Medulloblastoma is the most common malignant brain tumor of childhood. Group 3 medulloblastoma, the most aggressive molecular subtype, frequently disseminates through the leptomeningeal cerebral spinal fluid (CSF) spaces in the brain and spinal cord. The mechanism of dissemination through the CSF remains poorly understood, and the molecular pathways involved in medulloblastoma metastasis and self-renewal are largely unknown. Here we show that NOTCH1 signaling pathway regulates both the initiation of metastasis and the self-renewal of medulloblastoma. We identify a mechanism in which NOTCH1 activates BMI1 through the activation of TWIST1. NOTCH1 expression and activity are directly related to medulloblastoma metastasis and decreased survival rate of tumor-bearing mice. Finally, medulloblastoma-bearing mice intrathecally treated with anti-NRR1, a NOTCH1 blocking antibody, present lower frequency of spinal metastasis and higher survival rate. These findings identify NOTCH1 as a pivotal driver of Group 3 medulloblastoma metastasis and self-renewal, supporting the development of therapies targeting this pathway.

Cranial radiotherapy improves survival of the most common childhood cancers, including brain tumors and leukemia. Unfortunately, long-term survivors are faced with consequences of secondary neoplasia, including radiation-induced meningiomas (RIMs). We characterized 31 RIMs with exome/NF2 intronic sequencing, RNA sequencing and methylation profiling, and found NF2 gene rearrangements in 12/31 of RIMs, an observation previously unreported in sporadic meningioma (SM). Additionally, known recurrent mutations characteristic of SM, including AKT1, KLF4, TRAF7 and SMO, were not observed in RIMs. Combined losses of chromosomes 1p and 22q were common in RIMs (16/18 cases) and overall, chromosomal aberrations were more complex than that observed in SM. Patterns of DNA methylation profiling supported similar cell of origin between RIMs and SMs. The findings indicate that the mutational landscape of RIMs is distinct from SMs, and have significant therapeutic implications for survivors of childhood cranial radiation and the elucidation of the molecular pathogenesis of meningiomas.Radiation-induced meningiomas are often more aggressive than sporadic ones. In this study, the authors perform an exome, methylation and RNA-seq analysis of 31 cases of radiation-induced meningioma and show NF2 rearrangement, an observation previously unreported in the sporadic tumors.

Sarcomas are malignant soft tissue and bone tumours affecting adults, adolescents and children. They represent a morphologically heterogeneous class of tumours and some entities lack defining histopathological features. Therefore, the diagnosis of sarcomas is burdened with a high inter-observer variability and misclassification rate. Here, we demonstrate classification of soft tissue and bone tumours using a machine learning classifier algorithm based on array-generated DNA methylation data. This sarcoma classifier is trained using a dataset of 1077 methylation profiles from comprehensively pre-characterized cases comprising 62 tumour methylation classes constituting a broad range of soft tissue and bone sarcoma subtypes across the entire age spectrum. The performance is validated in a cohort of 428 sarcomatous tumours, of which 322 cases were classified by the sarcoma classifier. Our results demonstrate the potential of the DNA methylation-based sarcoma classification for research and future diagnostic applications.

Targeting oncogenic pathways holds promise for brain tumor treatment, but inhibition of Sonic Hedgehog (SHH) signaling has failed in SHH-driven medulloblastoma. Cellular diversity within tumors and reduced lineage commitment can undermine targeted therapy by increasing the probability of treatment-resistant populations. Using single-cell RNA-seq and lineage tracing, we analyzed cellular diversity in medulloblastomas in transgenic, medulloblastoma-prone mice, and responses to the SHH-pathway inhibitor vismodegib. In untreated tumors, we find expected stromal cells and tumor-derived cells showing either a spectrum of neural progenitor-differentiation states or glial and stem cell markers. Vismodegib reduces the proliferative population and increases differentiation. However, specific cell types in vismodegib-treated tumors remain proliferative, showing either persistent SHH-pathway activation or stem cell characteristics. Our data show that even in tumors with a single pathway-activating mutation, diverse mechanisms drive tumor growth. This diversity confers early resistance to targeted inhibitor therapy, demonstrating the need to target multiple pathways simultaneously.

Pineoblastoma is a rare pediatric cancer induced by germline mutations in the tumor suppressors RB1 or DICER1. Presence of leptomeningeal metastases is indicative of poor prognosis. Here we report that inactivation of Rb plus p53 via a WAP-Cre transgene, commonly used to target the mammary gland during pregnancy, induces metastatic pineoblastoma resembling the human disease with 100% penetrance. A stabilizing mutation rather than deletion of p53 accelerates metastatic dissemination. Deletion of Dicer1 plus p53 via WAP-Cre also predisposes to pineoblastoma, albeit with lower penetrance. In silico analysis predicts tricyclic antidepressants such as nortriptyline as potential therapeutics for both pineoblastoma models. Nortriptyline disrupts the lysosome, leading to accumulation of non-functional autophagosome, cathepsin B release and pineoblastoma cell death. Nortriptyline further synergizes with the antineoplastic drug gemcitabine to effectively suppress pineoblastoma in our preclinical models, offering new modality for this lethal childhood malignancy.

Metastatic breast-cancer is a major cause of death in women worldwide, yet the relationship between oncogenic drivers that promote metastatic versus primary cancer is still contentious. To elucidate this relationship in treatment-naive animals, we hereby describe mammary-specific transposon-mutagenesis screens in female mice together with loss-of-function Rb, which is frequently inactivated in breast-cancer. We report gene-centric common insertion-sites (gCIS) that are enriched in primary-tumors, in metastases or shared by both compartments. Shared-gCIS comprise a major MET-RAS network, whereas metastasis-gCIS form three additional hubs: Rho-signaling, Ubiquitination and RNA-processing. Pathway analysis of four clinical cohorts with paired primary-tumors and metastases reveals similar organization in human breast-cancer with subtype-specific shared-drivers (e.g. RB1-loss, TP53-loss, high MET, RAS, ER), primary-enriched (EGFR, TGFβ and STAT3) and metastasis-enriched (RHO, PI3K) oncogenic signaling. Inhibitors of RB1-deficiency or MET plus RHO-signaling cooperate to block cell migration and drive tumor cell-death. Thus, targeting shared- and metastasis- but not primary-enriched derivers offers a rational avenue to prevent metastatic breast-cancer.

Many genes that drive normal cellular development also contribute to oncogenesis. Medulloblastoma (MB) tumors likely arise from neuronal progenitors in the cerebellum, and we hypothesized that the heterogeneity observed in MBs with sonic hedgehog (SHH) activation could be due to differences in developmental pathways. To investigate this question, here we perform single-nucleus RNA sequencing on highly differentiated SHH MBs with extensively nodular histology and observed malignant cells resembling each stage of canonical granule neuron development. Through innovative computational approaches, we connect these results to published datasets and find that some established molecular subtypes of SHH MB appear arrested at different developmental stages. Additionally, using multiplexed proteomic imaging and MALDI imaging mass spectrometry, we identify distinct histological and metabolic profiles for highly differentiated tumors. Our approaches are applicable to understanding the interplay between heterogeneity and differentiation in other cancers and can provide important insights for the design of targeted therapies.

Infant gliomas have paradoxical clinical behavior compared to those in children and adults: low-grade tumors have a higher mortality rate, while high-grade tumors have a better outcome. However, we have little understanding of their biology and therefore cannot explain this behavior nor what constitutes optimal clinical management. Here we report a comprehensive genetic analysis of an international cohort of clinically annotated infant gliomas, revealing 3 clinical subgroups. Group 1 tumors arise in the cerebral hemispheres and harbor alterations in the receptor tyrosine kinases ALK, ROS1, NTRK and MET. These are typically single-events and confer an intermediate outcome. Groups 2 and 3 gliomas harbor RAS/MAPK pathway mutations and arise in the hemispheres and midline, respectively. Group 2 tumors have excellent long-term survival, while group 3 tumors progress rapidly and do not respond well to chemoradiation. We conclude that infant gliomas comprise 3 subgroups, justifying the need for specialized therapeutic strategies.

Sonic hedgehog medulloblastoma encompasses a clinically and molecularly diverse group of cancers of the developing central nervous system. Here, we use unbiased sequencing of the transcriptome across a large cohort of 250 tumors to reveal differences among molecular subtypes of the disease, and demonstrate the previously unappreciated importance of non-coding RNA transcripts. We identify alterations within the cAMP dependent pathway (GNAS, PRKAR1A) which converge on GLI2 activity and show that 18% of tumors have a genetic event that directly targets the abundance and/or stability of MYCN. Furthermore, we discover an extensive network of fusions in focally amplified regions encompassing GLI2, and several loss-of-function fusions in tumor suppressor genes PTCH1, SUFU and NCOR1. Molecular convergence on a subset of genes by nucleotide variants, copy number aberrations, and gene fusions highlight the key roles of specific pathways in the pathogenesis of Sonic hedgehog medulloblastoma and open up opportunities for therapeutic intervention.

The most common events in breast cancer (BC) involve chromosome arm losses and gains. Here we describe identification of 1089 gene-centric common insertion sites (gCIS) from transposon-based screens in 8 mouse models of BC. Some gCIS are driver-specific, others driver non-specific, and still others associated with tumor histology. Processes affected by driver-specific and histology-specific mutations include well-known cancer pathways. Driver non-specific gCIS target the Mediator complex, Ca++ signaling, Cyclin D turnover, RNA-metabolism among other processes. Most gCIS show single allele disruption and many map to genomic regions showing high-frequency hemizygous loss in human BC. Two gCIS, Nf1 and Trps1, show synthetic haploinsufficient tumor suppressor activity. Many gCIS act on the same pathway responsible for tumor initiation, thereby selecting and sculpting just enough and just right signaling. These data highlight ~1000 genes with predicted conditional haploinsufficient tumor suppressor function and the potential to promote chromosome arm loss in BC.

Ependymoma is a tumor of the brain or spinal cord. The two most common and aggressive molecular groups of ependymoma are the supratentorial ZFTA-fusion associated and the posterior fossa ependymoma group A. In both groups, tumors occur mainly in young children and frequently recur after treatment. Although molecular mechanisms underlying these diseases have recently been uncovered, they remain difficult to target and innovative therapeutic approaches are urgently needed. Here, we use genome-wide chromosome conformation capture (Hi-C), complemented with CTCF and H3K27ac ChIP-seq, as well as gene expression and DNA methylation analysis in primary and relapsed ependymoma tumors, to identify chromosomal conformations and regulatory mechanisms associated with aberrant gene expression. In particular, we observe the formation of new topologically associating domains ('neo-TADs') caused by structural variants, group-specific 3D chromatin loops, and the replacement of CTCF insulators by DNA hyper-methylation. Through inhibition experiments, we validate that genes implicated by these 3D genome conformations are essential for the survival of patient-derived ependymoma models in a group-specific manner. Thus, this study extends our ability to reveal tumor-dependency genes by 3D genome conformations even in tumors that lack targetable genetic alterations.

OTX2 is a potent oncogene that promotes tumor growth in Group 3 medulloblastoma. However, the mechanisms by which OTX2 represses neural differentiation are not well characterized. Here, we perform extensive multiomic analyses to identify an OTX2 regulatory network that controls Group 3 medulloblastoma cell fate. OTX2 silencing modulates the repressive chromatin landscape, decreases levels of PRC2 complex genes and increases the expression of neurodevelopmental transcription factors including PAX3 and PAX6. Expression of PAX3 and PAX6 is significantly lower in Group 3 medulloblastoma patients and is correlated with reduced survival, yet only PAX3 inhibits self-renewal in vitro and increases survival in vivo. Single cell RNA sequencing of Group 3 medulloblastoma tumorspheres demonstrates expression of an undifferentiated progenitor program observed in primary tumors and characterized by translation/elongation factor genes. Identification of mTORC1 signaling as a downstream effector of OTX2-PAX3 reveals roles for protein synthesis pathways in regulating Group 3 medulloblastoma pathogenesis.

Human cerebral cancers are known to contain cell types resembling the varying stages of neural development. However, the basis of this association remains unclear. Here, we map the development of mouse cerebrum across the developmental time-course, from embryonic day 12.5 to postnatal day 365, performing single-cell transcriptomics on >100,000 cells. By comparing this reference atlas to single-cell data from >100 glial tumours of the adult and paediatric human cerebrum, we find that tumour cells have an expression signature that overlaps with temporally restricted, embryonic radial glial precursors (RGPs) and their immediate sublineages. Further, we demonstrate that prenatal transformation of RGPs in a genetic mouse model gives rise to adult cerebral tumours that show an embryonic/juvenile RGP identity. Together, these findings implicate the acquisition of embryonic-like states in the genesis of adult glioma, providing insight into the origins of human glioma, and identifying specific developmental cell types for therapeutic targeting.

Most lncRNAs display species-specific expression patterns suggesting that animal models of cancer may only incompletely recapitulate the regulatory crosstalk between lncRNAs and oncogenic pathways in humans. Among these pathways, Sonic Hedgehog (SHH) signaling is aberrantly activated in several human cancer entities. We unravel that aberrant expression of the primate-specific lncRNA HedgeHog Interacting Protein-AntiSense 1 (HHIP-AS1) is a hallmark of SHH-driven tumors including medulloblastoma and atypical teratoid/rhabdoid tumors. HHIP-AS1 is actively transcribed from a bidirectional promoter shared with SHH regulator HHIP. Knockdown of HHIP-AS1 induces mitotic spindle deregulation impairing tumorigenicity in vitro and in vivo. Mechanistically, HHIP-AS1 binds directly to the mRNA of cytoplasmic dynein 1 intermediate chain 2 (DYNC1I2) and attenuates its degradation by hsa-miR-425-5p. We uncover that neither HHIP-AS1 nor the corresponding regulatory element in DYNC1I2 are evolutionary conserved in mice. Taken together, we discover an lncRNA-mediated mechanism that enables the pro-mitotic effects of SHH pathway activation in human tumors.

Pediatric high-grade gliomas of the subclass MYCN (HGG-MYCN) are highly aggressive tumors frequently carrying MYCN amplifications, TP53 mutations, or both alterations. Due to their rarity, such tumors have only recently been identified as a distinct entity, and biological as well as clinical characteristics have not been addressed specifically. To gain insights into tumorigenesis and molecular profiles of these tumors, and to ultimately suggest alternative treatment options, we generated a genetically engineered mouse model by breeding hGFAP-cre::Trp53Fl/Fl::lsl-MYCN mice. All mice developed aggressive forebrain tumors early in their lifetime that mimic human HGG-MYCN regarding histology, DNA methylation, and gene expression. Single-cell RNA sequencing revealed a high intratumoral heterogeneity with neuronal and oligodendroglial lineage signatures. High-throughput drug screening using both mouse and human tumor cells finally indicated high efficacy of Doxorubicin, Irinotecan, and Etoposide as possible therapy options that children with HGG-MYCN might benefit from.