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While molecular subgrouping has revolutionized medulloblastoma classification, the extent of heterogeneity within subgroups is unknown. Similarity network fusion (SNF) applied to genome-wide DNA methylation and gene expression data across 763 primary samples identifies very homogeneous clusters of patients, supporting the presence of medulloblastoma subtypes. After integration of somatic copy-number alterations, and clinical features specific to each cluster, we identify 12 different subtypes of medulloblastoma. Integrative analysis using SNF further delineates group 3 from group 4 medulloblastoma, which is not as readily apparent through analyses of individual data types. Two clear subtypes of infants with Sonic Hedgehog medulloblastoma with disparate outcomes and biology are identified. Medulloblastoma subtypes identified through integrative clustering have important implications for stratification of future clinical trials., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Background: Patients with incomplete surgical resection of medulloblastoma are controversially regarded as having a marker of high-risk disease, which leads to patients undergoing aggressive surgical resections, so-called second-look surgeries, and intensified chemoradiotherapy. All previous studies assessing the clinical importance of extent of resection have not accounted for molecular subgroup. We analysed the prognostic value of extent of resection in a subgroup-specific manner., Methods: We retrospectively identified patients who had a histological diagnosis of medulloblastoma and complete data about extent of resection and survival from centres participating in the Medulloblastoma Advanced Genomics International Consortium. We collected from resections done between April, 1997, and February, 2013, at 35 international institutions. We established medulloblastoma subgroup affiliation by gene expression profiling on frozen or formalin-fixed paraffin-embedded tissues. We classified extent of resection on the basis of postoperative imaging as gross total resection (no residual tumour), near-total resection (<1·5 cm(2) tumour remaining), or sub-total resection (≥1·5 cm(2) tumour remaining). We did multivariable analyses of overall survival and progression-free survival using the variables molecular subgroup (WNT, SHH, group 4, and group 3), age (<3 vs ≥3 years old), metastatic status (metastases vs no metastases), geographical location of therapy (North America/Australia vs rest of the world), receipt of chemotherapy (yes vs no) and receipt of craniospinal irradiation (<30 Gy or >30 Gy vs no craniospinal irradiation). The primary analysis outcome was the effect of extent of resection by molecular subgroup and the effects of other clinical variables on overall and progression-free survival., Findings: We included 787 patients with medulloblastoma (86 with WNT tumours, 242 with SHH tumours, 163 with group 3 tumours, and 296 with group 4 tumours) in our multivariable Cox models of progression-free and overall survival. We found that the prognostic benefit of increased extent of resection for patients with medulloblastoma is attenuated after molecular subgroup affiliation is taken into account. We identified a progression-free survival benefit for gross total resection over sub-total resection (hazard ratio [HR] 1·45, 95% CI 1·07-1·96, p=0·16) but no overall survival benefit (HR 1·23, 0·87-1·72, p=0·24). We saw no progression-free survival or overall survival benefit for gross total resection compared with near-total resection (HR 1·05, 0·71-1·53, p=0·8158 for progression-free survival and HR 1·14, 0·75-1·72, p=0·55 for overall survival). No significant survival benefit existed for greater extent of resection for patients with WNT, SHH, or group 3 tumours (HR 1·03, 0·67-1·58, p=0·89 for sub-total resection vs gross total resection). For patients with group 4 tumours, gross total resection conferred a benefit to progression-free survival compared with sub-total resection (HR 1·97, 1·22-3·17, p=0·0056), especially for those with metastatic disease (HR 2·22, 1·00-4·93, p=0·050). However, gross total resection had no effect on overall survival compared with sub-total resection in patients with group 4 tumours (HR 1·67, 0·93-2·99, p=0·084)., Interpretation: The prognostic benefit of increased extent of resection for patients with medulloblastoma is attenuated after molecular subgroup affiliation is taken into account. Although maximum safe surgical resection should remain the standard of care, surgical removal of small residual portions of medulloblastoma is not recommended when the likelihood of neurological morbidity is high because there is no definitive benefit to gross total resection compared with near-total resection., Funding: Canadian Cancer Society Research Institute, Terry Fox Research Institute, Canadian Institutes of Health Research, National Institutes of Health, Pediatric Brain Tumor Foundation, and the Garron Family Chair in Childhood Cancer Research., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

The development of targeted anti-cancer therapies through the study of cancer genomes is intended to increase survival rates and decrease treatment-related toxicity. We treated a transposon-driven, functional genomic mouse model of medulloblastoma with 'humanized' in vivo therapy (microneurosurgical tumour resection followed by multi-fractionated, image-guided radiotherapy). Genetic events in recurrent murine medulloblastoma exhibit a very poor overlap with those in matched murine diagnostic samples (<5%). Whole-genome sequencing of 33 pairs of human diagnostic and post-therapy medulloblastomas demonstrated substantial genetic divergence of the dominant clone after therapy (<12% diagnostic events were retained at recurrence). In both mice and humans, the dominant clone at recurrence arose through clonal selection of a pre-existing minor clone present at diagnosis. Targeted therapy is unlikely to be effective in the absence of the target, therefore our results offer a simple, proximal, and remediable explanation for the failure of prior clinical trials of targeted therapy.

Genomic characterization of medulloblastoma has improved molecular risk classification but struggles to define functional biological processes, particularly for the most aggressive subgroups. We present here a novel proteomic approach to this problem using a reference library of stable isotope labeled medulloblastoma-specific proteins as a spike-in standard for accurate quantification of the tumor proteome. Utilizing high-resolution mass spectrometry, we quantified the tumor proteome of group 3 medulloblastoma cells and demonstrate that high-risk MYC amplified tumors can be segregated based on protein expression patterns. We cross-validated the differentially expressed protein candidates using an independent transcriptomic data set and further confirmed them in a separate cohort of medulloblastoma tissue samples to identify the most robust proteogenomic differences. Interestingly, highly expressed proteins associated with MYC-amplified tumors were significantly related to glycolytic metabolic pathways via alternative splicing of pyruvate kinase (PKM) by heterogeneous ribonucleoproteins (HNRNPs). Furthermore, when maintained under hypoxic conditions, these MYC-amplified tumors demonstrated increased viability compared to non-amplified tumors within the same subgroup. Taken together, these findings highlight the power of proteomics as an integrative platform to help prioritize genetic and molecular drivers of cancer biology and behavior.

Medulloblastoma is the most common malignant brain tumor of childhood. Novel therapeutic strategies are urgently needed to overcome cytotoxic resistance. We hypothesized that antiapoptotic signals contribute to resistance and that treatment with proapoptotic agents could increase the efficacy of conventional therapies. A PCR array was used to assess the status of the apoptotic signaling pathway in medulloblastoma cells after treatment with cytotoxic chemotherapy. Treatment with cisplatin led to the upregulation of antiapoptotic signals, including inhibitor of apoptosis proteins (IAP), in medulloblastoma cells. We subsequently investigated the synergistic effect of a small-molecule IAP inhibitor, LBW242, in combination with cisplatin and/or radiotherapy in three human medulloblastoma cell lines and 5 short term primary patient medulloblastoma cultures. The addition of LBW242 to chemotherapy resulted in significantly increased antitumor activity with a similar effect observed in combination with radiotherapy. Measurement of caspase-8 and -9 activity indicated that the synergy resulted from induction of both the intrinsic and extrinsic apoptotic pathways. Apoptosis was confirmed by Annexin V staining and activation of caspases 3/7. Xenograft models were used to evaluate the mechanism of action and efficacy in vivo. The combination therapy significantly reduced the tumor burden in a medulloblastoma xenograft model and TUNEL analysis in a medulloblastoma orthograft confirmed in vivo induction of apoptosis. These findings support the strategy of targeting IAPs in combination with cytotoxic therapy as a novel treatment strategy for patients with medulloblastoma.

Current treatment for medulloblastoma is successful in more than half of all cases but results in substantial disability in survivors. Accordingly, there is considerable interest in drugs that may target specific signaling pathways activated in the tumors, with inhibitors of both the Hedgehog and Notch pathways currently proposed as possible therapeutics. Here, we tested the hypothesis that Notch pathway inhibition in vivo may block the formation of Hedgehog-dependent medulloblastoma. We took the general approach of using a cre recombinase under the control of the GFAP promoter to generate medulloblastoma in mice carrying a conditional Ptc1 allele and introduced a conditional RBP-J allele to ablate canonical Notch signaling. Loss of RBP-J from the developing cerebellum led to a modest loss of stem cells and an overall developmental delay. These phenotypes could be partially compensated by activation of the Hedgehog pathway. Hedgehog-dependent medulloblastoma were not blocked by loss of RBP-J, indicating that canonical Notch signaling is not required for tumor initiation and growth in this model.

Toward the goal of generating a mouse medulloblastoma model with increased tumor incidence, we developed a homozygous version of our ND2:SmoA1 model. Medulloblastomas form in 94% of homozygous Smo/Smo mice by 2 months of age. Tumor formation is, thus, predictable by age, before the symptomatic appearance of larger lesions. This high incidence and early onset of tumors is ideal for preclinical studies because mice can be enrolled before symptom onset and with a greater latency period before late-stage disease. Smo/Smo tumors also display leptomeningeal dissemination of neoplastic cells to the brain and spine, which occurs in many human cases. Despite an extended proliferation of granule neuron precursors (GNP) in the postnatal external granular layer (EGL), the internal granular layer formed normally in Smo/Smo mice and tumor formation occurred only in localized foci on the superficial surface of the molecular layer. Thus, tumor formation is not simply the result of over proliferation of GNPs within the EGL. Moreover, Smo/Smo medulloblastomas were transplantable and serially passaged in vivo, demonstrating the aggressiveness of tumor cells and their transformation beyond a hyperplastic state. The Smo/Smo model is the first mouse medulloblastoma model to show leptomeningeal spread. The adherence to human pathology, high incidence, and early onset of tumors thus make Smo/Smo mice an efficient model for preclinical studies.

To develop a genetically faithful model of medulloblastoma with increased tumor incidence compared with the current best model we activated the Sonic Hedgehog (Shh) pathway by transgenically expressing a constitutively active form of Smoothened in mouse cerebellar granule neuron precursors (ND2:SmoA1 mice). This resulted in early cerebellar granule cell hyper-proliferation and a 48% incidence of medulloblastoma formation. Gene expression studies showed an increase in the known Shh targets Gli1 and Nmyc that correlated with increasing hyperplasia and tumor formation. Notch2 and the Notch target gene, HES5, were also significantly elevated in Smoothened-induced tumors showing that Shh pathway activation is sufficient to induce Notch pathway signaling. In human medulloblastomas reverse transcription-PCR for Shh and Notch targets revealed activation of both of these pathways in most tumors when compared with normal cerebellum. Notch pathway inhibition with soluble Delta ligand or gamma secretase inhibitors resulted in a marked reduction of viable cell numbers in medulloblastoma cell lines and primary tumor cultures. Treatment of mice with D283 medulloblastoma xenografts with a gamma secretase inhibitor resulted in decreased proliferation and increased apoptosis, confirming that Notch signaling contributes to human medulloblastoma proliferation and survival. Medulloblastomas in ND2:SmoA1 mice and humans have concomitant increase in Shh and Notch pathway activities, both of which contribute to tumor survival.

The mechanisms of retinoid activity in tumors remain largely unknown. Here we establish that retinoids cause extensive apoptosis of medulloblastoma cells. In a xenograft model, retinoids largely abrogated tumor growth. Using receptor-specific retinoid agonists, we defined a subset of mRNAs that were induced by all active retinoids in retinoid-sensitive cell lines. We also identified bone morphogenetic protein-2 (BMP-2) as a candidate mediator of retinoid activity. BMP-2 protein induced medulloblastoma cell apoptosis, whereas the BMP-2 antagonist noggin blocked both retinoid and BMP-2-induced apoptosis. BMP-2 also induced p38 mitogen-activated protein kinase (MAPK), which is necessary for BMP-2- and retinoid-induced apoptosis. Retinoid-resistant medulloblastoma cells underwent apoptosis when treated with BMP-2 or when cultured with retinoid-sensitive medulloblastoma cells. Retinoid-induced expression of BMP-2 is thus necessary and sufficient for apoptosis of retinoid-responsive cells, and expression of BMP-2 by retinoid-sensitive cells is sufficient to induce apoptosis in surrounding retinoid-resistant cells.

Constitutive Hedgehog (Hh) pathway activity is associated with initiation of neoplasia, but its role in the continued growth of established tumors is unclear. Here, we investigate the therapeutic efficacy of the Hh pathway antagonist cyclopamine in preclinical models of medulloblastoma, the most common malignant brain tumor in children. Cyclopamine treatment of murine medulloblastoma cells blocked proliferation in vitro and induced changes in gene expression consistent with initiation of neuronal differentiation and loss of neuronal stem cell-like character. This compound also caused regression of murine tumor allografts in vivo and induced rapid death of cells from freshly resected human medulloblastomas, but not from other brain tumors, thus establishing a specific role for Hh pathway activity in medulloblastoma growth.