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Novel chromatin interactions by structural rearrangements and aberrant enhancer functions drive oncogenic programs in unfavorable neuroblastoma

Gartlgruber, Moritz Johannes

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Abstract

Neuroblastoma (NB) is an embryonal tumor derived from migrating neural crest cells and can occur within the whole developing sympathetic nervous system. Recently, several comprehensive sequencing studies in NB revealed a comparatively low mutation frequency and a heterogeneous mutation spectrum. Evidence accumulates that epigenetic deregulation play a prominent role in NB. Therefore, there is an urgent need to define entity-specific enhancer and super-enhancer (SE) profiles as regulatory elements control cell identity or oncogenes in a cancer context through cell type-specific gene expression. This will identify critical oncogenic driver genes essential for NB and help to devise a targeted therapy strategy. In the present study, whole genome sequencing (WGS) analysis of a 60 NB tumor cohort identified recurrent rearrangements in close proximity to the TERT gene in up to 24% of high-risk NB cases with poor clinical outcome. ChIP-sequencing analyses revealed that strong enhancers were juxtaposed to the TERT gene by these rearrangements likely driving increased TERT expression and TERT activity in the respective cases. In addition to TERT, discovery of recurrent repositioning of SE elements explained remarkably high expression levels of MYCN, MYC oncogenes in NB cell lines. Integrative analysis of WGS-based rearrangement data and RNA-seq based expression data in a cohort of 111 NB tumors allowed to identify highly upregulated genes in proximity to breakpoints. This approach located several other enhancer-hijacking candidate oncogenes including IGF2BP1 and ATOH1 in NB tumors and cell lines. Subsequent ChIP-seq analysis of the affected tumors and cell lines confirmed that highly active SE regions were juxtaposed to the oncogenes, which likely drives their high expression. Finally, physical interactions of juxtaposed enhancer elements with the oncogene promoters were confirmed by chromatin interaction analyses (4C-seq) in cell lines. Intriguingly, a lineage-specific SE region downstream of HAND2 and upstream of the FBXO8 gene locus was recurrently involved in the above mentioned rearrangements. In a set of 60 NB tumor specimen and 23 NB cell lines, the present study identified groups of tissue-specific SEs and associated target genes based on histone mark H3K27ac ChIP-seq data. In depth bioinformatic analysis of the enhancer data retrieved three gene signatures, associated with previously established clinico-biological association, namely MYCN-amplified, high-risk- and low-risk MYCN non-amplified. Intriguingly, a fourth SE-defined NB subgroup was resolved, as defined by a gene signature highly associated with mesenchymal (Mes) gene ontology terms. This subgroup was reproducibly recovered by an analogue and extended approach based on RNA-seq expression of SEs assigned signature genes and further proved its stability by its consistent presence in NB cell lines and tumors. Importantly, the Mes signature was associated with relapsed NB cases as well as with increased RAS and JUN/FOS signature expression. For the first time, integration of SE and expression data of primary tumors enabled the establishment of an NB entity and NB subgroup-specific regulome resulting in the definition and subsequent functional validation of subgroup-specific core-regulatory networks. Taken together, with the discovery of recurrent rearrangements of SE elements activating the TERT gene, the present study provides initial evidence for “enhancer hijacking” in NB tumors. The identification of further recurrent oncogene rearrangements involving known NB oncogenes MYC and MYCN in cell lines and as well as new candidates IGF2BP1 and ATOH1 in NB tumors demonstrates that recurrence of enhancer hijacking in vivo is not restricted to TERT. Importantly, the present study reveals the first entity and subgroup-specific SE landscape including assigned target genes and downstream core transcription factor networks based on ChIP-seq analyses in a large cohort of tumors. This study identifies several critical NB cancer genes, which may open a therapeutic window for selective inhibition of those dysregulated key genes.

Item Type: Dissertation
Supervisor: Höfer, Prof. Dr. Thomas
Date of thesis defense: 21 September 2018
Date Deposited: 05 Oct 2018 16:46
Date: 2018
Faculties / Institutes: The Faculty of Bio Sciences > Dean's Office of the Faculty of Bio Sciences
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