While molecular subgrouping has revolutionized medulloblastoma classification, the extent of heterogeneity within subgroups is unknown. Similarity network fusion (SNF) applied to genome-wide DNA methylation and gene expression data across 763 primary samples identifies very homogeneous clusters of patients, supporting the presence of medulloblastoma subtypes. After integration of somatic copy-number alterations, and clinical features specific to each cluster, we identify 12 different subtypes of medulloblastoma. Integrative analysis using SNF further delineates group 3 from group 4 medulloblastoma, which is not as readily apparent through analyses of individual data types. Two clear subtypes of infants with Sonic Hedgehog medulloblastoma with disparate outcomes and biology are identified. Medulloblastoma subtypes identified through integrative clustering have important implications for stratification of future clinical trials.

Ependymal tumors across age groups are currently classified and graded solely by histopathology. It is, however, commonly accepted that this classification scheme has limited clinical utility based on its lack of reproducibility in predicting patients' outcome. We aimed at establishing a uniform molecular classification using DNA methylation profiling. Nine molecular subgroups were identified in a large cohort of 500 tumors, 3 in each anatomical compartment of the CNS, spine, posterior fossa, supratentorial. Two supratentorial subgroups are characterized by prototypic fusion genes involving RELA and YAP1, respectively. Regarding clinical associations, the molecular classification proposed herein outperforms the current histopathological classification and thus might serve as a basis for the next World Health Organization classification of CNS tumors.

Malignant gliomas exhibit extensive heterogeneity and poor prognosis. Here we identify mitotic Olig2-expressing cells as tumor-propagating cells in proneural gliomas, elimination of which blocks tumor initiation and progression. Intriguingly, deletion of Olig2 resulted in tumors that grow, albeit at a decelerated rate. Genome occupancy and expression profiling analyses reveal that Olig2 directly activates cell-proliferation machinery to promote tumorigenesis. Olig2 deletion causes a tumor phenotypic shift from an oligodendrocyte precursor-correlated proneural toward an astroglia-associated gene expression pattern, manifest in downregulation of platelet-derived growth factor receptor-α and reciprocal upregulation of epidermal growth factor receptor (EGFR). Olig2 deletion further sensitizes glioma cells to EGFR inhibitors and extends the lifespan of animals. Thus, Olig2-orchestrated receptor signaling drives mitotic growth and regulates glioma phenotypic plasticity. Targeting Olig2 may circumvent resistance to EGFR-targeted drugs.

We recently reported that atypical teratoid rhabdoid tumors (ATRTs) comprise at least two transcriptional subtypes with different clinical outcomes; however, the mechanisms underlying therapeutic heterogeneity remained unclear. In this study, we analyzed 191 primary ATRTs and 10 ATRT cell lines to define the genomic and epigenomic landscape of ATRTs and identify subgroup-specific therapeutic targets. We found ATRTs segregated into three epigenetic subgroups with distinct genomic profiles, SMARCB1 genotypes, and chromatin landscape that correlated with differential cellular responses to a panel of signaling and epigenetic inhibitors. Significantly, we discovered that differential methylation of a PDGFRB-associated enhancer confers specific sensitivity of group 2 ATRT cells to dasatinib and nilotinib, and suggest that these are promising therapies for this highly lethal ATRT subtype.

Despite the histological similarity of ependymomas from throughout the neuroaxis, the disease likely comprises multiple independent entities, each with a distinct molecular pathogenesis. Transcriptional profiling of two large independent cohorts of ependymoma reveals the existence of two demographically, transcriptionally, genetically, and clinically distinct groups of posterior fossa (PF) ependymomas. Group A patients are younger, have laterally located tumors with a balanced genome, and are much more likely to exhibit recurrence, metastasis at recurrence, and death compared with Group B patients. Identification and optimization of immunohistochemical (IHC) markers for PF ependymoma subgroups allowed validation of our findings on a third independent cohort, using a human ependymoma tissue microarray, and provides a tool for prospective prognostication and stratification of PF ependymoma patients.

Glioblastoma (GBM) is a brain tumor that carries a dismal prognosis and displays considerable heterogeneity. We have recently identified recurrent H3F3A mutations affecting two critical amino acids (K27 and G34) of histone H3.3 in one-third of pediatric GBM. Here, we show that each H3F3A mutation defines an epigenetic subgroup of GBM with a distinct global methylation pattern, and that they are mutually exclusive with IDH1 mutations, which characterize a third mutation-defined subgroup. Three further epigenetic subgroups were enriched for hallmark genetic events of adult GBM and/or established transcriptomic signatures. We also demonstrate that the two H3F3A mutations give rise to GBMs in separate anatomic compartments, with differential regulation of transcription factors OLIG1, OLIG2, and FOXG1, possibly reflecting different cellular origins.

We discovered a high-level amplicon involving the chr19q13.41 microRNA (miRNA) cluster (C19MC) in 11/45 ( approximately 25%) primary CNS-PNET, which results in striking overexpression of miR-517c and 520g. Constitutive expression of miR-517c or 520g promotes in vitro and in vivo oncogenicity, modulates cell survival, and robustly enhances growth of untransformed human neural stem cells (hNSCs) in part by upregulating WNT pathway signaling and restricting differentiation of hNSCs. Remarkably, the C19MC amplicon, which is very rare in other brain tumors (1/263), identifies an aggressive subgroup of CNS-PNET with distinct gene-expression profiles, characteristic histology, and dismal survival. Our data implicate miR-517c and 520g as oncogenes and promising biological markers for CNS-PNET and provide important insights into oncogenic properties of the C19MC locus.

Pediatric low-grade gliomas (pLGG) are frequently driven by genetic alterations in the RAS-mitogen-activated protein kinase (RAS/MAPK) pathway yet show unexplained variability in their clinical outcome. To address this, we characterized a cohort of >1,000 clinically annotated pLGG. Eighty-four percent of cases harbored a driver alteration, while those without an identified alteration also often exhibited upregulation of the RAS/MAPK pathway. pLGG could be broadly classified based on their alteration type. Rearrangement-driven tumors were diagnosed at a younger age, enriched for WHO grade I histology, infrequently progressed, and rarely resulted in death as compared with SNV-driven tumors. Further sub-classification of clinical-molecular correlates stratified pLGG into risk categories. These data highlight the biological and clinical differences between pLGG subtypes and opens avenues for future treatment refinement.

We collated data from 157 unpublished cases of pediatric high-grade glioma and diffuse intrinsic pontine glioma and 20 publicly available datasets in an integrated analysis of >1,000 cases. We identified co-segregating mutations in histone-mutant subgroups including loss of FBXW7 in H3.3G34R/V, TOP3A rearrangements in H3.3K27M, and BCOR mutations in H3.1K27M. Histone wild-type subgroups are refined by the presence of key oncogenic events or methylation profiles more closely resembling lower-grade tumors. Genomic aberrations increase with age, highlighting the infant population as biologically and clinically distinct. Uncommon pathway dysregulation is seen in small subsets of tumors, further defining the molecular diversity of the disease, opening up avenues for biological study and providing a basis for functionally defined future treatment stratification.

Embryonal tumors with multilayered rosettes (ETMRs) are highly lethal infant brain cancers with characteristic amplification of Chr19q13.41 miRNA cluster (C19MC) and enrichment of pluripotency factor LIN28A. Here we investigated C19MC oncogenic mechanisms and discovered a C19MC-LIN28A-MYCN circuit fueled by multiple complex regulatory loops including an MYCN core transcriptional network and super-enhancers resulting from long-range MYCN DNA interactions and C19MC gene fusions. Our data show that this powerful oncogenic circuit, which entraps an early neural lineage network, is potently abrogated by bromodomain inhibitor JQ1, leading to ETMR cell death.

Progenitor heterogeneity and identities underlying tumor initiation and relapse in medulloblastomas remain elusive. Utilizing single-cell transcriptomic analysis, we demonstrated a developmental hierarchy of progenitor pools in Sonic Hedgehog (SHH) medulloblastomas, and identified OLIG2-expressing glial progenitors as transit-amplifying cells at the tumorigenic onset. Although OLIG2+ progenitors become quiescent stem-like cells in full-blown tumors, they are highly enriched in therapy-resistant and recurrent medulloblastomas. Depletion of mitotic Olig2+ progenitors or Olig2 ablation impeded tumor initiation. Genomic profiling revealed that OLIG2 modulates chromatin landscapes and activates oncogenic networks including HIPPO-YAP/TAZ and AURORA-A/MYCN pathways. Co-targeting these oncogenic pathways induced tumor growth arrest. Together, our results indicate that glial lineage-associated OLIG2+ progenitors are tumor-initiating cells during medulloblastoma tumorigenesis and relapse, suggesting OLIG2-driven oncogenic networks as potential therapeutic targets.