Project description:BackgroundCurrent clinical risk factors stratify patients with neuroblastoma (NB) for appropriate treatments, yet patients with similar clinical behaviors evoke variable responses. MYCN amplification is one of the established drivers of NB and, when combined with high-risk displays, worsens outcomes. Growing high-throughput transcriptomics studies suggest long noncoding RNA (lncRNA) dysregulation in cancers, including NB. However, expression-based lncRNA signatures are altered by MYCN amplification, which is associated with high-risk, and patient prognosis remains limited.MethodsWe investigated RNA-seq-based expression profiles of lncRNAs in MYCN status and risk status in a discovery cohort (n = 493) and validated them in three independent cohorts. In the discovery cohort, a prognostic association of lncRNAs was determined by univariate Cox regression and integrated into a signature using the risk score method. A novel risk score threshold selection criterion was developed to stratify patients into risk groups. Outcomes by risk group and clinical subgroup were assessed using Kaplan-Meier survival curves and multivariable Cox regression. The performance of lncRNA signatures was evaluated by receiver operating characteristic curve. All statistical tests were two-sided.ResultsIn the discovery cohort, 16 lncRNAs that were differentially expressed (fold change ≥ 2 and adjusted P ≤ 0.01) integrated into a prognostic signature. A high risk score group of lncRNA signature had poor event-free survival (EFS; P < 1E-16). Notably, lncRNA signature was independent of other clinical risk factors when predicting EFS (hazard ratio = 3.21, P = 5.95E-07). The findings were confirmed in independent cohorts (P = 2.86E-02, P = 6.18E-03, P = 9.39E-03, respectively). Finally, the lncRNA signature had higher accuracy for EFS prediction (area under the curve = 0.788, 95% confidence interval = 0.746 to 0.831).ConclusionsHere, we report the first (to our knowledge) RNA-seq 16-lncRNA prognostic signature for NB that may contribute to precise clinical stratification and EFS prediction.

Project description:Purpose: Neuroblastoma is the most common extracranial solid tumor in childhood. We previously showed that circulating cell-free DNA (cfDNA) and tumor biopsy derived 5-hydroxymethylcytosime (5-hmC) profiles identified patients with neuroblastoma who experienced subsequent relapse. Here, we hypothesized that 5-hmC modifications selectively enriched in cfDNA compared with tumor biopsy samples would identify epigenetic changes associated with aggressive tumor behavior and identify novel biomarkers of outcome in patients with high- risk neuroblastoma. Methods: 5-hmC profiles from cfDNA (n = 64) and tumor biopsies (n = 48) were compared. Two neuroblastoma cell lines underwent chromatin immunopre- cipitation followed by sequencing (ChIP-Seq) for H3K27me3, H3K4me3, and H3K27ac; kethoxal-associated single-stranded DNA sequencing; hmC-Seal for 5-hmC; and RNA-sequencing (RNA-Seq). Genes enriched for both H3K27me3 and H3K4me3 in the included cell lines were defined as bivalent. Using bivalent genes defined in vitro, a bivalent signature was established in three publicly available cohorts of patients with neuroblastoma through gene set variation analysis. Differences between tumors with high or low bivalent signatures were assessed by the Kaplan-Meier method and Cox proportional hazards models. Results: In cfDNA compared with tumor biopsy derived 5-hmC profiles, we found in- creased 5-hmC deposition on Polycomb Repressive Complex 2 target genes, a finding previously described in the context of bivalent genes. We identified 313 genes that bore bivalent chromatin marks, were enriched for mediators of neuronal differentiation, and were transcriptionally repressed across a panel of heterogeneous neuroblastoma cell lines. In three distinct clinical cohorts, low bivalent signature was significantly and independently associated with worse clinical outcome in patients with high-risk neuroblastoma. Conclusion: Low expression of bivalent genes is a biomarker of worse outcome in patients with high-risk neuroblastoma.

Project description:Neuroblastoma is a pediatric tumor of the peripheral sympathetic nervous system with diverse clinical behaviors. Even with multimodal therapies, high-risk neuroblastoma has an unfavorable outcome irrespective of MYCN amplification, a well-established oncogenic driver in neuroblastoma pathogenesis, and its genetic heterogeneity has largely impeded efforts to correlate molecular targets with biological consequences for more effective treatment strategies. Here, using a gene expression-based approach, we identified the FDA-approved anthelmintic niclosamide as a potential anti-neuroblastoma drug. By combining the gene expression signature associated with high-risk neuroblastoma and the recurrent drug−transcript relationships inferred from up to one million perturbational gene expression profiles, our algorithm predicted effective therapeutic candidates by evaluating the extent to which a given compound or their combinations could ‘reverse’ the high-risk signature. Furthermore, we performed quantitative polymerase chain reaction (qPCR) to validate top five candidate reverse genes which are involved in DNA replication, including cyclin A2 (CCNA2), minichromosome maintenance 10 replication initiation factor (MCM10), ERCC excision repair 6 like, spindle assembly checkpoint helicase (ERCC6L), kinesin family member 20A (KIF20A), and RuvB like AAA ATPase 1 (RUVBL1). Indeed, those five genes were downregulated in niclosamide-treated cells, indicating niclosamide suppressed DNA replication and then inhibited cell proliferation. Using cell proliferation and clonogenic assays as well as flow cytometry, we determined the cytotoxic effects of niclosamide in MYCN-amplified SK-N-DZ and non-amplified SK-N-AS cells. The results showed that niclosamide could effectively reduce not only cell proliferation and colony formation but also trigger cell cycle arrest and apoptosis. Moreover, we conducted human tumor xenografts in a nude mice model to evaluate the in vivo efficacy of niclosamide and found that it significantly suppressed tumor growth and prolonged survival rate, but doesn’t cause organ damage and change body weight. To explore the molecular mechanism of niclosamide, stable-isotope dimethyl labeling strategy for quantitative proteomics was performed on both cell-based or xenograft-based MYCN-amplified SK-N-DZ and MYCN-nonamplified SK-N-AS models. We confirmed niclosamide not only mediated the function of mitochondrial electron transport chain but also the other functions in high risk neuroblastoma cell lines and xenografts. The results suggest that our developed expression-based strategy is useful for drug discovery and provides the possibility of repurposing the anthelminthic drug niclosamide for treating high-risk neuroblastoma therapy.

Project description:Purpose: Neuroblastoma is the most common extracranial solid tumor in childhood. We previously showed that circulating cell-free DNA (cfDNA) and tumor biopsy derived 5-hydroxymethylcytosime (5-hmC) profiles identified patients with neuroblastoma who experienced subsequent relapse. Here, we hypothesized that 5-hmC modifications selectively enriched in cfDNA compared with tumor biopsy samples would identify epigenetic changes associated with aggressive tumor behavior and identify novel biomarkers of outcome in patients with high- risk neuroblastoma. Methods: 5-hmC profiles from cfDNA (n = 64) and tumor biopsies (n = 48) were compared. Two neuroblastoma cell lines underwent chromatin immunopre- cipitation followed by sequencing (ChIP-Seq) for H3K27me3, H3K4me3, and H3K27ac; kethoxal-associated single-stranded DNA sequencing; hmC-Seal for 5-hmC; and RNA-sequencing (RNA-Seq). Genes enriched for both H3K27me3 and H3K4me3 in the included cell lines were defined as bivalent. Using bivalent genes defined in vitro, a bivalent signature was established in three publicly available cohorts of patients with neuroblastoma through gene set variation analysis. Differences between tumors with high or low bivalent signatures were assessed by the Kaplan-Meier method and Cox proportional hazards models. Results: In cfDNA compared with tumor biopsy derived 5-hmC profiles, we found in- creased 5-hmC deposition on Polycomb Repressive Complex 2 target genes, a finding previously described in the context of bivalent genes. We identified 313 genes that bore bivalent chromatin marks, were enriched for mediators of neuronal differentiation, and were transcriptionally repressed across a panel of heterogeneous neuroblastoma cell lines. In three distinct clinical cohorts, low bivalent signature was significantly and independently associated with worse clinical outcome in patients with high-risk neuroblastoma. Conclusion: Low expression of bivalent genes is a biomarker of worse outcome in patients with high-risk neuroblastoma.

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A p53 drug response signature identifies prognostic genes in high-risk neuroblastoma.

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A p53 drug response signature identifies prognostic genes in high-risk neuroblastoma.

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