Genomic analysis of diffuse intrinsic pontine gliomas identifies three molecular subgroups and recurrent activating ACVR1 mutations

Abstract

Diffuse intrinsic pontine glioma (DIPG) is a fatal brain cancer that arises in the brainstem of children, with no effective treatment and near 100% fatality. The failure of most therapies can be attributed to the delicate location of these tumors and to the selection of therapies on the basis of assumptions that DIPGs are molecularly similar to adult disease. Recent studies have unraveled the unique genetic makeup of this brain cancer, with nearly 80% found to harbor a p.Lys27Met histone H3.3 or p.Lys27Met histone H3.1 alteration. However, DIPGs are still thought of as one disease, with limited understanding of the genetic drivers of these tumors. To understand what drives DIPGs, we integrated whole-genome sequencing with methylation, expression and copy number profiling, discovering that DIPGs comprise three molecularly distinct subgroups (H3-K27M, silent and MYCN) and uncovering a new recurrent activating mutation affecting the activin receptor gene ACVR1 in 20% of DIPGs. Mutations in ACVR1 were constitutively activating, leading to SMAD phosphorylation and increased expression of the downstream activin signaling targets ID1 and ID2. Our results highlight distinct molecular subgroups and novel therapeutic targets for this incurable pediatric cancer.

Figures

Figure 1. Methylation profiling reveals three molecular subgroups of DIPG

(a) Heat map of methylation levels in three DIPG subgroups identified by unsupervised hierarchical clustering and supported by (b) principal components analysis, (c), non-negative matrix factorization (cophenetic coefficient = 0.9934, k=3) and (d) consensus clustering represented by cumulative distribution function and change in Gini.

Figure 2. Molecular subgroups of DIPG share common clinical features and recurrent genomic events

(a) Clinical and genomic features such as gender, histology, frequency of recurrent mutations, alternative lengthening of telomeres and copy number alterations are represented in a DIPG subgroup specific manner. (b) Probability of two mutational or structural features of DIPG co-occurring based on odds ratio suggests statistically significant association between K27M-H3.3 and PDGFRA amplifications (OR = 8.0, p = 0.0127) and between K27M-H3.1 and ACVR1 mutations (OR = 15.8, p < 0.001). (C) Probability of mutations or structural event of DIPG occurring with a clinical feature such as gender or tumor histology based on odds ratio shows statistically significant correlation between P53 mutations and GBM histology (OR = 10.8, p < 0.005), among others.

Figure 3. ACVR1 mutations constitutively activate BMP signaling in vitro and in ACVR1 mutant DIPG

(a) Four mutations (R206H, Q207E, G328E and G328V) were detected in 12/61 DIPG patients. The R206H and Q207E mutations occur in the GS domain and the G328-mutations occur in the protein kinase domain. (b) Human DIPG with ACVR1 mutations have increased pSMAD1/5 expression compared with ACVR1 wild-type DIPG. (c) Western blot showing increased pSMAD1/5 in ACVR1 mutant NHA and DIPG58 cells transfected with G328V-ACVR1 as compared to control cells. (d) Real-time PCR in NHA transfected with empty vector, K27M-H3.3, G328V-ACVR1 or a combination of K27M-H3.3 and G328V-ACVR1 shows increase in ID1 and ID2 gene expression as compared to empty vector control. Error bars represent standard deviation. (e) Mutant G328V-ACVR1 expressing NHA cells have an increased growth rate as compared to empty vector controls (p = 0.0034). (f) Compared to WT-ACVR1 murine brainstem progenitor cultures, mutant ACVR1 has significantly higher BrdU incorporation suggesting increased proliferation (* p<0.05). Error bars represent standard error of the mean.