Project description:High-risk neuroblastoma is often distinguished by amplification of MYCN and loss of differentiation potential with tumors refractory to retinoic acid differentiation based therapies. Here, we leverage high-throughput drug screening of epigenetic targeted therapies across a large and diverse tumor cell line panel to uncover the hypersensitivity of neuroblastoma cells to GSK-J4, a small molecule dual inhibitor of H3K27 demethylases UTX and JMJD3. Mechanistically, GSK-J4 induced neuroblastoma differentiation and ER stress with accompanying upregulation of PUMA and apoptosis induction. Retinoic acid (RA)-resistant neuroblastoma cells were sensitive to GSK-J4. Additionally, GSK-J4 was effective at blocking the growth of chemorefractory and patient-derived xenograft models of high-risk neuroblastoma in vivo. Further, GSK-J4 and RA combined to induce differentiation, ER-stress and limit the growth of neuroblastomas resistant to either drug alone. In MYCN-amplified neuroblastoma, which is the most prevalent driver gene alteration in the refractory population, PUMA induction by GSK-J4 sensitized tumors to the BCL-2 inhibitor venetoclax, demonstrating that epigenetic targeted therapies and BH3 mimetics can be rationally combined to treat high-risk subset of neuroblastoma. Therefore, H3K27 demethylation inhibition is a promising therapeutic target to treat high-risk neuroblastoma, and H3K27 demethylation can be part of rational combination therapies to induce robust anti-neuroblastoma activity. Overall design: Four neuroblastoma cell lines were untreated or treated with GSK-J4 for 72 hours. Each cell line and condition was performed in triplicate.
Project description:Neuroblastoma cell lines are an important and cost-effective model used to study oncogenic drivers of the disease. While many of these cell lines have been previously characterized with SNP, methylation, and/or expression microarrays, there has not been an effort to comprehensively sequence these cell lines. Here, we present raw whole transcriptome data generated by RNA sequencing of 39 commonly-used neuroblastoma cell lines. This data can be used to perform differential expression analysis based on a genetic aberration or phenotype in neuroblastoma (eg: MYCN amplification status, ALK mutation status, 11q status, sensitivity to pharmacological pertubation). Additionally, we designed this experiment to enable structural variant and/or long-noncoding RNA analysis across these cell lines. Finally, as more DNase/ATAC and histone/transcription factor ChIP sequencing is performed in these cell lines, our RNA-Seq data will be an important complement to inform transcriptional targets as well as regulatory (enhancer or repressor) elements in neuroblastoma. Overall design: We sequenced neuroblastoma cell lines (N=39) with varying genomic characteristics, disease stages, and phases of therapy. We also sequenced the hTERT-immortalized retinal pigmented epithelial cell line, RPE-1 (N=1), which is commonly used as a non-neuroblastoma control, as well as RNA from the fetal brain (N=1), which can serve as a non-neuroblastoma neural-cell derived control.
Project description:Intra-tumour heterogeneity is increasingly appreciated as a determinant of tumour recurrence. Several tumour types were recently found to include phenotypically divergent cell types, reflecting lineage development stages (1,2,3). Lineage identity has been proposed to ensue super-enhancer (SE)-associated transcription factor (TF) networks (4,5), but their role in intra-tumour heterogeneity is unknown. Neuroblastoma is a paediatric tumour of the adrenergic differentiation lineage. Here we show that most neuroblastoma tumors include two types of tumor cells with highly diverging gene expression profiles. The undifferentiated mesenchymal cells and more differentiated adrenergic cells can interconvert and may relate to normal lineage differentiation stages. ChIP-seq analysis of isogenic pairs of mesenchymal and adrenergic neuroblastoma cells revealed a distinct, highly consistent super-enhancer landscape for each cell type. Two SE-associated TF networks emerged that potentially master each cell type. Accordingly, the mesenchymal TF PRRX1 could reprogram the SE- and mRNA-profiles of adrenergic cells towards a mesenchymal state. To assess the clinical relevance of this bi-phasic system, we investigated chemo-sensitivity of both cell types. Mesenchymal cells were more resistant in vitro and were enriched in post-therapy and relapsed neuroblastoma in patients. Intra-tumor heterogeneity in neuroblastoma is therefore structured according to distinct SE-associated transcriptional programs that mediate a dynamic bi-phasic structure. Overall design: Cultured cells were fixed with 1% formaldehyde. Nuclei were isolated and the DNA was sheared to 200 to 300 bp fragments. Histon-bound DNA was precipitated using antibodies H3K27Ac (#4729, Abcam) and H3K4me3 (#04-745, Millipore). De-crosslinked DNA was purified using Qiagen PCR purification kit (Qiagen) and quantified with Quant-IT Picogreen (Invitrogen). The DNA was used to generate sequencing libraries according to the manufactures procedure (Life Technologies): The DNA was end-polished, dA-tailed and adaptors with barcodes were ligated. The fragments were amplified (8 cycles) and quantified with a Bioanalyzer (Agilent). The libraries were prepped with the 5500W Flowchip v2 kit (Life Technologies) and sequenced on the SOLiD Wildfire (Illumina) resulting in 50 bp reads. Alternatively, the libraries were sequenced using the HiSeq PE cluster kit v4 (Illumina) with the HiSeq2500 (Illumina) resulting in 125 bp reads.
Project description:Expression profiling in Neuroblastoma Primary tumors and Cell lines Overall design: We studied the expression profiles of Neuroblastoma Primary tumors and Cell lines using Illumina Human 6 version 2 expression l bead chip.
Project description:This SuperSeries is composed of the following subset Series: GSE31229: Neuroblastoma cell lines treated with DAC (2'-deoxy-5-azacytidine), a DNA-methylation inhibitor GSE31353: Methylation map of 8 neuroblastoma cell lines: NGS after MBD2-capture Refer to individual Series
Project description:Human neuroblatoma cell lines (N=25) and retinal pigmented epithelium cell lines (N=4) were analyzed for gene expression under untreated/baseline growth conditions. Expression profiling and characterization of a set of Neuroblastoma and retinal pigmented epithelium cell lines