Medulloblastoma Development Research Articles

Abstract 889: Metformin treatment inhibits proliferation and induces apoptosis in medulloblastoma cells

Abstract Medulloblastoma is the most common malignant brain tumor in children that typically requires a patient specific combination of surgical resection, chemotherapy, and radiation. Up to 80% of medulloblastoma patients respond to treatment and survive long-term. However, survivors often suffer from severe long term sequelae secondary to treatment that affects their neurocognitive and growth potential. Therefore, there is an urgency to develop less toxic, more focused treatment strategies that target specific pathways or molecules involved in medulloblastoma development and progression. Metformin is a widely used non-toxic anti-diabetic drug that has mild, generally well-tolerated side effects. Studies have shown that it can reduce cancer risk and improve the prognosis of certain malignancies. However, the mechanism underlying its anti-cancer effect is still unclear and varies depending on the type of cancer. There are reports that metformin can target the specificity protein 1 (Sp1) transcription factor. Sp1 regulates the expression of a variety of proteins involved in regulation of cancer cell proliferation including survivin, an Inhibitor of Apoptosis Protein family member. Expression of survivin is increased in medulloblastoma cells and often correlates with poor prognosis. We studied the anti-cancer activity of metformin on medulloblastoma using DAOY and D283 cells. Metformin treatment resulted in a dose and time dependent reduction in cell proliferation in both cell lines. This growth inhibition was accompanied by the induction of apoptosis as determined by annexin-V staining, PARP cleavage, and activation of caspase3/7. Metformin also induced cell cycle arrest in G0/G1 phase. Western blot analysis using extracts from cells treated with metformin revealed that the anti-proliferative and apoptotic effects of metformin in medulloblastoma were accompanied by a reduction in the expression of survivin protein. Overexpression of survivin is reported to be associated with chemo- and radio-resistance. This study suggests the use of metformin as a novel anticancer agent in combination with standard therapy for the treatment of medulloblastoma. Citation Format: Kristen C. Payne, Abigail Hunter, W Paul Bowman, Jeffrey C. Murray, Riyaz Basha, Umesh T. Sankpal. Metformin treatment inhibits proliferation and induces apoptosis in medulloblastoma cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 889.

Abstract 5092: Establishment of an induction protocol for medulloblastoma during prenatal stages of a floxed-cre inducible model

Abstract Advances in mouse genetics in the last decades have improved the site-specific and conditional control of specific genes in vivo. Despite the availability of different recombinases, the most popular and best-characterized model is the Cre-loxP system. To achieve the recombination in specific tissues or cell types, Cre expression is regulated by a cell-specific promoter and it is also possible to use a tamoxifen-inducible form of Cre for temporal control of the recombination process. Different models have been described using this technology. However, the recombination in the central nervous system tissues is still problematic. Here, we describe a pilot study to induce the recombination process in the brain at prenatal stages in a Cre-loxP system. To test the temporal control of the recombination in the brain, we used a Ptch1fl/fl mouse line in combination with the previously described Math1-creERT2 mouse line that can be induced by tamoxifen administration. All experimental procedures on animals, together with mouse breeding, were performed in accordance with the guidelines of the animal ethics committee of the Karolinska Institutet. The detection of the floxed allele and the cre gene was carried out by PCR at each breeding step. In order to control the specific time point of the induction, the presence of the males for mating was controlled for two days. After this, females were monitored and the induction of the model was carried out at E14.5 (embryonic days) for two days by oral administration of tamoxifen. Issues due to tamoxifen administrations as problems in delivery and mortality/toxicity in pups were not detected. The litters obtained were genotyped to detect the correct combination, Ptch1fl/fl; Math1creERT2 +/-. Mice with the correct genotype developed neurological symptoms between 7 and 9 weeks old, and once the brain was dissected, an abnormal size of the cerebellum was detected. Cerebellum was dissected and the cells obtained were grown and maintained in different specific culture media. The deleted allele of Ptch1 was detected by PCR in the tumoral tissue and in the cell cultures. Here, we described a protocol to induce a Cre-loxP system during embryonic stages. The present protocol avoids the problems described for extensive tamoxifen administration. We also described new culture media to grow and expand the tumor cells obtained. Our findings could help to improve the induction of this kind of systems and study mutations during embryonic stages related with the development and progression of medulloblastoma. The culture of cells derived from these systems, can be used to establish therapeutic mouse models and to evaluate novel treatment strategies for this tumor. Citation Format: Gabriel Gallo-Oller, Ani Azatyan, Ninib Baryawno, Per Kogner, John Inge Johnsen. Establishment of an induction protocol for medulloblastoma during prenatal stages of a floxed-cre inducible model [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5092.

The FDA approved PI3K inhibitor GDC-0941 enhances invitro the anti-neoplastic efficacy of Axitinib against c-myc-amplified high-risk medulloblastoma.

Aberrant receptor kinase signalling and tumour neovascularization are hallmarks of medulloblastoma development and are both considered valuable therapeutic targets. In addition to VEGFR1/2, expression of PDGFR α/β in particular has been documented as characteristic of metastatic disease correlating with poor prognosis. Therefore, we have been suggested that the clinically approved multi‐kinase angiogenesis inhibitor Axitinib, which specifically targets these kinases, might constitute a promising option for medulloblastoma treatment. Indeed, our results delineate anti‐neoplastic activity of Axitinib in medulloblastoma cell lines modelling the most aggressive c‐myc‐amplified Non‐WNT/Non‐SHH and SHH‐TP53‐mutated tumours. Exposure of medulloblastoma cell lines to Axitinib results in marked inhibition of proliferation and profound induction of cell death. The differential efficacy of Axitinib is in line with target expression of medulloblastoma cells identifying VEGFR 1/2, PDGFR α/β and c‐kit as potential markers for drug application. The high specificity of Axitinib and the consequential low impact on the haematopoietic and immune system render this drug ideal multi‐modal treatment approaches. In this context, we demonstrate that the clinically available PI3K inhibitor GDC‐0941 enhances the anti‐neoplastic efficacy of Axitinib against c‐myc‐amplified medulloblastoma. Our findings provide a rational to further evaluate Axitinib alone and in combination with other therapeutic agents for the treatment of most aggressive medulloblastoma subtypes.

Jak2-mediated phosphorylation of Atoh1 is critical for medulloblastoma growth

Treatment for medulloblastoma, the most common malignant brain tumor in children, remains limited to surgical resection, radiation, and traditional chemotherapy; with long-term survival as low as 50-60% for Sonic Hedgehog (Shh)-type medulloblastoma. We have shown that the transcription factor Atonal homologue 1 (Atoh1) is required for Shh-type medulloblastoma development in mice. To determine whether reducing either Atoh1 levels or activity in tumors after their development is beneficial, we studied Atoh1 dosage and modifications in Shh-type medulloblastoma. Heterozygosity of Atoh1 reduced tumor occurrence and prolonged survival. We discovered tyrosine 78 of Atoh1 is phosphorylated by a Jak2-mediated pathway only in tumor-initiating cells and in human SHH-type medulloblastoma. Phosphorylation of tyrosine 78 stabilizes Atoh1, increases Atoh1's transcriptional activity, and is independent of canonical Jak2 signaling. Importantly, inhibition of Jak2 impairs tyrosine 78 phosphorylation and tumor growth in vivo. Taken together, inhibiting Jak2-mediated tyrosine 78 phosphorylation could provide a viable therapy for medulloblastoma.

Abstract 1945: PPM1D/Wip1, promising new target in childhood cancers neuroblastoma and medulloblastoma

Abstract Background: The most common cytogenetic lesions in the embryonal neural tumors medulloblastoma (MB) and neuroblastoma (NB) affect chromosome 17, with 17q+ or isochromosome 17q, in approximately one-third of MB with these aberrations being a significant indicator of poor clinical outcome. Similarly, in NB gain of 17q is the most powerful genetic predictor of adverse clinical outcome. 17q+ correlates with poor survival in our population-based material where we found aberrations of chromosome 17 in 85% of primary neuroblastomas, specifically, gain of PPM1D/Wip1 at 17q23. Wip1 is a serine/threonine phosphatase encoded by the gene PPM1D, described as a gatekeeper in the Mdm2-p53 regulatory loop involved in genetic stability, inflammation and a potential oncogene contributing to carcinogenesis. Methods: Comparative genomic hybridization (CGH), immunostaining, mRNA arrays, qPCR, exome- and RNA-sequencing was used to examine PPM1D/Wip1 in neuroblastoma and medulloblastoma. Genetic and pharmacological inhibition was used to analyze the function of Wip1 in preclinical neuroblastoma and medulloblastoma models. Results: CGH-array analysis detected PPM1D/Wip1 extra copies in all tumors and cell lines containing 17q-gain. Expression arrays and immunostaining showed high expression of Wip1 in neuroblastoma corresponding to poor survival. RNA sequencing confirmed PPM1D-gain and revealed truncated isoforms with oncogenic potential. Exome-sequencing detected a mutation leading to constitutive PPM1D/Wip1 activation in an aggressive metastatic infant neuroblastoma. Wip1 knockdown experiments showed significant decrease of cell viability, proliferation and colony formation as well as substantial increase of DNA-damage response in neuroblastoma and medulloblastoma cells. Tumor neuroblastoma xenograft development was significantly delayed showing median tumor development (0.10 mL) to be more than doubled (median 15 days, vs. 33 days, p<0.001) after Wip1 downregulation compared to scrambled controls. A novel Wip1 inhibitor was highly potent in cytotoxic/cytostatic effect in neuroblastoma and medulloblastoma cell lines. Furthermore, this Wip1 inhibitor significantly inhibited growth of established human neuroblastomas and medulloblastomas in nude mice after treatment (P<0.01). Conclusions: Our results show that PPM1D/Wip1 is oncogenic in neuroblastoma and medulloblastoma development. We propose three different ways on how PPM1D/Wip1 is activated: due to chromosomal gain, alternative RNA-isoforms and/or DNA-mutation. PPM1D/Wip1 provides a novel therapeutic target in neuroblastoma and medulloblastoma. Citation Format: Jelena Milosevic, Nina Eissler, Diana Treis, Malin Wicktröm, Susanne Fransson, Baldur Sveinbjornsson, Ninib Baryawno, Subazini Kosalai, Chandrasekhar Kanduri, Kazuyasu Sakaguchi, Tommy Martinsson, John Inge Johnsen, Per Kogner. PPM1D/Wip1, promising new target in childhood cancers neuroblastoma and medulloblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1945. doi:10.1158/1538-7445.AM2017-1945

Design, Synthesis, and Structure-Activity Relationship of Tetrahydropyrido[4,3-d]pyrimidine Derivatives as Potent Smoothened Antagonists with in Vivo Activity.

Medulloblastoma is one of the most prevalent brain tumors in children. Aberrant hedgehog (Hh) pathway signaling is thought to be involved in the initiation and development of medulloblastoma. Vismodegib, the first FDA-approved cancer therapy based on inhibition of aberrant hedgehog signaling, targets smoothened (Smo), a G-protein coupled receptor (GPCR) central to the Hh pathway. Although vismodegib exhibits promising therapeutic efficacy in tumor treatment, concerns have been raised from its nonlinear pharmacokinetic (PK) profiles at high doses partly due to low aqueous solubility. Many patients experience adverse events such as muscle spasms and weight loss. In addition, drug resistance often arises among tumor cells during treatment with vismodegib. There is clearly an urgent need to explore novel Smo antagonists with improved potency and efficacy. Through a scaffold hopping strategy, we have identified a series of novel tetrahydropyrido[4,3-d]pyrimidine derivatives, which exhibited effective inhibition of Hh signaling. Among them, compound 24 is three times more potent than vismodegib in the NIH3T3-GRE-Luc reporter gene assay. Compound 24 has a lower melting point and much greater solubility compared with vismodegib, resulting in linear PK profiles when dosed orally at 10, 30, and 100 mg/kg in rats. Furthermore, compound 24 showed excellent PK profiles with a 72% oral bioavailability in beagle dogs. Compound 24 demonstrated overall favorable in vitro safety profiles with respect to CYP isoform and hERG inhibition. Finally, compound 24 led to significant regression of subcutaneous tumor generated by primary Ptch1-deficient medulloblastoma cells in SCID mouse. In conclusion, tetrahydropyrido[4,3-d]pyrimidine derivatives represent a novel set of Smo inhibitors that could potentially be utilized to treat medulloblastoma and other Hh pathway related malignancies.

MEDU-05. THE ROLE OF GABA METABOLISM IN MEDULLOBLASTOMA

Abstract BACKGROUND: Brain tumors are the most common cause of childhood oncological death, and medulloblastoma (originating in the cerebellum) is the most common malignant pediatric brain tumor. In the microenvironment of the brain, especially the cerebellum, variables related to GABA, the major inhibitory neurotransmitter in the nervous system, are particularly prominent. Abnormal GABAergic Receptor activation has been described in in aggressive MYC-driven Group 3 medulloblastoma. However these studies did not look at the metabolic contribution of GABA for the development of medulloblastomas. In addition to its role in neurotransmission through GABA receptor, GABA can act as a trophic factor during nervous system development to influence cellular events including proliferation, migration, differentiation, synapse maturation, and cell death. Under conditions that inhibit the tricarboxylic acid cycle (TCA), impair respiration, and enhance the accumulation of reactive oxygen intermediates, GABA can be used as an NADH energy source for growth through the GABA-shunt pathway regulators (ABAT, SSADH, GAT-1, GAT-3). Therefore, we hypothesize that blocking GABA-metabolic-shunt will lead to growth suppression and invasiveness of medulloblastoma in the cerebellar GABA-rich microenvironment. RESULTS: Our results show RNA microarray from patient medulloblastoma tissue have high expression of GABA-shunt regulators with ~3-fold increase in the expression of ABAT in MYC amplified versus non-amplified MYC tumors. When medulloblastomas were supplemented with GABA, there was a significant fold change in expression of GABA-shunt mediators and induction of large and stable tumor spheres with Epithelial-Mesenchymal Transition gene expression signature. We next investigated whether a novel perrilyl alcohol-based small molecule NEO216 targeted the GABA-shunt metabolic pathway. NEO216 administration significantly reduced GABA-mediated NADH levels, reversed EMT-profiling, leading to loss of sphere formation. CONCLUSION: Thus, the expression of GABA-metabolic shunt in medulloblastoma could be a malignant microenvironmental adaptation for growth and metastasis that could potentially be exploited through targeted therapy for patients benefit.

Comprehensive analysis of a microRNA expression profile in pediatric medulloblastoma.

Medulloblastoma is the most common malignant brain tumor of the central nervous system among children. Medulloblastoma is an embryonal tumor, of which little is known about the pathogenesis. Several efforts have been made to understand the molecular aspects of its tumorigenic pathways; however, these are poorly understood. microRNA (miRNA), a type of non-coding short RNA, has been proven to be associated with a number of physiological processes and pathological processes of serious diseases, including brain tumors. Differentially expressed miRNAs serve an important role in numerous types of malignancy. The present study aims to define a differentially expressed set of miRNAs in medulloblastoma tumor tissue, compared with normal samples, to improve the understanding of the tumorigenesis. It was identified that 22 miRNAs were upregulated and 26 miRNAs were downregulated in the tumor tissue compared with the normal group. However, when the medulloblastoma tissue was compared with normal cerebellum tissue, 9 miRNAs were identified to be up or downregulated in the tumor samples. The differentially expressed miRNAs in the tumor tissue were identified in order to clarify the networks and pathways of tumorigenesis using Ingenuity Pathway Analysis. Subsequently, key regulatory genes associated with the development of medulloblastoma were identified, including tumor protein p53, insulin like growth factor 1 receptor, argonaute 2, mitogen-activated protein kinases 1 and 3, sirtuin 1 and Y box binding protein 1.

Sensitive Detection of Radiation-Induced Medulloblastomas after Acute or Protracted Gamma-Ray Exposures in Ptch1 Heterozygous Mice Using a Radiation-Specific Molecular Signature.

Recently reported studies have led to a heightened awareness of the risks of cancer induced by diagnostic radiological imaging, and in particular, the risk of brain cancer after childhood CT scans. One feature of Ptch1+/- mice is their sensitivity to radiation-induced medulloblastomas (an embryonic cerebellar tumor) during a narrow window of time centered on the days around birth. Little is known about the dynamics of how dose protraction interacts with such narrow windows of sensitivity in individual tissues. Using medulloblastomas from irradiated Ptch1+/- mice with a hybrid C3H × C57BL/6 F1 genetic background, we previously showed that the alleles retained on chromosome 13 (which harbors the Ptch1 gene) reveal two major mechanisms of loss of the wild-type allele. The loss of parental alleles from the telomere extending up to or past the Ptch1 locus by recombination (spontaneous type) accounts for almost all medulloblastomas in nonirradiated mice, while tumors in irradiated mice often exhibited interstitial deletions, which start downstream of the wild-type Ptch1 and extend up varying lengths towards the centromere (radiation type). In this study, Ptch1+/- mice were exposed to an acute dose of either 100 or 500 mGy gamma rays in utero or postnatally, or the same radiation doses protracted over a four-day period, and were monitored for medulloblastoma development. The results showed dose- and age-dependent radiation-induced type tumors. Furthermore, the size of the radiation-induced deletion differed with the dose rate. The results of this work suggest that tumor latency may be related to the size of the deletion. In this study, 500 mGy exposure produced radiation-induced type tumors at all ages and dose rates, while 100 mGy exposure did not significantly produce radiation-induced type tumors. The radiation signature allows for unique mechanistic insight into the action of radiation to induce DNA lesions with known causal relationship to a specific tumor type, particularly for doses and dose rates that are relevant to both diagnostic and accidental radiological exposures.

Nestin Mediates Hedgehog Pathway Tumorigenesis.

The intermediate filament protein Nestin serves as a biomarker for stem cells and has been used to identify subsets of cancer stem-like cells. However, the mechanistic contributions of Nestin to cancer pathogenesis are not understood. Here, we report that Nestin binds the hedgehog pathway transcription factor Gli3 to mediate the development of medulloblastomas of the hedgehog subtype. In a mouse model system, Nestin levels increased progressively during medulloblastoma formation, resulting in enhanced tumor growth. Conversely, loss of Nestin dramatically inhibited proliferation and promoted differentiation. Mechanistic investigations revealed that the tumor-promoting effects of Nestin were mediated by binding to Gli3, a zinc finger transcription factor that negatively regulates hedgehog signaling. Nestin binding to Gli3 blocked Gli3 phosphorylation and its subsequent proteolytic processing, thereby abrogating its ability to negatively regulate the hedgehog pathway. Our findings show how Nestin drives hedgehog pathway-driven cancers and uncover in Gli3 a therapeutic target to treat these malignancies. Cancer Res; 76(18); 5573-83. ©2016 AACR.

SCF (Fbxl17) ubiquitylation of Sufu regulates Hedgehog signaling and medulloblastoma development.

Skp1‐Cul1‐F‐box protein (SCF) ubiquitin ligases direct cell survival decisions by controlling protein ubiquitylation and degradation. Sufu (Suppressor of fused) is a central regulator of Hh (Hedgehog) signaling and acts as a tumor suppressor by maintaining the Gli (Glioma‐associated oncogene homolog) transcription factors inactive. Although Sufu has a pivotal role in Hh signaling, the players involved in controlling Sufu levels and their role in tumor growth are unknown. Here, we show that Fbxl17 (F‐box and leucine‐rich repeat protein 17) targets Sufu for proteolysis in the nucleus. The ubiquitylation of Sufu, mediated by Fbxl17, allows the release of Gli1 from Sufu for proper Hh signal transduction. Depletion of Fbxl17 leads to defective Hh signaling associated with an impaired cancer cell proliferation and medulloblastoma tumor growth. Furthermore, we identify a mutation in Sufu, occurring in medulloblastoma of patients with Gorlin syndrome, which increases Sufu turnover through Fbxl17‐mediated polyubiquitylation and leads to a sustained Hh signaling activation. In summary, our findings reveal Fbxl17 as a novel regulator of Hh pathway and highlight the perturbation of the Fbxl17–Sufu axis in the pathogenesis of medulloblastoma.

SMARCA4/Brg1 coordinates genetic and epigenetic networks underlying Shh-type medulloblastoma development.

Recent large-scale genomic studies have classified medulloblastoma into four subtypes: Wnt, Shh, Group 3 and Group 4. Each is characterized by specific mutations and distinct epigenetic states. Previously, we showed that a chromatin regulator SMARCA4/Brg1 is required for Gli-mediated transcription activation in Sonic hedgehog (Shh) signaling. We report here that Brg1 controls a transcriptional program that specifically regulates Shh-type medulloblastoma growth. Using a mouse model of Shh-type medulloblastoma, we deleted Brg1 in precancerous progenitors and primary or transplanted tumors. Brg1 deletion significantly inhibited tumor formation and progression. Genome-wide expression analyses and binding experiments indicate that Brg1 specifically coordinates with key transcription factors including Gli1, Atoh1 and REST to regulate the expression of both oncogenes and tumor suppressors that are required for medulloblastoma identity and proliferation. Shh-type medulloblastoma displays distinct H3K27me3 properties. We demonstrate that Brg1 modulates activities of H3K27me3 modifiers to regulate the expression of medulloblastoma genes. Brg1-regulated pathways are conserved in human Shh-type medulloblastoma, and Brg1 is important for the growth of a human medulloblastoma cell line. Thus, Brg1 coordinates a genetic and epigenetic network that regulates the transcriptional program underlying the Shh-type medulloblastoma development.

Abstract IA19: Genome-epigenome interplay in medulloblastoma

Abstract The recent application of genomics to large series of primary human medulloblastomas has dramatically improved our perspective of the genes, pathways, and molecular processes underpinning this highly malignant pediatric brain tumor. Transcriptionally defined molecular subgroups have emerged, changing the fundamental definition of medulloblastoma from a single histologically recognized entity, to four distinct subtypes: WNT, SHH, Group3, and Group4. These subgroups exhibit highly discriminatory genomic landscapes, patient demographics, and clinical phenotypes. Next-generation sequencing has identified and implicated recurrently altered somatic drivers, many of which are affected in a subgroup-restricted manner. In contrast, somatic variants affecting chromatin-modifying genes, including histone writers, erasers, and readers are prevalent in medulloblastoma and occur across subgroups, suggesting that deregulation of the epigenome is an essential step during medulloblastoma development. Integrative omics-based strategies have begun to dissect the fascinating genome-epigenome interplay underlying medulloblastoma, revealing previously undisclosed oncogenic mechanisms and drivers of disease. Furthermore, new insights into the biology and putative origins of the poorly understood Group3 and Group4 medulloblastomas have likewise been revealed. This talk will summarize these recent discoveries afforded through genomics and discuss their potential implications, both in the lab and in the clinic, going forward. Citation Format: Paul Northcott. Genome-epigenome interplay in medulloblastoma. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Pediatric Cancer Research: From Mechanisms and Models to Treatment and Survivorship; 2015 Nov 9-12; Fort Lauderdale, FL. Philadelphia (PA): AACR; Cancer Res 2016;76(5 Suppl):Abstract nr IA19.

High expression of DNA methyltransferases in primary human medulloblastoma.

Epigenetic alterations have been implicated in cancer development. DNA methylation modulates gene expression, which is catalyzed by DNA methyltransferases (DNMTs). The objective of our study was to evaluate expression of DNMTs in medulloblastoma and analyze its correlation with clinical features. Nuclear expression of DNMT1, DNMT3A and DNMT3B was analyzed in human primary medulloblastoma of 44 patients using immunohistochemistry. Correlation of expression of DNMT levels with classical histological subtypes, novel molecular subgroups and survival of patients was analyzed. Elevated expression of DNMT1, DNMT3A and DNMT3B was observed in 63.64%, 68.18% and 72.73% of all cases, respectively. None of them showed a correlation with classical histology or survival. Concerning molecular subtypes, significantly higher expression of DNMT1 was observed in the SHH group compared to non-SHH samples (p = 0.02), but without significant difference in DNMT3A or DNMT3B levels between any subtypes. In conclusion, DNMT1, DNMT3A and DNMT3B are highly expressed in human medulloblastoma samples, suggesting that promoter hypermethylation may play a role in medulloblastoma development. Demethylation of tumor suppressor gene promoters may be considered as a possible future target in therapy of medulloblastoma.

Abstract IA17: Genome-epigenome interplay in medulloblastoma

Abstract The recent application of genomics to large series of primary human medulloblastomas has dramatically improved our perspective of the genes, pathways, and molecular processes underpinning this highly malignant pediatric brain tumor. Transcriptionally defined molecular subgroups have emerged, changing the fundamental definition of medulloblastoma from a single histologically recognized entity, to four distinct subtypes: WNT, SHH, Group3, and Group4. These subgroups exhibit highly discriminatory genomic landscapes, patient demographics, and clinical phenotypes. Next-generation sequencing has identified and implicated recurrently altered somatic drivers, many of which are affected in a subgroup-restricted manner. In contrast, somatic variants affecting chromatin-modifying genes, including histone “writers,” “erasers,” and “readers” are prevalent in medulloblastoma and occur across subgroups, suggesting that deregulation of the epigenome is an essential step during medulloblastoma development. Integrative “omics”-based strategies have begun to dissect the fascinating genome-epigenome interplay underlying medulloblastoma, revealing previously undisclosed oncogenic mechanisms and drivers of disease. Furthermore, new insights into the biology and putative origins of the poorly understood Group3 and Group4 medulloblastomas have likewise been revealed. This talk will summarize these recent discoveries afforded through genomics and discuss their potential implications, both in the lab and in the clinic, going forward. Citation Format: Paul A. Northcott. Genome-epigenome interplay in 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 IA17.

TMOD-01BCOR IS A NOVEL TUMOR SUPPRESSOR GENE IN SHH-DRIVEN MEDULLOBLASTOMA

Sonic hedgehog (SHH) medulloblastoma is the most well characterized molecular subgroup of this malignant embryonal brain tumor, accounting for 1/3rd of all medulloblastoma diagnoses. Germline/somatic alterations affecting genes in the SHH pathway occur in most cases, resulting in constitutively aberrant activation of the SHH signaling cascade. Pharmacological inhibitors of SHH signaling have shown encouraging albeit transient efficacy in treating patients with SHH subgroup disease, with most patients developing resistance to conventional inhibitors. Therefore, understanding the genetic events cooperating with aberrant SHH pathway activation in medulloblastoma is of paramount importance for the development of rational, more effective combination therapies that improve cure rates for this patient subgroup. Next-generation sequencing studies have identified the BCL6 corepressor, BCOR, as among the most frequently mutated genes in SHH subgroup patients, with the majority of observed variants inferred to result in BCOR loss-of-function. Somatic mutations and homozygous deletions of BCOR are restricted to SHH subgroup patients, suggesting that deregulation of this gene plays a critical role in potentiating SHH subgroup malignancy. To validate BCOR as a novel tumor suppressor in SHH medulloblastoma, we generated Ptch1 + /−; Atoh1-Cre; Bcorfl/fl/Bcorfl/y mice to specifically inactivate Bcor in cerebellar granule neuron progenitors in vivo, presumed cells-of-origin for this medulloblastoma subgroup. Excitingly, Ptch1 + /−; Atoh1-Cre; Bcorfl/fl/Bcorfl/y mice rapidly develop ∼100% penetrant (n = 16/16) medulloblastomas (median latency = 74 days), compared to ∼36% of mice (n = 4/11) that develop medulloblastoma (average latency = 224 days) due to loss of Ptch1 + /− alone. Histologically, tumors resulting from the Bcor null genotype are highly similar to prototypical Ptch1 + /− medulloblastomas, despite exhibiting considerable gene expression differences as determined by Affymetrix expression profiling. Studies aimed at elucidating the molecular mechanism(s) associated with the development of Bcor-deficient SHH medulloblastoma are ongoing and will be essential for extrapolation of these findings into improved treatment options for SHH medulloblastoma patients in the future.

CCND2, CTNNB1, DDX3X, GLI2, SMARCA4, MYC, MYCN, PTCH1, TP53, and MLL2 gene variants and risk of childhood medulloblastoma.

Recent studies have described a number of genes that are frequently altered in medulloblastoma tumors and that have putative key roles in the development of the disease. We hypothesized that common germline genetic variations in these genes may be associated with medulloblastoma development. Based on recent publications, we selected 10 genes that were frequently altered in medulloblastoma: CCND2, CTNNB1, DDX3X, GLI2, SMARCA4, MYC, MYCN, PTCH1, TP53, and MLL2 (now renamed as KMT2D). Common genetic variants (single nucleotide polymorphisms) annotating these genes (n = 221) were genotyped in germline DNA (neonatal dried blood spot samples) from 243 childhood medulloblastoma cases and 247 control subjects from Sweden and Denmark. Eight genetic variants annotating three genes in the sonic hedgehog signaling pathway; CCND2, PTCH1, and GLI2, were found to be associated with the risk of medulloblastoma (Pcombined < 0.05). The findings were however not statistically significant following correction for multiple testing by the very stringent Bonferroni method. The results do not support our hypothesis that common germline genetic variants in the ten studied genes are associated with the risk of developing medulloblastoma.Electronic supplementary materialThe online version of this article (doi:10.1007/s11060-015-1891-1) contains supplementary material, which is available to authorized users.

Abstract 503: The PPM1D encoded phosphatase Wip1 is a novel oncogene and potential therapeutic target in neuroblastoma and medulloblastoma

Abstract Background: The most common cytogenetic lesions in the embryonal neural tumors medulloblastoma (MB) and neuroblastoma (NB) affect chromosome 17, with 17q+ or isochromosome 17q, in approximately one-third of MB with these aberrations being a significant indicator of poor clinical outcome. Similarly, in NB gain of 17q is the most powerful genetic predictor of adverse clinical outcome. 17q+ correlates with poor survival in our population-based material where we found aberrations of chromosome 17 in 85% of primary neuroblastomas, specifically, gain of PPM1D/Wip1 at 17q23. Wip1 is a serine/threonine phosphatase encoded by the gene PPM1D, described as a gatekeeper in the Mdm2-p53 regulatory loop involved in genetic stability, inflammation and a potential oncogene contributing to carcinogenesis. Methods: Comparative genomic hybridization (CGH) was used to examine PPM1D/Wip1 in neuroblastoma and medulloblastoma tumors and cell lines. Stable Wip1 knockdown SK-N-BE(2) cells were generated by shRNA transfections and tested in vivo in tumor xenografts. Pharmacological inhibition with the p53-mdm2 modulating inhibitors RITA, Nutlin-3 and a new PPM1D/Wip1 inhibitor, was used to evaluate the function of PPM1D/Wip1 in preclinical neuroblastoma and medulloblastoma models. Results: CGH-array analysis detected PPM1D/Wip1 extra copies in all tumors and cell lines containing 17q-gain. Tumor neuroblastoma xenograft development was significantly delayed showing median tumor development (0.10 mL) to be more than doubled (median 15 days, vs. 33 days, p&amp;lt;0.001) after Wip1 downregulation compared to scrambled controls. A novel Wip1 inhibitor was highly potent in cytotoxic/cytostatic effect in a variety of neuroblastoma and medulloblastoma cell lines. Furthermore, this Wip1 inhibitor significantly inhibited growth of established human neuroblastoma- and medulloblastoma tumors in nude mice after treatment (P&amp;lt;0.01). Conclusions: Our results show that PPM1D/Wip1 is oncogenic in neuroblastoma and medulloblastoma development and provides a novel therapeutic target in these two childhood cancers of the nervous system. More studies investigating the effects of PPM1D/Wip1 inhibition are needed to evaluate its clinical significance. Citation Format: Jelena Milosevic, Diana Treis, Malin Wickstrom, Susanne Fransson, Nina Eissler, Baldur Sveinbjörnsson, Ninib Baryawno, Keiji Tanino, Galina Selivanova, Kazuyasu Sakaguchi, Tommy Martinsson, John Inge Johnsen, Per Kogner. The PPM1D encoded phosphatase Wip1 is a novel oncogene and potential therapeutic target in neuroblastoma and medulloblastoma. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 503. doi:10.1158/1538-7445.AM2015-503

Dicer Is Required for Normal Cerebellar Development and to Restrain Medulloblastoma Formation

Dicer, a ribonuclease III enzyme, is required for the maturation of microRNAs. To assess its role in cerebellar and medulloblastoma development, we genetically deleted Dicer in Nestin-positive neural progenitors and in mice lacking one copy for the Sonic Hedgehog receptor, Patched 1. We found that conditional loss of Dicer in mouse neural progenitors induced massive Trp53-independent apoptosis in all proliferative zones of the brain and decreased proliferation of cerebellar granule progenitors at embryonic day 15.5 leading to abnormal cerebellar development and perinatal lethality. Loss of one copy of Dicer significantly accelerated the formation of mouse medulloblastoma of the Sonic Hedgehog subgroup in Patched1-heterozygous mice. We conclude that Dicer is required for proper cerebellar development, and to restrain medulloblastoma formation.

Somatic CRISPR/Cas9-mediated tumour suppressor disruption enables versatile brain tumour modelling.

In vivo functional investigation of oncogenes using somatic gene transfer has been successfully exploited to validate their role in tumorigenesis. For tumour suppressor genes this has proven more challenging due to technical aspects. To provide a flexible and effective method for investigating somatic loss-of-function alterations and their influence on tumorigenesis, we have established CRISPR/Cas9-mediated somatic gene disruption, allowing for in vivo targeting of TSGs. Here we demonstrate the utility of this approach by deleting single (Ptch1) or multiple genes (Trp53, Pten, Nf1) in the mouse brain, resulting in the development of medulloblastoma and glioblastoma, respectively. Using whole-genome sequencing (WGS) we characterized the medulloblastoma-driving Ptch1 deletions in detail and show that no off-targets were detected in these tumours. This method provides a fast and convenient system for validating the emerging wealth of novel candidate tumour suppressor genes and the generation of faithful animal models of human cancer.