RESUMEN
Childhood neuroblastomas exhibit plasticity between an undifferentiated neural crest-like mesenchymal cell state and a more differentiated sympathetic adrenergic cell state. These cell states are governed by autoregulatory transcriptional loops called core regulatory circuitries (CRCs), which drive the early development of sympathetic neuronal progenitors from migratory neural crest cells during embryogenesis. The adrenergic cell identity of neuroblastoma requires LMO1 as a transcriptional cofactor. Both LMO1 expression levels and the risk of developing neuroblastoma in children are associated with a single nucleotide polymorphism, G/T, that affects a GATA motif in the first intron of LMO1. Here, we showed that WT zebrafish with the GATA genotype developed adrenergic neuroblastoma, while knock-in of the protective TATA allele at this locus reduced the penetrance of MYCN-driven tumors, which were restricted to the mesenchymal cell state. Whole genome sequencing of childhood neuroblastomas demonstrated that TATA/TATA tumors also exhibited a mesenchymal cell state and were low risk at diagnosis. Thus, conversion of the regulatory GATA to a TATA allele in the first intron of LMO1 reduced the neuroblastoma-initiation rate by preventing formation of the adrenergic cell state. This mechanism was conserved over 400 million years of evolution, separating zebrafish and humans.
Asunto(s)
Predisposición Genética a la Enfermedad , Neuroblastoma , Animales , Niño , Humanos , Pez Cebra/genética , Pez Cebra/metabolismo , Adrenérgicos , Genotipo , Neuroblastoma/patología , Proteína Proto-Oncogénica N-Myc/genéticaRESUMEN
Childhood neuroblastomas exhibit plasticity between an undifferentiated neural crest-like "mesenchymal" cell state and a more differentiated sympathetic "adrenergic" cell state. These cell states are governed by autoregulatory transcriptional loops called core regulatory circuitries (CRCs), which drive the early development of sympathetic neuronal progenitors from migratory neural crest cells during embryogenesis. The adrenergic cell identity of neuroblastoma requires LMO1 as a transcriptional co-factor. Both LMO1 expression levels and the risk of developing neuroblastoma in children are associated with a single nucleotide polymorphism G/T that affects a G ATA motif in the first intron of LMO1. Here we show that wild-type zebrafish with the G ATA genotype develop adrenergic neuroblastoma, while knock-in of the protective T ATA allele at this locus reduces the penetrance of MYCN-driven tumors, which are restricted to the mesenchymal cell state. Whole genome sequencing of childhood neuroblastomas demonstrates that T ATA/ T ATA tumors also exhibit a mesenchymal cell state and are low risk at diagnosis. Thus, conversion of the regulatory G ATA to a T ATA allele in the first intron of LMO1 reduces the neuroblastoma initiation rate by preventing formation of the adrenergic cell state, a mechanism that is conserved over 400 million years of evolution separating zebrafish and humans.
RESUMEN
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.
Asunto(s)
Anemia Hemolítica Autoinmune , Betacoronavirus/metabolismo , Infecciones por Coronavirus , Pandemias , Neumonía Viral , Adolescente , Corticoesteroides/administración & dosificación , Anemia Hemolítica Autoinmune/sangre , Anemia Hemolítica Autoinmune/diagnóstico , Anemia Hemolítica Autoinmune/patología , Anemia Hemolítica Autoinmune/terapia , Benzoatos/administración & dosificación , COVID-19 , Infecciones por Coronavirus/sangre , Infecciones por Coronavirus/diagnóstico , Infecciones por Coronavirus/patología , Infecciones por Coronavirus/terapia , Transfusión de Eritrocitos , Humanos , Hidrazinas/administración & dosificación , Masculino , Ácido Micofenólico/administración & dosificación , Oxígeno/administración & dosificación , Neumonía Viral/sangre , Neumonía Viral/diagnóstico , Neumonía Viral/patología , Neumonía Viral/terapia , Pirazoles/administración & dosificación , SARS-CoV-2RESUMEN
A heritable polymorphism within regulatory sequences of the LMO1 gene is associated with its elevated expression and increased susceptibility to develop neuroblastoma, but the oncogenic pathways downstream of the LMO1 transcriptional co-regulatory protein are unknown. Our ChIP-seq and RNA-seq analyses reveal that a key gene directly regulated by LMO1 and MYCN is ASCL1, which encodes a basic helix-loop-helix transcription factor. Regulatory elements controlling ASCL1 expression are bound by LMO1, MYCN and the transcription factors GATA3, HAND2, PHOX2B, TBX2 and ISL1-all members of the adrenergic (ADRN) neuroblastoma core regulatory circuitry (CRC). ASCL1 is required for neuroblastoma cell growth and arrest of differentiation. ASCL1 and LMO1 directly regulate the expression of CRC genes, indicating that ASCL1 is a member and LMO1 is a coregulator of the ADRN neuroblastoma CRC.
Asunto(s)
Adrenérgicos/metabolismo , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Proteínas de Unión al ADN/metabolismo , Redes Reguladoras de Genes , Proteínas con Dominio LIM/metabolismo , Proteína Proto-Oncogénica N-Myc/metabolismo , Neuroblastoma/metabolismo , Factores de Transcripción/metabolismo , Diferenciación Celular , Línea Celular Tumoral , Regulación Neoplásica de la Expresión Génica , Humanos , Neuroblastoma/genética , Proteínas Proto-Oncogénicas c-ret/genética , Proteínas Proto-Oncogénicas c-ret/metabolismo , Análisis de SupervivenciaRESUMEN
Childhood high-risk neuroblastomas with MYCN gene amplification are difficult to treat effectively1. This has focused attention on tumor-specific gene dependencies that underlie tumorigenesis and thus provide valuable targets for the development of novel therapeutics. Using unbiased genome-scale CRISPR-Cas9 approaches to detect genes involved in tumor cell growth and survival2-6, we identified 147 candidate gene dependencies selective for MYCN-amplified neuroblastoma cell lines, compared to over 300 other human cancer cell lines. We then used genome-wide chromatin-immunoprecipitation coupled to high-throughput sequencing analysis to demonstrate that a small number of essential transcription factors-MYCN, HAND2, ISL1, PHOX2B, GATA3, and TBX2-are members of the transcriptional core regulatory circuitry (CRC) that maintains cell state in MYCN-amplified neuroblastoma. To disable the CRC, we tested a combination of BRD4 and CDK7 inhibitors, which act synergistically, in vitro and in vivo, with rapid downregulation of CRC transcription factor gene expression. This study defines a set of critical dependency genes in MYCN-amplified neuroblastoma that are essential for cell state and survival in this tumor.
Asunto(s)
Regulación Neoplásica de la Expresión Génica , Proteína Proto-Oncogénica N-Myc/genética , Neuroblastoma/genética , Transcripción Genética , Animales , Sistemas CRISPR-Cas , Línea Celular Tumoral , Modelos Animales de Enfermedad , Regulación hacia Abajo , Femenino , Amplificación de Genes , Humanos , Ratones , Ratones Desnudos , Factores de Transcripción/genéticaRESUMEN
High-risk neuroblastomas show a paucity of recurrent somatic mutations at diagnosis. As a result, the molecular basis for this aggressive phenotype remains elusive. Recent progress in regulatory network analysis helped us elucidate disease-driving mechanisms downstream of genomic alterations, including recurrent chromosomal alterations. Our analysis identified three molecular subtypes of high-risk neuroblastomas, consistent with chromosomal alterations, and identified subtype-specific master regulator proteins that were conserved across independent cohorts. A 10-protein transcriptional module-centered around a TEAD4-MYCN positive feedback loop-emerged as the regulatory driver of the high-risk subtype associated with MYCN amplification. Silencing of either gene collapsed MYCN-amplified (MYCNAmp) neuroblastoma transcriptional hallmarks and abrogated viability in vitro and in vivo Consistently, TEAD4 emerged as a robust prognostic marker of poor survival, with activity independent of the canonical Hippo pathway transcriptional coactivators YAP and TAZ. These results suggest novel therapeutic strategies for the large subset of MYCN-deregulated neuroblastomas.Significance: Despite progress in understanding of neuroblastoma genetics, little progress has been made toward personalized treatment. Here, we present a framework to determine the downstream effectors of the genetic alterations sustaining neuroblastoma subtypes, which can be easily extended to other tumor types. We show the critical effect of disrupting a 10-protein module centered around a YAP/TAZ-independent TEAD4-MYCN positive feedback loop in MYCNAmp neuroblastomas, nominating TEAD4 as a novel candidate for therapeutic intervention. Cancer Discov; 8(5); 582-99. ©2018 AACR.This article is highlighted in the In This Issue feature, p. 517.
Asunto(s)
Proteínas de Unión al ADN/genética , Regulación Neoplásica de la Expresión Génica , Proteínas Musculares/genética , Proteína Proto-Oncogénica N-Myc/genética , Neuroblastoma/genética , Neuroblastoma/metabolismo , Secuencias Reguladoras de Ácidos Nucleicos , Factores de Transcripción/genética , Aciltransferasas , Proteínas de Ciclo Celular , Línea Celular Tumoral , Biología Computacional/métodos , Proteínas de Unión al ADN/metabolismo , Perfilación de la Expresión Génica , Humanos , Proteínas Musculares/metabolismo , Proteína Proto-Oncogénica N-Myc/metabolismo , Estadificación de Neoplasias , Neuroblastoma/diagnóstico , Proteínas Nucleares/metabolismo , Complejo de la Endopetidasa Proteasomal/metabolismo , Interferencia de ARN , Factores de Transcripción de Dominio TEA , Factores de Transcripción/metabolismo , Activación TranscripcionalRESUMEN
The amplified MYCN gene serves as an oncogenic driver in approximately 20% of high-risk pediatric neuroblastomas. Here, we show that the family member MYC is a potent transforming gene in a separate subset of high-risk neuroblastoma cases (â¼10%), based on (i) its upregulation by focal enhancer amplification or genomic rearrangements leading to enhancer hijacking, and (ii) its ability to transform neuroblastoma precursor cells in a transgenic animal model. The aberrant regulatory elements associated with oncogenic MYC activation include focally amplified distal enhancers and translocation of highly active enhancers from other genes to within topologically associating domains containing the MYC gene locus. The clinical outcome for patients with high levels of MYC expression is virtually identical to that of patients with amplification of the MYCN gene, a known high-risk feature of this disease. Together, these findings establish MYC as a bona fide oncogene in a clinically significant group of high-risk childhood neuroblastomas.Significance: Amplification of the MYCN oncogene is a recognized hallmark of high-risk pediatric neuroblastoma. Here, we demonstrate that MYC is also activated as a potent oncogene in a distinct subset of neuroblastoma cases through either focal amplification of distal enhancers or enhancer hijacking mediated by chromosomal translocation. Cancer Discov; 8(3); 320-35. ©2017 AACR.This article is highlighted in the In This Issue feature, p. 253.
Asunto(s)
Elementos de Facilitación Genéticos , Neuroblastoma/genética , Proteínas Proto-Oncogénicas c-myc/genética , Animales , Animales Modificados Genéticamente , Línea Celular Tumoral , Niño , Amplificación de Genes , Regulación Neoplásica de la Expresión Génica , Humanos , Proteínas Luminiscentes/genética , Proteína Proto-Oncogénica N-Myc/genética , Neoplasias Experimentales/genética , Neuroblastoma/mortalidad , Neuroblastoma/patología , Análisis de Supervivencia , Translocación Genética , Pez Cebra/genética , Proteína Fluorescente RojaRESUMEN
A genome-wide association study identified LMO1, which encodes an LIM-domain-only transcriptional cofactor, as a neuroblastoma susceptibility gene that functions as an oncogene in high-risk neuroblastoma. Here we show that dßh promoter-mediated expression of LMO1 in zebrafish synergizes with MYCN to increase the proliferation of hyperplastic sympathoadrenal precursor cells, leading to a reduced latency and increased penetrance of neuroblastomagenesis. The transgenic expression of LMO1 also promoted hematogenous dissemination and distant metastasis, which was linked to neuroblastoma cell invasion and migration, and elevated expression levels of genes affecting tumor cell-extracellular matrix interaction, including loxl3, itga2b, itga3, and itga5. Our results provide in vivo validation of LMO1 as an important oncogene that promotes neuroblastoma initiation, progression, and widespread metastatic dissemination.
Asunto(s)
Carcinogénesis/patología , Proteínas de Unión al ADN/metabolismo , Proteínas con Dominio LIM/metabolismo , Proteína Proto-Oncogénica N-Myc/metabolismo , Neuroblastoma/metabolismo , Neuroblastoma/patología , Factores de Transcripción/metabolismo , Animales , Animales Modificados Genéticamente , Carcinogénesis/metabolismo , Línea Celular Tumoral , Movimiento Celular/genética , Matriz Extracelular/metabolismo , Regulación Neoplásica de la Expresión Génica , Humanos , Hiperplasia , Modelos Biológicos , Invasividad Neoplásica , Metástasis de la Neoplasia , Neuroblastoma/genética , Transducción de Señal/genética , Transgenes , Pez CebraRESUMEN
This corrects the article DOI: 10.1038/ncomms14385.
RESUMEN
The non-coding regions of tumour cell genomes harbour a considerable fraction of total DNA sequence variation, but the functional contribution of these variants to tumorigenesis is ill-defined. Among these non-coding variants, somatic insertions are among the least well characterized due to challenges with interpreting short-read DNA sequences. Here, using a combination of Chip-seq to enrich enhancer DNA and a computational approach with multiple DNA alignment procedures, we identify enhancer-associated small insertion variants. Among the 102 tumour cell genomes we analyse, small insertions are frequently observed in enhancer DNA sequences near known oncogenes. Further study of one insertion, somatically acquired in primary leukaemia tumour genomes, reveals that it nucleates formation of an active enhancer that drives expression of the LMO2 oncogene. The approach described here to identify enhancer-associated small insertion variants provides a foundation for further study of these abnormalities across human cancers.
Asunto(s)
Elementos de Facilitación Genéticos , Genoma Humano , Mutagénesis Insercional/genética , Oncogenes , Adolescente , Adulto , Secuencia de Bases , Línea Celular Tumoral , Niño , Preescolar , Regulación Leucémica de la Expresión Génica , Humanos , Lactante , Leucemia-Linfoma de Células T del Adulto/genética , Reproducibilidad de los Resultados , Adulto JovenRESUMEN
Neuroblastoma is a paediatric malignancy that typically arises in early childhood, and is derived from the developing sympathetic nervous system. Clinical phenotypes range from localized tumours with excellent outcomes to widely metastatic disease in which long-term survival is approximately 40% despite intensive therapy. A previous genome-wide association study identified common polymorphisms at the LMO1 gene locus that are highly associated with neuroblastoma susceptibility and oncogenic addiction to LMO1 in the tumour cells. Here we investigate the causal DNA variant at this locus and the mechanism by which it leads to neuroblastoma tumorigenesis. We first imputed all possible genotypes across the LMO1 locus and then mapped highly associated single nucleotide polymorphism (SNPs) to areas of chromatin accessibility, evolutionary conservation and transcription factor binding sites. We show that SNP rs2168101 G>T is the most highly associated variant (combined P = 7.47 × 10(-29), odds ratio 0.65, 95% confidence interval 0.60-0.70), and resides in a super-enhancer defined by extensive acetylation of histone H3 lysine 27 within the first intron of LMO1. The ancestral G allele that is associated with tumour formation resides in a conserved GATA transcription factor binding motif. We show that the newly evolved protective TATA allele is associated with decreased total LMO1 expression (P = 0.028) in neuroblastoma primary tumours, and ablates GATA3 binding (P < 0.0001). We demonstrate allelic imbalance favouring the G-containing strand in tumours heterozygous for this SNP, as demonstrated both by RNA sequencing (P < 0.0001) and reporter assays (P = 0.002). These findings indicate that a recently evolved polymorphism within a super-enhancer element in the first intron of LMO1 influences neuroblastoma susceptibility through differential GATA transcription factor binding and direct modulation of LMO1 expression in cis, and this leads to an oncogenic dependency in tumour cells.