Recurrent somatic mutations in ACVR1 in pediatric midline high-grade astrocytoma

Abstract

Pediatric midline high-grade astrocytomas (mHGAs) are incurable with few treatment targets identified. Most tumors harbor mutations encoding p.Lys27Met in histone H3 variants. In 40 treatment-naive mHGAs, 39 analyzed by whole-exome sequencing, we find additional somatic mutations specific to tumor location. Gain-of-function mutations in ACVR1 occur in tumors of the pons in conjunction with histone H3.1 p.Lys27Met substitution, whereas FGFR1 mutations or fusions occur in thalamic tumors associated with histone H3.3 p.Lys27Met substitution. Hyperactivation of the bone morphogenetic protein (BMP)-ACVR1 developmental pathway in mHGAs harboring ACVR1 mutations led to increased levels of phosphorylated SMAD1, SMAD5 and SMAD8 and upregulation of BMP downstream early-response genes in tumor cells. Global DNA methylation profiles were significantly associated with the p.Lys27Met alteration, regardless of the mutant histone H3 variant and irrespective of tumor location, supporting the role of this substitution in driving the epigenetic phenotype. This work considerably expands the number of potential treatment targets and further justifies pretreatment biopsy in pediatric mHGA as a means to orient therapeutic efforts in this disease.

Figure 1. Genomic landscape of pediatric midline high-grade astrocytomas

Distribution of mutations and alterations in 40 pediatric midline high-grade astrocytomas (Midline HGAs) and 42 cortical high-grade astrocytomas and high-grade gliomas (Cortical HGA-HGGs) described in the study. Mutations (mut) were identified with whole-exome sequencing (WES) where available and are indicated by coloured boxes according to the legend in the right panel. Amplifications (amp) and losses were identified utilizing DNA methylation profile derived copy number variant (Methyl-CNV) analysis and are indicated where available by coloured boxes according to the right panel legend. SC = spinal cord, 4^th V = 4^th ventricle. Boxes coloured in light grey indicate samples for which data is not available. Detailed information regarding tumor samples included herein can be found in Table S1, with specific variants and transcript accessions presented in Table S6.

Figure 2. Increased levels of phospho-SMAD1/5/8 in ACVR1 mutant pediatric HGAs

Immunohistochemical analysis of midline pediatric HGAs harbouring ACVR1 mutations identified in this study (n=4, left panels) demonstrate increased nuclear positivity of phospho-SMAD1/5/8 compared to pediatric midline HGAs wild-type for ACVR1 mutation (n=3) and control brain samples (right panels), with total-SMAD1 staining shown in each case as positive control. Scale bars indicate 20X=20μm. Clinicopathologic and molecular characteristics of tumor samples are presented in Table S1.

Figure 3. Clustering analysis of global DNA methylation profiles of 98 pediatric high-grade astrocytomas

Global DNA methylation clustering analysis of pediatric HGAs distributed across the brain demonstrates similar impact on epigenomic dysregulation caused by K27M mutation regardless of age, brain location, associated mutation or the particular histone 3 variant affected. The top 10,000 most variable normalized methylation β-values were utilized for UPGMA clustering with colour key and scale in top left panel, and indicated mutations of interest in inset left panel. Robustness was assessed utilizing multiscale bootstrapping in Figure S6. Detailed information regarding sample clinicopathologic characteristics are included in Table S1 with methylation-derived CNVs presented in Tables S4–5.

Figure 4. Mutations identified in ACVR1 are associated with activation of downstream SMAD signaling pathways

a, Distribution of mutations identified in ACVR1 (n=5) demonstrating their impact of amino acid substitutions on the kinase domain of the protein. b, Immunoblotting analysis of phospho-SMAD1/5/8 levels in ACVR1 wild-type NHA cells grown in 10% FBS, or NHA and DIPGIV (ACVR1 G328V) serum starved for 1 hour in serum/growth factor-free media (0%). c, Quantitative PCR (qPCR) analysis of downstream BMP effectors ID1, ID2, ID3 and SNAI1 expression in ACVR1 mutant (DIPGIV) and wild-type GBM cell line (KNS42). Values represented are fold changes calculated using the 2^−ΔΔCt method, normalized to ACTB expression in calibrator NHA cells. P-values calculated using two-tailed T-test for significance, with error bars indicated as standard deviation from two replicates.