RESUMEN
Neuroblastoma is a childhood tumour of the peripheral sympathetic nervous system. The pathogenesis has for a long time been quite enigmatic, as only very few gene defects were identified in this often lethal tumour. Frequently detected gene alterations are limited to MYCN amplification (20%) and ALK activations (7%). Here we present a whole-genome sequence analysis of 87 neuroblastoma of all stages. Few recurrent amino-acid-changing mutations were found. In contrast, analysis of structural defects identified a local shredding of chromosomes, known as chromothripsis, in 18% of high-stage neuroblastoma. These tumours are associated with a poor outcome. Structural alterations recurrently affected ODZ3, PTPRD and CSMD1, which are involved in neuronal growth cone stabilization. In addition, ATRX, TIAM1 and a series of regulators of the Rac/Rho pathway were mutated, further implicating defects in neuritogenesis in neuroblastoma. Most tumours with defects in these genes were aggressive high-stage neuroblastomas, but did not carry MYCN amplifications. The genomic landscape of neuroblastoma therefore reveals two novel molecular defects, chromothripsis and neuritogenesis gene alterations, which frequently occur in high-risk tumours.
Asunto(s)
Cromosomas Humanos/genética , Neuritas/metabolismo , Neuroblastoma/genética , Neuroblastoma/patología , Envejecimiento/genética , Análisis por Conglomerados , ADN Helicasas/genética , Análisis Mutacional de ADN , Regulación Neoplásica de la Expresión Génica , Genoma Humano/genética , Conos de Crecimiento/metabolismo , Conos de Crecimiento/patología , Factores de Intercambio de Guanina Nucleótido/genética , Humanos , Mutación , Estadificación de Neoplasias , Neuroblastoma/diagnóstico , Neuroblastoma/metabolismo , Proteínas Nucleares/genética , Pronóstico , Proteína 1 de Invasión e Inducción de Metástasis del Linfoma-T , Proteína Nuclear Ligada al Cromosoma X , Proteínas de Unión al GTP rac/metabolismo , Proteínas de Unión al GTP rho/metabolismoRESUMEN
The tightly controlled network of cell cycle genes consists of a core of cyclin dependent kinases (CDKs) that are activated by periodically expressed cyclins. The activity of the cyclin-CDK complexes is regulated by cyclin dependent kinase inhibitors (CDKIs) and multiple signal transduction routes that converge on the cell cycle. Neuroblastoma are pediatric tumors that belong to the group of small round blue cell tumors, characterized by a fast proliferation. Here, we present high throughput analyses of cell cycle regulating genes in neuroblastoma. We analyzed a series of 82 neuroblastomas by comparative genomic hybridization arrays, single nucleotide polymorphism arrays, and Affymetrix expression arrays and analyzed the datasets in parallel with the R2 bioinformatic tool (http://r2.amc.nl). About 30% of the tumors had genomic amplifications, gains, or losses with shortest regions of overlap that suggested implication of a series of G1 cell cycle regulating genes. CCND1 (cyclin D1) and CDK4 were amplified or gained and the chromosomal regions containing the CDKN2 (INK4) group of CDKIs were frequently deleted. Cluster analysis showed that tumors with genomic aberrations in G1 regulating genes over-expressed E2F target genes, which regulate S and G2/M phase progression. These tumors have a poor prognosis. Our findings suggest that pharmacological inhibition of cell cycle genes might bear therapeutic promises for patients with high risk neuroblastoma.
Asunto(s)
Factores de Transcripción E2F/metabolismo , Fase G1/genética , Dosificación de Gen , Genes cdc , Neuroblastoma/genética , Aberraciones Cromosómicas , Análisis por Conglomerados , Ciclina D1/genética , Proteínas Inhibidoras de las Quinasas Dependientes de la Ciclina/genética , Quinasas Ciclina-Dependientes/genética , Expresión Génica , Humanos , Neuroblastoma/diagnóstico , Pronóstico , ARN Mensajero/genéticaRESUMEN
Intronic single-nucleotide polymorphisms (SNPs) in FOXO3A are associated with human longevity. Currently, it is unclear how these SNPs alter FOXO3A functionality and human physiology, thereby influencing lifespan. Here, we identify a primate-specific FOXO3A transcriptional isoform, FOXO3A-Short (FOXO3A-S), encoding a major longevity-associated SNP, rs9400239 (C or T), within its 5' untranslated region. The FOXO3A-S mRNA is highly expressed in the skeletal muscle and has very limited expression in other tissues. We find that the rs9400239 variant influences the stability and functionality of the primarily nuclear protein(s) encoded by the FOXO3A-S mRNA. Assessment of the relationship between the FOXO3A-S polymorphism and peripheral glucose clearance during insulin infusion (Rd clamp) in a cohort of Danish twins revealed that longevity T-allele carriers have markedly faster peripheral glucose clearance rates than normal lifespan C-allele carriers. In vitro experiments in human myotube cultures utilizing overexpression of each allele showed that the C-allele represses glycolysis independently of PI3K signaling, while overexpression of the T-allele represses glycolysis only in a PI3K-inactive background. Supporting this finding inducible knockdown of the FOXO3A-S C-allele in cultured myotubes increases the glycolytic rate. We conclude that the rs9400239 polymorphism acts as a molecular switch which changes the identity of the FOXO3A-S-derived protein(s), which in turn alters the relationship between FOXO3A-S and insulin/PI3K signaling and glycolytic flux in the skeletal muscle. This critical difference endows carriers of the FOXO3A-S T-allele with consistently higher insulin-stimulated peripheral glucose clearance rates, which may contribute to their longer and healthier lifespans.
Asunto(s)
Glucosa , Longevidad , Animales , Humanos , Proteína Forkhead Box O3/genética , Factores de Transcripción Forkhead/genética , Factores de Transcripción Forkhead/metabolismo , Insulina/genética , Insulina/metabolismo , Longevidad/genética , Fosfatidilinositol 3-Quinasas/genética , ARN MensajeroRESUMEN
BACKGROUND: Neuroblastoma are pediatric tumors of the sympathetic nervous system with a poor prognosis. Apoptosis is often deregulated in cancer cells, but only a few defects in apoptotic routes have been identified in neuroblastoma. METHODS: Here we investigated genomic aberrations affecting genes of the intrinsic apoptotic pathway in neuroblastoma. We analyzed DNA profiling data (CGH and SNP arrays) and mRNA expression data of 31 genes of the intrinsic apoptotic pathway in a dataset of 88 neuroblastoma tumors using the R2 bioinformatic platform ( http://r2.amc.nl). BIRC6 was selected for further analysis as a tumor driving gene. Knockdown experiments were performed using BIRC6 lentiviral shRNA and phenotype responses were analyzed by Western blot and MTT-assays. In addition, DIABLO levels and interactions were investigated with immunofluorescence and co-immunoprecipitation. RESULTS: We observed frequent gain of the BIRC6 gene on chromosome 2, which resulted in increased mRNA expression. BIRC6 is an inhibitor of apoptosis protein (IAP), that can bind and degrade the cytoplasmic fraction of the pro-apoptotic protein DIABLO. DIABLO mRNA expression was exceptionally high in neuroblastoma but the protein was only detected in the mitochondria. Upon silencing of BIRC6 by shRNA, DIABLO protein levels increased and cells went into apoptosis. Co-immunoprecipitation confirmed direct interaction between DIABLO and BIRC6 in neuroblastoma cell lines. CONCLUSION: Our findings indicate that BIRC6 may have a potential oncogenic role in neuroblastoma by inactivating cytoplasmic DIABLO. BIRC6 inhibition may therefore provide a means for therapeutic intervention in neuroblastoma.
Asunto(s)
Proteínas Inhibidoras de la Apoptosis/genética , Neuroblastoma/genética , Apoptosis/genética , Proteínas Reguladoras de la Apoptosis , Caspasa 9/genética , Hibridación Genómica Comparativa , Citoplasma/metabolismo , Perfilación de la Expresión Génica , Técnicas de Silenciamiento del Gen , Humanos , Proteínas Inhibidoras de la Apoptosis/metabolismo , Péptidos y Proteínas de Señalización Intracelular/genética , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Proteínas Mitocondriales/genética , Proteínas Mitocondriales/metabolismo , Terapia Molecular Dirigida/métodos , Neuroblastoma/metabolismo , Polimorfismo de Nucleótido Simple , ARN Interferente Pequeño/genética , SurvivinRESUMEN
Two genes have a synthetically lethal relationship when the silencing or inhibiting of 1 gene is only lethal in the context of a mutation or activation of the second gene. This situation offers an attractive therapeutic strategy, as inhibition of such a gene will only trigger cell death in tumor cells with an activated second oncogene but spare normal cells without activation of the second oncogene. Here we present evidence that CDK2 is synthetically lethal to neuroblastoma cells with MYCN amplification and over-expression. Neuroblastomas are childhood tumors with an often lethal outcome. Twenty percent of the tumors have MYCN amplification, and these tumors are ultimately refractory to any therapy. Targeted silencing of CDK2 by 3 RNA interference techniques induced apoptosis in MYCN-amplified neuroblastoma cell lines, but not in MYCN single copy cells. Silencing of MYCN abrogated this apoptotic response in MYCN-amplified cells. Inversely, silencing of CDK2 in MYCN single copy cells did not trigger apoptosis, unless a MYCN transgene was activated. The MYCN induced apoptosis after CDK2 silencing was accompanied by nuclear stabilization of P53, and mRNA profiling showed up-regulation of P53 target genes. Silencing of P53 rescued the cells from MYCN-driven apoptosis. The synthetic lethality of CDK2 silencing in MYCN activated neuroblastoma cells can also be triggered by inhibition of CDK2 with a small molecule drug. Treatment of neuroblastoma cells with roscovitine, a CDK inhibitor, at clinically achievable concentrations induced MYCN-dependent apoptosis. The synthetically lethal relationship between CDK2 and MYCN indicates CDK2 inhibitors as potential MYCN-selective cancer therapeutics.
Asunto(s)
Quinasa 2 Dependiente de la Ciclina/antagonistas & inhibidores , Neuroblastoma/terapia , Proteínas Nucleares/genética , Proteínas Oncogénicas/genética , Apoptosis/efectos de los fármacos , Línea Celular Tumoral , Quinasa 2 Dependiente de la Ciclina/genética , Amplificación de Genes , Humanos , Proteína Proto-Oncogénica N-Myc , Neuroblastoma/genética , Neuroblastoma/patología , Purinas/farmacología , Interferencia de ARN , Roscovitina , Proteína p53 Supresora de Tumor/fisiologíaRESUMEN
Despite intensive therapy, children with high-risk neuroblastoma are at risk of treatment failure. We applied a multiomic system approach to evaluate metabolic vulnerabilities in human neuroblastoma. We combined metabolomics, CRISPR screening, and transcriptomic data across more than 700 solid tumor cell lines and identified dihydroorotate dehydrogenase (DHODH), a critical enzyme in pyrimidine synthesis, as a potential treatment target. Of note, DHODH inhibition is currently under clinical investigation in patients with hematologic malignancies. In neuroblastoma, DHODH expression was identified as an independent risk factor for aggressive disease, and high DHODH levels correlated to worse overall and event-free survival. A subset of tumors with the highest DHODH expression was associated with a dismal prognosis, with a 5-year survival of less than 10%. In xenograft and transgenic neuroblastoma mouse models treated with the DHODH inhibitor brequinar, tumor growth was dramatically reduced, and survival was extended. Furthermore, brequinar treatment was shown to reduce the expression of MYC targets in 3 neuroblastoma models in vivo. A combination of brequinar and temozolomide was curative in the majority of transgenic TH-MYCN neuroblastoma mice, indicating a highly active clinical combination therapy. Overall, DHODH inhibition combined with temozolomide has therapeutic potential in neuroblastoma, and we propose this combination for clinical testing.
Asunto(s)
Neuroblastoma , Oxidorreductasas actuantes sobre Donantes de Grupo CH-CH , Animales , Dihidroorotato Deshidrogenasa , Humanos , Ratones , Neuroblastoma/tratamiento farmacológico , Neuroblastoma/genética , Oxidorreductasas actuantes sobre Donantes de Grupo CH-CH/genética , Oxidorreductasas actuantes sobre Donantes de Grupo CH-CH/metabolismo , Pronóstico , TemozolomidaRESUMEN
Cancer therapy frequently fails due to the emergence of resistance. Many tumors include phenotypically immature tumor cells, which have been implicated in therapy resistance. Neuroblastoma cells can adopt a lineage-committed adrenergic (ADRN) or an immature mesenchymal (MES) state. They differ in epigenetic landscape and transcription factors, and MES cells are more resistant to chemotherapy. Here we analyzed the response of MES cells to targeted drugs. Activating anaplastic lymphoma kinase (ALK) mutations are frequently found in neuroblastoma and ALK inhibitors (ALKi) are in clinical trials. ALKi treatment of ADRN neuroblastoma cells with a tumor-driving ALK mutation induced cell death. Conversely, MES cells did not express either mutant or wild-type ALK and were resistant to ALKi, and MES cells formed tumors that progressed under ALKi therapy. In assessing the role of MES cells in relapse development, TRAIL was identified to specifically induce apoptosis in MES cells and to suppress MES tumor growth. Addition of TRAIL to ALKi treatment of neuroblastoma xenografts delayed relapses in a subset of the animals, suggesting a role for MES cells in relapse formation. While ADRN cells resembled normal embryonal neuroblasts, MES cells resembled immature precursor cells, which also lacked ALK expression. Resistance to targeted drugs can therefore be an intrinsic property of immature cancer cells based on their resemblance to developmental precursors. SIGNIFICANCE: In neuroblastoma, mesenchymal tumor cells lack expression of the tumor-driving ALK oncogene and are resistant to ALKi, but dual treatment with ALKi and mesenchymal cell-targeting TRAIL delays tumor relapse.
Asunto(s)
Quinasa de Linfoma Anaplásico/antagonistas & inhibidores , Neuroblastoma/genética , Línea Celular Tumoral , Humanos , Neuroblastoma/patologíaRESUMEN
RNAi efficiency is influenced by local RNA structure of the target sequence. We studied this structure-based resistance in detail by targeting a perfect RNA hairpin and subsequently destabilized its tight structure by mutation, thereby gradually exposing the target sequence. Although the tightest RNA hairpins were completely resistant to RNAi, we observed an inverse correlation between the overall target hairpin stability and RNAi efficiency within a specific thermodynamic stability (DeltaG) range. Increased RNAi efficiency was shown to be caused by improved binding of the siRNA to the destabilized target RNA hairpins. The mutational effects vary for different target regions. We find an accessible target 3' end to be most important for RNAi-mediated inhibition. However, these 3' end effects cannot be reproduced in siRNA-target RNA-binding studies in vitro, indicating the important role of RISC components in the in vivo RNAi reaction. The results provide a more detailed insight into the impact of target RNA structure on RNAi and we discuss several possible implications. With respect to lentiviral-mediated delivery of shRNA expression cassettes, we present a DeltaG window to destabilize the shRNA insert for vector improvement, while avoiding RNAi-mediated self-targeting during lentiviral vector production.
Asunto(s)
Interferencia de ARN , ARN Mensajero/química , Secuencia de Bases , Línea Celular Tumoral , Humanos , Conformación de Ácido Nucleico , ARN Interferente Pequeño/química , ARN Interferente Pequeño/metabolismoRESUMEN
Transition between differentiation states in development occurs swift but the mechanisms leading to epigenetic and transcriptional reprogramming are poorly understood. The pediatric cancer neuroblastoma includes adrenergic (ADRN) and mesenchymal (MES) tumor cell types, which differ in phenotype, super-enhancers (SEs) and core regulatory circuitries. These cell types can spontaneously interconvert, but the mechanism remains largely unknown. Here, we unravel how a NOTCH3 intracellular domain reprogrammed the ADRN transcriptional landscape towards a MES state. A transcriptional feed-forward circuitry of NOTCH-family transcription factors amplifies the NOTCH signaling levels, explaining the swift transition between two semi-stable cellular states. This transition induces genome-wide remodeling of the H3K27ac landscape and a switch from ADRN SEs to MES SEs. Once established, the NOTCH feed-forward loop maintains the induced MES state. In vivo reprogramming of ADRN cells shows that MES and ADRN cells are equally oncogenic. Our results elucidate a swift transdifferentiation between two semi-stable epigenetic cellular states.
Asunto(s)
Neuronas Adrenérgicas/patología , Reprogramación Celular/genética , Células Madre Mesenquimatosas/patología , Neuroblastoma/patología , Receptor Notch3/fisiología , Neuronas Adrenérgicas/metabolismo , Línea Celular Tumoral , Epigénesis Genética , Retroalimentación Fisiológica , Perfilación de la Expresión Génica , Regulación Neoplásica de la Expresión Génica , Humanos , Células Madre Mesenquimatosas/metabolismo , Neuroblastoma/metabolismo , Receptor Notch3/genética , Receptor Notch3/metabolismoRESUMEN
BACKGROUND: The latent HIV-1 reservoir in treated patients primarily consists of resting memory CD4+ T cells. Stimulating the T-cell receptor (TCR), which facilitates transition of resting into effector T cells, is the most effective strategy to purge these latently infected cells. Here we supply evidence that TCR-stimulated effector T cells still frequently harbor latent HIV-1. METHODS: Primary HIV-1 infected cells were used in a latency assay with or without dendritic cells (DCs) and reversion of HIV-1 latency was determined, in the presence or absence of specific pathway inhibitors. FINDINGS: Renewed TCR-stimulation or subsequent activation with latency reversing agents (LRAs) did not overcome latency. However, interaction of infected effector cells with DCs triggered further activation of latent HIV-1. When compared to TCR-stimulation only, CD4+ T cells from aviremic patients receiving TCRâ¯+â¯DC-stimulation reversed latency more frequently. Such a "one-two punch" strategy seems ideal for purging the reservoir. We determined that DC contact activates the PI3K-Akt-mTOR pathway in CD4+ T cells. INTERPRETATION: This insight could facilitate the development of a novel class of potent LRAs that purge latent HIV beyond levels reached by T-cell activation.
Asunto(s)
Infecciones por VIH/metabolismo , Infecciones por VIH/virología , VIH-1/fisiología , Fosfatidilinositol 3-Quinasas/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Receptores de Antígenos de Linfocitos T/metabolismo , Serina-Treonina Quinasas TOR/metabolismo , Latencia del Virus , Adulto , Anciano , Linfocitos T CD4-Positivos/efectos de los fármacos , Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD4-Positivos/metabolismo , Linfocitos T CD4-Positivos/virología , Células Dendríticas/inmunología , Células Dendríticas/metabolismo , Femenino , Infecciones por VIH/tratamiento farmacológico , Infecciones por VIH/inmunología , VIH-1/efectos de los fármacos , Humanos , Activación de Linfocitos/inmunología , Masculino , Persona de Mediana Edad , Modelos Biológicos , FN-kappa B/metabolismo , Fosforilación , Unión Proteica , Proteínas Proto-Oncogénicas c-fos/química , Proteínas Proto-Oncogénicas c-fos/metabolismo , Proteínas Proto-Oncogénicas c-jun/química , Proteínas Proto-Oncogénicas c-jun/metabolismo , Transducción de Señal , Latencia del Virus/inmunologíaRESUMEN
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.
Asunto(s)
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
BACKGROUND: Eradication of HIV-1 from an infected individual cannot be achieved by current drug regimens. Viral reservoirs established early during the infection remain unaffected by anti-retroviral therapy and are able to replenish systemic infection upon interruption of the treatment. Therapeutic targeting of viral latency will require a better understanding of the basic mechanisms underlying the establishment and long-term maintenance of HIV-1 in resting memory CD4 T cells, the most prominent reservoir of transcriptional silent provirus. However, the molecular mechanisms that permit long-term transcriptional control of proviral gene expression in these cells are still not well understood. Exploring the molecular details of viral latency will provide new insights for eventual future therapeutics that aim at viral eradication. RESULTS: We set out to develop a new in vitro HIV-1 latency model system using the doxycycline (dox)-inducible HIV-rtTA variant. Stable cell clones were generated with a silent HIV-1 provirus, which can subsequently be activated by dox-addition. Surprisingly, only a minority of the cells was able to induce viral gene expression and a spreading infection, eventhough these experiments were performed with the actively dividing SupT1 T cell line. These latent proviruses are responsive to TNFalpha treatment and alteration of the DNA methylation status with 5-Azacytidine or genistein, but not responsive to the regular T cell activators PMA and IL2. Follow-up experiments in several T cell lines and with wild-type HIV-1 support these findings. CONCLUSION: We describe the development of a new in vitro model for HIV-1 latency and discuss the advantages of this system. The data suggest that HIV-1 proviral latency is not restricted to resting T cells, but rather an intrinsic property of the virus.
Asunto(s)
Regulación Viral de la Expresión Génica , VIH-1/fisiología , Provirus/fisiología , Linfocitos T/citología , Linfocitos T/virología , Latencia del Virus/fisiología , División Celular , Línea Celular , VIH-1/genética , Humanos , ARN Viral/análisis , ARN Viral/genética , Activación Viral , Latencia del Virus/genética , Productos del Gen tat del Virus de la Inmunodeficiencia Humana/metabolismoRESUMEN
HIV-1 replication can be efficiently inhibited by intracellular expression of an siRNA targeting the viral RNA. However, HIV-1 escape variants emerged after prolonged culturing. These RNAi-resistant viruses contain nucleotide substitutions or deletions in or near the targeted sequence. We observed an inverse correlation between the level of resistance and the stability of the siRNA/target-RNA duplex. However, two escape variants showed a higher level of resistance than expected based on the duplex stability. We demonstrate that these mutations induce alternative folding of the RNA such that the target sequence is occluded from binding to the siRNA, resulting in reduced RNAi efficiency. HIV-1 can thus escape from RNAi-mediated inhibition not only through nucleotide substitutions or deletions in the siRNA target sequence, but also through mutations that alter the local RNA secondary structure. The results highlight the enormous genetic flexibility of HIV-1 and provide detailed molecular insight into the sequence specificity of RNAi and the impact of target RNA secondary structure.
Asunto(s)
Fármacos Anti-VIH/farmacología , VIH-1/genética , Interferencia de ARN , ARN Interferente Pequeño/farmacología , ARN Viral/química , Fármacos Anti-VIH/química , Fármacos Anti-VIH/metabolismo , Secuencia de Bases , Línea Celular , Productos del Gen nef/antagonistas & inhibidores , Productos del Gen nef/genética , Genoma Viral , Humanos , Datos de Secuencia Molecular , Mutación , Conformación de Ácido Nucleico , Estabilidad del ARN , ARN Interferente Pequeño/química , ARN Interferente Pequeño/metabolismo , ARN Viral/efectos de los fármacos , ARN Viral/genética , Productos del Gen nef del Virus de la Inmunodeficiencia HumanaRESUMEN
Neuroblastoma and other pediatric tumors show a paucity of gene mutations, which has sparked an interest in their epigenetic regulation. Several tumor types include phenotypically divergent cells, resembling cells from different lineage development stages. It has been proposed that super-enhancer-associated transcription factor (TF) networks underlie lineage identity, but the role of these enhancers in intratumoral heterogeneity is unknown. Here we show that most neuroblastomas include two types of tumor cells with divergent gene expression profiles. Undifferentiated mesenchymal cells and committed adrenergic cells can interconvert and resemble cells from different lineage differentiation stages. ChIP-seq analysis of isogenic pairs of mesenchymal and adrenergic cells identified a distinct super-enhancer landscape and super-enhancer-associated TF network for each cell type. Expression of the mesenchymal TF PRRX1 could reprogram the super-enhancer and mRNA landscapes of adrenergic cells toward a mesenchymal state. Mesenchymal cells were more chemoresistant in vitro and were enriched in post-therapy and relapse tumors. Two super-enhancer-associated TF networks, which probably mediate lineage control in normal development, thus dominate epigenetic control of neuroblastoma and shape intratumoral heterogeneity.
Asunto(s)
Diferenciación Celular/genética , Epigénesis Genética , Neuroblastoma/genética , Neuroblastoma/patología , Antígeno AC133/genética , Neuronas Adrenérgicas/citología , Línea Celular Tumoral , Linaje de la Célula , Proteínas de Homeodominio/genética , Humanos , Mesodermo/citología , Factores de Transcripción/metabolismo , TranscriptomaRESUMEN
BACKGROUND: RNA interference (RNAi) has proven to be a powerful tool to suppress gene expression and can be used as a therapeutic strategy against human pathogenic viruses such as human immunodeficiency virus type 1 (HIV-1). Theoretically, RNAi-mediated inhibition can occur at two points in the replication cycle, upon viral entry before reverse transcription of the RNA genome, and on the newly transcribed viral RNA transcripts. There have been conflicting results on whether RNAi can target the RNA genome of infecting HIV-1 particles. We have addressed this issue with HIV-1-based lentiviral vectors. RESULTS: We determined the transduction efficiency of a lentiviral vector, as measured by GFP expressing cells, which reflects the number of successful integration events in a cell line stably expressing shNef. We did not observe a difference in the transduction efficiency comparing lentiviral vectors with or without the Nef target sequence in their genome. The results were similar with particles pseudotyped with either the VSV-G or HIV-1 envelope. Additionally, no reduced transduction efficiencies were observed with multiple other shRNAs targeting the vector genome or with synthetic siNef when transiently transfected prior to transduction. CONCLUSION: Our findings indicate that the incoming HIV-1 RNA genome is not targeted by RNAi, probably due to inaccessibility to the RNAi machinery. Thus, therapeutic RNAi strategies aimed at preventing proviral integration should be targeting cellular receptors or co-factors involved in pre-integration events.
Asunto(s)
VIH-1/genética , Interferencia de ARN , ARN Viral/genética , Virión/genética , Clonación Molecular , Productos del Gen nef/genética , Terapia Genética , Vectores Genéticos/genética , Genoma Viral , Proteínas Fluorescentes Verdes/biosíntesis , Proteínas Fluorescentes Verdes/genética , Infecciones por VIH/terapia , Humanos , Lentivirus/genética , ARN Interferente Pequeño/genética , ARN Viral/antagonistas & inhibidores , Linfocitos T/fisiología , Linfocitos T/virología , Transducción Genética , Productos del Gen nef del Virus de la Inmunodeficiencia HumanaRESUMEN
The anti-apoptotic protein B cell lymphoma/leukaemia 2 (BCL-2) is highly expressed in neuroblastoma and plays an important role in oncogenesis. In this study, the selective BCL-2 inhibitor ABT199 was tested in a panel of neuroblastoma cell lines with diverse expression levels of BCL-2 and other BCL-2 family proteins. ABT199 caused apoptosis more potently in neuroblastoma cell lines expressing high BCL-2 and BIM/BCL-2 complex levels than low expressing cell lines. Effects on cell viability correlated with effects on BIM displacement from BCL-2 and cytochrome c release from the mitochondria. ABT199 treatment of mice with neuroblastoma tumors expressing high BCL-2 levels only resulted in growth inhibition, despite maximum BIM displacement from BCL-2 and the induction of a strong apoptotic response. We showed that neuroblastoma cells might survive ABT199 treatment due to its acute upregulation of the anti-apoptotic BCL-2 family protein myeloid cell leukaemia sequence 1 (MCL-1) and BIM sequestration by MCL-1. In vitro inhibition of MCL-1 sensitized neuroblastoma cell lines to ABT199, confirming the pivotal role of MCL-1 in ABT199 resistance. Our findings suggest that neuroblastoma patients with high BCL-2 and BIM/BCL-2 complex levels might benefit from combination treatment with ABT199 and compounds that inhibit MCL-1 expression.
Asunto(s)
Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Compuestos Bicíclicos Heterocíclicos con Puentes/uso terapéutico , Proteína 1 de la Secuencia de Leucemia de Células Mieloides/metabolismo , Neuroblastoma/tratamiento farmacológico , Proteínas Proto-Oncogénicas c-bcl-2/antagonistas & inhibidores , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Sulfonamidas/uso terapéutico , Animales , Protocolos de Quimioterapia Combinada Antineoplásica/farmacología , Apoptosis/efectos de los fármacos , Proteína 11 Similar a Bcl2/metabolismo , Compuestos Bicíclicos Heterocíclicos con Puentes/farmacología , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Citocromos c/metabolismo , Evaluación Preclínica de Medicamentos , Femenino , Humanos , Concentración 50 Inhibidora , Ratones , Mitocondrias/metabolismo , Proteína 1 de la Secuencia de Leucemia de Células Mieloides/antagonistas & inhibidores , Neuroblastoma/patología , Sulfonamidas/farmacología , Resultado del Tratamiento , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
A quantitative bioanalytical liquid chromatography-tandem mass spectrometric (LC-MS/MS) assay for GSK2126458, a dual PI3K/mTOR inhibitor, was developed and validated. Plasma and tumor homogenate samples were pre-treated using protein precipitation with acetonitrile containing dabrafenib as internal standard. After dilution with water, the extract was directly injected into the reversed-phase liquid chromatographic system. The eluate was transferred into the electrospray interface with positive ionization and compounds were detected in the selected reaction monitoring mode of a triple quadrupole mass spectrometer. The assay was completely validated for plasma in a 4-4000 ng/ml calibration range with r(2)=0.9996±0.0003 using double logarithmic calibration (n=5). Within-run precisions (n=6) were 2.0-5.3% and between-run (3 runs; n=18) precisions 2.7-5.8%. Accuracies were between 101 and 105% for the whole calibration range. The drug was sufficiently stable under all relevant analytical conditions. Finally, the assay was successfully applied to determine plasma and tumor drug levels after oral administration of GSK2126458 to mice with AMC711T neuroblastoma xenografts.
Asunto(s)
Neoplasias/química , Inhibidores de las Quinasa Fosfoinosítidos-3 , Plasma/química , Inhibidores de Proteínas Quinasas/sangre , Quinolinas/sangre , Quinolinas/química , Sulfonamidas/sangre , Sulfonamidas/química , Serina-Treonina Quinasas TOR/antagonistas & inhibidores , Animales , Calibración , Cromatografía Liquida/métodos , Femenino , Ratones , Neuroblastoma/tratamiento farmacológico , Neuroblastoma/metabolismo , Inhibidores de Proteínas Quinasas/química , Inhibidores de Proteínas Quinasas/farmacología , Piridazinas , Quinolinas/farmacología , Reproducibilidad de los Resultados , Espectrometría de Masa por Ionización de Electrospray/métodos , Sulfonamidas/farmacología , Espectrometría de Masas en Tándem/métodosRESUMEN
The majority of patients with neuroblastoma have tumors that initially respond to chemotherapy, but a large proportion will experience therapy-resistant relapses. The molecular basis of this aggressive phenotype is unknown. Whole-genome sequencing of 23 paired diagnostic and relapse neuroblastomas showed clonal evolution from the diagnostic tumor, with a median of 29 somatic mutations unique to the relapse sample. Eighteen of the 23 relapse tumors (78%) showed mutations predicted to activate the RAS-MAPK pathway. Seven of these events were detected only in the relapse tumor, whereas the others showed clonal enrichment. In neuroblastoma cell lines, we also detected a high frequency of activating mutations in the RAS-MAPK pathway (11/18; 61%), and these lesions predicted sensitivity to MEK inhibition in vitro and in vivo. Our findings provide a rationale for genetic characterization of relapse neuroblastomas and show that RAS-MAPK pathway mutations may function as a biomarker for new therapeutic approaches to refractory disease.
Asunto(s)
Sistema de Señalización de MAP Quinasas/genética , Proteínas Quinasas Activadas por Mitógenos/genética , Mutación , Recurrencia Local de Neoplasia/genética , Neuroblastoma/genética , Proteínas ras/genética , Quinasa de Linfoma Anaplásico , Animales , Bencimidazoles/farmacología , Western Blotting , Línea Celular Tumoral , Niño , Preescolar , Aberraciones Cromosómicas , Inhibidor p16 de la Quinasa Dependiente de Ciclina/genética , Inhibidor p16 de la Quinasa Dependiente de Ciclina/metabolismo , Femenino , Regulación Neoplásica de la Expresión Génica , Células HEK293 , Humanos , Lactante , Masculino , Ratones SCID , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Neuroblastoma/tratamiento farmacológico , Neuroblastoma/patología , Fosforilación/efectos de los fármacos , Proteínas Tirosina Quinasas Receptoras/genética , Proteínas Tirosina Quinasas Receptoras/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Ensayos Antitumor por Modelo de Xenoinjerto , Proteínas ras/metabolismoRESUMEN
Neuroblastoma is a pediatric tumor of the peripheral sympathetic nervous system with a highly variable prognosis. Activation of the phosphoinositide 3-kinase (PI3K)/AKT pathway in neuroblastoma is correlated with poor patient prognosis, but the precise downstream effectors mediating this effect have not been determined. Here we identify the forkhead transcription factor FOXO3a as a key target of the PI3K/AKT pathway in neuroblastoma. FOXO3a expression was elevated in low-stage neuroblastoma tumors and normal embryonal neuroblasts, but reduced in late-stage neuroblastoma. Inactivation of FOXO3a by AKT was essential for neuroblastoma cell survival. Treatment of neuroblastoma cells with the dual PI3K/mTOR inhibitor PI-103 activated FOXO3a and triggered apoptosis. This effect was rescued by FOXO3a silencing. Conversely, apoptosis induced by PI-103 or the AKT inhibitor MK-2206 was potentiated by FOXO3a overexpression. Furthermore, levels of total or phosphorylated FOXO3a correlated closely with apoptotic sensitivity to MK-2206. In clinical specimens, there was an inverse relationship between gene expression signatures regulated by PI3K signaling and FOXO3a transcriptional activity. Moreover, high PI3K activity and low FOXO3a activity were each associated with an extremely poor prognosis. Our work indicates that expression of FOXO3a and its targets offer useful prognostic markers as well as biomarkers for PI3K/AKT inhibitor efficacy in neuroblastoma.