Project description:Neuroblastoma is a pediatric tumor of the sympathetic nervous system. MYCN (V-myc myelocytomatosis viral-related oncogene, neuroblastoma derived [avian]) is amplified in 20% of neuroblastomas, and these tumors carry a poor prognosis. However, tumors without MYCN amplification also may have a poor outcome. Here, we identified downstream targets of MYCN by shRNA-mediated silencing MYCN in neuroblastoma cells. From these targets, 157 genes showed an expression profile correlating with MYCN mRNA levels in NB88, a series of 88 neuroblastoma tumors, and therefore represent in vivo relevant MYCN pathway genes. This 157-gene signature identified very poor prognosis tumors in NB88 and independent neuroblastoma cohorts and was more powerful than MYCN amplification or MYCN expression alone. Remarkably, this signature also identified poor outcome of a group of tumors without MYCN amplification. Most of these tumors have low MYCN mRNA levels but high nuclear MYCN protein levels, suggesting stabilization of MYCN at the protein level. One tumor has an MYC amplification and high MYC expression. Chip-on-chip analyses showed that most genes in this signature are directly regulated by MYCN. MYCN induces genes functioning in cell cycle and DNA repair while repressing neuronal differentiation genes. The functional MYCN-157 signature recognizes classical neuroblastoma with MYCN amplification, as well as a newly identified group marked by MYCN protein stabilization.
Project description:Amplification of the MYCN oncogene predicts treatment resistance in childhood neuroblastoma. Using a MYC target gene signature that predicts poor neuroblastoma prognosis we identified the histone chaperone, FAcilitates Chromatin Transcription (FACT), as a crucial mediator of the MYC signal and a therapeutic target in the disease. FACT and MYCN expression created a forward feedback loop in neuroblastoma cells that was essential for maintaining mutual high expression. FACT inhibition by the small molecule Curaxin compound, CBL0137, markedly reduced tumor initiation and progression in vivo. CBL0137 exhibited strong synergy with chemotherapy in standard use by blocking repair of DNA damage caused by genotoxic drugs, thus creating a synthetic lethal environment in MYCN amplified neuroblastoma cells and a treatment strategy for MYCN-driven neuroblastoma
Project description:Amplification of the MYCN oncogene predicts treatment resistance in childhood neuroblastoma. Using a MYC target gene signature that predicts poor neuroblastoma prognosis we identified the histone chaperone, FAcilitates Chromatin Transcription (FACT), as a crucial mediator of the MYC signal and a therapeutic target in the disease. FACT and MYCN expression created a forward feedback loop in neuroblastoma cells that was essential for maintaining mutual high expression. FACT inhibition by the small molecule Curaxin compound, CBL0137, markedly reduced tumor initiation and progression in vivo. CBL0137 exhibited strong synergy with chemotherapy in standard use by blocking repair of DNA damage caused by genotoxic drugs, thus creating a synthetic lethal environment in MYCN amplified neuroblastoma cells and a treatment strategy for MYCN-driven neuroblastoma
Project description:Amplification of the MYCN oncogene predicts treatment resistance in childhood neuroblastoma. Using a MYC target gene signature that predicts poor neuroblastoma prognosis we identified the histone chaperone, FAcilitates Chromatin Transcription (FACT), as a crucial mediator of the MYC signal and a therapeutic target in the disease. FACT and MYCN expression created a forward feedback loop in neuroblastoma cells that was essential for maintaining mutual high expression. FACT inhibition by the small molecule Curaxin compound, CBL0137, markedly reduced tumor initiation and progression in vivo. CBL0137 exhibited strong synergy with chemotherapy in standard use by blocking repair of DNA damage caused by genotoxic drugs, thus creating a synthetic lethal environment in MYCN amplified neuroblastoma cells and a treatment strategy for MYCN-driven neuroblastoma
Project description:Amplification of the MYCN oncogene predicts treatment resistance in childhood neuroblastoma. Using a MYC target gene signature that predicts poor neuroblastoma prognosis we identified the histone chaperone, FAcilitates Chromatin Transcription (FACT), as a crucial mediator of the MYC signal and a therapeutic target in the disease. FACT and MYCN expression created a forward feedback loop in neuroblastoma cells that was essential for maintaining mutual high expression. FACT inhibition by the small molecule Curaxin compound, CBL0137, markedly reduced tumor initiation and progression in vivo. CBL0137 exhibited strong synergy with chemotherapy in standard use by blocking repair of DNA damage caused by genotoxic drugs, thus creating a synthetic lethal environment in MYCN amplified neuroblastoma cells and a treatment strategy for MYCN-driven neuroblastoma
Project description:<p>Neuroblastoma is the most common extra-cranial solid tumor in children. It represents 8% to 10% of all childhood cancers. Stage 4 Neuroblastoma is characterized by its clinical heterogeneous outcome. The special category, stage 4S tumors (2-5% of all NB) are chemo-sensitive, and the patients show spontaneous regression. On the other hand, MYCN amplification (25-30% of all NB) is associated with poor outcome of neuroblastoma, thus we further categorize stage 4 neuroblastoma into MYCN non-amplified and MYCN amplified group. Here we use transcriptome sequencing to characterize the transcriptome in 29 stage 4 Neuroblastoma samples.</p>
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:The stabilization of MYCN by FOXR2 represents an alternative mechanism to MYCN amplification to increase MYCN protein levels. As such, FOXR2 expression identifies another subset of neuroblastoma patients with unfavorable clinical outcome. Background: Clinical outcomes of neuroblastoma patients range from spontaneous tumor regression to fatality. Hence, understanding the mechanisms that cause tumor progression are crucial for the treatment of patients. In this study, we show that FOXR2 activation identifies a subset of neuroblastoma tumors with unfavorable outcome and we investigate the mechanism how FOXR2 relates to poor outcome in patients.
Project description:MYCN amplification in neuroblastoma leads to aberrant expression of MYCN oncoprotein, which binds active genes promoting transcriptional amplification. Yet how MYCN coordinates transcription elongation to meet productive transcriptional amplification and which elongation machinery represents MYCN-driven vulnerability remain to be identified. We conducted a targeted screen of transcription elongation factors and identified the super elongation complex (SEC) as a unique vulnerability in MYCN-amplified neuroblastomas. MYCN directly binds EAF1 and recruits SEC to enhance processive transcription elongation. Depletion of EAF1 or AFF1/AFF4, another core subunit of SEC, leads to a global reduction in transcription elongation and elicits selective apoptosis of MYCN-amplified neuroblastoma cells. A combination screen reveals SEC inhibition synergistically potentiates the therapeutic efficacies of FDA-approved BCL2 antagonist ABT-199, in part due to suppression of MCL1 expression, both in MYCN-amplified neuroblastoma cells and in patient-derived xenografts. These findings identify disruption of the MYCN-SEC regulatory axis as a promising therapeutic strategy in neuroblastoma.
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