Diffuse intrinsic pontine glioma (DIPG) is a fatal childhood cancer. We performed a chemical screen in patient-derived DIPG cultures along with RNA-seq analyses and integrated computational modeling to identify potentially effective therapeutic strategies. The multi-histone deacetylase inhibitor panobinostat demonstrated therapeutic efficacy both in vitro and in DIPG orthotopic xenograft models. Combination testing of panobinostat and the histone demethylase inhibitor GSK-J4 revealed that the two had synergistic effects. Together, these data suggest a promising therapeutic strategy for DIPG.

Recurrent medulloblastoma is a therapeutic challenge because it is almost always fatal. Studies have confirmed that medulloblastoma consists of at least four distinct subgroups. We sought to delineate subgroup-specific differences in medulloblastoma recurrence patterns.

Telomerase reverse transcriptase (TERT) promoter mutations were recently shown to drive telomerase activity in various cancer types, including medulloblastoma. However, the clinical and biological implications of TERT mutations in medulloblastoma have not been described. Hence, we sought to describe these mutations and their impact in a subgroup-specific manner. We analyzed the TERT promoter by direct sequencing and genotyping in 466 medulloblastomas. The mutational distributions were determined according to subgroup affiliation, demographics, and clinical, prognostic, and molecular features. Integrated genomics approaches were used to identify specific somatic copy number alterations in TERT promoter-mutated and wild-type tumors. Overall, TERT promoter mutations were identified in 21 % of medulloblastomas. Strikingly, the highest frequencies of TERT mutations were observed in SHH (83 %; 55/66) and WNT (31 %; 4/13) medulloblastomas derived from adult patients. Group 3 and Group 4 harbored this alteration in <5 % of cases and showed no association with increased patient age. The prognostic implications of these mutations were highly subgroup-specific. TERT mutations identified a subset with good and poor prognosis in SHH and Group 4 tumors, respectively. Monosomy 6 was mostly restricted to WNT tumors without TERT mutations. Hallmark SHH focal copy number aberrations and chromosome 10q deletion were mutually exclusive with TERT mutations within SHH tumors. TERT promoter mutations are the most common recurrent somatic point mutation in medulloblastoma, and are very highly enriched in adult SHH and WNT tumors. TERT mutations define a subset of SHH medulloblastoma with distinct demographics, cytogenetics, and outcomes.

Posterior fossa ependymoma comprises two distinct molecular variants termed EPN_PFA and EPN_PFB that have a distinct biology and natural history. The therapeutic value of cytoreductive surgery and radiation therapy for posterior fossa ependymoma after accounting for molecular subgroup is not known.

The Pax6 transcription factor is expressed in cerebellar granule cells and when mutated, as in the Sey/Sey mouse, produces granule cells with disturbed survival and migration and with defects in neurite extension. The impact of Pax6 on other genes in the context of cerebellar development has not been identified. In this study, we performed transcriptome comparisons between wildtype and Pax6-null whole cerebellar tissue at embryonic day (E) 13.5, 15.5 and 18.5 using Affymetrix arrays (U74Av2). Statistical analyses identified 136 differentially regulated transcripts (FDR 0.05, 1.2-fold change cutoff) over time in Pax6-null cerebellar tissue. In parallel we examined the Math1-null granuloprival cerebellum and identified 228 down-regulated transcripts (FDR 0.05, 1.2-fold change cutoff). The intersection of these two microarray datasets produced a total of 21 differentially regulated transcripts. For a subset of the identified transcripts, we used qRT-PCR to validate the microarray data and demonstrated the expression in the rhombic lip lineage and differential expression in Pax6-null cerebellum with in situ hybridisation analysis. The candidate genes identified in this way represent direct or indirect Pax6-downstream genes involved in cerebellar development.

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.

Diffuse intrinsic pontine glioma (DIPG) is a poor-prognosis pediatric brain tumor. No effective curative therapy is currently available and no therapeutic advances have been made in several decades. BMI-1 is a member of the multimeric protein complex Polycomb repressor complex 1. It is highly expressed in a number of diseases and malignancies and has been implicated in self-renewal of normal and cancer cells, and in DNA damage signaling. The role of BMI-1 in DIPG is largely unknown. Here, we show that BMI-1 is highly expressed in tumor tissue samples of DIPG patients and in patient-derived cancer stem-like cells. BMI-1 downregulation leads to the inhibition of DIPG patient-derived neurosphere cell proliferation, cell cycle signaling, self-renewal, telomerase expression and activity, and suppresses DIPG cell migration. Moreover, targeted inhibition of BMI-1 sensitizes DIPG cells to radiomimetic drug-induced DNA damage. Together, our data validate BMI-1 as a potential therapeutic target to treat children with DIPG.

DEAD-Box Helicase 3 X-Linked (DDX3X) is frequently mutated in the Wingless (WNT) and Sonic hedghog (SHH) subtypes of medulloblastoma-the commonest malignant childhood brain tumor, but whether DDX3X functions as a medulloblastoma oncogene or tumor suppressor gene is not known. Here, we show that Ddx3x regulates hindbrain patterning and development by controlling Hox gene expression and cell stress signaling. In mice predisposed to Wnt- or Shh medulloblastoma, Ddx3x sensed oncogenic stress and suppressed tumor formation. WNT and SHH medulloblastomas normally arise only in the lower and upper rhombic lips, respectively. Deletion of Ddx3x removed this lineage restriction, enabling both medulloblastoma subtypes to arise in either germinal zone. Thus, DDX3X is a medulloblastoma tumor suppressor that regulates hindbrain development and restricts the competence of cell lineages to form medulloblastoma subtypes.

Math1 is the defining molecule of the cerebellar rhombic lip and Pax6 is downstream in the Math1 pathway. In the present study, we discover that Wntless (Wls) is a novel molecular marker of the cells in the interior face of the rhombic lip throughout normal mouse cerebellar development. Wls expression is found complementary to the expression of Math1 and Pax6, which are localized to the exterior face of the rhombic lip. To determine the interaction between these molecules, we examine the loss-of-Math1 or loss-of-Pax6 in the cerebellum, i.e., the Math1-null and Pax6-null (Sey) mutant cerebella. The presence of Wls-positive cells in the Math1-null rhombic lip indicates that Wls expression is independent of Math1. In the Sey mutant cerebellum, there is an expansion of Wls-expressing cells into regions that are normally colonized by Pax6-expressing cells. The ectopic expression of Wls in the Pax6-null cerebellum suggests a negative interaction between Wls-expressing cells and Pax6-positive cells. These findings suggest that the rhombic lip is dynamically patterned by the expression of Wls, Math1, and Pax6. We also examine five rhombic lip cell markers (Wls, Math1, Pax6, Lmx1a, and Tbr2) to identify four molecularly distinct compartments in the rhombic lip during cerebellar development. The existence of spatial compartmentation in the rhombic lip and the interplay between Wls, Math1, and Pax6 in the rhombic lip provides novel views of early cerebellar development.

Choroid plexus carcinomas (CPCs) are poorly understood and frequently lethal brain tumors with few treatment options. Using a mouse model of the disease and a large cohort of human CPCs, we performed a cross-species, genome-wide search for oncogenes within syntenic regions of chromosome gain. TAF12, NFYC, and RAD54L co-located on human chromosome 1p32-35.3 and mouse chromosome 4qD1-D3 were identified as oncogenes that are gained in tumors in both species and required for disease initiation and progression. TAF12 and NFYC are transcription factors that regulate the epigenome, whereas RAD54L plays a central role in DNA repair. Our data identify a group of concurrently gained oncogenes that cooperate in the formation of CPC and reveal potential avenues for therapy.

Wntless (Wls) is implicated in the Wnt signaling pathway by regulating the secretion of Wnt molecules. During brain development, Wls is expressed in the isthmic organizer (ISO) and rhombic lip (RL). Wls regulates Wnt1 secretion at the ISO which is required to induce midbrain-hindbrain structures. However, Wls function in the RL is not known. Here, we employed Nestin-cre to delete Wls specifically in the RL during mid-gestation. The loss-of-Wls leads to an abnormal RL during development and cerebellar vermis hypoplasia at birth. The Wls conditional knockout (cKO) has rudimentary foliation with an absence of Bergmann glia fibers in the external germinal layer (EGL). The Wls-cKO cerebellum also exhibits ectopia of several cell types and aberrations in granule cell organization. Finally, there is a loss of 85% of unipolar brush cells. From these findings, Wls-expressing cells in the rhombic lip are implicated in the orchestration of cerebellar development.

The work of the FANTOM5 Consortium has brought forth a new level of understanding of the regulation of gene transcription and the cellular processes involved in creating diversity of cell types. In this study, we extended the analysis of the FANTOM5 Cap Analysis of Gene Expression (CAGE) transcriptome data to focus on understanding the genetic regulators involved in mouse cerebellar development.

Diffuse intrinsic pontine glioma (DIPG) and midline high-grade glioma (mHGG) are lethal childhood brain tumors. Spatial genomic heterogeneity has been well-described in adult HGG but has not been comprehensively characterized in pediatric HGG. We performed whole exome sequencing on 38-matched primary, contiguous, and metastatic tumor sites from eight children with DIPG (n = 7) or mHGG (n = 1) collected using a unique MRI-guided autopsy protocol. Validation was performed using Sanger sequencing, Droplet Digital polymerase-chain reaction, immunohistochemistry, and fluorescent in-situ hybridization.

The development of the brain requires precise coordination of molecular processes across many cell-types. Underpinning these events are gene expression programs which require intricate regulation by non-coding regulatory sequences known as enhancers. In the context of the developing brain, transcribed enhancers (TEs) regulate temporally-specific expression of genes critical for cell identity and differentiation. Transcription of non-coding RNAs at active enhancer sequences, known as enhancer RNAs (eRNAs), is tightly associated with enhancer activity and has been correlated with target gene expression. TEs have been characterized in a multitude of developing tissues, however their regulatory role has yet to be described in the context of embryonic and early postnatal brain development. In this study, eRNA transcription was analyzed to identify TEs active during cerebellar development, as a proxy for the developing brain. Cap Analysis of Gene Expression followed by sequencing (CAGE-seq) was conducted at 12 stages throughout embryonic and early postnatal cerebellar development.

ROR-alpha is an orphan nuclear receptor, inactivation of which cell-autonomously blocks differentiation of cerebellar Purkinje cells with a secondary loss of granule neurons. As part of our ENU mutagenesis screen we isolated the recessive tmgc26 mouse mutant, characterized by early-onset progressive ataxia, cerebellar degeneration and juvenile lethality. Detailed analysis of the tmgc26-/- cerebella revealed Purkinje cell and granule cell abnormalities, and defects in molecular layer interneurons and radial glia. Chimera studies suggested a cell-autonomous effect of the tmgc26 mutation in Purkinje cells and molecular layer interneurons, and a non-cell-autonomous effect in granule cells. The mutation was mapped to a 13-Mb interval on chromosome 9, a region that contains the ROR-alpha gene. Sequencing of genomic DNA revealed a T-to-A transition in exon 5 of the ROR-alpha gene, resulting in a nonsense mutation C257X and severe truncation of the ROR-alpha protein. Together, our data identify new roles for ROR-alpha in molecular layer interneurons and radial glia development and suggest tmgc26 as a novel ROR-alpha allele that may be used to further delineate the molecular mechanisms of ROR-alpha action.

Cancer stem cells are remarkably similar to normal stem cells: both self-renew, are multipotent and express common surface markers, for example, prominin 1 (PROM1, also called CD133). What remains unclear is whether cancer stem cells are the direct progeny of mutated stem cells or more mature cells that reacquire stem cell properties during tumour formation. Answering this question will require knowledge of whether normal stem cells are susceptible to cancer-causing mutations; however, this has proved difficult to test because the identity of most adult tissue stem cells is not known. Here, using an inducible Cre, nuclear LacZ reporter allele knocked into the Prom1 locus (Prom1(C-L)), we show that Prom1 is expressed in a variety of developing and adult tissues. Lineage-tracing studies of adult Prom1(+/C-L) mice containing the Rosa26-YFP reporter allele showed that Prom1(+) cells are located at the base of crypts in the small intestine, co-express Lgr5 (ref. 2), generate the entire intestinal epithelium, and are therefore the small intestinal stem cell. Prom1 was reported recently to mark cancer stem cells of human intestinal tumours that arise frequently as a consequence of aberrant wingless (Wnt) signalling. Activation of endogenous Wnt signalling in Prom1(+/C-L) mice containing a Cre-dependent mutant allele of beta-catenin (Ctnnb1(lox(ex3))) resulted in a gross disruption of crypt architecture and a disproportionate expansion of Prom1(+) cells at the crypt base. Lineage tracing demonstrated that the progeny of these cells replaced the mucosa of the entire small intestine with neoplastic tissue that was characterized by focal high-grade intraepithelial neoplasia and crypt adenoma formation. Although all neoplastic cells arose from Prom1(+) cells in these mice, only 7% of tumour cells retained Prom1 expression. Our data indicate that Prom1 marks stem cells in the adult small intestine that are susceptible to transformation into tumours retaining a fraction of mutant Prom1(+) tumour cells.

Children with high-grade glioma, including diffuse intrinsic pontine glioma (DIPG), have a poor prognosis despite multimodal therapy. Identifying novel therapeutic targets is critical to improve their outcome. We evaluated prognostic roles of telomere maintenance mechanisms in children with HGG, including DIPG. A multi-institutional retrospective study was conducted involving 50 flash-frozen HGG (35 non-brainstem; 15 DIPG) tumors from 45 children (30 non-brainstem; 15 DIPG). Telomerase activity, expression of hTERT mRNA (encoding telomerase catalytic component) and TERC (telomerase RNA template) and alternative lengthening of telomeres (ALT) mechanism were assayed. Cox Proportional Hazard regression analyses assessed association of clinical and pathological variables, TERC and hTERT levels, telomerase activity, and ALT use with progression-free or overall survival (OS). High TERC and hTERT expression was detected in 13/28 non-brainstem HGG samples as compared to non-neoplastic controls. High TERC and hTERT expression was identified in 13/15 and 11/15 DIPG samples, respectively, compared to controls. Evidence of ALT was noted in 3/11 DIPG and 10/19 non-brainstem HGG specimens. ALT and telomerase use were identified in 4/19 non-brainstem HGG and 2/11 DIPG specimens. In multivariable analyses, increased TERC and hTERT levels were associated with worse OS in patients with non-brainstem HGG, after controlling for tumor grade or resection extent. Children with HGG and DIPG, have increased hTERT and TERC expression. In children with non-brainstem HGG, increased TERC and hTERT expression levels are associated with a worse OS, making telomerase a promising potential therapeutic target in pediatric HGG.

The iPrEx study demonstrated that combination oral emtricitabine and tenofovir disoproxil fumarate (FTC/TDF) as preexposure prophylaxis (PrEP) protects against HIV acquisition in men who have sex with men and transgender women. Selection for drug resistance could offset PrEP benefits.

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.