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Neuroblastomas are childhood tumors with frequent fatal relapses after induction treatment, which is related to tumor evolution with additional genomic events. Our whole-genome sequencing data analysis revealed a high frequency of somatic cytosine > adenine (C > A) substitutions in primary neuroblastoma tumors, which was associated with poor survival. We showed that increased levels of C > A substitutions correlate with copy number loss (CNL) of OGG1 or MUTYH Both genes encode DNA glycosylases that recognize 8-oxo-guanine (8-oxoG) lesions as a first step of 8-oxoG repair. Tumor organoid models with CNL of OGG1 or MUTYH show increased 8-oxoG levels compared to wild-type cells. We used CRISPR-Cas9 genome editing to create knockout clones of MUTYH and OGG1 in neuroblastoma cells. Whole-genome sequencing of single-cell OGG1 and MUTYH knockout clones identified an increased accumulation of C > A substitutions. Mutational signature analysis of these OGG1 and MUTYH knockout clones revealed enrichment for C > A signatures 18 and 36, respectively. Clustering analysis showed that the knockout clones group together with tumors containing OGG1 or MUTYH CNL. In conclusion, we demonstrate that defects in 8-oxoG repair cause accumulation of C > A substitutions in neuroblastoma, which contributes to mutagenesis and tumor evolution.
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Reparación del ADN/genética , Guanosina/análogos & derivados , Neuroblastoma/genética , Adenina/metabolismo , Niño , Citosina/metabolismo , Daño del ADN , ADN Glicosilasas/genética , ADN Glicosilasas/metabolismo , Femenino , Guanina/metabolismo , Guanosina/genética , Guanosina/metabolismo , Humanos , Masculino , Mutagénesis , Recurrencia Local de Neoplasia/genética , Neuroblastoma/metabolismo , Estrés Oxidativo , Polimorfismo de Nucleótido Simple/genéticaRESUMEN
BACKGROUND: Pediatric cancer is the leading cause of disease-related death in children and the need for better therapeutic options remains urgent. Due to the limited number of patients, target and drug development for pediatrics is often supplemented by data from studies focused on adult cancers. Recent evidence shows that pediatric cancers possess different vulnerabilities that should be explored independently from adult cancers. METHODS: Using the publicly available Genomics of Drug Sensitivity in Cancer database, we explore therapeutic targets and biomarkers specific to the pediatric solid malignancies Ewing sarcoma, medulloblastoma, neuroblastoma, osteosarcoma, and rhabdomyosarcoma. Results are validated using cell viability assays and high-throughput drug screens are used to identify synergistic combinations. RESULTS: Using published drug screening data, PARP is identified as a drug target of interest across multiple different pediatric malignancies. We validate these findings, and we show that efficacy can be improved when combined with conventional chemotherapeutics, namely topoisomerase inhibitors. Additionally, using gene set enrichment analysis, we identify ribosome biogenesis as a potential biomarker for PARP inhibition in pediatric cancer cell lines. CONCLUSION: Collectively, our results provide evidence to support the further development of PARP inhibition and the combination with TOP1 inhibition as a therapeutic approach in solid pediatric malignancies. Additionally, we propose ribosome biogenesis as a component to PARP inhibitor sensitivity that should be further investigated to help maximize the potential utility of PARP inhibition and combinations across pediatric solid malignancies.
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Antineoplásicos , Neoplasias Cerebelosas , Neuroblastoma , Sarcoma de Ewing , Humanos , Niño , Inhibidores de Poli(ADP-Ribosa) Polimerasas/uso terapéutico , Antineoplásicos/uso terapéutico , Sarcoma de Ewing/tratamiento farmacológico , Neuroblastoma/patología , Neoplasias Cerebelosas/tratamiento farmacológico , Línea Celular TumoralRESUMEN
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.
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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
Introduction: Mutations affecting the RAS-MAPK pathway occur frequently in relapsed neuroblastoma tumors and are associated with response to MEK inhibition in vitro. However, these inhibitors alone do not lead to tumor regression in vivo, indicating the need for combination therapy. Methods and results: Via high-throughput combination screening, we identified that the MEK inhibitor trametinib can be combined with BCL-2 family member inhibitors, to efficiently inhibit growth of neuroblastoma cell lines with RAS-MAPK mutations. Suppressing the RAS-MAPK pathway with trametinib led to an increase in pro-apoptotic BIM, resulting in more BIM binding to anti-apoptotic BCL-2 family members. By favoring the formation of these complexes, trametinib treatment enhances sensitivity to compounds targeting anti-apoptotic BCL-2 family members. In vitro validation studies confirmed that this sensitizing effect is dependent on an active RAS-MAPK pathway. In vivo combination of trametinib with BCL-2 inhibitors led to tumor inhibition in NRAS-mutant and NF1-deleted xenografts. Conclusion: Together, these results show that combining MEK inhibition with BCL-2 family member inhibition could potentially improve therapeutic outcomes for RAS-MAPK-mutated neuroblastoma patients.
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Neuroblastoma is the most common extracranial solid tumor in children. A subgroup of high-risk patients is characterized by aberrations in the chromatin remodeller ATRX that is encoded by 35 exons. In contrast to other pediatric cancer where ATRX point mutations are most frequent, multi-exon deletions (MEDs) are the most frequent type of ATRX aberrations in neuroblastoma. 75% of these MEDs are predicted to produce in-frame fusion proteins, suggesting a potential gain-of-function effect compared to nonsense mutations. For neuroblastoma there are only a few patient-derived ATRX aberrant models. Therefore, we created isogenic ATRX aberrant models using CRISPR-Cas9 in several neuroblastoma cell lines and one tumoroid and performed total RNA-sequencing on these and the patient-derived models. Gene set enrichment analysis (GSEA) showed decreased expression of genes related to both ribosome biogenesis and several metabolic processes in our isogenic ATRX exon 2-10 MED model systems, the patient-derived MED models and in tumor data containing two patients with an ATRX exon 2-10 MED. In sharp contrast, these same processes showed an increased expression in our isogenic ATRX knock-out and exon 2-13 MED models. Our validations confirmed a role of ATRX in the regulation of ribosome homeostasis. The two distinct molecular expression patterns within ATRX aberrant neuroblastomas that we identified imply that there might be a need for distinct treatment regimens.
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Neuroblastoma , Niño , Humanos , Proteína Nuclear Ligada al Cromosoma X/genética , Neuroblastoma/genética , Neuroblastoma/patología , Cromatina , Línea Celular , Expresión GénicaRESUMEN
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.
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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
Neuroblastoma is the most common extracranial solid tumor found in children and despite intense multi-modal therapeutic approaches, low overall survival rates of high-risk patients persist. Tumors with heterozygous loss of chromosome 11q and MYCN amplification are two genetically distinct subsets of neuroblastoma that are associated with poor patient outcome. Using an isogenic 11q deleted model system and high-throughput drug screening, we identify checkpoint kinase 1 (CHK1) as a potential therapeutic target for 11q deleted neuroblastoma. Further investigation reveals MYCN amplification as a possible additional biomarker for CHK1 inhibition, independent of 11q loss. Overall, our study highlights the potential power of studying chromosomal aberrations to guide preclinical development of novel drug targets and combinations. Additionally, our study builds on the growing evidence that DNA damage repair and replication stress response pathways offer therapeutic vulnerabilities for the treatment of neuroblastoma.
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iTHER is a Dutch prospective national precision oncology program aiming to define tumour molecular profiles in children and adolescents with primary very high-risk, relapsed, or refractory paediatric tumours. Between April 2017 and April 2021, 302 samples from 253 patients were included. Comprehensive molecular profiling including low-coverage whole genome sequencing (lcWGS), whole exome sequencing (WES), RNA sequencing (RNA-seq), Affymetrix, and/or 850k methylation profiling was successfully performed for 226 samples with at least 20% tumour content. Germline pathogenic variants were identified in 16% of patients (35/219), of which 22 variants were judged causative for a cancer predisposition syndrome. At least one somatic alteration was detected in 204 (90.3%), and 185 (81.9%) were considered druggable, with clinical priority very high (6.1%), high (21.3%), moderate (26.0%), intermediate (36.1%), and borderline (10.5%) priority. iTHER led to revision or refinement of diagnosis in 8 patients (3.5%). Temporal heterogeneity was observed in paired samples of 15 patients, indicating the value of sequential analyses. Of 137 patients with follow-up beyond twelve months, 21 molecularly matched treatments were applied in 19 patients (13.9%), with clinical benefit in few. Most relevant barriers to not applying targeted therapies included poor performance status, as well as limited access to drugs within clinical trial. iTHER demonstrates the feasibility of comprehensive molecular profiling across all ages, tumour types and stages in paediatric cancers, informing of diagnostic, prognostic, and targetable alterations as well as reportable germline variants. Therefore, WES and RNA-seq is nowadays standard clinical care at the Princess Máxima Center for all children with cancer, including patients at primary diagnosis. Improved access to innovative treatments within biology-driven combination trials is required to ultimately improve survival.
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Neoplasias , Adolescente , Niño , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Oncología Médica , Mutación , Neoplasias/tratamiento farmacológico , Neoplasias/genética , Medicina de Precisión , Estudios Prospectivos , Secuenciación del ExomaRESUMEN
BACKGROUND: Despite intensive treatment protocols and recent advances, neuroblastomas still account for approximately 15% of all childhood cancer deaths. In contrast with adult cancers, p53 pathway inactivation in neuroblastomas is rarely caused by p53 mutation but rather by altered MDM2 or p14ARF expression. Moreover, neuroblastomas are characterised by high proliferation rates, frequently triggered by pRb pathway dysfunction due to aberrant expression of cyclin D1, CDK4 or p16INK4a. Simultaneous disturbance of these pathways can occur via co-amplification of MDM2 and CDK4 or homozygous deletion of CDKN2A, which encodes both p14ARF and p16INK4a. METHODS AND RESULTS: We examined whether both single and combined inhibition of MDM2 and CDK4/6 is effective in reducing neuroblastoma cell viability. In our panel of ten cell lines with a spectrum of aberrations in the p53 and pRb pathway, idasanutlin and abemaciclib were the most potent MDM2 and CDK4/6 inhibitors, respectively. No correlation was observed between the genetic background and response to the single inhibitors. We confirmed this lack of correlation in isogenic systems overexpressing MDM2 and/or CDK4. In addition, combined inhibition did not result in synergistic effects. Instead, abemaciclib diminished the pro-apoptotic effect of idasanutlin, leading to slightly antagonistic effects. In vivo treatment with idasanutlin and abemaciclib led to reduced tumour growth compared with single drug treatment, but no synergistic response was observed. CONCLUSION: We conclude that p53 and pRb pathway aberrations cannot be used as predictive biomarkers for neuroblastoma sensitivity to MDM2 and/or CDK4/6 inhibitors. Moreover, we advise to be cautious with combining these inhibitors in neuroblastomas.
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Neuroblastoma/genética , Medicina de Precisión/métodos , Proteína p53 Supresora de Tumor/metabolismo , Animales , Humanos , Ratones , Neuroblastoma/patologíaRESUMEN
Neuroblastoma tumors frequently overexpress the anti-apoptotic protein B-cell lymphoma/leukemia 2 (BCL-2). We previously showed that treating BCL-2-dependent neuroblastoma cells with the BCL-2 inhibitor venetoclax results in apoptosis, but unfortunately partial therapy resistance is observed. The current study describes the identification of drugs capable of resensitizing venetoclax-resistant neuroblastoma cells to venetoclax. To examine these effects, venetoclax resistance was induced in BCL-2-dependent neuroblastoma cell lines KCNR and SJNB12 by continuous exposure to high venetoclax concentrations. Non-resistant and venetoclax-resistant neuroblastoma cell lines were exposed to a 209-compound library in the absence and presence of venetoclax to identify compounds that were more effective in the venetoclax-resistant cell lines under venetoclax pressure. Top hits were further validated in combination with venetoclax using BCL-2-dependent neuroblastoma model systems. Overall, high-throughput drug screening identified the MDM2 inhibitor idasanutlin as a promising resensitizing agent for venetoclax-resistant neuroblastoma cell lines. Idasanutlin treatment induced BAX-mediated apoptosis in venetoclax-resistant neuroblastoma cells in the presence of venetoclax, whereas it caused p21-mediated growth arrest in control cells. In vivo combination treatment showed tumor regression and superior efficacy over single-agent therapies in a BCL-2-dependent neuroblastoma cell line xenograft and a patient-derived xenograft. However, xenografts less dependent on BCL-2 were not sensitive to venetoclax-idasanutlin combination therapy. This study demonstrates that idasanutlin can overcome resistance to the BCL-2 inhibitor venetoclax in preclinical neuroblastoma model systems, which supports clinical development of a treatment strategy combining the two therapies.
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Ensayos Analíticos de Alto Rendimiento/métodos , Neuroblastoma/tratamiento farmacológico , Proteínas Proto-Oncogénicas c-mdm2/uso terapéutico , Pirrolidinas/uso terapéutico , para-Aminobenzoatos/uso terapéutico , Animales , Línea Celular Tumoral , Modelos Animales de Enfermedad , Humanos , Ratones , Proteínas Proto-Oncogénicas c-mdm2/farmacología , Pirrolidinas/farmacología , para-Aminobenzoatos/farmacologíaRESUMEN
Cancer immunotherapy has transformed the landscape of adult cancer treatment and holds a great promise to treat paediatric malignancies. However, in vitro test coculture systems to evaluate the efficacy of immunotherapies on representative paediatric tumour models are lacking. Here, we describe a detailed procedure for the establishment of an ex vivo test coculture system of paediatric tumour organoids and immune cells that enables assessment of different immunotherapy approaches in paediatric tumour organoids. We provide a step-by-step protocol for an efficient generation of patient-derived diffuse intrinsic pontine glioma (DIPG) and neuroblastoma organoids stably expressing eGFP-ffLuc transgenes using defined serum-free medium. In contrast to the chromium-release assay, the new platform allows for visualization, monitoring and robust quantification of tumour organoid cell cytotoxicity using a non-radioactive assay in real-time. To evaluate the utility of this system for drug testing in the paediatric immuno-oncology field, we tested our in vitro assay using a clinically used immunotherapy strategy for children with high-risk neuroblastoma, dinutuximab (anti-GD2 monoclonal antibody), on GD2 proficient and deficient patient-derived neuroblastoma organoids. We demonstrated the feasibility and sensitivity of our ex vivo coculture system using human immune cells and paediatric tumour organoids as ex vivo tumour models. Our study provides a novel platform for personalized testing of potential anticancer immunotherapies for aggressive paediatric cancers such as neuroblastoma and DIPG.
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Mutations affecting the RAS-MAPK pathway frequently occur in relapsed neuroblastoma tumors, which suggests that activation of this pathway is associated with a more aggressive phenotype. To explore this hypothesis, we generated several model systems to define a neuroblastoma RAS-MAPK pathway signature. Activation of this pathway in primary tumors indeed correlated with poor survival and was associated with known activating mutations in ALK and other RAS-MAPK pathway genes. Integrative analysis showed that mutations in PHOX2B, CIC, and DMD were also associated with an activated RAS-MAPK pathway. Mutation of PHOX2B and deletion of CIC in neuroblastoma cell lines induced activation of the RAS-MAPK pathway. This activation was independent of phosphorylated ERK in CIC knockout systems. Furthermore, deletion of CIC caused a significant increase in tumor growth in vivo These results show that the RAS-MAPK pathway is involved in tumor progression and establish CIC as a powerful tumor suppressor that functions downstream of this pathway in neuroblastoma.Significance: This work identifies CIC as a powerful tumor suppressor affecting the RAS-MAPK pathway in neuroblastoma and reinforces the importance of mutation-driven activation of this pathway in cancer. Cancer Res; 78(21); 6297-307. ©2018 AACR.
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Sistema de Señalización de MAP Quinasas , Neuroblastoma/genética , Proteínas Represoras/genética , Animales , Línea Celular Tumoral , Análisis por Conglomerados , Progresión de la Enfermedad , Femenino , Perfilación de la Expresión Génica , Regulación Neoplásica de la Expresión Génica , Genes ras , Genoma Humano , Genómica , Proteínas de Homeodominio/metabolismo , Humanos , Ratones , Ratones Noqueados , Ratones Desnudos , Mutación , Recurrencia Local de Neoplasia/genética , Trasplante de Neoplasias , Neuroblastoma/patología , Fenotipo , Fosforilación , Pronóstico , Proteínas Represoras/metabolismo , Transducción de Señal , Factores de Transcripción/metabolismo , Resultado del TratamientoRESUMEN
Purpose: mTORC1 inhibitors are promising agents for neuroblastoma therapy; however, they have shown limited clinical activity as monotherapy, thus rational drug combinations need to be explored to improve efficacy. Importantly, neuroblastoma maintains both an active p53 and an aberrant mTOR signaling.Experimental Design: Using an orthotopic xenograft model and modulating p53 levels, we investigated the antitumor effects of the mTORC1 inhibitor temsirolimus in neuroblastoma expressing normal, decreased, or mutant p53, both as single agent and in combination with first- and second-generation MDM2 inhibitors to reactivate p53.Results: Nongenotoxic p53 activation suppresses mTOR activity. Moreover, p53 reactivation via RG7388, a second-generation MDM2 inhibitor, strongly enhances the in vivo antitumor activity of temsirolimus. Single-agent temsirolimus does not elicit apoptosis, and tumors rapidly regrow after treatment suspension. In contrast, our combination therapy triggers a potent apoptotic response in wild-type p53 xenografts and efficiently blocks tumor regrowth after treatment completion. We also found that this combination uniquely led to p53-dependent suppression of survivin whose ectopic expression is sufficient to rescue the apoptosis induced by our combination.Conclusions: Our study supports a novel highly effective strategy that combines RG7388 and temsirolimus in wild-type p53 neuroblastoma, which warrants testing in early-phase clinical trials. Clin Cancer Res; 23(21); 6629-39. ©2017 AACR.
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Neuroblastoma/tratamiento farmacológico , Proteínas Proto-Oncogénicas c-mdm2/genética , Serina-Treonina Quinasas TOR/genética , Proteína p53 Supresora de Tumor/genética , Animales , Protocolos de Quimioterapia Combinada Antineoplásica/administración & dosificación , Apoptosis/efectos de los fármacos , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Resistencia a Antineoplásicos/genética , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Humanos , Diana Mecanicista del Complejo 1 de la Rapamicina/antagonistas & inhibidores , Ratones , Neuroblastoma/genética , Neuroblastoma/patología , Proteínas Proto-Oncogénicas c-mdm2/antagonistas & inhibidores , Pirrolidinas/administración & dosificación , Sirolimus/administración & dosificación , Sirolimus/análogos & derivados , Serina-Treonina Quinasas TOR/antagonistas & inhibidores , Ensayos Antitumor por Modelo de Xenoinjerto , para-Aminobenzoatos/administración & dosificaciónRESUMEN
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.
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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
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.
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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
Recently protocols have been devised for the culturing of cell lines from fresh tumours under serum-free conditions in defined neural stem cell medium. These cells, frequently called tumour initiating cells (TICs) closely retained characteristics of the tumours of origin. We report the isolation of eight newly-derived neuroblastoma TICs from six primary neuroblastoma tumours and two bone marrow metastases. The primary tumours from which these TICs were generated have previously been fully typed by whole genome sequencing (WGS). Array comparative genomic hybridisation (aCGH) analysis showed that TIC lines retained essential characteristics of the primary tumours and exhibited typical neuroblastoma chromosomal aberrations such as MYCN amplification, gain of chromosome 17q and deletion of 1p36. Protein analysis showed expression for neuroblastoma markers MYCN, NCAM, CHGA, DBH and TH while haematopoietic markers CD19 and CD11b were absent. We analysed the growth characteristics and confirmed tumour-forming potential using sphere-forming assays, subcutaneous and orthotopic injection of these cells into immune-compromised mice. Affymetrix mRNA expression profiling of TIC line xenografts showed an expression pattern more closely mimicking primary tumours compared to xenografts from classical cell lines. This establishes that these neuroblastoma TICs cultured under serum-free conditions are relevant and useful neuroblastoma tumour models.
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Línea Celular Tumoral , Neuroblastoma/genética , Neuroblastoma/patología , Animales , Biomarcadores de Tumor/genética , Biomarcadores de Tumor/metabolismo , Niño , Preescolar , Medio de Cultivo Libre de Suero , Genotipo , Humanos , Lactante , Ratones , Ratones Desnudos , Neuroblastoma/metabolismo , Fenotipo , ARN Mensajero/genética , ARN Mensajero/metabolismoRESUMEN
The BIRC5 (Survivin) gene is located at chromosome 17q in the region that is frequently gained in high risk neuroblastoma. BIRC5 is strongly over expressed in neuroblastoma tumour samples, which correlates to a poor prognosis. We recently validated BIRC5 as a potential therapeutic target by showing that targeted knock down with shRNA's triggers an apoptotic response through mitotic catastrophe. We now tested YM155, a novel small molecule selective BIRC5 suppressant that is currently in phase I/II clinical trials. Drug response curves showed IC50 values in the low nM range (median: 35 nM, range: 0.5-> 10,000 nM) in a panel of 23 neuroblastoma cell lines and four TIC-lines, which resulted from an apoptotic response. Nine out of 23 cell lines were relatively resistant to YM155 with IC50 values > 200 nM, although in the same cells shRNA mediated knock down of BIRC5 caused massive apoptosis. Analysis of differentially expressed genes between five most sensitive and five most resistant cell lines using Affymetrix mRNA expression data revealed ABCB1 (MDR1) as the most predictive gene for resistance to YM155. Inhibition of the multi-drug resistance pump ABCB1 with cyclosporine or knockdown with shRNA prior to treatment with YM155 demonstrated that cell lines with ABCB1 expression became 27-695 times more sensitive to YM155 treatment. We conclude that most neuroblastoma cell lines are sensitive to YM155 in the low nM range and that resistant cells can be sensitised by ABCB1 inhibitors. Therefore YM155 is a promising novel compound for treatment of neuroblastoma with low ABCB1 expression.
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Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/biosíntesis , Apoptosis/efectos de los fármacos , Imidazoles/farmacología , Proteínas Inhibidoras de la Apoptosis/antagonistas & inhibidores , Naftoquinonas/farmacología , Neuroblastoma/tratamiento farmacológico , Neuroblastoma/metabolismo , Subfamilia B de Transportador de Casetes de Unión a ATP , Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/antagonistas & inhibidores , Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/genética , Apoptosis/genética , Línea Celular Tumoral , Resistencia a Antineoplásicos/genética , Silenciador del Gen , Células HEK293 , Humanos , Proteínas Inhibidoras de la Apoptosis/biosíntesis , Proteínas Inhibidoras de la Apoptosis/genética , Neuroblastoma/genética , Neuroblastoma/patología , ARN Interferente Pequeño/administración & dosificación , ARN Interferente Pequeño/genética , Survivin , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Genomic aberrations of key regulators of the apoptotic pathway have hardly been identified in neuroblastoma. We detected high BCL2 mRNA and protein levels in the majority of neuroblastoma tumours by Affymetrix expression profiling and Tissue Micro Array analysis. This BCL2 mRNA expression is strongly elevated compared to normal tissues and other malignancies. Most neuroblastoma cell lines lack this high BCL2 expression. Only two neuroblastoma cell lines (KCNR and SJNB12) show BCL2 expression levels representative for neuroblastoma tumours. To validate BCL2 as a therapeutic target in neuroblastoma we employed lentivirally mediated shRNA. Silencing of BCL2 in KCNR and SJNB12 resulted in massive apoptosis, while cell lines with low BCL2 expression were insensitive. Identical results were obtained by treatment of the neuroblastoma cell lines with the small molecule BCL2 inhibitor ABT263, which is currently being clinically evaluated. Combination assays of ABT263 with most classical cytostatics showed strong synergistic responses. Subcutaneous xenografts of a neuroblastoma cell line with high BCL2 expression in NMRI nu/nu mice showed a strong response to ABT263. These findings establish BCL2 as a promising drug target in neuroblastoma and warrant further evaluation of ABT263 and other BCL2 inhibiting drugs.
Asunto(s)
Compuestos de Anilina/farmacología , Antineoplásicos/farmacología , Terapia Molecular Dirigida , Neuroblastoma/tratamiento farmacológico , Proteínas Proto-Oncogénicas c-bcl-2/antagonistas & inhibidores , Sulfonamidas/farmacología , Animales , Protocolos de Quimioterapia Combinada Antineoplásica/farmacología , Apoptosis/efectos de los fármacos , Línea Celular Tumoral , Relación Dosis-Respuesta a Droga , Sinergismo Farmacológico , Perfilación de la Expresión Génica/métodos , Regulación Neoplásica de la Expresión Génica , Células HEK293 , Humanos , Ratones , Ratones Desnudos , Neuroblastoma/genética , Neuroblastoma/metabolismo , Neuroblastoma/patología , Análisis de Secuencia por Matrices de Oligonucleótidos , Proteínas Proto-Oncogénicas c-bcl-2/genética , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Interferencia de ARN , ARN Mensajero/metabolismo , Factores de Tiempo , Transfección , Carga Tumoral/efectos de los fármacos , Regulación hacia Arriba , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
BIRC5 (survivin) is one of the genes located on chromosome arm 17q in the region that is often gained in neuroblastoma. BIRC5 is a protein in the intrinsic apoptotic pathway that interacts with XIAP and DIABLO leading to caspase-3 and caspase-9 inactivation. BIRC5 is also involved in stabilizing the microtubule-kinetochore dynamics. Based on the Affymetrix mRNA expression data, we here show that BIRC5 expression is strongly upregulated in neuroblastoma compared with normal tissues, adult malignancies, and non-malignant fetal adrenal neuroblasts. The over-expression of BIRC5 correlates with an unfavorable prognosis independent of the presence of 17q gain. Silencing of BIRC5 in neuroblastoma cell lines by various antisense molecules resulted in massive apoptosis as measured by PARP cleavage and FACS analysis. As both the intrinsic apoptotic pathway and the chromosomal passenger complex can be therapeutically targeted, we investigated in which of them BIRC5 exerted its essential anti-apoptotic role. Immunofluorescence analysis of neuroblastoma cells after BIRC5 silencing showed formation of multinucleated cells indicating mitotic catastrophe, which leads to apoptosis via P53 and CASP2. We show that BIRC5 silencing indeed resulted in activation of P53 and we could rescue apoptosis by CASP2 inhibition. We conclude that BIRC5 stabilizes the microtubules in the chromosomal passenger complex in neuroblastoma and that the apoptotic response results from mitotic catastrophe, which makes BIRC5 an interesting target for therapy.