Pediatric high-grade glioma (HGG) is a devastating disease with a two-year survival of less than 20%1. We analyzed 127 pediatric HGGs, including diffuse intrinsic pontine gliomas (DIPGs) and non-brainstem HGGs (NBS-HGGs) by whole genome, whole exome, and/or transcriptome sequencing. We identified recurrent somatic mutations in ACVR1 exclusively in DIPG (32%), in addition to the previously reported frequent somatic mutations in histone H3, TP53 and ATRX in both DIPG and NBS-HGGs2-5. Structural variants generating fusion genes were found in 47% of DIPGs and NBS-HGGs, with recurrent fusions involving the neurotrophin receptor genes NTRK1, 2, or 3 in 40% of NBS-HGGs in infants. Mutations targeting receptor tyrosine kinase/RAS/PI3K signaling, histone modification or chromatin remodeling, and cell cycle regulation were found in 68%, 73% and 59%, respectively, of pediatric HGGs, including DIPGs and NBS-HGGs. This comprehensive analysis provides insights into the unique and shared pathways driving pediatric HGG within and outside the brainstem.

A B S T R A C T PurposeTo define copy number alterations and gene expression signatures underlying pediatric high-grade glioma (HGG). Patients and MethodsWe conducted a high-resolution analysis of genomic imbalances in 78 de novo pediatric HGGs, including seven diffuse intrinsic pontine gliomas, and 10 HGGs arising in children who received cranial irradiation for a previous cancer using single nucleotide polymorphism microarray analysis. Gene expression was analyzed with gene expression microarrays for 53 tumors. Results were compared with publicly available data from adult tumors. ResultsSignificant differences in copy number alterations distinguish childhood and adult glioblastoma. PDGFRA was the predominant target of focal amplification in childhood HGG, including diffuse intrinsic pontine gliomas, and gene expression analyses supported an important role for deregulated PDGFR␣ signaling in pediatric HGG. No IDH1 hotspot mutations were found in pediatric tumors, highlighting molecular differences with adult secondary glioblastoma. Pediatric and adult glioblastomas were clearly distinguished by frequent gain of chromosome 1q (30% v 9%, respectively) and lower frequency of chromosome 7 gain (13% v 74%, respectively) and 10q loss (35% v 80%, respectively). PDGFRA amplification and 1q gain occurred at significantly higher frequency in irradiation-induced tumors, suggesting that these are initiating events in childhood gliomagenesis. A subset of pediatric HGGs showed minimal copy number changes. ConclusionIntegrated molecular profiling showed substantial differences in the molecular features underlying pediatric and adult HGG, indicating that findings in adult tumors cannot be simply extrapolated to younger patients. PDGFR␣ may be a useful target for pediatric HGG, including diffuse pontine gliomas.

A B S T R A C T PurposeLong-term survival for children with diffuse intrinsic pontine glioma (DIPG) is less than 10%, and new therapeutic targets are urgently required. We evaluated a large cohort of DIPGs to identify recurrent genomic abnormalities and gene expression signatures underlying DIPG. Patients and MethodsSingle-nucleotide polymorphism arrays were used to compare the frequencies of genomic copy number abnormalities in 43 DIPGs and eight low-grade brainstem gliomas with data from adult and pediatric (non-DIPG) glioblastomas, and expression profiles were evaluated using gene expression arrays for 27 DIPGs, six low-grade brainstem gliomas, and 66 nonbrainstem low-grade gliomas. ResultsFrequencies of specific large-scale and focal imbalances varied significantly between DIPGs and nonbrainstem pediatric glioblastomas. Focal amplifications of genes within the receptor tyrosine kinase-Ras-phosphoinositide 3-kinase signaling pathway were found in 47% of DIPGs, the most common of which involved PDGFRA and MET. Thirty percent of DIPGs contained focal amplifications of cell-cycle regulatory genes controlling retinoblastoma protein (RB) phosphorylation, and 21% had concurrent amplification of genes from both pathways. Some tumors showed heterogeneity in amplification patterns. DIPGs showed distinct gene expression signatures related to developmental processes compared with nonbrainstem pediatric high-grade gliomas, whereas expression signatures of low-grade brainstem and nonbrainstem gliomas were similar. ConclusionDIPGs comprise a molecularly related but distinct subgroup of pediatric gliomas. Genomic studies suggest that targeted inhibition of receptor tyrosine kinases and RB regulatory proteins may be useful therapies for DIPG.

The potent bioactive sphingolipid mediator, sphingosine-1-phosphate (S1P), is produced by 2 sphingosine kinase isoenzymes, SphK1 and SphK2. Expression of SphK1 is up-regulated in cancers, including leukemia, and associated with cancer progression. A screen of sphingosine analogs identified (2R,3S,4E)-N-methyl-5-(4-pentylphenyl)-2-aminopent-4-ene-1,3-diol, designated SK1-I (BML-258), as a potent, water-soluble, isoenzymespecific inhibitor of SphK1. In contrast to pan-SphK inhibitors, SK1-I did not inhibit SphK2, PKC, or numerous other protein kinases. SK1-I decreased growth and survival of human leukemia U937 and Jurkat cells, and enhanced apoptosis and cleavage of Bcl-2. Lethality of SK1-I was reversed by caspase inhibitors and by expression of Bcl-2. SK1-I not only decreased S1P levels but concomitantly increased levels of its proapoptotic precursor ceramide. Conversely, S1P protected against SK1-I-induced apoptosis. SK1-I also induced multiple perturbations in activation of signaling and survival-related proteins, including diminished phosphorylation of ERK1/2 and IntroductionSphingosine-1-phosphate (S1P), a potent lipid mediator produced from sphingosine by sphingosine kinases (SphKs), regulates many processes important for cancer progression, including cell growth and survival. 1 In contrast to S1P, its precursors, sphingosine and ceramide, are associated with growth arrest and induction of apoptosis. 2 Thus, the balance between these interconvertible sphingolipid metabolites has been viewed as a cellular rheostat determining cell fate. 3 Numerous studies have shown that perturbations in the S1P/ceramide rheostat are involved in the regulation of resistance to chemotherapy and radiation therapy of neoplastic cells, including those of hematopoietic origin. 2,4,5 Two SphK isoenzymes, SphK1 and SphK2, have been described that, although sharing many features, 6,7 exhibit distinct functions. SphK1 promotes cell growth and survival, [8][9][10][11] whereas SphK2, when overexpressed, has opposite effects. 12,13 SphK1 is a key enzyme that regulates the S1P/ceramide rheostat. 12,14,15 Indeed, S1P and SphK1 have long been implicated in resistance of both primary leukemic cells and leukemia cell lines to apoptosis induced by commonly used cytotoxic agents. 3,[16][17][18] Non-isoenzyme-specific inhibitors of SphKs, such as L-threo-dihydrosphingosine (safingol) and N,N-dimethylsphingosine (DMS), are cytotoxic to leukemia cells. 18,19 Interestingly, multidrugresistant HL-60 myelogenous leukemia cells were more sensitive to DMS than the parental cells. 18 Moreover, SphK1 activity was lower in HL-60 cells sensitive to doxorubicin or etoposide than in MDR (multidrug resistance protein)-1-or MRP1 (multidrug resistance protein 1)-positive HL-60 cells. Enforced expression of SphK1 in sensitive HL-60 cells blocked apoptosis, whereas down-regulation of SphK1 overcame chemoresistance by inducing mitochondria-dependent apoptosis. 10 These observations take on added significance in light of evidence that MDR expression is a strong p...

Graphical Abstract Highlights d H3.3 K27M mutation enhances neural stem cell self-renewal d Neonatal PDGFRa activation and Trp53 loss induces supratentorial and brainstem glioma d H3.3 K27M preferentially accelerates hindbrain tumorigenesis d H3.3 K27M drives bivalent gene activation associated with neurodevelopment in DIPG SUMMARYDiffuse intrinsic pontine gliomas (DIPGs) are incurable childhood brainstem tumors with frequent histone H3 K27M mutations and recurrent alterations in PDGFRA and TP53. We generated genetically engineered inducible mice and showed that H3.3 K27M enhanced neural stem cell self-renewal while preserving regional identity. Neonatal induction of H3.3 K27M cooperated with activating platelet-derived growth factor receptor a (PDGFRa) mutant and Trp53 loss to accelerate development of diffuse brainstem gliomas that recapitulated human DIPG gene expression signatures and showed global changes in H3K27 posttranslational modifications, but relatively restricted gene expression changes. Genes upregulated in H3.3 K27M tumors were enriched for those associated with neural development where H3K27me3 loss released the poised state of apparently bivalent promoters, whereas downregulated genes were enriched for those encoding homeodomain transcription factors.

The outcome for children with high-grade gliomas (HGG) remains dismal, with a two-year survival rate of only 10–30%. Diffuse intrinsic pontine glioma (DIPG) comprise a subset of HGG that arise in brainstem almost exclusively in children. Genome-wide analyses of copy number imbalances previously showed that platelet derived growth factor receptor alpha (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% (13/90) of non-brainstem pediatric HGGs and 4.7% (2/43) of DIPGs, including missense mutations and in-frame deletions and insertions not previously described. 40% of tumors with mutation showed concurrent amplification, while 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 non-brainstem pediatric HGG and 57 DIPGs. Thus, a distinct spectrum of mutations confers constitutive receptor activation and oncogenic activity to PDGFRα in childhood HGG.

Purpose Malignant astrocytomas (MAs) are aggressive central nervous system tumors with poor prognosis. Activating mutation of BRAF (BRAFV600E) has been reported in a subset of these tumors, especially in children. We have investigated the incidence of BRAFV600E in additional pediatric patient cohorts, and examined the effects of BRAF blockade in preclinical models of BRAFV600E and wild-type BRAF MA. Experimental Design BRAFV600E mutation status was examined in two pediatric MA patient cohorts. For functional studies, BRAFV600E MA cell lines were used to investigate the effects of BRAF shRNA knockdown in vitro, and to investigate BRAF pharmacologic inhibition, in vitro and in vivo. Results BRAFV600E mutations were identified in 11 and 10 percent of MAs from two distinct series of tumors (6 of 58 cases total). BRAF was expressed in all MA cell lines examined, among which BRAFV600E was identified in four instances. Using the BRAFV600E specific inhibitor PLX4720, pharmacologic blockade of BRAF revealed preferential anti-proliferative activity against BRAFV600E mutant cells in vitro, in contrast to the use of shRNA-mediated knockdown of BRAF, which inhibited cell growth of glioma cell lines regardless of BRAF mutation status. Using orthotopic MA xenografts, we demonstrate that PLX4720 treatment decreases tumor growth and increases overall survival in mice bearing BRAFV600E mutant xenografts, while being ineffective, and possibly tumor promoting, against xenografts with wild-type BRAF. Conclusions Our results indicate a 10% incidence of activating BRAFV600E among pediatric MAs. With regard to implications for therapy, our results support evaluation of BRAFV600E specific inhibitors for treating BRAFV600E MA patients.

Chronic inflammation and inflammatory cytokines have recently been implicated in the development and progression of various types of cancer. In the brain, neuroinflammatory cytokines affect the growth and differentiation of both normal and malignant glial cells, with interleukin 1 (IL-1) shown to be secreted by the majority of glioblastoma cells. Recently, elevated levels of sphingosine kinase 1 (SphK1), but not SphK2, were correlated with a shorter survival prognosis for patients with glioblastoma multiforme. SphK1 is a lipid kinase that produces the pro-growth, anti-apoptotic sphingosine 1-phosphate, which can induce invasion of glioblastoma cells. Here, we show that the expression of IL-1 correlates with the expression of SphK1 in glioblastoma cells, and neutralizing anti-IL-1 antibodies inhibit both the growth and invasion of glioblastoma cells. Furthermore, IL-1 up-regulates SphK1 mRNA levels, protein expression, and activity in both primary human astrocytes and various glioblastoma cell lines; however, it does not affect SphK2 expression. The IL-1-induced SphK1 up-regulation can be blocked by the inhibition of JNK, the overexpression of the dominant-negative c-Jun(TAM67), and the down-regulation of c-Jun expression by small interference RNA. Activation of SphK1 expression by IL-1 occurs on the level of transcription and is mediated via a novel AP-1 element located within the first intron of the sphk1 gene. In summary, our results suggest that SphK1 expression is transcriptionally regulated by IL-1 in glioblastoma cells, and this pathway may be important in regulating survival and invasiveness of glioblastoma cells.