Unknown,Transcriptomics,Genomics,Proteomics

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Network-based, cross-cohort discovery of transcriptional mechanisms presiding over maintenance of high-risk neuroblastoma subtype state

ABSTRACT: Network-based analysis of neuroblastoma samples from two large cohorts identified master regulator proteins controlling the transcriptional state of three high-risk molecular subtypes. In particular, a TEAD4-MYCN positive feedback loop emerged as the core regulatory motif of a small protein module presiding over implementation and stability of the subtype associated with MYCN amplification. Specifically, MYCN transcriptionally activates TEAD4, which in turn activates MYCN both transcriptionally and post-translationally. The resulting MYCN-TEAD4 positive feedback loop plays a critical role in maintaining aberrant activity of a 10-protein regulatory module that causally regulates the transcriptional state of this subtype. Consistently, loss of TEAD4 activity induces core module activity collapse and abrogates neuroblastoma cell viability in vitro and in vivo, thus suggesting novel therapeutic strategies for this important childhood cancer. Study of the transcriptional control by TEAD4 and MYCN positive feedback loop using RNA-seq profiles of TEAD4 and MYCN shRNA knockdowns in neuroblastoma BE2 cells. ChIP-Seq analysis using TEAD4 antibody in BE2 cells.

ORGANISM(S): Homo sapiens

SUBMITTER: Gonzalo Lopez

PROVIDER: E-GEOD-84389 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:In neuroblastoma, amplification of the oncogenic basic helix-loop-helix (bHLH) transcription factor (TF) MYCN is the defining prognosticator of high-risk disease, occurs in one-third of neuroblastoma, and drastically reduces overall survival rates. As a proto-oncogene, targeted MYCN overexpression in peripheral neural crest is sufficient to initiate disease in mouse models. In MYCN amplified neuroblastoma, elevated expression of the factor is crucial to maintain tumor stemness and is associated with increased proliferation and aberrant cell cycle progression, as these tumors lack the ability to arrest in G1 in response to irradiation. MYCN down-regulation broadly reverses these oncogenic phenotypes in a variety of neuroblastoma models and recent thereapeutic strategies to indirectly target MYCN production or protein stability have reduced tumor growth in vivo. These observations motivate an investigation of MYCN binding in MYCN amplified tumors as it remains fundamentally unclear how elevated levels of the factor occupy the genome and alter transcriptional programs in neuroblastoma. Here we present the first dynamic chromatin and transcriptional landscape of direct MYCN perturbation in neuroblastoma. We find that at oncogenic levels, MYCN associates with E-box (CANNTG) binding motifs in an affinity dependent manner across most active cis-regulatory promoters and enhancers. MYCN shutdown globally reduces histone acetylation and transcription, consistent with prior descriptions of MYC proteins as non-linear amplifiers of gene expression. We establish that MYCN load at the promoter and proximal enhancers predicts transcriptional responsiveness to MYCN shutdown and that MYCN enhancer binding occurs prominently at the most strongly occupied and down-regulated genes, suggesting a role for these tissue specific elements in predicating MYCN responsive âtargetâ genes. At these invaded enhancers, we identify the lineage specific bHLH TWIST1 as a key collaborator and dependency of oncogenic MYCN. These data suggest that MYCN enhancer invasion helps shape transcriptional amplification of the neuroblastoma gene expression program to promote tumorigenesis. ChIP-Seq in SHEP21, BE2C, KELLY, and NGP neuroblastoma cell lines for H3K27ac, H3K4me3, RNA PolII, MYCN, BRD4, or TWIST1

Project description:In neuroblastoma, amplification of the oncogenic basic helix-loop-helix (bHLH) transcription factor (TF) MYCN is the defining prognosticator of high-risk disease, occurs in one-third of neuroblastoma, and drastically reduces overall survival rates1,2. As a proto-oncogene, targeted MYCN overexpression in peripheral neural crest is sufficient to initiate disease in mouse models3. In MYCN amplified neuroblastoma, elevated expression of the factor is crucial to maintain tumor stemness4,5 and is associated with increased proliferation and aberrant cell cycle progression, as these tumors lack the ability to arrest in G1 in response to irradiation6-9. MYCN down-regulation broadly reverses these oncogenic phenotypes in a variety of neuroblastoma models10-12 and recent thereapeutic strategies to indirectly target MYCN production or protein stability have reduced tumor growth in vivo13-15. These observations motivate an investigation of MYCN binding in MYCN amplified tumors as it remains fundamentally unclear how elevated levels of the factor occupy the genome and alter transcriptional programs in neuroblastoma. Here we present the first dynamic chromatin and transcriptional landscape of direct MYCN perturbation in neuroblastoma. We find that at oncogenic levels, MYCN associates with E-box (CANNTG) binding motifs in an affinity dependent manner across most active cis-regulatory promoters and enhancers. MYCN shutdown globally reduces histone acetylation and transcription, consistent with prior descriptions of MYC proteins as non-linear amplifiers of gene expression. We establish that MYCN load at the promoter and proximal enhancers predicts transcriptional responsiveness to MYCN shutdown and that MYCN enhancer binding occurs prominently at the most strongly occupied and down-regulated genes, suggesting a role for these tissue specific elements in predicating MYCN responsive â??targetâ?? genes. At these invaded enhancers, we identify the lineage specific bHLH TWIST1 as a key collaborator and dependency of oncogenic MYCN. These data suggest that MYCN enhancer invasion helps shape transcriptional amplification of the neuroblastoma gene expression program to promote tumorigenesis. ATAC-Seq in SHEP21, BE2C, KELLY, NGP, and MM1S cell lines

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