Unique genetic and epigenetic mechanisms driving paediatric diffuse high-grade glioma

Abstract

Diffuse high-grade gliomas (HGGs) of childhood are a devastating spectrum of disease with no effective cures. The two-year survival for paediatric HGG ranges from 30%, for tumours arising in the cerebral cortex, to less than 10% for diffuse intrinsic pontine gliomas (DIPGs), which arise in the brainstem. Recent genome-wide studies provided abundant evidence that unique selective pressures drive HGG in children compared to adults, identifying novel oncogenic mutations connecting tumorigenesis and chromatin regulation, as well as developmental signalling pathways. These new genetic findings give insights into disease pathogenesis and the challenges and opportunities for improving patient survival in these mostly incurable childhood brain tumours.

Figure 1. Brain regions and selective mutations distinguish subgroups of paediatric HGG

High-grade gliomas in children frequently arise in the midline as DIPG in the pons, or involving the thalamus, or much less frequently, the cerebellum or spinal cord. Childhood HGGs also arise in the cerebral cortex, the region where the majority of HGGs in adults originate. Mutation frequencies indicate differences in selective pressures between different regions of developing brain. For example, histone H3.3 G34R/V mutations and BRAF V600E mutations are predominantly found in cortical tumours, histone H3 K27M mutations are found in all midline locations with highest frequency in DIPG, ACVR1 mutations are restricted to DIPG, and FGFR1 mutations are found in thalamic HGGs.

Figure 2. Mutational burden of paediatric HGG

Somatic non-synonymous coding mutations per sample are plotted on a log scale for a variety of paediatric and adult cancer types, and ordered by increasing median (red bar)^{, , , ,} . Infant HGG (shaded orange) have the lowest number of somatic mutations per sample other than paediatric low grade glioma, with a median of 2. Paediatric HGG (pink) and DIPG (red) have very similar profiles, with a median of 15 non-synonymous coding mutations per sample for both, considerably lower than for adult GBM (61 mutations per sample, p<0.001 for both pHGG and DIPG, Mann-Whitney U test). There was no significant difference between pre-treatment DIPG samples taken at biopsy and post-treatment samples at autopsy. Untreated paediatric HGG classed as ‘hypermutator’ (dark red) harboured the highest mutational burden of all, with a median of 6,810 (range 262–18,369) non-synonymous coding mutations per sample. AML: acute myeloid leukaemia. CML: chronic lymphocytic leukaemia. LGG: low grade glioma.

Figure 3. Recurrent mutations activate PI3K and MAPK signaling pathways in paediatric HGG

Growth factor binding to receptor tyrosine kinases (RTKs) activates downstream signaling through the PI3 kinase and MAP kinase signaling pathways. PI3 kinase alpha is comprised of the p85 regulatory subunit and the p110α catalytic subunit, encoded by PIK3R1 and PIK3CA, respectively. PI3K phosphorylates the signaling lipid PIP2 to generate PIP3, leading to subsequent activation of downstream cascades, including the serine/threonine kinase AKT. The PTEN tumour suppressor is a protein and lipid phosphatase that directly reverses the action of PI3K. Growth factor binding to RTKs can activate both RAS and PI3K through different adapter proteins, or through direct interaction with PI3K, depending on the specific receptor. RAS is a small G-protein which is activated when in the GTP-bound state. The NF1 tumour suppressor is a GTP-ase activating protein that negatively regulates RAS activity. RAS participates in PI3K pathway signaling as well as downstream signaling through the serine/threonine kinase BRAF to additional downstream targets in the MAP kinase pathway. The frequency of mutations for the most recurrently mutated components of these pathways in paediatric high-grade glioma are indicated^–. Some mutations show different frequencies in cortical (Cort) versus thalamic/midline HGGs or DIPGs. Of note, PTEN and EGFR mutations, which are frequent in adults, are not highly recurrent in childhood HGGs. mut: mutated, amp: amplified

Figure 4. Histone and chromatin modifier mutations in paediatric high-grade and diffuse intrinsic pontine glioma

(a) Specific recurrent mutations in the genes encoding the histone variants H3.3 (H3F3A) and H3.1 (HIST1H3B and HIST1H3C). K27M mutations in H3.3 and H3.1 result in a global reduction of H3K27me3, leading to derepression of targets of the polycomb repressive complex 2 (PRC2). H3.3 K27M mutations are found in nearly two-thirds of DIPG, half of thalamic HGG, and other midline tumours. H3.1 K27M mutations are found in over 20% of DIPG and 5% of midline HGGs, and are strongly associated with younger patient age^–. H3.3 G34R/V mutations are found in 15% paediatric HGG and are restricted to cerebral hemispheric tumours of adolescents and young adults, and confer differential distribution of the activating H3K36me3 mark. (b) Mutations in other chromatin modifiers. Numerous writers (top) and erasers (bottom) of histone H3 lysine marks are mutated in paediatric HGG and DIPG, with an apparent exception of lysine 27. Lysines in the H3 tail that are modified by trimethylation (me marks) or acetylation (ac) are indicated. In addition, H3 chaperones such as ATRX or DAXX are mutated in 15–20% of supratentorial tumours, along with a variety of other chromatin remodellers in tumours of all locations^–.

Figure 5. ACVR1 mutations in diffuse intrinsic pontine glioma

(a) Cartoon showing somatic mutations in the glycine/serine-rich domain inhibitory domain (GS) and kinase domains of ACVR1 in 46/195 (24%) DIPG^– samples. No mutations were identified in the extracellular (ECD) or transmembrane (TM) domains. All residues targeted are common to those observed in the germlines of fibrodysplasia ossificans progressiva (FOP) patients, with the same amino acid substitutions except for R258G and G328V, which are to date unique to DIPG. (b) BMP signalling pathway. Upon ligand binding, a type II receptor such as BMPRII heterodimerises with and phosphorylates ACVR1, a serine threonine kinase, which in turn phosphorylates the SMADs 1/5/8 transcription factors, causing binding to SMAD4, translocation to the nucleus, and transcription of target genes such as ID1 and ID2. The ACVR1 mutations in DIPG and FOP enhance the kinase function and/or disrupt binding of the negative regulator FKBP12 to ACVR1, conferring activation of this pathway.