Medulloblastoma (MB) is the most common malignant brain tumor in children, where one-third of patients succumb to their disease. This SnapShot describes the classification of MB subgroups, historically by histopathology and currently based on genomic information. Genomics-based classification has identified four major subgroups and provides greater opportunity for developing targeted therapies more successful than current conventional therapy.

Two recurrent mutations, K27M and G34R/V, within histone variant H3.3 were recently identified in ∼50% of pHGGs. Both mutations define clinically and biologically distinct subgroups of pHGGs. Here, we provide further insight about the dominant-negative effect of K27M mutant H3.3, leading to a global reduction of the repressive histone mark H3K27me3. We demonstrate that this is caused by aberrant recruitment of the PRC2 complex to K27M mutant H3.3 and enzymatic inhibition of the H3K27me3-establishing methyltransferase EZH2. By performing chromatin immunoprecipitation followed by next-generation sequencing and whole-genome bisulfite sequencing in primary pHGGs, we show that reduced H3K27me3 levels and DNA hypomethylation act in concert to activate gene expression in K27M mutant pHGGs.

Medulloblastoma (MB) is the most common malignant brain tumor in children. Patients whose tumors exhibit overexpression or amplification of the MYC oncogene (c-MYC) usually have an extremely poor prognosis, but there are no animal models of this subtype of the disease. Here, we show that cerebellar stem cells expressing Myc and mutant Trp53 (p53) generate aggressive tumors following orthotopic transplantation. These tumors consist of large, pleiomorphic cells and resemble human MYC-driven MB at a molecular level. Notably, antagonists of PI3K/mTOR signaling, but not Hedgehog signaling, inhibit growth of tumor cells. These findings suggest that cerebellar stem cells can give rise to MYC-driven MB and identify a novel model that can be used to test therapies for this devastating disease.

Tumors of the same histologic type often comprise clinically and molecularly distinct subgroups; however, the etiology of these subgroups is unknown. Here, we report that histologically identical, but genetically distinct, ependymomas exhibit patterns of gene expression that recapitulate those of radial glia cells in the corresponding region of the central nervous system. Cancer stem cells isolated from ependymomas displayed a radial glia phenotype and formed tumors when orthotopically transplanted in mice. These findings identify restricted populations of radial glia cells as candidate stem cells of the different subgroups of ependymoma, and they support a general hypothesis that subgroups of the same histologic tumor type are generated by different populations of progenitor cells in the tissues of origin.

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.

The current consensus recognizes four main medulloblastoma subgroups (wingless, Sonic hedgehog, group 3 and group 4). While medulloblastoma subgroups have been characterized extensively at the (epi-)genomic and transcriptomic levels, the proteome and phosphoproteome landscape remain to be comprehensively elucidated. Using quantitative (phospho)-proteomics in primary human medulloblastomas, we unravel distinct posttranscriptional regulation leading to highly divergent oncogenic signaling and kinase activity profiles in groups 3 and 4 medulloblastomas. Specifically, proteomic and phosphoproteomic analyses identify aberrant ERBB4-SRC signaling in group 4. Hence, enforced expression of an activated SRC combined with p53 inactivation induces murine tumors that resemble group 4 medulloblastoma. Therefore, our integrative proteogenomics approach unveils an oncogenic pathway and potential therapeutic vulnerability in the most common medulloblastoma subgroup.

High-grade gliomas defined by histone 3 K27M driver mutations exhibit global loss of H3K27 trimethylation and reciprocal gain of H3K27 acetylation, respectively shaping repressive and active chromatin landscapes. We generated tumor-derived isogenic models bearing this mutation and show that it leads to pervasive H3K27ac deposition across the genome. In turn, active enhancers and promoters are not created de novo and instead reflect the epigenomic landscape of the cell of origin. H3K27ac is enriched at repeat elements, resulting in their increased expression, which in turn can be further amplified by DNA demethylation and histone deacetylase inhibitors providing an exquisite therapeutic vulnerability. These agents may therefore modulate anti-tumor immune responses as a therapeutic modality for this untreatable disease.

Functional heterogeneity within tumors presents a significant therapeutic challenge. Here we show that quiescent, therapy-resistant Sox2(+) cells propagate sonic hedgehog subgroup medulloblastoma by a mechanism that mirrors a neurogenic program. Rare Sox2(+) cells produce rapidly cycling doublecortin(+) progenitors that, together with their postmitotic progeny expressing NeuN, comprise tumor bulk. Sox2(+) cells are enriched following anti-mitotic chemotherapy and Smoothened inhibition, creating a reservoir for tumor regrowth. Lineage traces from Sox2(+) cells increase following treatment, suggesting that this population is responsible for relapse. Targeting Sox2(+) cells with the antineoplastic mithramycin abrogated tumor growth. Addressing functional heterogeneity and eliminating Sox2(+) cells presents a promising therapeutic paradigm for treatment of sonic hedgehog subgroup medulloblastoma.

Gain-of-function mutations in histone 3 (H3) variants are found in a substantial proportion of pediatric high-grade gliomas (pHGG), often in association with TP53 loss and platelet-derived growth factor receptor alpha (PDGFRA) amplification. Here, we describe a somatic mouse model wherein H3.3K27M and Trp53 loss alone are sufficient for neoplastic transformation if introduced in utero. H3.3K27M-driven lesions are clonal, H3K27me3 depleted, Olig2 positive, highly proliferative, and diffusely spreading, thus recapitulating hallmark molecular and histopathological features of pHGG. Addition of wild-type PDGFRA decreases latency and increases tumor invasion, while ATRX knockdown is associated with more circumscribed tumors. H3.3K27M-tumor cells serially engraft in recipient mice, and preliminary drug screening reveals mutation-specific vulnerabilities. Overall, we provide a faithful H3.3K27M-pHGG model which enables insights into oncohistone pathogenesis and investigation of future therapies.

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.

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.

Glioblastomas display hierarchies with self-renewing cancer stem-like cells (CSCs). RNA sequencing and enhancer mapping revealed regulatory programs unique to CSCs causing upregulation of the iron transporter transferrin, the top differentially expressed gene compared with tissue-specific progenitors. Direct interrogation of iron uptake demonstrated that CSCs potently extract iron from the microenvironment more effectively than other tumor cells. Systematic interrogation of iron flux determined that CSCs preferentially require transferrin receptor and ferritin, two core iron regulators, to propagate and form tumors in vivo. Depleting ferritin disrupted CSC mitotic progression, through the STAT3-FoxM1 regulatory axis, revealing an iron-regulated CSC pathway. Iron is a unique, primordial metal fundamental for earliest life forms, on which CSCs have an epigenetically programmed, targetable dependence.

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.

The proto-oncogene MYCN is mis-expressed in various types of human brain tumors. To clarify how developmental and regional differences influence transformation, we transduced wild-type or mutationally stabilized murine N-myc(T58A) into neural stem cells (NSCs) from perinatal murine cerebellum, brain stem, and forebrain. Transplantation of N-myc(WT) NSCs was insufficient for tumor formation. N-myc(T58A) cerebellar and brain stem NSCs generated medulloblastoma/primitive neuroectodermal tumors, whereas forebrain NSCs developed diffuse glioma. Expression analyses distinguished tumors generated from these different regions, with tumors from embryonic versus postnatal cerebellar NSCs demonstrating Sonic Hedgehog (SHH) dependence and SHH independence, respectively. These differences were regulated in part by the transcription factor SOX9, activated in the SHH subclass of human medulloblastoma. Our results demonstrate context-dependent transformation of NSCs in response to a common oncogenic signal.

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.

We developed an RNA-sequencing-based pipeline to discover differentially expressed cell-surface molecules in neuroblastoma that meet criteria for optimal immunotherapeutic target safety and efficacy. Here, we show that GPC2 is a strong candidate immunotherapeutic target in this childhood cancer. We demonstrate high GPC2 expression in neuroblastoma due to MYCN transcriptional activation and/or somatic gain of the GPC2 locus. We confirm GPC2 to be highly expressed on most neuroblastomas, but not detectable at appreciable levels in normal childhood tissues. In addition, we demonstrate that GPC2 is required for neuroblastoma proliferation. Finally, we develop a GPC2-directed antibody-drug conjugate that is potently cytotoxic to GPC2-expressing neuroblastoma cells. Collectively, these findings validate GPC2 as a non-mutated neuroblastoma oncoprotein and candidate immunotherapeutic target.

Medulloblastoma (MB) is a highly malignant pediatric brain tumor. Despite aggressive therapy, many patients succumb to the disease, and survivors experience severe side effects from treatment. MYC-driven MB has a particularly poor prognosis and would greatly benefit from more effective therapies. We used an animal model of MYC-driven MB to screen for drugs that decrease viability of tumor cells. Among the most effective compounds were histone deacetylase inhibitors (HDACIs). HDACIs potently inhibit survival of MYC-driven MB cells in vitro, in part by inducing expression of the FOXO1 tumor suppressor gene. HDACIs also synergize with phosphatidylinositol 3-kinase inhibitors to inhibit tumor growth in vivo. These studies identify an effective combination therapy for the most aggressive form of MB.