases reveals a H3K27-methylator phenotype (K27+) occurring exclusively in Group 3 and Group 4 medulloblastomas across 103 primary medulloblastomas. c Discovery and validation transcriptome cohorts comprising 290 tumors demonstrate a statistically significant enrichment of an H3K27-methylator phenotype (K27+) in Group 3 and Group 4 MB. d Transcriptome-wide analysis of significantly (P < 0.05) and differentially (>2-fold) expressed genes in H3K27-methylator phenotype (K27+) versus non-K27+ medulloblastomas. Molecular Signature (MSigDB) analysis reveals extensive deregulation of PRC2-target genes in the discovery (40 %) and validation (10 %) cohorts. e Comparison of significantly (P < 0.05) and differentially (>2-fold) expressed genes across the discovery and validation transcriptome cohort reveals a small number of overlapping genes. Many of the overlapping genes (50 %, 3/5) have previously been implicated as H3K27me3-targets. f Dot plot analysis of genes putatively regulated by H3K27me3 methylation across both the discovery and validation transcriptome cohorts highlights the significantly differential fold changes and possible utility for future PRC2-therapy-related diagnostics

Fig. 5

Schematic representation of normal developmental Trithorax (trxG) and Polycomb (PcG) group proteins and the effects of MLL2 and KDM6A mutations on chromatin state and transcriptional activation. a Representation of the balancing act of bivalent domains in which Trithorax (trxG) and Polycomb (PcG) group proteins counteract each other to maintain genes poised for activation. Activation of gene expression through a trxG-mediated shift towards H3K4-trimethylation and repressed transcription via PcG-regulated H3K27me3 induce differentiation. b Wild-type and pathogenic roles of MLL2 and KDM6A on chromatin state and transcription. MLL2 functions as H3K4 methyltransferase shunting promoters towards an active state. MLL2 mutations in medulloblastoma largely disrupt the coding sequence through nonsense and frameshift mutations causing premature truncation of the transcript and ablation of the methyltransferase activity preventing the normal activation of gene expression. KDM6A, a H3K27-demethylase, functions as a trxG-protein to inhibit PcG-mediate H3K27me3 marks. KDM6A mutations in medulloblastomas result in the accumulation of H3K27me3, normally removed by the wild-type gene. In Group 3 and 4 medulloblastomas an H3K27me3-enriched phenotype (K27+) is observed, in which overexpression of the H3K27-methyltransferase EZH2 with concomitant down-regulation of both KDM6A and KDM6B demethylases results in a strong shift towards increased H3K27me3