Abstract
MYC-driven Group 3 (G3) medulloblastoma (MB) is the most aggressive of four molecular subgroups classified by transcriptome, genomic landscape and clinical outcomes. Mouse models that recapitulate human G3 MB all rely on retroviral vector-induced Myc expression driven by viral regulatory elements (Retro-Myc tumors). We used nuclease-deficient CRISPR/dCas9-based gene activation with combinatorial single guide RNAs (sgRNAs) to enforce transcription of endogenous Myc in Trp53-null neurospheres that were orthotopically transplanted into the brains of naïve animals. Three combined sgRNAs linked to dCas9-VP160 induced cellular Myc expression and large cell anaplastic MBs (CRISPR-Myc tumors) which recapitulated the molecular characteristics of mouse and human G3 MBs. The BET inhibitor JQ1 suppressed MYC expression in a human G3 MB cell line (HD-MB03) and CRISPR-Myc, but not in Retro-Myc MBs. This G3 MB mouse model in which Myc expression is regulated by its own promoter will facilitate pre-clinical studies with drugs that regulate Myc transcription.
Highlights
Medulloblastoma (MB), the most common malignant pediatric brain tumor originating from the cerebellum, is classified into four major distinct molecular subgroups, including Wingless (WNT), Sonic Hedgehog (SHH), Group 3 (G3) and Group 4 (G4)[1,2]
Using previous H3K27acetylation chromatin immunoprecipitation sequencing (ChIP-Seq) and ATAC-Seq data from purified mouse G3 Retro-Myc MBs8, we located a ~1.2 Kb open chromatin region corresponding to the cellular Myc dual P1 and P2 promoter region (Supplementary Fig. S1) to which we designed a series of CRISPR guide RNAs
We fused sequences encoding 4X or 10X tandem repeats of the transactivation domain of Herpes simplex virus protein VP16 (VP64 or VP160, respectively) to the C-terminus of nuclease-deficient dCas[9] (D10A, H840A) and fused these to T2A-GFP in a lentivirus backbone or transposon vector[23] (Fig. 1a)
Summary
Medulloblastoma (MB), the most common malignant pediatric brain tumor originating from the cerebellum, is classified into four major distinct molecular subgroups, including Wingless (WNT), Sonic Hedgehog (SHH), Group 3 (G3) and Group 4 (G4)[1,2]. Several G3 mouse models have been developed by various methods including orthotopic transplantation of Trp53-null granule neural progenitors (GNPs) infected with retroviruses encoding Myc[7] (hereafter referred as Retro-Myc); wild type GNPs infected with retroviruses expressing Myc and Gfi[18]; wild type embryonic neural stem cells co-transduced with retrovirally expressed Myc and a dominant-negative (DN) form of Trp[539] or Gfi[18,10]; or delivery of vectors expressing a conditional form of Myc and DN Trp[53] into the embryonic cerebellum by in utero electroporation[7,9,11] All these mouse models fully recapitulate human G3 MBs identified by cross-species gene expression analysis. We demonstrate the ability of the CRISPR-dCas9-VP160 system to modulate endogenous Myc expression in Trp53-null neurosphere cells to generate an orthotopic mouse model of G3 MB amenable to BETi treatment
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