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Medulloblastoma and central nervous system primitive neuroectodermal tumors (CNS-PNET) are aggressive, poorly differentiated brain tumors with limited effective therapies. Using Sleeping Beauty (SB) transposon mutagenesis, we identified novel genetic drivers of medulloblastoma and CNS-PNET. Cross-species gene expression analyses classified SB-driven tumors into distinct medulloblastoma and CNS-PNET subgroups, indicating they resemble human Sonic hedgehog and group 3 and 4 medulloblastoma and CNS neuroblastoma with FOXR2 activation. This represents the first genetically induced mouse model of CNS-PNET and a rare model of group 3 and 4 medulloblastoma. We identified several putative proto-oncogenes including Arhgap36, Megf10, and Foxr2. Genetic manipulation of these genes demonstrated a robust impact on tumorigenesis in vitro and in vivo. We also determined that FOXR2 interacts with N-MYC, increases C-MYC protein stability, and activates FAK/SRC signaling. Altogether, our study identified several promising therapeutic targets in medulloblastoma and CNS-PNET. Significance: A transposon-induced mouse model identifies several novel genetic drivers and potential therapeutic targets in medulloblastoma and CNS-PNET.

Genomic sequencing has driven precision-based oncology therapy; however, the genetic drivers of many malignancies remain unknown or non-targetable, so alternative approaches to the identification of therapeutic leads are necessary. Ependymomas are chemotherapy-resistant brain tumours, which, despite genomic sequencing, lack effective molecular targets. Intracranial ependymomas are segregated on the basis of anatomical location (supratentorial region or posterior fossa) and further divided into distinct molecular subgroups that reflect differences in the age of onset, gender predominance and response to therapy1,2,3. 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 but have favourable clinical outcomes1,3. More than 70% of supratentorial ependymomas are defined by highly recurrent gene fusions in the NF-κB subunit gene RELA (ST-EPN-RELA), and a smaller number 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 supratetorial and posterior fossa compartments, and account for the majority of tumours in the molecular subgroups ST-EPN-SE and PF-EPN-SE. Here we describe mapping of 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 depend. Enhancer regions revealed putative oncogenes, molecular targets and pathways; inhibition of these targets with small molecule inhibitors or short hairpin RNA diminished the 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 that lack known genetic drivers and are therefore difficult to treat., This work was supported by an Alex's Lemonade Stand Young Investigator Award (S.C.M.), The CIHR Banting Fellowship (S.C.M.), The Cancer Prevention Research Institute of Texas (S.C.M., RR170023), Sibylle Assmus Award for Neurooncology (K.W.P.), the DKFZ-MOST (Ministry of Science, Technology & Space, Israel) program in cancer research (H.W.), James S. McDonnell Foundation (J.N.R.) and NIH grants: CA154130 (J.N.R.), R01 CA169117 (J.N.R.), R01 CA171652 (J.N.R.), R01 NS087913 (J.N.R.) and R01 NS089272 (J.N.R.). R.C.G. is supported by NIH grants T32GM00725 and F30CA217065. M.D.T. is supported by The Garron Family Chair in Childhood Cancer Research, and grants from the Pediatric Brain Tumour Foundation, Grand Challenge Award from CureSearch for Children’s Cancer, the National Institutes of Health (R01CA148699, R01CA159859), The Terry Fox Research Institute and Brainchild. M.D.T. is also supported by a Stand Up To Cancer St. Baldrick’s Pediatric Dream Team Translational Research Grant (SU2C-AACR-DT1113).

microRNAs (miRNAs) have wide-ranging effects on large-scale gene regulation. As such, they play a vital role in dictating normal development, and their aberrant expression has been implicated in cancer. There has been a large body of research on the role of miRNAs in medulloblastoma, the most common malignant brain tumor of childhood. The identification of the 4 molecular subgroups with distinct biological, genetic, and transcriptional features has revolutionized the field of medulloblastoma research over the past 5 years. Despite this, the growing body of research on miRNAs in medulloblastoma has largely focused on the clinical entity of a single disease rather than the molecular subgroups. This review begins by highlighting the role of miRNAs in development and progresses to explore their myriad of implications in cancer. Medulloblastoma is characterized by increased proliferation, inhibition of apoptosis, and maintenance of stemness programs—features that are inadvertently regulated by altered expression patterns in miRNAs. This review aims to contextualize the large body of work on miRNAs within the framework of medulloblastoma subgroups. The goal of this review is to stimulate new areas of research, including potential therapeutics, within a rapidly growing field.

Spatial heterogeneity of transcriptional and genetic markers between physically isolated biopsies of a single tumor poses major barriers to the identification of biomarkers and the development of targeted therapies that will be effective against the entire tumor. We analyzed the spatial heterogeneity of multiregional biopsies from 35 patients, using a combination of transcriptomic and genomic profiles. Medulloblastomas (MBs), but not high-grade gliomas (HGGs), demonstrated spatially homogeneous transcriptomes, which allowed for accurate subgrouping of tumors from a single biopsy. Conversely, somatic mutations that affect genes suitable for targeted therapeutics demonstrated high levels of spatial heterogeneity in MB, malignant glioma, and renal cell carcinoma (RCC). Actionable targets found in a single MB biopsy were seldom clonal across the entire tumor, which brings the efficacy of monotherapies against a single target into question. Clinical trials of targeted therapies for MB should first ensure the spatially ubiquitous nature of the target mutation.

Posterior fossa A (PFA) ependymomas are lethal malignancies of the hindbrain in infants and toddlers. Lacking highly recurrent somatic mutations, PFA ependymomas are proposed to be epigenetically driven tumors for which model systems are lacking. Here we demonstrate that PFA ependymomas are maintained under hypoxia, associated with restricted availability of specific metabolites to diminish histone methylation, and increase histone demethylation and acetylation at histone 3 lysine 27 (H3K27). PFA ependymomas initiate from a cell lineage in the first trimester of human development that resides in restricted oxygen. Unlike other ependymomas, transient exposure of PFA cells to ambient oxygen induces irreversible cellular toxicity. PFA tumors exhibit a low basal level of H3K27me3, and, paradoxically, inhibition of H3K27 methylation specifically disrupts PFA tumor growth. Targeting metabolism and/or the epigenome presents a unique opportunity for rational therapy for infants with PFA ependymoma. Hypoxia reprograms the cellular metabolome and epigenome to promote growth of the most lethal ependymomas.

Recurrent medulloblastoma and ependymoma are universally lethal, with no approved targeted therapies and few candidates presently under clinical evaluation. Nearly all recurrent medulloblastomas and posterior fossa group A (PFA) ependymomas are located adjacent to and bathed by the cerebrospinal fluid, presenting an opportunity for locoregional therapy, bypassing the blood–brain barrier. We identify three cell-surface targets, EPHA2, HER2 and interleukin 13 receptor α2, expressed on medulloblastomas and ependymomas, but not expressed in the normal developing brain. We validate intrathecal delivery of EPHA2, HER2 and interleukin 13 receptor α2 chimeric antigen receptor T cells as an effective treatment for primary, metastatic and recurrent group 3 medulloblastoma and PFA ependymoma xenografts in mouse models. Finally, we demonstrate that administration of these chimeric antigen receptor T cells into the cerebrospinal fluid, alone or in combination with azacytidine, is a highly effective therapy for multiple metastatic mouse models of group 3 medulloblastoma and PFA ependymoma, thereby providing a rationale for clinical trials of these approaches in humans.

© The Author(s), under exclusive licence to Springer Nature Limited 2019, In cancer, recurrent somatic single-nucleotide variants-which are rare in most paediatric cancers-are confined largely to protein-coding genes1-3. Here we report highly recurrent hotspot mutations (r.3A>G) of U1 spliceosomal small nuclear RNAs (snRNAs) in about 50% of Sonic hedgehog (SHH) medulloblastomas. These mutations were not present across other subgroups of medulloblastoma, and we identified these hotspot mutations in U1 snRNA in only

Brain tumours are the main cause of cancer-related death during childhood and medulloblastoma an aggressive embryonal tumour that arises in the posterior fossa – is the most common malignant tumour in this age group. Chemotherapy is a cornerstone of the postsurgical treatment, particularly in younger children in whom craniospinal irradiation is omitted due to the devastating side effects in the developing brain. Medulloblastoma often progresses or recurs after chemotherapy with a dismal prognosis. We used the Sleeping Beauty (SB) transposon-driven Ptch+/−/Math1-SB11/T2Onc sonic hedgehog (SHH) medulloblastoma murine model as a functional genomic tool to perform a genome-wide screen and identify genes and pathways that promote resistance to chemotherapy. After sub-total resection of the primary tumours, the mice were treated with repeated cycles of chemotherapy (cisplatin 5 mg/kg IP once on day 1 followed by cyclophosphamide 150 mg/kg IP daily from day 2 – 5) every 2 weeks for up to 3 cycles and monitored for tumour recurrence. The primary tumours (pre-treatment) and the tumours and metastasis that regrew after chemotherapy were deep sequenced to determine the transposon insertion sites. We identified recurrence-specific clonally selected insertions that promoted tumour growth despite therapy, including p53 (recurrently mutated in human tumours at relapse) and several other genes involved in DNA repair. Using cerebellar orthotopic models of p53-mutated SHH medulloblastoma, we observed a significant improvement in survival when the ATM inhibitor AZ32 was added to the chemotherapy backbone. This provides a rationale for developing therapeutic approaches targeting DNA repair in combination with conventional chemotherapy to prevent chemoresistance and medulloblastoma recurrence.

Study of the origin and development of cerebellar tumours has been hampered by the complexity and heterogeneity of cerebellar cells that change over the course of development. Here we use single-cell transcriptomics to study more than 60,000 cells from the developing mouse cerebellum and show that different molecular subgroups of childhood cerebellar tumours mirror the transcription of cells from distinct, temporally restricted cerebellar lineages. The Sonic Hedgehog medulloblastoma subgroup transcriptionally mirrors the granule cell hierarchy as expected, while group 3 medulloblastoma resembles Nestin+ stem cells, group 4 medulloblastoma resembles unipolar brush cells, and PFA/PFB ependymoma and cerebellar pilocytic astrocytoma resemble the prenatal gliogenic progenitor cells. Furthermore, single-cell transcriptomics of human childhood cerebellar tumours demonstrates that many bulk tumours contain a mixed population of cells with divergent differentiation. Our data highlight cerebellar tumours as a disorder of early brain development and provide a proximate explanation for the peak incidence of cerebellar tumours in early childhood. Sequencing data from the developing cerebellum are compared with bulk sequencing data from paediatric tumours, providing insights into their potential origins and suggesting that many cerebellar tumours have their origins early in utero.

SummaryThe study of the origin and development of cerebellar tumours 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 Group 3 medulloblastoma resemble Nestin+ve stem cells, Group 4 medulloblastomas resemble unipolar brush cells, and PFA/PFB ependymoma and cerebellar pilocytic astrocytoma resemble the prenatal 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.

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

While the preponderance of morbidity and mortality in medulloblastoma patients are due to metastatic disease, most research focuses on the primary tumor due to a dearth of metastatic tissue samples and model systems. Medulloblastoma metastases are found almost exclusively on the leptomeningeal surface of the brain and spinal cord; dissemination is therefore thought to occur through shedding of primary tumor cells into the cerebrospinal fluid followed by distal re-implantation on the leptomeninges. We present evidence for medulloblastoma circulating tumor cells (CTCs) in therapy-naive patients and demonstrate in vivo, through flank xenografting and parabiosis, that medulloblastoma CTCs can spread through the blood to the leptomeningeal space to form leptomeningeal metastases. Medulloblastoma leptomeningeal metastases express high levels of the chemokine CCL2, and expression of CCL2 in medulloblastoma in vivo is sufficient to drive leptomeningeal dissemination. Hematogenous dissemination of medulloblastoma offers a new opportunity to diagnose and treat lethal disseminated medulloblastoma. In addition to disseminating through the cerebrospinal fluid, medulloblastoma can metastasize through circulating tumor cells in the blood, thereby providing a different angle for clinical diagnosis and treatment development.

While medulloblastoma, a pediatric tumor of the cerebellum, is characterized by aberrations in developmental pathways, the majority of genetic determinants remain unknown. An unbiased Sleeping Beauty transposon screen revealed MyoD as a putative medulloblastoma tumor suppressor. This was unexpected, as MyoD is a muscle differentiation factor and not previously known to be expressed in cerebellum or medulloblastoma. In response to deletion of one allele of MyoD, two other Sonic hedgehog-driven mouse medulloblastoma models showed accelerated tumor formation and death, confirming MyoD as a tumor suppressor in these models. In normal cerebellum, MyoD was expressed in the proliferating granule neuron progenitors that are thought to be precursors to medulloblastoma. Similar to some other tumor suppressors that are induced in cancer, MyoD was expressed in proliferating medulloblastoma cells in three mouse models and in human medulloblastoma cases. This suggests that although expression of MyoD in a proliferating tumor is insufficient to prevent tumor progression, its expression in the cerebellum hinders medulloblastoma genesis. Cancer Res; 73(22); 6828–37. ©2013 AACR.

Medulloblastomas are malignant brain tumors that arise in the cerebellum in children and disseminate via the cerebrospinal fluid to the leptomeningeal spaces of the brain and spinal cord. Challenged by the poor prognosis for patients with metastatic dissemination, pediatric oncologists have developed aggressive treatment protocols, combining surgery, craniospinal radiation, and high-dose chemotherapy, that often cause disabling neurotoxic effects in long-term survivors. Insights into the genetic control of medulloblastoma dissemination have come from transposon insertion mutagenesis studies. Mobilizing the Sleeping Beauty transposon in cerebellar neural progenitor cells caused widespread dissemination of typically nonmetastatic medulloblastomas in Patched+/− mice, in which Shh signaling is hyperactive. Candidate metastasis genes were identified by sequencing the insertion sites and then mapping these sequences back to the mouse genome. To determine whether genes located at transposon insertion sites directly caused medulloblastomas to disseminate, we overexpressed candidate genes in Nestin+ neural progenitors in the cerebella of mice by retroviral transfer in combination with Shh. We show here that ectopic expression of Eras, Lhx1, Ccrk, and Akt shifted the in vivo growth characteristics of Shh-induced medulloblastomas from a localized pattern to a disseminated pattern in which tumor cells seeded the leptomeningeal spaces of the brain and spinal cord. Cancer Res; 72(19); 4944–53. ©2012 AACR.

Metastatic medulloblastoma is a high-risk disease, because the majority of children dying of medulloblastoma expire from metastases rather than recurrence of the primary tumor. Current dogma dictates that medulloblastoma cells are shed from the primary tumor into the CSF, float through the spinal fluid and establish metastatic colonies on the leptomeningeal surface and spinal cord. Empirical evidence for this mechanism is lacking in the literature. Whole genome sequencing on matched trios of human primary tumor, metastases, and peripheral blood showed somatic mutations from the medulloblastoma at low clonal frequency in the blood samples — consistent with circulating tumor cells. In addition, we identified circulating tumor cells (CTC) in the blood of transgenic mice bearing metastatic medulloblastoma, and in mice intra-cranially implanted with patient derived MB. Surprisingly, when we implanted human or mouse medulloblastoma cells in the flank of immunocompromised mice, the MB cells colonized the leptomeningeal space causing death. As these MB xenografts are not in continuity with the CSF, they must have arisen through hematogenous dissemination. Importantly, flow cytometry shows that MB CTCs express CD34, the cell-surface marker used clinically in children to collect peripheral blood stem cells; suggesting that autologous bone marrow transplants from children with medulloblastoma are likely contaminated with CTCs. Finally we established a parabiosis model where two immunocompromised, sibling mice are surgically joined, and a common blood circulation is established. After parabiosis, one mouse is intra-cranially implanted with MB, and the mice are subsequently separated when the xenografted twin becomes symptomatic. The surviving twin goes on to die from leptomeningeal metastases. Our data challenge the existing paradigm of CSF mediated dissemination of medulloblastoma by demonstrating the capacity of medulloblastoma to spread through the blood system. Identification of a systemic route for MB metastases offers new avenues for the diagnosis and therapy of metastatic medulloblastoma.

Tissue homeostasis in normal prostate and two-compartment nonmalignant prostate tumors depends on harmonious two-way communications between epithelial and stromal compartments. Within the fibroblast growth factor (FGF) family, signaling to an epithelial cell-specific FGF receptor (FGFR) 2IIIb-heparan sulfate complex from stromal-specific FGF7 and FGF10 delivers directionally specific instruction from stroma to epithelium without autocrine interference. Using a two-compartment transplantable prostate tumor model in which survival of stromal cells in vivo depends on epithelial cells, we show that signaling from epithelial FGF9 to stromal FGFR3 potentially mediates epithelial-to-stromal communication that also is directionally specific. FGF9 mRNA was expressed exclusively in the epithelial cells derived from well-differentiated, two-compartment Dunning R3327 rat prostate tumors. In contrast, FGFR3 was expressed at functionally significant levels only in the derived stromal cells. Competition binding and immunoprecipitation assays revealed that FGF9 only bound to an FGFR on the stromal cells. FGF9 also failed to covalently cross-link to clonal lines of stromal cells devoid of FGFR3 that expressed FGFR1 and FGFR2IIIc. Furthermore, FGF9 specifically stimulated DNA synthesis in stromal cells expressing FGFR3. These results demonstrate a directionally specific paracrine signaling from epithelial FGF9 and stromal FGFR3. Similar to the FGF7/FGF10 to FGFR2IIIb signaling from the stroma to the epithelium, the directional specificity from epithelium to stroma appears set by a combination of cell-specific expression of isoforms and cell-context specificity of FGFR isotypes for FGF.

BACKGROUND: (blind field). METHODS: Expression profiling, molecular subgrouping and analysis of somatic copy number alterations were conducted on multiple independent cohorts of patient tumour samples to examine intermediates of the MET signaling pathway in medulloblastoma. To examine the in vitro and in vivo effects of foretinib treatment; MET signalling biochemical analysis; migration and invasion assays; and foretinib pharmacokinetic studies were performed. Medulloblastoma xenografts and transgenic mouse models were used to evaluate foretinib treatment in vivo. RESULTS: We analyzed three large non-overlapping cohorts of medulloblastoma patients (discovery cohort, n = 199; validation cohort 1, n = 439; validation cohort 2, n = 285) and demonstrated that cMET, known to be involved in tumor progression and dissemination, is a marker of sonic hedgehog (SHH) medulloblastoma. Importantly, immunohistochemical analysis of activated cMET (phosphorylated cMET) in another independent patient cohort (n = 385) revealed that cMET activation correlates with increased tumor relapse and a poor survival in pediatric patients with SHH medulloblastomas, thus defining a subset of patients that may benefit from cMET targeted therapy. We show that foretinib, an FDA approved inhibitor of cMET, suppresses cMET activation, decreases proliferation and induces apoptosis, both in medulloblastoma cell lines and in SHH medulloblastoma xenografts. Furthermore foretinib penetrates the blood-brain barrier and is effective both in the primary and in the metastatic compartments. Treatment of mouse xenografts and of an aggressive transgenic model of metastatic SHH medulloblastoma with foretinib reduced primary medulloblastoma growth, decreased the incidence of metastases by 36% and increased survival by 45%. CONCLUSIONS: Our results provide strong rationale for advancing foretinib into clinical trials for SHH-driven medulloblastomas. SECONDARY CATEGORY: Tumor Biology.

Sleeping Beauty transposon mutagenesis in Shh medulloblastomas enabled us to identify that Pten was a common insertion site in both primary tumor and matched spinal metastases, suggesting PI3 kinase pathway is important for the progression of Shh medulloblastoma, in particular, for its metastasis. And PI3K-addiction could be one of mechanisms for the resistance to SHH signaling inhibitors seen in SHH medulloblastoma patients. To dissect the role of PI3 kinase pathway during the development of metastatic Shh medulloblastoma, and to establish an in vivo model for pre-clinical trials for PI3K pathway inhibitors on metastatic SHH medulloblastoma, we recently generate a novel targeted mutant mouse model Rosa-Tetoff-lsl-Pik3CAH1047R –t2a-Fluc. In this mouse model, a Dox-off and Cre-LoxP dual system mediates the spatial-temporal specific expression of Pik3CAH1047R, and the tumor cells could be visualized by bioluminescent imaging (BLI). We have crossed Rosa-Tetoff-lsl-Pik3CAH1047R –t2a-Fluc mouse with Math1Cre and Ptc heterozygous deletion mice to generate Pik3CAH1047R/Math1Cre/Ptc triple mice. Preliminary results showed that in the absence of Doxycycline the triple mice developed massive metastases at the frontal brain and spinal cord. More mice are generated and aged for detail experiments. In summary, we established a novel mouse model to express Pik3CAH1047R in a spatial-temporal specific manner, and the mutant Pik3CA greatly promoted Shh medulloblastoma metastasis to the spinal cord and frontal brain.

Ependymomas are common childhood brain tumours that occur throughout the nervous system, but are most common in the paediatric hindbrain. Current standard therapy comprises surgery and radiation, but not cytotoxic chemotherapy as it does not further increase survival. Whole-genome and whole-exome sequencing of 47 hindbrain ependymomas reveals an extremely low mutation rate, and zero significant recurrent somatic single nucleotide variants. Although devoid of recurrent single nucleotide variants and focal copy number aberrations, poor-prognosis hindbrain ependymomas exhibit a CpG island methylator phenotype. Transcriptional silencing driven by CpG methylation converges exclusively on targets of the Polycomb repressive complex 2 which represses expression of differentiation genes through trimethylation of H3K27. CpG island methylator phenotype-positive hindbrain ependymomas are responsive to clinical drugs that target either DNA or H3K27 methylation both in vitro and in vivo. We conclude that epigenetic modifiers are the first rational therapeutic candidates for this deadly malignancy, which is epigenetically deregulated but genetically bland.

Purpose Medulloblastoma comprises four distinct molecular subgroups: WNT, SHH, Group 3, and Group 4. Current medulloblastoma protocols stratify patients based on clinical features: patient age, metastatic stage, extent of resection, and histologic variant. Stark prognostic and genetic differences among the four subgroups suggest that subgroup-specific molecular biomarkers could improve patient prognostication. Patients and Methods Molecular biomarkers were identified from a discovery set of 673 medulloblastomas from 43 cities around the world. Combined risk stratification models were designed based on clinical and cytogenetic biomarkers identified by multivariable Cox proportional hazards analyses. Identified biomarkers were tested using fluorescent in situ hybridization (FISH) on a nonoverlapping medulloblastoma tissue microarray (n = 453), with subsequent validation of the risk stratification models. Results Subgroup information improves the predictive accuracy of a multivariable survival model compared with clinical biomarkers alone. Most previously published cytogenetic biomarkers are only prognostic within a single medulloblastoma subgroup. Profiling six FISH biomarkers (GLI2, MYC, chromosome 11 [chr11], chr14, 17p, and 17q) on formalin-fixed paraffin-embedded tissues, we can reliably and reproducibly identify very low-risk and very high-risk patients within SHH, Group 3, and Group 4 medulloblastomas. Conclusion Combining subgroup and cytogenetic biomarkers with established clinical biomarkers substantially improves patient prognostication, even in the context of heterogeneous clinical therapies. The prognostic significance of most molecular biomarkers is restricted to a specific subgroup. We have identified a small panel of cytogenetic biomarkers that reliably identifies very high-risk and very low-risk groups of patients, making it an excellent tool for selecting patients for therapy intensification and therapy de-escalation in future clinical trials.

Medulloblastoma, the most common malignant paediatric brain tumour, arises in the cerebellum and disseminates through the cerebrospinal fluid in the leptomeningeal space to coat the brain and spinal cord(1). Dissemination, a marker of poor prognosis, is found in up to 40% of children at diagnosis and inmost children at the time of recurrence. Affected children therefore are treated with radiation to the entire developing brain and spinal cord, followed by high-dose chemotherapy, with the ensuing deleterious effects on the developing nervous system(2). The mechanisms of dissemination through the cerebrospinal fluid are poorly studied, and medulloblastoma metastases have been assumed to be biologically similar to the primary tumour(3,4). Here we show that in both mouse and human medulloblastoma, the metastases from an individual are extremely similar to each other but are divergent from the matched primary tumour. Clonal genetic events in the metastases can be demonstrated in a restricted subclone of the primary tumour, suggesting that only rare cells within the primary tumour have the ability to metastasize. Failure to account for the bicompartmental nature of metastatic medulloblastoma could be a major barrier to the development of effective targeted therapies

Evolution of unresponsiveness to homeostasis-promoting signals from the microenvironment is a hallmark of malignant tumor cells. In Dunning R3327 model rat prostate tumors that are comprised of distinct stromal and epithelial compartments, progression from non-malignant, androgen-responsive tumors to malignancy is characterized by loss of compartmentation coincident with a loss of resident epithelial cell FGFR2IIIb that receives instructive signals from stromal FGF7 and FGF10. Restoration of FGFR2IIIb to malignant tumor cells restores responsiveness to stromal cells, restores distinct stromal and epithelial compartments, and retards malignant progression. Cultured stromal cells from two-compartment tumors are comprised of smooth muscle α-actin-positive cells that express predominantly FGFR3 and fibroblast-like cells devoid of α-actin and FGFR3. Here, we show that it is primarily the smooth muscle cell-like α-actin-expressing stromal cells that survive, morphologically differentiate, and delay tumor incidence and size in the presence of malignant cells in which FGFR2IIIb has been restored. Expression of FGFR3 by transfection in the fibroblast-like stromal cells conferred ability to respond similar to the smooth muscle cell-like stromal cells in which FGFR3 is normally resident. These results highlight the importance of the two-way communication back and forth between stroma and epithelium that is mediated by signaling within the FGFR family during progression to malignancy.

Recurrence of the childhood brain cancer medulloblastoma is almost universally fatal. The current strategy for clinical trials is to test novel therapies at the time of recurrence, using targets discovered at diagnosis. To define the relationship between naïve and post-treatment medulloblastoma, we developed a functional genomic transposon-driven mouse-model. Deep sequencing of the commonly inserted genes in diagnostic and recurrent tumors revealed dramatic divergence post-therapy, with recurrences demonstrating a convergence on the TP53 pathway. Loss of function insertions in Tp53 are clonal in the recurrence, result in loss of expression of the Tp53 target gene P21, and arise through selection of minor subclones present before therapy. The majority of insertions identified at diagnosis are no longer present at recurrence, and therefore represent futile targets for therapy. To validate these findings, we studied 36 pairs of human primary/recurrent tumors by whole genome sequencing. When comparing SNVs, indels or structural aberrations, we found minimal genetic overlap between the dominant clones at diagnosis versus recurrence. Recurrent tumors have more genomic aberrations than their paired diagnostic sample, and commonly display alterations in the TP53 (SHH), or the TP53 and TGF-β pathways (Group 3/4 tumors). Clonal dynamics between primary and recurrent tumors was measured using ultra-deep sequencing (1,500X). Overall, recurrent tumors share little genetic identity with their diagnostic tumor, arise through clonal selection, target specific signaling pathways to avoid cell death, and assume clinical dominance after therapy. Our findings undermine the key assumption that diagnostic medulloblastoma samples provide rational targets for therapy in recurrent disease, and provide a simple reason for the failure of prior trials of targeted therapy. Repeated genetic convergence on specific signaling pathways at the time of recurrence suggests that it may be possible to develop ‘anticipatory therapy’ delivered up front to prevent the later emergence of minor resistant clones driving recurrence.

We used high-resolution SNP genotyping to identify regions of genomic gain and loss in the genomes of 212 medulloblastomas, malignant pediatric brain tumors. We found focal amplifications of 15 known oncogenes and focal deletions of 20 known tumor suppressor genes (TSG), most not previously implicated in medulloblastoma. Notably, we identified previously unknown amplifications and homozygous deletions, including recurrent, mutually exclusive, highly focal genetic events in genes targeting histone lysine methylation, particularly that of histone 3, lysine 9 (H3K9). Post-translational modification of histone proteins is critical for regulation of gene expression, can participate in determination of stem cell fates and has been implicated in carcinogenesis. Consistent with our genetic data, restoration of expression of genes controlling H3K9 methylation greatly diminishes proliferation of medulloblastoma in vitro. Copy number aberrations of genes with critical roles in writing, reading, removing and blocking the state of histone lysine methylation, particularly at H3K9, suggest that defective control of the histone code contributes to the pathogenesis of medulloblastoma.

Human medulloblastoma (MB) consists of four molecular subgroups. In spite of enormous effort majority of current MB mouse models are SHH subgroup, represented by Patched heterozygous mice (designated as SHH-A). GTML MB is the only published spontaneous Group3 MB mouse model (designated as G3), which was induced by expressing MycN under the regulation of GLT1 promoter. By using Sleeping Beauty transposon system we have created and published two SHH mouse models: Math1SB11/T2Onc/Ptch+/- (designated as SHH-B) and Math1SB11/T2Onc/P53mut (designated as SHH-C). To dissect the molecular mechanism of SHH and Group3 MB during tumor evolution (initiation, maintenance and maturation), we profiled transcriptomes on SHH-A, SHH-B, SHH-C and G3 MBs along with normal mouse cerebellum (CB) of different developmental stage (E15, E18, P0, P60, P95 and P122). Fetal and/or adult human CBs are routinely used as normal controls for human pediatric MBs. One major advantage of profiling mouse tumor samples over human tumor samples is the availability of age-matched normal mouse CB counterparts. This is particularly true in case of developmental diseases such as pediatric MBs. Indeed, our study showed that transcriptomes of normal mouse CB from E15, E18 and P0 (designated as EdCB, representing early developmental CB), are similar to each other but distinct from mouse CB at P60, P95 and P122 (designated as LdCB, representing late developmental CB). Importantly P60 to P120 are the ages at which most of spontaneous MBs are harvested from a tumor-bearing mouse. We therefore analyzed global gene expression level of MBs and EdCB in comparison with LdCB, the mouse MB's normal counterparts. As a developmental disease, MB is presumably initiated from a single cell or a single cell clone that is transformed at an early developmental stage of a normal CB. In fact, it is well known that a SHH MB is initiated from transformation of granule cell precursors at EGL, the external granule layer. However, it is unclear about the initiation of Group3 MB. In this study we showed that compared to a normal LdCB, a particular MB transriptome harbors four major types of abnormally expressed genes: 1. Genes not only specific to one subgroup but also shared with EdCB; representing tumor initiation events. 2. Genes specific to a subgroup only; representing subgroup-specific tumor promotion events. 3. Genes shared not only by both subgroups but also by EdCB, representing non-subgroup specific tumor maintenance events; 4. Genes shared by both subgroups only; representing non-subgroup specific late tumor maturation events. We confirmed that SHH MBs from SHH-A, B, and C are all initiated at Math1/Atoh1 expressing cells, which are further transformed by up-regulation of Olig3 and MycN among other genes. Rxrg-expressing cells are identified as Group3 MB initiating cells, which are further transformed by over-expression of Crx (Otx3) along with other genes such as Otx2, and down-regulation of cerebellum-specific Zic1 and others such as Lgi1. Both subgroups of MBs may be maintained and mature as genes associated with cell cycle, mitosis and chromosome integrity, such as KIFs, CDK1, PLK1/4, AURKA/B, CENP family and MCMs are abnormally overexpressed. Our study reveals distinct molecular mechanisms underlying tumor evolution of SHH and Group3 MBs. We propose a MB evolution model, illustrating how a normal cell at specific cell of origin can be transformed to a fully developed subgroup specific MB. Furthermore, our results demonstrate that despite the fact that they are initiated from distinct cell of origin, different subgroup of MBs do share common abnormal molecular events for tumor maintenance and maturation, suggesting that subgroup specific target therapies alone may be not as effective as their combination with therapies targeting abnormalities common to different subgroup of MBs. Citation Format: Xiaochong Wu, Adrian Dubuc, William A. Weiss, Robert J. Wechsler-Reya, Michael D. Taylor. Transcriptome analysis on tumor and its normal cerebellar counterpart from mouse models reveals molecular mechanisms underlying evolution of SHH and Group3 medulloblastoma. [abstract]. In: Proceedings of the AACR Special Conference: Advances in Brain Cancer Research; May 27-30, 2015; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2015;75(23 Suppl):Abstract nr A46.

Medulloblastoma (MB) is the most common malignant brain tumor in children. Current treatment includes surgery, craniospinal radiation, and high-dose cytotoxic chemotherapy. Despite these aggressive therapies, one-third of patients still succumb to their disease, and survivors suffer devastating side effects, including cognitive deficits, endocrine disorders, and increased incidence of secondary cancers later in life. More effective and less toxic therapies are desperately needed for MB.

Ependymoma is the third most common pediatric brain tumor and remains incurable in 45% of patients. It arises in the spinal cord, supratentorial brain, and most commonly in children, the posterior fossa (PF). We recently reported the identification of two molecularly and clinically distinct subgroups of PF ependymoma, which we named Group A and B. While patients with Group B tumors harbor a large number of gross chromosomal gains and losses (approx. 17 arm events per tumor) and have favorable prognoses (5 year PFS = 92%), patients with Group A tumors have balanced genomic profiles (approx. 1 arm event per tumor) with poor clinical outcomes (5 year PFS = 24%). We hypothesized that aberrant DNA methylation could be a mechanism driving the tumorigenesis of Group A PF ependymoma. To this end, we isolated methylated DNA in 92 ependymomas by Methyl Binding Domain 2 protein assisted recovery, and hybridized enriched DNA to promoter tiling arrays (Nimblegen). Using unsupervised hierarchical clustering we determined that the DNA methylation profiles of ependymoma were regionally specified, dividing tumors into subgroups according to their anatomical origin. Using both gene expression and DNA methylation platforms, we identified a subset of PF ependymoma, which clustered with spinal tumors, supporting the vast molecular differences between Group A and B PF ependymoma. We next compared the number of methylated genes identified in Group A versus B, and observed that Group A tumors exhibited a greater number of methylated genes at specific CpG islands, a feature described as a CpG island methylator phenotype (CIMP) in glioma, colon cancer, and breast cancer. We validated these findings in a non-overlapping cohort of 48 PF ependymomas, analyzed using a different array technology (Illumina Infinium 450K). Using various unsupervised clustering methods (HCL, K-MEANS, NMF, and SOM), we verified that Group A and B exhibited highly distinct DNA methylation profiles. Further, we confirmed that Group A tumours were defined by a greater overall number of methylated genes (A: 855, B: 233; Wilcoxon-Rank Sum Test), and a greater number of methylated genes per tumour (A: 511, B: 425; Wilcoxon-Rank Sum Test). We performed Gene Set Enrichment analysis and observed that many genes methylated in Group A exhibited a significant overlap with genes marked by the polycomb repressor (PRC2) complex in embryonic stem cells (p Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1428. doi:1538-7445.AM2012-1428

Medulloblastoma metastasizes through the CSF in the leptomeningeal space to cover the brain and spinal cord. Mechanisms driving metastasis are currently unknown. It had been assumed that the primary tumor and its metastases share very similar biology. The Sleeping Beauty (SB) system is a novel functional genomics tool for cancer gene discovery. Ptch +/− mice develop localized medulloblastoma in 15% of cases, while ≈100% of Math1-SB11, SB transposon donor, Ptch +/− mice develop metastatic medulloblastoma. We sequenced over 158,000 insertion sites (Roche 454) from a series of >140 SB-induced primary medulloblastomas, as well as matched spinal and frontal lobe leptomeningeal metastases. Statistical analysis identified 359 commonly inserted genes (CIGs) in the primary tumors and 285 CIGs in the leptomeningeal metastases. Although primary tumors and their metastases always demonstrate a common transformed ancestor through sharing of identical clonal insertion sites, less than 10% of CIGs were found in both primary tumor and matched metastases. Matched spinal and frontal lobe metastases shared identical clonal insertions that were very highly subclonal in the primary tumor, suggesting that leptomeningeal dissemination arises only once, or from a single small subclonal population in the primary tumor. These data support the clonal selection model of metastasis, and suggest that MET-CIG insertions are acting as metastasis virulence genes. Similarity between metastases supports a model in which medulloblastoma is a bicompartmental disease (primary vs. metastases) that arises through parallel evolution in the two compartments. We validated our mouse model through copy number profiling of three matched trios of human medullolblastoma (primary and two metastases). In each case we demonstrate highly clonal regions of chromosomal gain/loss that are present in both metastatic samples, but are not apparent in the matched primary tumor. Similarly, profiling of promotor CpG island methylation in human primary medulloblastomas and matched metastases shows that within a given child the metastases are very similar to each other, but distinct from the primary tumor. PCR amplification was used to demonstrate a clonal 179 Kb deletion in a pair of human metastases, which was present in an extremely small subclone of the primary tumor, strongly supporting the clonal selection model during dissemination of human medulloblastoma. Our results support a model in which individual medulloblastomas metastases are similar to each other, but distinct from the primary tumor. This ‘bicompartmental model’ suggests a proximate reason for failure of current therapies, and that future clinical trials may need to address each compartment individually. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4350.

Leptomeningeal dissemination (LMD), the metastatic spread of tumor cells via the cerebrospinal fluid to the brain and spinal cord, is an ominous prognostic sign for patients with the pediatric brain tumor medulloblastoma. The need to reduce the risk of LMD has driven the development of aggressive treatment regimens, which cause disabling neurotoxic side effects in long-term survivors. Transposon-mediated mutagenesis studies in mice have revealed numerous candidate metastasis genes. Understanding how these genes drive LMD will require functional assessment using in vivo and cell culture models of medulloblastoma. We analyzed two genes that were sites of frequent transposon insertion and highly expressed in human medulloblastomas: Arnt (aryl hydrocarbon receptor nuclear translocator) and Gdi2 (GDP dissociation inhibitor 2). Here we show that ectopic expression of Arnt and Gdi2 promoted LMD in mice bearing Sonic hedgehog (Shh)-induced medulloblastomas. We overexpressed Arnt and Gdi2 in a human medulloblastoma cell line (DAOY) and an immortalized, nontransformed cell line derived from mouse granule neuron precursors (SHH-NPD) and quantified migration, invasiveness, and anchorage-independent growth, cell traits that are associated with metastatic competence in carcinomas. In SHH-NPD cells. Arnt and Gdi2 stimulated all three traits. In DAOY cells, Arnt had the same effects, but Gdi2 stimulated invasiveness only. These results support a mechanism whereby Arnt and Gdi2 cause cells to detach from the primary tumor mass by increasing cell motility and invasiveness. By conferring to tumor cells the ability to proliferate without surface attachment, Arnt and Gdi2 favor the formation of stable colonies of cells capable of seeding the leptomeninges. Electronic supplementary material The online version of this article (doi:10.1186/s40478-014-0085-y) contains supplementary material, which is available to authorized users.