Unknown,Transcriptomics,Genomics,Proteomics

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An Animal Model of Myc-driven medulloblastoma

ABSTRACT: Medulloblastoma (MB) is the most common malignant brain tumor in children. Patients whose tumors exhibit overexpression or amplification of the MYC oncogene (c-MYC) usually have an extremely poor prognosis, but there are no animal models of this subtype of the disease. Here we show that cerebellar stem cells expressing Myc and mutant Trp53 (p53) generate aggressive tumors following orthotopic transplantation. These tumors consist of large, pleiomorphic cells and resemble human MYC-driven MB at a molecular level. Notably, antagonists of PI3K/mTOR signaling, but not Hedgehog signaling, inhibit growth of tumor cells. These findings suggest that cerebellar stem cells can give rise to MYC-driven MB, and identify a novel model that can be used to test therapies for this devastating disease. To gain insight into the pathways that control growth of MYC-driven MB, we compared gene expression profiles of murine Myc/DNp53 (MP) tumor cells to those of freshly isolated cerebellar stem cells (Prom1+Lin- cells) and of tumors from Ptch1 mutant mice (a model for Sonic Hedgehog-associated MB). RNA was isolated from stem cells and tumor cells using the RNAqueous kit (Ambion). RNA was labeled and hybridized to Affymetrix Mouse Genome 430 2.0 arrays. 19 mouse cell samples (stem cells and tumor cells) were analyzed. There are four groups of samples, three with five biological replicates and the last with four (one outlier was removed). To gain insight into the mechanisms of transformation into tumors, we compared the gene expression profiles of MP tumor cells derived from stem cells (Myc/DNp53-infected Prom1+Lin- cells, designated MP-pl) or progenitors (Myc/DNp53-infected Prom1+ cells, designated MP-p) to gene expression profiles of uninfected stem cells (designated NSC) and profiles from a distinct model of medulloblastoma, the patched mutant mouse (designated ptch1).

ORGANISM(S): Mus musculus

SUBMITTER: Alexey Eroshkin

PROVIDER: E-GEOD-34126 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:We examined the transformation susceptibility of different cerebellar stem/progenitors by developing several new Group3 medulloblastoma murine models using orthotopic transplantation and in utero electroporation (EP)-based in vivo gene transfer with Cre/LoxP-mediated conditional Myc gene activation and loss of Trp53 function. We used microarrays to compared the transcriptome of these novel Group3 medulloblastoma mouse models to existing mouse models of medulloblastoma subgroups and used cross-species analysis to compare these models to human medulloblastoma subgroups This study aimed to investigate the cell of origin of Group3 MB using our orthotopical MYC model followed by a novel electroporation approach. Orthotopic cell-lineage specific MYC tumors were generated by enforced Myc expression in P6 GNPs isolated from P0-1 tamoxifen treated [Atoh1-CreER;Trp53fl/-] and [Prom1-CreER;Trp53fl/-] mice followed by cortical implants in immunocompromised mice. These tumors are referred to as Atoh1ER-MYC [dka072-075], Prom1-CreER [dka077-081]. The first Group3 MB models in which tumors developed in situ were generated by electroporation of plasmids containing Myc and dominant negative Trp53 flanked by loxP sites into the fourth ventricle of E13.5 Blbp-Cre [dka081, 087, 089, 090, 091 and blm121], Gad2-IRES-Cre [blm128-130 and blm134] and Ptf1a-Cre [blm135-137] mouse embryos. The gene expression profile of these tumors were compared to our previously published Group3 MB model as well as SHH and WNT models of medulloblastoma. For SHH subgroup medulloblastoma, [dka001-005, 009, 033 and 034] and [dka050-057], spontaneous medulloblastomas from [Cdkn2c-/-; Trp53Fl/Fl; Nestin-Cre] and [Cdkn2c-/-; Ptch1+/-] (Uziel et al.,2005 Genes Dev) were used, respectively. For Group3 medulloblastomas, [dka013-16, 049 and 065-067], in which Myc was overexpressed in Cdkn2c-/-, Trp53-/- cerebellar cells and implanted into the cortices of immunocompromised nude mice prior to tumor isolation. For WNT subgroup medulloblastomas [pgr003, 016 and 066], spontaneously developed tumors from CTNNB1+/lox (ex3); BLBP-Cre; Trp53Fl/Fl (Gibson et al., 2010, Nature) were removed for RNA extraction.

Project description:Medulloblastomas (MBs) are cerebellar tumors that can be classified into molecularly distinct subgroups that differ in pathology and prognosis. The mechanisms that underlie subgroup specification are largely unknown. While human SHH MBs express MYCN, Group3 (G3) MBs are associated with c-MYC (MYC) overexpression and often show metastasis that confers a poor prognosis. Although MYC proteins are thought to be functionally exchangeable, ectopic expression of Myc or N-myc in Trp53-/-;Cdkn2c-/- cerebellar granule neuron progenitors (GNPs) induces G3 and SHH MBs, respectively, demonstrating that each Myc protein has distinct biological properties. We now show that Myc and N-myc differ in their affinity to Miz1 and that Myc, but not N-myc, effectively recruits Miz1 to its target sites on chromatin. The interaction of Myc with Miz1 is required for the genesis of G3 MB. Myc suppresses ciliogenesis and âreprogramsâ the transcriptome of SHH-dependent GNPs to stem-like cells by repressing genes highly expressed in SHH MB via Miz1. Consistently, target genes of Myc/Miz1 are repressed in human G3 MBs but not in other MB subgroups. Collectively, the data show that the interaction of Myc with Miz1 is a defining hallmark of G3 MB development. In this study, we show that Myc and N-myc differ in their affinity for Miz1, and Myc/Miz1 interaction is required for Group3 medulloblastoma (MB). The gene expression profiles of these tumors were compared to our previously published Group3 MB model as well as SHH model of MB (Kawachi et al., 2012, Cancer Cell). Cerebellar granule neuron progenitors (GNPs) [dka220-222] from postnatal (P) 6 Math1-GFP;Trp53-/-;Cdkn2c-/-. For SHH medulloblastomas, [dka204-206] and [blm015-017 or dka050-dka051], spontaneous medulloblastomas from Cdkn2c-/-;Trp53Fl/Fl;Nestin-Cre (Kawachi et al., 2012, Cancer Cell) and Ptch1+/-;Cdkn2c-/- (Uziel et al., 2005, Genes Dev) were used, respectively. Myc- [dka201-203] and MycV394D (termed MycVD thereafter)- [bvo017-bvo023] tumors were from Trp53-/-;Cdkn2c-/- cerebella of P6-P7 pups. Myc/ÎPOZ tumors [bvo002-bvo006] were obtained from Trp5Fl/Fl;Miz1ÎPOZ/POZ;Nestin-Cre cerebella of P6-P7 pups.

Project description:Medulloblastoma (MB) is the most common malignant brain tumor. MB is a cerebellar tumor that occurs mostly in children between the ages of 3-7 years but also in adults. Human MBs are classified into four subgroups: Wingless (WNT), Sonic Hedgehog (SHH), Group 3 (G3) and G4, each of which with distinct molecular signatures. SHH MBs with MYCN amplification and TP53 mutations and G3 MBs characterized by C-MYC (MYC) overexpression in ~17% of cases from gene amplification with stem like properties, are the most aggressive and least curable with current therapeutic regimen. Using an orthotopic transplant approach, we found that enforced expression of MYCN in cerebellar granule neural progenitors (CGNPs) from 5-7 days old Trp53-null mice induced SHH MBs after transfer in the cortices or cerebella of naive recipient mice. In contrast, overexpression of MYC induced G3 MBs. Because MYCN and MYC bind to the same E-box DNA sequences, we hypothesized that the difference between MYC and MYCN-induced gene expression and tumor identity might be due to their interaction with different partners in CGNPs. In this study, we investigated the role of the Myc interacting zinc finger protein 1 (MIZ1). We found that MIZ1 binds with higher affinity to MYC than to MYCN and that MIZ1 and MYC co-occupy thousands of promoters in G3 MB. Remarkably, enforced expression of a MYC mutant (MYCV394D) that no longer binds to MIZ1 in Trp53-null CGNPs or expression of wild type MYC in CGNPs that lack functional Miz1 resulted in massive changes of the resulting tumorâ??s transcriptional program. Tumors differed from both SHH and G3 medulloblastoma and had a later time of onset compared to MYC-driven G3 medulloblastoma. Our data demonstrate that interaction of MYC with MIZ1 is required for the development of G3 medulloblastoma. ChIP-Seq experiments for Miz1, c-Myc and NMyc from murine medulloblastoma material. Either sorted tumor cells (Miz1 and c-Myc) or spheres from tumor cells (Miz1 and NMyc) were used. Input-samples were sequenced as controls. RNA-Seq from G3 medulloblastomas after restoration of Atoh1 expression.

Project description:Pediatric medulloblastoma (MB) is the most common solid malignant brain neoplasm, with group 3 (G3) MB representing the most aggressive subgroup associated with a poor prognosis and a remarkable ability to resist upfront multimodal therapy. Despite a low mutational burden of disease and MYC amplification identified as a independent factor associated with poor survivorship, efforts to target the MYC has met with limited therapeutic success. Consequently alternative mediators associated with the aggressive phenotype of G3 MB continues as a common goal within the MB community. Here we show how the neural stem cell determinant Musashi 1 (MSI1) is a central and vital moderator of G3 MB in both a MYC amplified mouse model of G3 MB and patient derived xenografts (PDX). Specifically, we modified the MYC amplified and p53 mutated (MP) mouse model of G3 MB to generate Msi1 conditional knockout mice (Msi1flox/flox), which led to the observation that MSI1 is required for tumor initiation of MP tumors. To identify the translational potential of these findings, we employed shRNA against Msi1 in multiple PDX lines, observing a striking deficit in multiple key stem cell features including self-renewal, proliferation, and failure to progress through the cell cycle. Notably, Msi1 inhibition resulted in a failure of tumor initiation, translating to a significantly prolonged survival, reaffirming the essential role for MSI1 in G3 MB. To determine how MSI1 symphonizes the anarchic post-transcriptional landscape of G3MB, we differentially analyzed the MSI1 binding sites in normal neural stem cells and G3 MB and subsequently compared the MSI1-binding transcriptome, and proteome following Msi1 inhibition. Comparative analysis suggested an integral role for post-transcriptional regulators, such as MSI1 and its binding prey mRNA, as a therapeutic target. Here we propose the neural RNA binding protein MSI1, as a master regulator, hijacked from its normal neural developmental function, orchestrating the aberrant translational landscape of G3 MB.

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An Animal Model of Myc-driven medulloblastoma

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