Human neural stem cell cultures provide progenitor cells that are potential cells of origin for brain cancers. However, the extent to which genetic predisposition to tumor formation can be faithfully captured in stem cell lines is uncertain. Here, we evaluated neuroepithelial stem (NES) cells, representative of cerebellar progenitors. We transduced NES cells with MYCN, observing medulloblastoma upon orthotopic implantation in mice. Significantly, transcriptomes and patterns of DNA methylation from xenograft tumors were globally more representative of human medulloblastoma compared to a MYCN-driven genetically engineered mouse model. Orthotopic transplantation of NES cells generated from Gorlin syndrome patients, who are predisposed to medulloblastoma due to germline-mutated PTCH1, also generated medulloblastoma. We engineered candidate cooperating mutations in Gorlin NES cells, with mutation of DDX3X or loss of GSE1 both accelerating tumorigenesis. These findings demonstrate that human NES cells provide a potent experimental resource for dissecting genetic causation in medulloblastoma.

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.

Posterior fossa type A (PFA) ependymomas exhibit very low H3K27 methylation and express high levels of EZHIP (Enhancer of Zeste Homologs Inhibitory Protein, also termed CXORF67). Here we find that a conserved sequence in EZHIP is necessary and sufficient to inhibit PRC2 catalytic activity in vitro and in vivo. EZHIP directly contacts the active site of the EZH2 subunit in a mechanism similar to the H3 K27M oncohistone. Furthermore, expression of H3 K27M or EZHIP in cells promotes similar chromatin profiles: loss of broad H3K27me3 domains, but retention of H3K27me3 at CpG islands. We find that H3K27me3-mediated allosteric activation of PRC2 substantially increases the inhibition potential of EZHIP and H3 K27M, providing a mechanism to explain the observed loss of H3K27me3 spreading in tumors. Our data indicate that PFA ependymoma and DIPG are driven in part by the action of peptidyl PRC2 inhibitors, the K27M oncohistone and the EZHIP 'oncohistone-mimic', that dysregulate gene silencing to promote tumorigenesis.

Extensive molecular analyses of ependymal tumors have revealed that supratentorial and posterior fossa ependymomas have distinct molecular profiles and are likely to be different diseases. The presence of C11orf95-RELA fusion genes in a subset of supratentorial ependymomas (ST-EPN) indicated the existence of molecular subgroups. However, the pathogenesis of RELA fusion-negative ependymomas remains elusive. To investigate the molecular pathogenesis of these tumors and validate the molecular classification of ependymal tumors, we conducted thorough molecular analyses of 113 locally diagnosed ependymal tumors from 107 patients in the Japan Pediatric Molecular Neuro-Oncology Group. All tumors were histopathologically reviewed and 12 tumors were re-classified as non-ependymomas. A combination of RT-PCR, FISH, and RNA sequencing identified RELA fusion in 19 of 29 histologically verified ST-EPN cases, whereas another case was diagnosed as ependymoma RELA fusion-positive via the methylation classifier (68.9%). Among the 9 RELA fusion-negative ST-EPN cases, either the YAP1 fusion, BCOR tandem duplication, EP300-BCORL1 fusion, or FOXO1-STK24 fusion was detected in single cases. Methylation classification did not identify a consistent molecular class within this group. Genome-wide methylation profiling successfully sub-classified posterior fossa ependymoma (PF-EPN) into PF-EPN-A (PFA) and PF-EPN-B (PFB). A multivariate analysis using Cox regression confirmed that PFA was the sole molecular marker which was independently associated with patient survival. A clinically applicable pyrosequencing assay was developed to determine the PFB subgroup with 100% specificity using the methylation status of 3 genes, CRIP1, DRD4 and LBX2. Our results emphasized the significance of molecular classification in the diagnosis of ependymomas. RELA fusion-negative ST-EPN appear to be a heterogeneous group of tumors that do not fall into any of the existing molecular subgroups and are unlikely to form a single category.

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.

Over the past decade, wingless-activated (WNT) medulloblastoma has been identified as a candidate for therapy de-escalation based on excellent survival; however, a paucity of relapses has precluded additional analyses of markers of relapse. To address this gap in knowledge, an international cohort of 93 molecularly confirmed WNT MB was assembled, where 5-year progression-free survival is 0.84 (95%, 0.763-0.925) with 15 relapsed individuals identified. Maintenance chemotherapy is identified as a strong predictor of relapse, with individuals receiving high doses of cyclophosphamide or ifosphamide having only one very late molecularly confirmed relapse (p = 0.032). The anatomical location of recurrence is metastatic in 12 of 15 relapses, with 8 of 12 metastatic relapses in the lateral ventricles. Maintenance chemotherapy, specifically cumulative cyclophosphamide doses, is a significant predictor of relapse across WNT MB. Future efforts to de-escalate therapy need to carefully consider not only the radiation dose but also the chemotherapy regimen and the propensity for metastatic relapses.

Histone H3.3 lysine-to-methionine substitutions K27M and K36M impair the deposition of opposing chromatin marks, H3K27me3/me2 and H3K36me3/me2. We show that these mutations induce hypotrophic and disorganized eyes in Drosophila eye primordia. Restriction of H3K27me3 spread in H3.3K27M and its redistribution in H3.3K36M result in transcriptional deregulation of PRC2-targeted eye development and of piRNA biogenesis genes, including krimp. Notably, both mutants promote redistribution of H3K36me2 away from repetitive regions into active genes, which associate with retrotransposon de-repression in eye discs. Aberrant expression of krimp represses LINE retrotransposons but does not contribute to the eye phenotype. Depletion of H3K36me2 methyltransferase ash1 in H3.3K27M, and of PRC2 component E(z) in H3.3K36M, restores the expression of eye developmental genes and normal eye growth, showing that redistribution of antagonistic marks contributes to K-to-M pathogenesis. Our results implicate a novel function for H3K36me2 and showcase convergent downstream effects of oncohistones that target opposing epigenetic marks.

Ependymomas encompass a heterogeneous group of central nervous system (CNS) neoplasms that occur along the entire neuroaxis. In recent years, extensive (epi-)genomic profiling efforts have identified several molecular groups of ependymoma that are characterized by distinct molecular alterations and/or patterns. Based on unsupervised visualization of a large cohort of genome-wide DNA methylation data, we identified a highly distinct group of pediatric-type tumors (n = 40) forming a cluster separate from all established CNS tumor types, of which a high proportion were histopathologically diagnosed as ependymoma. RNA sequencing revealed recurrent fusions involving the pleomorphic adenoma gene-like 1 (PLAGL1) gene in 19 of 20 of the samples analyzed, with the most common fusion being EWSR1:PLAGL1 (n = 13). Five tumors showed a PLAGL1:FOXO1 fusion and one a PLAGL1:EP300 fusion. High transcript levels of PLAGL1 were noted in these tumors, with concurrent overexpression of the imprinted genes H19 and IGF2, which are regulated by PLAGL1. Histopathological review of cases with sufficient material (n = 16) demonstrated a broad morphological spectrum of tumors with predominant ependymoma-like features. Immunohistochemically, tumors were GFAP positive and OLIG2- and SOX10 negative. In 3/16 of the cases, a dot-like positivity for EMA was detected. All tumors in our series were located in the supratentorial compartment. Median age of the patients at the time of diagnosis was 6.2 years. Median progression-free survival was 35 months (for 11 patients with data available). In summary, our findings suggest the existence of a novel group of supratentorial neuroepithelial tumors that are characterized by recurrent PLAGL1 fusions and enriched for pediatric patients.

How abnormal neurodevelopment relates to the tumour aggressiveness of medulloblastoma (MB), the most common type of embryonal tumour, remains elusive. Here we uncover a neurodevelopmental epigenomic programme that is hijacked to induce MB metastatic dissemination. Unsupervised analyses of integrated publicly available datasets with our newly generated data reveal that SMARCD3 (also known as BAF60C) regulates Disabled 1 (DAB1)-mediated Reelin signalling in Purkinje cell migration and MB metastasis by orchestrating cis-regulatory elements at the DAB1 locus. We further identify that a core set of transcription factors, enhancer of zeste homologue 2 (EZH2) and nuclear factor I X (NFIX), coordinates with the cis-regulatory elements at the SMARCD3 locus to form a chromatin hub to control SMARCD3 expression in the developing cerebellum and in metastatic MB. Increased SMARCD3 expression activates Reelin-DAB1-mediated Src kinase signalling, which results in a MB response to Src inhibition. These data deepen our understanding of how neurodevelopmental programming influences disease progression and provide a potential therapeutic option for patients with MB.

Cancers arising from germline DNA mismatch repair deficiency or polymerase proofreading deficiency (MMRD and PPD) in children harbour the highest mutational and microsatellite insertion-deletion (MS-indel) burden in humans. MMRD and PPD cancers are commonly lethal due to the inherent resistance to chemo-irradiation. Although immune checkpoint inhibitors (ICIs) have failed to benefit children in previous studies, we hypothesized that hypermutation caused by MMRD and PPD will improve outcomes following ICI treatment in these patients. Using an international consortium registry study, we report on the ICI treatment of 45 progressive or recurrent tumors from 38 patients. Durable objective responses were observed in most patients, culminating in a 3 year survival of 41.4%. High mutation burden predicted response for ultra-hypermutant cancers (>100 mutations per Mb) enriched for combined MMRD + PPD, while MS-indels predicted response in MMRD tumors with lower mutation burden (10-100 mutations per Mb). Furthermore, both mechanisms were associated with increased immune infiltration even in 'immunologically cold' tumors such as gliomas, contributing to the favorable response. Pseudo-progression (flare) was common and was associated with immune activation in the tumor microenvironment and systemically. Furthermore, patients with flare who continued ICI treatment achieved durable responses. This study demonstrates improved survival for patients with tumors not previously known to respond to ICI treatment, including central nervous system and synchronous cancers, and identifies the dual roles of mutation burden and MS-indels in predicting sustained response to immunotherapy.

BRAF genomic alterations are the most common oncogenic drivers in pediatric low-grade glioma (pLGG). Arm 1 (n = 77) of the ongoing phase 2 FIREFLY-1 (PNOC026) trial investigated the efficacy of the oral, selective, central nervous system-penetrant, type II RAF inhibitor tovorafenib (420 mg m-2 once weekly; 600 mg maximum) in patients with BRAF-altered, relapsed/refractory pLGG. Arm 2 (n = 60) is an extension cohort, which provided treatment access for patients with RAF-altered pLGG after arm 1 closure. Based on independent review, according to Response Assessment in Neuro-Oncology High-Grade Glioma (RANO-HGG) criteria, the overall response rate (ORR) of 67% met the arm 1 prespecified primary endpoint; median duration of response (DOR) was 16.6 months; and median time to response (TTR) was 3.0 months (secondary endpoints). Other select arm 1 secondary endpoints included ORR, DOR and TTR as assessed by Response Assessment in Pediatric Neuro-Oncology Low-Grade Glioma (RAPNO) criteria and safety (assessed in all treated patients and the primary endpoint for arm 2, n = 137). The ORR according to RAPNO criteria (including minor responses) was 51%; median DOR was 13.8 months; and median TTR was 5.3 months. The most common treatment-related adverse events (TRAEs) were hair color changes (76%), elevated creatine phosphokinase (56%) and anemia (49%). Grade ≥3 TRAEs occurred in 42% of patients. Nine (7%) patients had TRAEs leading to discontinuation of tovorafenib. These data indicate that tovorafenib could be an effective therapy for BRAF-altered, relapsed/refractory pLGG. ClinicalTrials.gov registration: NCT04775485 .

Recent studies suggest that long non-coding RNAs (lncRNAs) contribute to medulloblastoma (MB) formation and progression. We have identified an lncRNA, lnc-HLX-2-7, as a potential therapeutic target in group 3 (G3) MBs. lnc-HLX-2-7 RNA specifically accumulates in the promoter region of HLX, a sense-overlapping gene of lnc-HLX-2-7, which activates HLX expression by recruiting multiple factors, including enhancer elements. RNA sequencing and chromatin immunoprecipitation reveal that HLX binds to and activates the promoters of several oncogenes, including TBX2, LIN9, HOXM1, and MYC. Intravenous treatment with cerium-oxide-nanoparticle-coated antisense oligonucleotides targeting lnc-HLX-2-7 (CNP-lnc-HLX-2-7) inhibits tumor growth by 40%-50% in an intracranial MB xenograft mouse model. Combining CNP-lnc-HLX-2-7 with standard-of-care cisplatin further inhibits tumor growth and significantly prolongs mouse survival compared with CNP-lnc-HLX-2-7 monotherapy. Thus, the lnc-HLX-2-7-HLX-MYC axis is important for regulating G3 MB progression, providing a strong rationale for using lnc-HLX-2-7 as a therapeutic target for G3 MBs.

In 2012, an international consensus paper reported that medulloblastoma comprises four molecular subgroups (WNT, SHH, Group 3, and Group 4), each associated with distinct genomic features and clinical behavior. Independently, multiple recent reports have defined further intra-subgroup heterogeneity in the form of biologically and clinically relevant subtypes. However, owing to differences in patient cohorts and analytical methods, estimates of subtype number and definition have been inconsistent, especially within Group 3 and Group 4. Herein, we aimed to reconcile the definition of Group 3/Group 4 MB subtypes through the analysis of a series of 1501 medulloblastomas with DNA-methylation profiling data, including 852 with matched transcriptome data. Using multiple complementary bioinformatic approaches, we compared the concordance of subtype calls between published cohorts and analytical methods, including assessments of class-definition confidence and reproducibility. While the lowest complexity solutions continued to support the original consensus subgroups of Group 3 and Group 4, our analysis most strongly supported a definition comprising eight robust Group 3/Group 4 subtypes (types I-VIII). Subtype II was consistently identified across all component studies, while all others were supported by multiple class-definition methods. Regardless of analytical technique, increasing cohort size did not further increase the number of identified Group 3/Group 4 subtypes. Summarizing the molecular and clinico-pathological features of these eight subtypes indicated enrichment of specific driver gene alterations and cytogenetic events amongst subtypes, and identified highly disparate survival outcomes, further supporting their biological and clinical relevance. Collectively, this study provides continued support for consensus Groups 3 and 4 while enabling robust derivation of, and categorical accounting for, the extensive intertumoral heterogeneity within Groups 3 and 4, revealed by recent high-resolution subclassification approaches. Furthermore, these findings provide a basis for application of emerging methods (e.g., proteomics/single-cell approaches) which may additionally inform medulloblastoma subclassification. Outputs from this study will help shape definition of the next generation of medulloblastoma clinical protocols and facilitate the application of enhanced molecularly guided risk stratification to improve outcomes and quality of life for patients and their families.

Our ability to manage acute myeloid leukemia (AML) is limited by our incomplete understanding of the epigenetic disruption central to leukemogenesis, including improper histone methylation. Here we examine 16 histone H3 genes in 434 primary AML samples and identify Q69H, A26P, R2Q, R8H and K27M/I mutations (1.6%), with higher incidence in secondary AML (9%). These mutations occur in pre-leukemic hematopoietic stem cells (HSCs) and exist in the major leukemic clones in patients. They increase the frequency of functional HSCs, alter differentiation, and amplify leukemic aggressiveness. These effects are dependent on the specific mutation. H3K27 mutation increases the expression of genes involved in erythrocyte and myeloid differentiation with altered H3K27 tri-methylation and K27 acetylation. The functional impact of histone mutations is independent of RUNX1 mutation, although they at times co-occur. This study establishes that H3 mutations are drivers of human pre-cancerous stem cell expansion and important early events in leukemogenesis.

Lymphocyte functions triggered by antigen recognition and co-stimulation signals are associated with a rapid and intense cell division, and hence with metabolism adaptation. The nucleotide cytidine 5' triphosphate (CTP) is a precursor required for the metabolism of DNA, RNA and phospholipids. CTP originates from two sources: a salvage pathway and a de novo synthesis pathway that depends on two enzymes, the CTP synthases (or synthetases) 1 and 2 (CTPS1 with CTPS2); the respective roles of these two enzymes are not known. CTP synthase activity is a potentially important step for DNA synthesis in lymphocytes. Here we report the identification of a loss-of-function homozygous mutation (rs145092287) in CTPS1 in humans that causes a novel and life-threatening immunodeficiency, characterized by an impaired capacity of activated T and B cells to proliferate in response to antigen receptor-mediated activation. In contrast, proximal and distal T-cell receptor (TCR) signalling events and responses were only weakly affected by the absence of CTPS1. Activated CTPS1-deficient cells had decreased levels of CTP. Normal T-cell proliferation was restored in CTPS1-deficient cells by expressing wild-type CTPS1 or by addition of exogenous CTP or its nucleoside precursor, cytidine. CTPS1 expression was found to be low in resting T cells, but rapidly upregulated following TCR activation. These results highlight a key and specific role of CTPS1 in the immune system by its capacity to sustain the proliferation of activated lymphocytes during the immune response. CTPS1 may therefore represent a therapeutic target of immunosuppressive drugs that could specifically dampen lymphocyte activation.

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.

Medulloblastoma is a pediatric brain tumor with a variable prognosis due to clinical and genomic heterogeneity. Among the 4 major genomic sub-groups, patients with MYC amplified tumors have a particularly poor prognosis despite therapy with surgery, radiation and chemotherapy. Targeting the MYC oncogene has traditionally been problematic. Here we report that MYC driven medulloblastoma can be targeted by inhibition of the bromodomain protein BRD4. We show that bromodomain inhibition with JQ1 restricts c-MYC driven transcriptional programs in medulloblastoma, suppresses medulloblastoma cell growth and induces a cell cycle arrest. Importantly JQ1 suppresses stem cell associated signaling in medulloblastoma cells and inhibits medulloblastoma tumor cell self-renewal. Additionally JQ1 also promotes senescence in medulloblastoma cells by activating cell cycle kinase inhibitors and inhibiting activity of E2F1. Furthermore BRD4 inhibition displayed an anti-proliferative, pro-senescence effect in a medulloblastoma model in vivo. In clinical samples we found that transcriptional programs suppressed by JQ1 are associated with adverse risk in medulloblastoma patients. Our work indicates that BRD4 inhibition attenuates stem cell signaling in MYC driven medulloblastoma and demonstrates the feasibility BET domain inhibition as a therapeutic approach in vivo.

No abstract available

Knowledge of marine ecosystems that grow and reside on and around subsea oil and gas infrastructure is required to understand impacts of this offshore industry on the marine environment and inform decommissioning decisions. This study used baited remote underwater stereo-video systems (stereo-BRUVs) to compare species richness, fish abundance and size along 42.3 km of subsea pipeline and in adjacent areas of varying habitats. The pipeline is laid in an onshore-offshore direction enabling surveys to encompass a range of depths from 9 m nearshore out to 140 m depth offshore. Surveys off the pipeline were performed across this depth range and in an array of natural habitats (sand, macroalgae, coral reef) between 1 km and 40 km distance from the pipeline. A total of 14,953 fish were observed comprising 240 species (131 on the pipeline and 225 off-pipeline) and 59 families (39 on the pipeline and 56 off-pipeline) and the length of 8,610 fish were measured. The fish assemblage on and off the pipeline was similar in depths of <80 m. In depths beyond 80 m, the predominant habitat off-pipeline was sand and differences between fish assemblages on and off-pipeline were more pronounced. The pipeline was characterised by higher biomass and abundances of larger-bodied, commercially important species such as: Pristipomoides multidens (goldband snapper), Lutjanus malabaricus (saddletail snapper) and Lutjanus russellii (Moses' snapper) among others, and possessed a catch value 2-3 times higher per stereo-BRUV deployment than that of fish observed off-pipeline. Adjacent natural seabed habitats possessed higher abundances of Atule mate (yellowtail scad), Nemipterus spp. (threadfin bream) and Terapon jarbua (crescent grunter), species of no or low commercial value. This is the first published study to use stereo-BRUVs to report on the importance of subsea infrastructure to commercially important fishes over a depth gradient and increases our knowledge of the fish assemblage associated with subsea infrastructure off north-west Australia. These results provide a greater understanding of ecological and fisheries implications of decommissioning subsea infrastructure on the north-west shelf, and will help better inform decision-making on the fate of infrastructure at different depths.

Pediatric brain cancer medulloblastoma (MB) standard-of-care results in numerous comorbidities. MB is comprised of distinct molecular subgroups. Group 3 molecular subgroup patients have the highest relapse rates and after standard-of-care have a 20% survival. Group 3 tumors have high expression of GABRA5, which codes for the α5 subunit of the γ-aminobutyric acid type A receptor (GABAAR). We are advancing a therapeutic approach for group 3 based on GABAAR modulation using benzodiazepine-derivatives.