RESUMEN
Neuroblastoma is a tumour that arises from the sympathoadrenal lineage occurring predominantly in children younger than five years. About half of the patients are diagnosed with high-risk tumours and undergo intensive multi-modal therapy. The success rate of current treatments for high-risk neuroblastoma is disappointingly low and survivors suffer from multiple therapy-related long-term side effects. Most chemotherapeutics drive cancer cells towards cell death or senescence. Senescence has long been considered to represent a terminal non-proliferative state and therefore an effective barrier against tumorigenesis. This dogma, however, has been challenged by recent observations that infer a much more dynamic and reversible nature for this process, which may have implications for the efficacy of therapy-induced senescence-oriented treatment strategies. Neuroblastoma cells in a dormant, senescent-like state may escape therapy, whilst their senescence-associated secretome may promote inflammation and invasiveness, potentially fostering relapse. Conversely, due to its distinct molecular identity, senescence may also represent an opportunity for the development of novel (combination) therapies. However, the limited knowledge on the molecular dynamics and diversity of senescence signatures demands appropriate models to study this process in detail. This review summarises the molecular knowledge about cellular senescence in neuroblastoma and investigates current and future options towards therapeutic exploration.
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Recurrencia Local de Neoplasia , Neuroblastoma , Transformación Celular Neoplásica , Senescencia Celular , Niño , Humanos , Neuroblastoma/genética , Neuroblastoma/patología , Neuroblastoma/terapiaRESUMEN
Human embryonic stem cells (hESCs) and embryonal tumors share a number of common features, including a compromised G1/S checkpoint. Consequently, these rapidly dividing hESCs and cancer cells undergo elevated levels of replicative stress, inducing genomic instability that drives chromosomal imbalances. In this context, it is of interest that long-term in vitro cultured hESCs exhibit a remarkable high incidence of segmental DNA copy number gains, some of which are also highly recurrent in certain malignancies such as 17q gain (17q+). The selective advantage of DNA copy number changes in these cells has been attributed to several underlying processes including enhanced proliferation. We hypothesized that these recurrent chromosomal imbalances become rapidly embedded in the cultured hESCs through a replicative stress driven Darwinian selection process. To this end, we compared the effect of hydroxyurea-induced replicative stress vs normal growth conditions in an equally mixed cell population of isogenic euploid and 17q + hESCs. We could show that 17q + hESCs rapidly overtook normal hESCs. Our data suggest that recurrent chromosomal segmental gains provide a proliferative advantage to hESCs under increased replicative stress, a process that may also explain the highly recurrent nature of certain imbalances in cancer.
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División Celular , Aberraciones Cromosómicas , Células Madre Embrionarias Humanas/citología , Selección Genética , Ciclo Celular/genética , Línea Celular Tumoral , Proliferación Celular , Cromosomas Humanos Par 17 , Variaciones en el Número de Copia de ADN , Humanos , Hidroxiurea , Estrés Fisiológico , TranscriptomaAsunto(s)
Proteínas de Homeodominio/genética , Poli A/genética , Leucemia-Linfoma Linfoblástico de Células T Precursoras/genética , Proteínas Proto-Oncogénicas/genética , ARN Largo no Codificante/genética , Análisis de Secuencia de ARN/métodos , Azepinas/farmacología , Línea Celular Tumoral , Perfilación de la Expresión Génica , Regulación Neoplásica de la Expresión Génica , Humanos , Interferencia de ARN , Linfocitos T/efectos de los fármacos , Linfocitos T/metabolismo , Triazoles/farmacologíaRESUMEN
The pediatric extra-cranial tumor neuroblastoma displays a low mutational burden while recurrent copy number alterations are present in most high-risk cases. Here, we identify SOX11 as a dependency transcription factor in adrenergic neuroblastoma based on recurrent chromosome 2p focal gains and amplifications, specific expression in the normal sympatho-adrenal lineage and adrenergic neuroblastoma, regulation by multiple adrenergic specific (super-)enhancers and strong dependency on high SOX11 expression in adrenergic neuroblastomas. SOX11 regulated direct targets include genes implicated in epigenetic control, cytoskeleton and neurodevelopment. Most notably, SOX11 controls chromatin regulatory complexes, including 10 SWI/SNF core components among which SMARCC1, SMARCA4/BRG1 and ARID1A. Additionally, the histone deacetylase HDAC2, PRC1 complex component CBX2, chromatin-modifying enzyme KDM1A/LSD1 and pioneer factor c-MYB are regulated by SOX11. Finally, SOX11 is identified as a core transcription factor of the core regulatory circuitry (CRC) in adrenergic high-risk neuroblastoma with a potential role as epigenetic master regulator upstream of the CRC.
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Neuroblastoma , Humanos , Niño , Neuroblastoma/genética , Factores de Transcripción/genética , Cromatina , Núcleo Celular , Aberraciones Cromosómicas , Adrenérgicos , ADN Helicasas , Proteínas Nucleares/genética , Factores de Transcripción SOXC/genética , Histona DemetilasasRESUMEN
Background: Real-time polymerase chain reaction (RT-PCR) testing on a nasopharyngeal swab is the current standard for SARS-CoV-2 virus detection. Since collection of this sample type is experienced uncomfortable by patients, saliva- and oropharyngeal swab collections should be considered as alternative specimens. Objectives: Evaluation of the relative performance of oropharyngeal swab, nasopharyngeal swab and saliva for the RT-PCR based SARS-CoV-2 Delta (B.1.617.2) and Omicron (B.1.1.529) variant detection. Study design: Nasopharyngeal swab, oropharyngeal swab and saliva were collected from 246 adult patients who presented for SARS-CoV-2 testing at the screening centre in Ypres (Belgium). RT-PCR SARS-CoV-2 detection was performed on all three sample types separately. Variant type was determined for each positive patient using whole genome sequencing or Allplex SARS-CoV-2 variants I and II Assay. Results and conclusions: Saliva is superior compared to nasopharyngeal swab for the detection of the Omicron variant. For the detection of the Delta variant, nasopharyngeal swab and saliva can be considered equivalent specimens. Oropharyngeal swab is the least sensitive sample type and shows little added value when collected in addition to a single nasopharyngeal swab.
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High-risk neuroblastoma, a pediatric tumor originating from the sympathetic nervous system, has a low mutation load but highly recurrent somatic DNA copy number variants. Previously, segmental gains and/or amplifications allowed identification of drivers for neuroblastoma development. Using this approach, combined with gene dosage impact on expression and survival, we identified ribonucleotide reductase subunit M2 (RRM2) as a candidate dependency factor further supported by growth inhibition upon in vitro knockdown and accelerated tumor formation in a neuroblastoma zebrafish model coexpressing human RRM2 with MYCN. Forced RRM2 induction alleviates excessive replicative stress induced by CHK1 inhibition, while high RRM2 expression in human neuroblastomas correlates with high CHK1 activity. MYCN-driven zebrafish tumors with RRM2 co-overexpression exhibit differentially expressed DNA repair genes in keeping with enhanced ATR-CHK1 signaling activity. In vitro, RRM2 inhibition enhances intrinsic replication stress checkpoint addiction. Last, combinatorial RRM2-CHK1 inhibition acts synergistic in high-risk neuroblastoma cell lines and patient-derived xenograft models, illustrating the therapeutic potential.
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Neuroblastoma is a pediatric tumor arising from the sympatho-adrenal lineage and a worldwide leading cause of childhood cancer-related deaths. About half of high-risk patients die from the disease while survivors suffer from multiple therapy-related side-effects. While neuroblastomas present with a low mutational burden, focal and large segmental DNA copy number aberrations are highly recurrent and associated with poor survival. It can be assumed that the affected chromosomal regions contain critical genes implicated in neuroblastoma biology and behavior. More specifically, evidence has emerged that several of these genes are implicated in tumor dependencies thus potentially providing novel therapeutic entry points. In this review, we briefly review the current status of recurrent DNA copy number aberrations in neuroblastoma and provide an overview of the genes affected by these genomic variants for which a direct role in neuroblastoma has been established. Several of these genes are implicated in networks that positively regulate MYCN expression or stability as well as cell cycle control and apoptosis. Finally, we summarize alternative approaches to identify and prioritize candidate copy-number driven dependency genes for neuroblastoma offering novel therapeutic opportunities.
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MYCN is an oncogenic driver in neural crest-derived neuroblastoma and medulloblastoma. To better understand the early effects of MYCN activation in a neural-crest lineage context, we profiled the transcriptome of immortalized human retina pigment epithelial cells with inducible MYCN activation. Gene signatures associated with elevated MYC/MYCN activity were induced after 24 h of MYCN activation, which attenuated but sustained at later time points. Unexpectedly, MYCN activation was accompanied by reduced cell growth. Gene set enrichment analysis revealed a senescence-like signature with strong induction of p53 and p21 but in the absence of canonical hallmarks of senescence such as ß-galactosidase positivity, suggesting incomplete cell fate commitment. When scrutinizing the putative drivers of this growth attenuation, differential gene expression analysis identified several regulators of nucleolar stress. This process was also reflected by phenotypic correlates such as cytoplasmic granule accrual and nucleolar coalescence. Hence, we propose that the induction of MYCN congests the translational machinery, causing nucleolar stress and driving cells into a transient pre-senescent state. Our findings shed new light on the early events induced by MYCN activation and may help unravelling which factors are required for cells to tolerate unscheduled MYCN overexpression during early malignant transformation.
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Proteína Proto-Oncogénica N-Myc , Neuroblastoma , Ciclo Celular , Diferenciación Celular , Línea Celular Tumoral , Proliferación Celular , Humanos , Cresta Neural/metabolismo , Proteínas Proto-Oncogénicas c-mycRESUMEN
The migrational propensity of neuroblastoma is affected by cell identity, but the mechanisms behind the divergence remain unknown. Using RNAi and time-lapse imaging, we show that ADRN-type NB cells exhibit RAC1- and kalirin-dependent nucleokinetic (NUC) migration that relies on several integral components of neuronal migration. Inhibition of NUC migration by RAC1 and kalirin-GEF1 inhibitors occurs without hampering cell proliferation and ADRN identity. Using three clinically relevant expression dichotomies, we reveal that most of up-regulated mRNAs in RAC1- and kalirin-GEF1-suppressed ADRN-type NB cells are associated with low-risk characteristics. The computational analysis shows that, in a context of overall gene set poverty, the upregulomes in RAC1- and kalirin-GEF1-suppressed ADRN-type cells are a batch of AU-rich element-containing mRNAs, which suggests a link between NUC migration and mRNA stability. Gene set enrichment analysis-based search for vulnerabilities reveals prospective weak points in RAC1- and kalirin-GEF1-suppressed ADRN-type NB cells, including activities of H3K27- and DNA methyltransferases. Altogether, these data support the introduction of NUC inhibitors into cancer treatment research.
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Factores de Intercambio de Guanina Nucleótido/metabolismo , Neuroblastoma/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Proteína de Unión al GTP rac1/metabolismo , Neuronas Adrenérgicas/metabolismo , Línea Celular Tumoral , Movimiento Celular/genética , Células Cultivadas , Preescolar , Bases de Datos Genéticas , Femenino , Factores de Intercambio de Guanina Nucleótido/fisiología , Humanos , Masculino , Neuroblastoma/patología , Estudios Prospectivos , Proteínas Serina-Treonina Quinasas/fisiología , Proteína de Unión al GTP rac1/fisiologíaRESUMEN
Roughly half of all high-risk neuroblastoma patients present with MYCN amplification. The molecular consequences of MYCN overexpression in this aggressive pediatric tumor have been studied for decades, but thus far, our understanding of the early initiating steps of MYCN-driven tumor formation is still enigmatic. We performed a detailed transcriptome landscaping during murine TH-MYCN-driven neuroblastoma tumor formation at different time points. The neuroblastoma dependency factor MEIS2, together with ASCL1, was identified as a candidate tumor-initiating factor and shown to be a novel core regulatory circuit member in adrenergic neuroblastomas. Of further interest, we found a KEOPS complex member (gm6890), implicated in homologous double-strand break repair and telomere maintenance, to be strongly upregulated during tumor formation, as well as the checkpoint adaptor Claspin (CLSPN) and three chromosome 17q loci CBX2, GJC1 and LIMD2. Finally, cross-species master regulator analysis identified FOXM1, together with additional hubs controlling transcriptome profiles of MYCN-driven neuroblastoma. In conclusion, time-resolved transcriptome analysis of early hyperplastic lesions and full-blown MYCN-driven neuroblastomas yielded novel components implicated in both tumor initiation and maintenance, providing putative novel drug targets for MYCN-driven neuroblastoma.
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Chromosome 17q gains are a common alteration in high-risk neuroblastomas with unknown functional significance. We identified a 17q super-enhancer regulated T-box Transcription Factor 2 (TBX2) as constituent of a core regulatory circuitry driving proliferation through enhancing V-myc myelocytomatosis viral-related oncogene, neuroblastoma derived (avian) (MYCN)/Forkhead box protein M1(FOXM1) reactivation of dimerization partner, RB-like, E2F and multi-vulval class B (DREAM) targets, which can be affected synergistically by combined cyclin-dependent kinase 7 and Bromo-domain inhibition.
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A correction to this article has been published and is linked from the HTML and PDF versions of this paper. The error has been fixed in the paper.
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Long intergenic non-coding RNAs (lincRNAs) are emerging as integral components of signaling pathways in various cancer types. In neuroblastoma, only a handful of lincRNAs are known as upstream regulators or downstream effectors of oncogenes. Here, we exploit RNA sequencing data of primary neuroblastoma tumors, neuroblast precursor cells, neuroblastoma cell lines and various cellular perturbation model systems to define the neuroblastoma lincRNome and map lincRNAs up- and downstream of neuroblastoma driver genes MYCN, ALK and PHOX2B. Each of these driver genes controls the expression of a particular subset of lincRNAs, several of which are associated with poor survival and are differentially expressed in neuroblastoma tumors compared to neuroblasts. By integrating RNA sequencing data from both primary tumor tissue and cancer cell lines, we demonstrate that several of these lincRNAs are expressed in stromal cells. Deconvolution of primary tumor gene expression data revealed a strong association between stromal cell composition and driver gene status, resulting in differential expression of these lincRNAs. We also explored lincRNAs that putatively act upstream of neuroblastoma driver genes, either as presumed modulators of driver gene activity, or as modulators of effectors regulating driver gene expression. This analysis revealed strong associations between the neuroblastoma lincRNAs MIAT and MEG3 and MYCN and PHOX2B activity or expression. Together, our results provide a comprehensive catalogue of the neuroblastoma lincRNome, highlighting lincRNAs up- and downstream of key neuroblastoma driver genes. This catalogue forms a solid basis for further functional validation of candidate neuroblastoma lincRNAs.
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Neuroblastoma/genética , ARN Largo no Codificante/genética , Línea Celular Tumoral , Tecnología de Genética Dirigida/métodos , Perfilación de la Expresión Génica/métodos , Humanos , Células-Madre Neurales/fisiología , Análisis de Secuencia de ARN/métodos , Transducción de Señal/genética , Factores de Transcripción/genéticaRESUMEN
A correction to this article has been published and is linked from the HTML and PDF versions of this paper. The error has been fixed in the paper.
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ALK mutations occur in 10% of primary neuroblastomas and represent a major target for precision treatment. In combination with MYCN amplification, ALK mutations infer an ultra-high-risk phenotype resulting in very poor patient prognosis. To open up opportunities for future precision drugging, a deeper understanding of the molecular consequences of constitutive ALK signaling and its relationship to MYCN activity in this aggressive pediatric tumor entity will be essential. We show that mutant ALK downregulates the 'HMG-box transcription factor 1' (HBP1) through the PI3K-AKT-FOXO3a signaling axis. HBP1 inhibits both the transcriptional activating and repressing activity of MYCN, the latter being mediated through PRC2 activity. HBP1 itself is under negative control of MYCN through miR-17~92. Combined targeting of HBP1 by PI3K antagonists and MYCN signaling by BET- or HDAC-inhibitors blocks MYCN activity and significantly reduces tumor growth, suggesting a novel targeted therapy option for high-risk neuroblastoma.
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Quinasa de Linfoma Anaplásico/genética , Proteínas del Grupo de Alta Movilidad/genética , Proteína Proto-Oncogénica N-Myc/genética , Neuroblastoma/genética , Proteínas Represoras/genética , Animales , Línea Celular Tumoral , Proliferación Celular/genética , Regulación hacia Abajo/genética , Proteína Forkhead Box O3/genética , Regulación Neoplásica de la Expresión Génica/genética , Humanos , Ratones , MicroARNs/genética , Mutación/genética , Fosfatidilinositol 3-Quinasas/genética , Proteínas Proto-Oncogénicas c-akt/genética , Transducción de Señal/genética , Activación Transcripcional/genéticaRESUMEN
Chemotherapy resistance is responsible for high mortality rates in neuroblastoma. MYCN, an oncogenic driver in neuroblastoma, controls pluripotency genes including LIN28B. We hypothesized that enhanced embryonic stem cell (ESC) gene regulatory programs could mark tumors with high pluripotency capacity and subsequently increased risk for therapy failure. An ESC miRNA signature was established based on publicly available data. In addition, an ESC mRNA signature was generated including the 500 protein coding genes with the highest positive expression correlation with the ESC miRNA signature score in 200 neuroblastomas. High ESC m(i)RNA expression signature scores were significantly correlated with poor neuroblastoma patient outcome specifically in the subgroup of MYCN amplified tumors and stage 4 nonamplified tumors. Further data-mining identified FOXM1, as the major predicted driver of this ESC signature, controlling a large set of genes implicated in cell cycle control and DNA damage response. Of further interest, re-analysis of published data showed that MYCN transcriptionally activates FOXM1 in neuroblastoma cells. In conclusion, a novel ESC m(i)RNA signature stratifies neuroblastomas with poor prognosis, enabling the identification of therapy-resistant tumors. The finding that this signature is strongly FOXM1 driven, warrants for drug design targeted at FOXM1 or key components controlling this pathway.
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Proteína Forkhead Box M1/metabolismo , Neuroblastoma/tratamiento farmacológico , Neuroblastoma/metabolismo , Transducción de Señal , Animales , Antineoplásicos/química , Antineoplásicos/uso terapéutico , Ciclo Celular/genética , Simulación por Computador , Daño del ADN/genética , Diseño de Fármacos , Células Madre Embrionarias/metabolismo , Células Madre Embrionarias/patología , Genes myc , Humanos , Ratones , Ratones Transgénicos , Células Madre Neoplásicas/metabolismo , Células Madre Neoplásicas/patología , Neuroblastoma/genética , Neuroblastoma/patología , PronósticoRESUMEN
Reverse transcription quantitative PCR (RT-qPCR) is the gold standard method for gene expression analysis on mRNA level. To remove experimental variation, expression levels of the gene of interest are typically normalized to the expression level of stably expressed endogenous reference genes. Identifying suitable reference genes and determining the optimal number of reference genes should precede each quantification study. Popular reference genes are not necessarily stably expressed in the examined conditions, possibly leading to inaccurate results. Stably and universally expressed repetitive elements (ERE) have previously been shown to be an excellent alternative for normalization using classic reference genes in human and zebrafish samples. Here, we confirm that in mouse tissues, EREs are broadly applicable reference targets for RT-qPCR normalization, provided that the RNA samples undergo a thorough DNase treatment. We identified Orr1a0, Rltr2aiap, and Rltr13a3 as the most stably expressed mouse EREs across six different experimental conditions. Therefore, we propose this set of ERE reference targets as good candidates for normalization of RT-qPCR data in a plethora of conditions. The identification of widely applicable stable mouse RT-qPCR reference targets for normalization has great potential to facilitate future murine gene expression studies and improve the validity of RT-qPCR data.
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Anomalías Cardiovasculares/genética , Leucemia Prolinfocítica de Células T/genética , Neuroblastoma/genética , Secuencias Repetitivas de Ácidos Nucleicos/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa/normas , Enfermedades de la Piel/genética , Animales , Anomalías Cardiovasculares/patología , Modelos Animales de Enfermedad , Femenino , Humanos , Leucemia Prolinfocítica de Células T/patología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Neuroblastoma/patología , Estándares de Referencia , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa/métodos , Enfermedades de la Piel/patología , Pez Cebra/metabolismoRESUMEN
Neuroblastoma is a pediatric cancer of the sympathetic nervous system where MYCN amplification is a key indicator of poor prognosis. However, mechanisms by which MYCN promotes neuroblastoma tumorigenesis are not fully understood. In this study, we analyzed global miRNA and mRNA expression profiles of tissues at different stages of tumorigenesis from TH-MYCN transgenic mice, a model of MYCN-driven neuroblastoma. On the basis of a Bayesian learning network model in which we compared pretumor ganglia from TH-MYCN+/+ mice to age-matched wild-type controls, we devised a predicted miRNA-mRNA interaction network. Among the miRNA-mRNA interactions operating during human neuroblastoma tumorigenesis, we identified miR-204 as a tumor suppressor miRNA that inhibited a subnetwork of oncogenes strongly associated with MYCN-amplified neuroblastoma and poor patient outcome. MYCN bound to the miR-204 promoter and repressed miR-204 transcription. Conversely, miR-204 directly bound MYCN mRNA and repressed MYCN expression. miR-204 overexpression significantly inhibited neuroblastoma cell proliferation in vitro and tumorigenesis in vivo Together, these findings identify novel tumorigenic miRNA gene networks and miR-204 as a tumor suppressor that regulates MYCN expression in neuroblastoma tumorigenesis.Significance: Network modeling of miRNA-mRNA regulatory interactions in a mouse model of neuroblastoma identifies miR-204 as a tumor suppressor and negative regulator of MYCN. Cancer Res; 78(12); 3122-34. ©2018 AACR.
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Regulación Neoplásica de la Expresión Génica , MicroARNs/metabolismo , Proteína Proto-Oncogénica N-Myc/genética , Neuroblastoma/genética , Neoplasias del Sistema Nervioso Periférico/genética , Animales , Carcinogénesis/genética , Línea Celular Tumoral , Proliferación Celular , Conjuntos de Datos como Asunto , Femenino , Redes Reguladoras de Genes , Genes Supresores de Tumor , Humanos , Masculino , Ratones , Ratones Desnudos , Ratones Transgénicos , MicroARNs/genética , Proteína Proto-Oncogénica N-Myc/metabolismo , Neuroblastoma/mortalidad , Neuroblastoma/patología , Oncogenes/genética , Neoplasias del Sistema Nervioso Periférico/patología , Regiones Promotoras Genéticas/genética , ARN Mensajero/genética , ARN Mensajero/metabolismo , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Chromosome 17q gains are almost invariably present in high-risk neuroblastoma cases. Here, we perform an integrative epigenomics search for dosage-sensitive transcription factors on 17q marked by H3K27ac defined super-enhancers and identify TBX2 as top candidate gene. We show that TBX2 is a constituent of the recently established core regulatory circuitry in neuroblastoma with features of a cell identity transcription factor, driving proliferation through activation of p21-DREAM repressed FOXM1 target genes. Combined MYCN/TBX2 knockdown enforces cell growth arrest suggesting that TBX2 enhances MYCN sustained activation of FOXM1 targets. Targeting transcriptional addiction by combined CDK7 and BET bromodomain inhibition shows synergistic effects on cell viability with strong repressive effects on CRC gene expression and p53 pathway response as well as several genes implicated in transcriptional regulation. In conclusion, we provide insight into the role of the TBX2 CRC gene in transcriptional dependency of neuroblastoma cells warranting clinical trials using BET and CDK7 inhibitors.