Summary Medulloblastoma, the most common malignant pediatric brain tumour, is currently treated with non-specific cytotoxic therapies including surgery, whole brain radiation, and aggressive chemotherapy. As medulloblastoma exhibits marked intertumoural heterogeneity, with at least four distinct molecular variants, prior attempts to identify targets for therapy have been underpowered due to small samples sizes. Here we report somatic copy number aberrations (SCNAs) in 1087 unique medulloblastomas. SCNAs are common in medulloblastoma, and are predominantly subgroup enriched. The most common region of focal copy number gain is a tandem duplication of the Parkinson’s disease gene SNCAIP, which is exquisitely restricted to Group 4α. Recurrent translocations of PVT1, including PVT1-MYC and PVT1-NDRG1 that arise through chromothripsis are restricted to Group 3. Numerous targetable SCNAs, including recurrent events targeting TGFβ signaling in Group 3, and NF-κB signaling in Group 4 suggest future avenues for rational, targeted therapy.

Summary: The study of the origin and development of cerebellar tumors has been hampered by the complexity and heterogeneity of cerebellar cells that change over the course of development. We used single-cell transcriptomics to study >60,000 cells from the developing murine cerebellum, and show that different molecular subgroups of childhood cerebellar tumors mirror the transcription of cells from distinct, temporally restricted cerebellar lineages. Sonic Hedgehog medulloblastoma transcriptionally mirrors the granule cell hierarchy as expected, whereas Group3 MB resemble Nestin+ve stem cells, Group 4 MB resemble unipolar brush cells, and PFA/PFB ependymoma and cerebellar pilocytic astrocytoma resemble the pre-natal gliogenic progenitor cells. Furthermore, single-cell transcriptomics of human childhood cerebellar tumors demonstrates that many bulk tumors contain a mixed population of cells with divergent differentiation. Our data highlight cerebellar tumors as a disorder of early brain development, and provide a proximate explanation for the peak incidence of cerebellar tumors in early childhood.

Of nine ependymoma molecular groups detected by DNA methylation profiling, the posterior fossa type A (PFA) is most prevalent. We used DNA methylation profiling to look for further molecular heterogeneity among 675 PFA ependymomas. Two major subgroups, PFA-1 and PFA-2, and nine minor subtypes were discovered. Transcriptome profiling suggested a distinct histogenesis for PFA-1 and PFA-2, but their clinical parameters were similar. In contrast, PFA subtypes differed with respect to age at diagnosis, gender ratio, outcome, and frequencies of genetic alterations. One subtype, PFA-1c, was enriched for 1q gain and had a relatively poor outcome, while patients with PFA-2c ependymomas showed an overall survival at 5 years of > 90%. Unlike other ependymomas, PFA-2c tumors express high levels of OTX2, a potential biomarker for this ependymoma subtype with a good prognosis. We also discovered recurrent mutations among PFA ependymomas. H3 K27M mutations were present in 4.2%, occurring only in PFA-1 tumors, and missense mutations in an uncharacterized gene, CXorf67, were found in 9.4% of PFA ependymomas, but not in other groups. We detected high levels of wildtype or mutant CXorf67 expression in all PFA subtypes except PFA-1f, which is enriched for H3 K27M mutations. PFA ependymomas are characterized by lack of H3 K27 trimethylation (H3 K27-me3), and we tested the hypothesis that CXorf67 binds to PRC2 and can modulate levels of H3 K27-me3. Immunoprecipitation/mass spectrometry detected EZH2, SUZ12, and EED, core components of the PRC2 complex, bound to CXorf67 in the Daoy cell line, which shows high levels of CXorf67 and no expression of H3 K27-me3. Enforced reduction of CXorf67 in Daoy cells restored H3 K27-me3 levels, while enforced expression of CXorf67 in HEK293T and neural stem cells reduced H3 K27-me3 levels. Our data suggest that heterogeneity among PFA ependymomas could have clinicopathologic utility and that CXorf67 may have a functional role in these tumors.

SUMMARY Genetic drivers of cancer can be dysregulated through epigenetic modifications of DNA. While the critical role of DNA 5-methylcytosine (5mC) in the regulation of transcription is recognized, the functions of other non-canonical DNA modifications remain obsure. Here, we report the identification of novel N(6)-methyladenine (N6-mA) DNA modifications in human tissues and implicate this epigenetic mark in human disease, specifically the highly malignant brain cancer, glioblastoma. Glioblastoma markedly upregulated N6-mA levels, which co-localized with heterochromatic histone modifications, predominantly H3K9me3. N6-mA levels were dynamically regulated by the DNA demethylase, ALKBH1, depletion of which led to transcriptional silencing of oncogenic pathways through decreasing chromatin accessibility. Targeting the N6-mA regulator, ALKBH1, in patient-derived human glioblastoma models inhibited tumor cell proliferation and extended survival of tumor-bearing mice, supporting this novel DNA modification as a potential therapeutic target for glioblastoma. Collectively, our results uncover a novel epigenetic node in cancer through the DNA modification, N6-mA.

SUMMARY Genomic sequencing has driven precision-based oncology therapy; however, genetic drivers remain unknown or non-targetable for many malignancies, demanding alternative approaches to identify therapeutic leads. Ependymomas are chemotherapy-resistant brain tumours, which, despite genomic sequencing, lack effective molecular targets. Intracranial ependymomas are segregated based on anatomical location – supratentorial region (ST) or posterior fossa (PF) – and further divided into distinct molecular subgroups that reflect differences in age of onset, gender predominance, and response to therapy1–3. The most common and aggressive subgroup, Posterior Fossa Ependymoma Group A (PF-EPN-A), occurs in young children and appears to lack recurrent somatic mutations2. Conversely, Posterior Fossa Ependymoma Group B (PF-EPN-B) tumours display frequent large-scale copy number gains and losses yet favourable clinical outcomes1,3. Greater than 70% of supratentorial ependymomas are defined by highly recurrent gene fusions in the NFκB subunit RELA (ST-EPN-RELA), and less frequently involve fusion of the gene encoding the transcriptional activator YAP1 (ST-EPN-YAP1).1,3,4 Subependymomas, a distinct histologic variant, can also be found within the ST and PF compartments accounting for the majority of tumours in the molecular subgroups ST-EPN-SE and PF-EPN-SE, respectively1. Here, we mapped active chromatin landscapes in 42 primary ependymomas in two non-overlapping primary ependymoma cohorts with the goal of identifying essential super enhancer associated genes on which tumour cells were dependent. Enhancer regions revealed putative oncogenes, molecular targets, and pathways, which when subjected to small molecule inhibitor or shRNA treatment, diminished proliferation of patient-derived neurospheres and increased survival in mouse models of ependymomas. Through profiling of transcriptional enhancers, our study provides a framework for target and drug discovery in other cancers recalcitrant to therapeutic development because of their lack of known genetic drivers.

Highlights d Enhancers active in the cell of origin are co-amplified with oncogenes d Circular extrachromosomal amplicons are associated with enhancer rewiring d Endogenous and new enhancers on amplicons contribute to cell proliferation d Skewed co-amplification that selects enhancers is found across several tumor types

Glioblastomas are highly lethal cancers, containing self-renewing glioblastoma stem cells (GSC). Here, we show that GSCs, differentiated glioblastoma cells (DGC), and nonmalignant brain cultures all displayed robust circadian rhythms, yet GSCs alone displayed exquisite dependence on core clock transcription factors, BMAL1 and CLOCK, for optimal cell growth. Downregulation of BMAL1 or CLOCK in GSCs induced cell-cycle arrest and apoptosis. Chromatin immunoprecipitation revealed that BMAL1 preferentially bound metabolic genes and was associated with active chromatin regions in GSCs compared with neural stem cells. Targeting BMAL1 or CLOCK attenuated mitochondrial metabolic function and reduced expression of tricarboxylic acid cycle enzymes. Small-molecule agonists of two independent BMAL1-CLOCK negative regulators, the cryptochromes and REV-ERBs, downregulated stem cell factors and reduced GSC growth. Combination of cryptochrome and REV-ERB agonists induced synergistic antitumor effi cacy. Collectively, these fi ndings show that GSCs co-opt circadian regulators beyond canonical circadian circuitry to promote stemness maintenance and metabolism, offering novel therapeutic paradigms. SIGNIFICANCE:Cancer stem cells are highly malignant tumor-cell populations. We demonstrate that GSCs selectively depend on circadian regulators, with increased binding of the regulators in active chromatin regions promoting tumor metabolism. Supporting clinical relevance, pharmacologic targeting of circadian networks specifi cally disrupted cancer stem cell growth and self-renewal.

Leukemia stem cells (LSCs) are thought to drive the genesis of acute myeloid leukemia (AML) as well as relapse following chemotherapy. Because of their unique biology, developing effective methods to eradicate LSCs has been a significant challenge. In the present study, we demonstrate that intrinsic overexpression of the mitochondrial dynamics regulator FIS1 mediates mitophagy activity that is essential for primitive AML cells. Depletion of FIS1 attenuates mitophagy and leads to inactivation of GSK3, myeloid differentiation, cell cycle arrest, and a profound loss of LSC self-renewal potential. Further, we report that the central metabolic stress regulator AMPK is also intrinsically activated in LSC populations and is upstream of FIS1. Inhibition of AMPK signaling recapitulates the biological effect of FIS1 loss. These data suggest a model in which LSCs co-opt AMPK/FIS1-mediated mitophagy as a means to maintain stem cell properties that may be otherwise compromised by the stresses induced by oncogenic transformation.