Novel oncogenic PDGFRA mutations in pediatric high-grade gliomas

Abstract

The outcome for children with high-grade gliomas (HGG) remains dismal, with a 2-year survival rate of only 10% to 30%. Diffuse intrinsic pontine glioma (DIPG) comprise a subset of HGG that arise in the brainstem almost exclusively in children. Genome-wide analyses of copy number imbalances previously showed that platelet-derived growth factor receptor α (PDGFRA) is the most frequent target of focal amplification in pediatric HGGs, including DIPGs. To determine whether PDGFRA is also targeted by more subtle mutations missed by copy number analysis, we sequenced all PDGFRA coding exons from a cohort of pediatric HGGs. Somatic-activating mutations were identified in 14.4% (13 of 90) of nonbrainstem pediatric HGGs and 4.7% (2 of 43) of DIPGs, including missense mutations and in-frame deletions and insertions not previously described. Forty percent of tumors with mutation showed concurrent amplification, whereas 60% carried heterozygous mutations. Six different mutations impacting different domains all resulted in ligand-independent receptor activation that was blocked by small molecule inhibitors of PDGFR. Expression of mutants in p53-null primary mouse astrocytes conferred a proliferative advantage in vitro and generated HGGs in vivo with complete penetrance when implanted into brain. The gene expression signatures of these murine HGGs reflected the spectrum of human diffuse HGGs. PDGFRA intragenic deletion of exons 8 and 9 were previously shown in adult HGG, but were not detected in 83 nonbrainstem pediatric HGG and 57 DIPGs. Thus, a distinct spectrum of mutations confers constitutive receptor activation and oncogenic activity to PDGFRα in childhood HGG.

Figure 1. PDGFRA somatic mutations identified in pediatric HGGs and DIPGs

Sanger sequencing of PDGFRA from genomic DNA revealed multiple mutations, including missense mutations, in-frame insertions and in-frame deletions. The schematic shows the location of the mutations and affected domains of PDGFRα. A star indicates samples with concomitant mutation and amplification of the PDGFRA locus. The signal peptide is designated by an orange oval and the transmembrane domain by a brown rectangle.

Figure 2. PDGFRα mutants are constitutively active and confer proliferative advantage

A) Western blot analysis of whole cell lysates from p53 null PMAs transduced with retroviruses expressing wild type PDGFRα (WT), PDGFRα mutants or empty vector. Cells were grown in serum free conditions overnight and then with or without PDGF-AA for 30 minutes. PDGFRα signaling activation of downstream targets was monitored using indicated antibodies in the presence and absence of PDGF-AA. B) Proliferation of p53 null PMAs transduced with retroviruses expressing wild type, PDGFRα mutants or empty vector was measured by an XTT assay in triplicate. Error bars show standard deviation. Shown is a representative figure of three independent experiments.

Figure 3. Wild-type and mutant PDGFRα are sensitive to small molecule inhibitors

A) Western blot analysis of whole cell lysates from p53 null PMAs transduced with retroviruses expressing wild type PDGFRα (WT), PDGFRα mutants or empty vector. Following overnight serum starvation, cells were pre-treated with Crenolanib (C), Dasatinib (D) or vehicle (−) for 3 hours and then stimulated with PDGF-AA for 30 minutes. Inhibition of PDGFRα signaling was analyzed using indicated antibodies. B) Growth of p53 null PMAs transduced with retroviruses expressing wild type, PDGFRα mutants or empty vector was measured with XTT assay in triplicate in the presence of crenolanib (left graph), dasatinib (right graph) or vehicle. Single dose of inhibitors (dashed lines) or vehicle (solid lines) was administered and growth was measured. Error bars show standard deviation. Shown are representative figures of three independent experiments.

Figure 4. PDGFRα mutants are tumorigenic in vivo

A) Kaplan–Meier curves showing time to morbidity of mice following intracranial implantation of p53 null PMAs expressing wild type PDGFRα (WT), PDGFRα mutants or empty vector. B) Diverse histologic phenotypes were generated in PDGFRα-driven HGGs. Most tumors diffusely infiltrated brain parenchyma, while also producing circumscribed masses. (a) Representative anaplastic astrocytoma, showing moderately pleomorphic tumor cells, some with astrocytic differentiation, diffusely infiltrating cerebral white matter (D842V; H&E x200), (b) Representative anaplastic oligoastrocytoma with admixed oligodendroglial and astrocytic phenotypes (C450ins; H&E x200), (c) Representative glioblastoma with focal giant cell phenotype (V544ins; H&E x200). C) Immunohistochemical analysis of a representative brain tumor stained for PDGFRα, p-4E-BP1 (T37/46), p-Akt (S473) and counterstained with hematoxylin. Scale bar is 50 μm. D) Western blot analysis of whole cell lysates from tissues of wild-type and mutant PDGFRα-driven brain tumors. Lysates from normal adult cortex (lanes N #1 and #2) were included as controls. Signaling pathway activation in PDGFRα-driven murine HGGs was assayed using the indicated antibodies.

Figure 5. Gene expression profiles of murine PDGFRα-driven HGGs resemble human HGGs

Heat map of single sample gene set enrichment analysis of representative murine brain tumors and untransduced p53-null PMA cultures using gene sets defining human HGG expression subgroups (PN-proneural, Pro-proliferative, Mes-mesenchymal) and murine cell type specific signatures (OPCs- oligodendrocyte progenitor cells, Oligo-oligodendrocyte, N- neurons, A- astrocytes and CA- cultured astroglia). The order of the tumor samples is arranged based on the unsupervised hierarchical clustering analysis using 1000 most variable probe sets, which reveled two major expression subgroups HC1 and HC2 (Figure S7). The histology of each tumor is indicated above the heat map: anaplastic astrocytoma (purple), anaplastic oligoastrocytomas (green), glioblastoma (brown), oligoastrocytoma (yellow) and white mark tumors with no diagnosis available. Untransduced p53-null PMA cultures are marked with black.