RESUMEN
Tumor cell heterogeneity in neuroblastoma, a pediatric cancer arising from neural crest-derived progenitor cells, poses a significant clinical challenge. In particular, unlike adrenergic (ADRN) neuroblastoma cells, mesenchymal (MES) cells are resistant to chemotherapy and retinoid therapy and thereby significantly contribute to relapses and treatment failures. Previous research suggested that overexpression or activation of miR-124, a neurogenic microRNA with tumor suppressor activity, can induce the differentiation of retinoic acid-resistant neuroblastoma cells. Leveraging our established screen for miRNA modulatory small molecules, we validated PP121, a dual inhibitor of tyrosine and phosphoinositide kinases, as a robust inducer of miR-124. A combination of PP121 and miR-132-inducing bufalin synergistically arrests proliferation, induces differentiation, and prolongs the survival of differentiated MES SK-N-AS cells for 8 weeks. RNA- seq and deconvolution analyses revealed a collapse of the ADRN core regulatory circuitry (CRC) and the emergence of novel CRCs associated with chromaffin cells and Schwann cell precursors. Using a similar protocol, we differentiated and maintained other MES neuroblastoma, as well as glioblastoma cells, over 16 weeks. In conclusion, our novel protocol suggests a promising treatment for therapy-resistant cancers of the nervous system. Moreover, these long-lived, differentiated cells provide valuable models for studying mechanisms underlying differentiation, maturation, and senescence.
RESUMEN
The N6-methyladenosine (m6A) RNA modification is an important regulator of gene expression. m6A is deposited by a methyltransferase complex that includes methyltransferase-like 3 (METTL3) and methyltransferase-like 14 (METTL14). High levels of METTL3/METTL14 drive the growth of many types of adult cancer, and METTL3/METTL14 inhibitors are emerging as new anticancer agents. However, little is known about the m6A epitranscriptome or the role of the METTL3/METTL14 complex in neuroblastoma, a common pediatric cancer. Here, we show that METTL3 knockdown or pharmacologic inhibition with the small molecule STM2457 leads to reduced neuroblastoma cell proliferation and increased differentiation. These changes in neuroblastoma phenotype are associated with decreased m6A deposition on transcripts involved in nervous system development and neuronal differentiation, with increased stability of target mRNAs. In preclinical studies, STM2457 treatment suppresses the growth of neuroblastoma tumors in vivo. Together, these results support the potential of METTL3/METTL14 complex inhibition as a therapeutic strategy against neuroblastoma.
Asunto(s)
Diferenciación Celular , Proliferación Celular , Metiltransferasas , Neuroblastoma , Metiltransferasas/metabolismo , Metiltransferasas/antagonistas & inhibidores , Neuroblastoma/patología , Neuroblastoma/metabolismo , Neuroblastoma/tratamiento farmacológico , Neuroblastoma/genética , Humanos , Diferenciación Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Línea Celular Tumoral , Animales , Ratones , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Adenosina/análogos & derivados , Adenosina/metabolismo , Adenosina/farmacologíaRESUMEN
Tumor MYCN amplification is seen in high-risk neuroblastoma, yet direct targeting of this oncogenic transcription factor has been challenging. Here, we take advantage of the dependence of MYCN-amplified neuroblastoma cells on increased protein synthesis to inhibit the activity of eukaryotic translation initiation factor 4A1 (eIF4A1) using an amidino-rocaglate, CMLD012824. Consistent with the role of this RNA helicase in resolving structural barriers in 5' untranslated regions (UTRs), CMLD012824 increased eIF4A1 affinity for polypurine-rich 5' UTRs, including that of the MYCN and associated transcripts with critical roles in cell proliferation. CMLD012824-mediated clamping of eIF4A1 spanned the full lengths of mRNAs, while translational inhibition was mediated through 5' UTR binding in a cap-dependent and -independent manner. Finally, CMLD012824 led to growth inhibition in MYCN-amplified neuroblastoma models without generalized toxicity. Our studies highlight the key role of eIF4A1 in MYCN-amplified neuroblastoma and demonstrate the therapeutic potential of disrupting its function.
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Regiones no Traducidas 5' , Factor 4A Eucariótico de Iniciación , Proteína Proto-Oncogénica N-Myc , Neuroblastoma , Animales , Humanos , Ratones , Regiones no Traducidas 5'/genética , Línea Celular Tumoral , Proliferación Celular , Factor 4A Eucariótico de Iniciación/metabolismo , Factor 4A Eucariótico de Iniciación/genética , Proteína Proto-Oncogénica N-Myc/metabolismo , Proteína Proto-Oncogénica N-Myc/genética , Neuroblastoma/genética , Neuroblastoma/patología , Neuroblastoma/metabolismo , Neuroblastoma/tratamiento farmacológico , ARN Mensajero/metabolismo , ARN Mensajero/genética , Femenino , Ratones Endogámicos C57BLRESUMEN
Solid tumors, especially those with aberrant MYCN activation, often harbor an immunosuppressive microenvironment to fuel malignant growth and trigger treatment resistance. Despite this knowledge, there are no effective strategies to tackle this problem. We found that chemokine-like factor (CKLF) is highly expressed by various solid tumor cells and transcriptionally up-regulated by MYCN. Using the MYCN-driven high-risk neuroblastoma as a model system, we demonstrated that as early as the premalignant stage, tumor cells secrete CKLF to attract CCR4-expressing CD4+ cells, inducing immunosuppression and tumor aggression. Genetic depletion of CD4+ T regulatory cells abolishes the immunorestrictive and protumorigenic effects of CKLF. Our work supports that disrupting CKLF-mediated cross-talk between tumor and CD4+ suppressor cells represents a promising immunotherapeutic approach to battling MYCN-driven tumors.
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Quimiocinas , Proteínas con Dominio MARVEL , Proteína Proto-Oncogénica N-Myc , Neuroblastoma , Humanos , Línea Celular Tumoral , Quimiocinas/metabolismo , Regulación Neoplásica de la Expresión Génica , Proteínas con Dominio MARVEL/metabolismo , Proteína Proto-Oncogénica N-Myc/metabolismo , Neuroblastoma/metabolismo , Neuroblastoma/patología , Neuroblastoma/terapia , Microambiente TumoralRESUMEN
BACKGROUND: Immunotherapy in high-risk neuroblastoma (HR-NBL) does not live up to its full potential due to inadequate (adaptive) immune engagement caused by the extensive immunomodulatory capacity of HR-NBL. We aimed to tackle one of the most notable immunomodulatory processes in neuroblastoma (NBL), absence of major histocompatibility complex class I (MHC-I) surface expression, a process greatly limiting cytotoxic T cell engagement. We and others have previously shown that MHC-I expression can be induced by cytokine-driven immune modulation. Here, we aimed to identify tolerable pharmacological repurposing strategies to upregulate MHC-I expression and therewith enhance T cell immunogenicity in NBL. METHODS: Drug repurposing libraries were screened to identify compounds enhancing MHC-I surface expression in NBL cells using high-throughput flow cytometry analyses optimized for adherent cells. The effect of positive hits was confirmed in a panel of NBL cell lines and patient-derived organoids. Compound-treated NBL cell lines and organoids were cocultured with preferentially expressed antigen of melanoma (PRAME)-reactive tumor-specific T cells and healthy-donor natural killer (NK) cells to determine the in vitro effect on T cell and NK cell cytotoxicity. Additional immunomodulatory effects of histone deacetylase inhibitors (HDACi) were identified by transcriptome and translatome analysis of treated organoids. RESULTS: Drug library screening revealed MHC-I upregulation by inhibitor of apoptosis inhibitor (IAPi)- and HDACi drug classes. The effect of IAPi was limited due to repression of nuclear factor kappa B (NFκB) pathway activity in NBL, while the MHC-I-modulating effect of HDACi was widely translatable to a panel of NBL cell lines and patient-derived organoids. Pretreatment of NBL cells with the HDACi entinostat enhanced the cytotoxic capacity of tumor-specific T cells against NBL in vitro, which coincided with increased expression of additional players regulating T cell cytotoxicity (eg, TAP1/2 and immunoproteasome subunits). Moreover, MICA and MICB, important in NK cell cytotoxicity, were also increased by entinostat exposure. Intriguingly, this increase in immunogenicity was accompanied by a shift toward a more mesenchymal NBL cell lineage. CONCLUSIONS: This study indicates the potential of combining (immuno)therapy with HDACi to enhance both T cell-driven and NKcell-driven immune responses in patients with HR-NBL.
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Células Asesinas Naturales , Neuroblastoma , Humanos , Linaje de la Célula , Neuroblastoma/tratamiento farmacológico , Neuroblastoma/genética , Antígenos de Histocompatibilidad Clase I , Linfocitos T Citotóxicos , Inhibidores de Histona Desacetilasas/farmacología , Inhibidores de Histona Desacetilasas/uso terapéutico , Epigénesis GenéticaRESUMEN
Apart from the anti-GD2 antibody, immunotherapy for neuroblastoma has had limited success due to immune evasion mechanisms, coupled with an incomplete understanding of predictors of response. Here, from bulk and single-cell transcriptomic analyses, we identify a subset of neuroblastomas enriched for transcripts associated with immune activation and inhibition and show that these are predominantly characterized by gene expression signatures of the mesenchymal lineage state. By contrast, tumors expressing adrenergic lineage signatures are less immunogenic. The inherent presence or induction of the mesenchymal state through transcriptional reprogramming or therapy resistance is accompanied by innate and adaptive immune gene activation through epigenetic remodeling. Mesenchymal lineage cells promote T cell infiltration by secreting inflammatory cytokines, are efficiently targeted by cytotoxic T and natural killer cells and respond to immune checkpoint blockade. Together, we demonstrate that distinct immunogenic phenotypes define the divergent lineage states of neuroblastoma and highlight the immunogenic potential of the mesenchymal lineage.
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Adrenérgicos , Neuroblastoma , Humanos , Linaje de la Célula/genética , Inhibidores de Puntos de Control Inmunológico , Neuroblastoma/genética , Citocinas/genética , FenotipoRESUMEN
Although activating mutations of the anaplastic lymphoma kinase (ALK) membrane receptor occur in â¼10% of neuroblastoma (NB) tumors, the role of the wild-type (WT) receptor, which is aberrantly expressed in most non-mutated cases, is unclear. Both WT and mutant proteins undergo extracellular domain (ECD) cleavage. Here, we map the cleavage site to Asn654-Leu655 and demonstrate that cleavage inhibition of WT ALK significantly impedes NB cell migration with subsequent prolongation of survival in mouse models. Cleavage inhibition results in the downregulation of an epithelial-to-mesenchymal transition (EMT) gene signature, with decreased nuclear localization and occupancy of ß-catenin at EMT gene promoters. We further show that cleavage is mediated by matrix metalloproteinase 9, whose genetic and pharmacologic inactivation inhibits cleavage and decreases NB cell migration. Together, our results indicate a pivotal role for WT ALK ECD cleavage in NB pathogenesis, which may be harnessed for therapeutic benefit.
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Quinasa de Linfoma Anaplásico/química , Quinasa de Linfoma Anaplásico/metabolismo , Movimiento Celular , Neuroblastoma/patología , Secuencia de Aminoácidos , Quinasa de Linfoma Anaplásico/antagonistas & inhibidores , Quinasa de Linfoma Anaplásico/genética , Animales , Secuencia de Bases , Línea Celular Tumoral , Membrana Celular/metabolismo , Modelos Animales de Enfermedad , Transición Epitelial-Mesenquimal/genética , Femenino , Perfilación de la Expresión Génica , Regulación Neoplásica de la Expresión Génica , Glicina/química , Células HEK293 , Humanos , Metaloproteinasa 9 de la Matriz/metabolismo , Ratones , Ratones Endogámicos C57BL , Mutación/genética , Células 3T3 NIH , Invasividad Neoplásica , Neuroblastoma/genética , Unión Proteica , Dominios ProteicosRESUMEN
Spatial transcriptomic and proteomic technologies have provided new opportunities to investigate cells in their native microenvironment. Here we present Giotto, a comprehensive and open-source toolbox for spatial data analysis and visualization. The analysis module provides end-to-end analysis by implementing a wide range of algorithms for characterizing tissue composition, spatial expression patterns, and cellular interactions. Furthermore, single-cell RNAseq data can be integrated for spatial cell-type enrichment analysis. The visualization module allows users to interactively visualize analysis outputs and imaging features. To demonstrate its general applicability, we apply Giotto to a wide range of datasets encompassing diverse technologies and platforms.
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Biología Computacional/métodos , Perfilación de la Expresión Génica/métodos , Hibridación in Situ , Programas Informáticos , Análisis de Datos , Procesamiento de Imagen Asistido por Computador , Inmunohistoquímica/métodos , Hibridación in Situ/métodos , Especificidad de Órganos/genética , Análisis Espacial , TranscriptomaRESUMEN
PURPOSE: Cyclin-dependent kinases (CDKs) that have critical roles in RNA polymerase II (Pol II)-mediated gene transcription are emerging as therapeutic targets in cancer. We have previously shown that THZ1, a covalent inhibitor of CDKs 7/12/13, leads to cytotoxicity in MYCN-amplified neuroblastoma through the downregulation of super-enhancer-associated transcriptional upregulation. Here we determined the effects of YKL-5-124, a novel covalent inhibitor with greater selectivity for CDK7 in neuroblastoma cells. EXPERIMENTAL DESIGN: We tested YKL-5-124 in MYCN-amplified and nonamplified neuroblastoma cells individually and in combination with other inhibitors in cell line and animal models. Cell viability, target validation, effects on cell cycle and transcription were analyzed. RESULTS: CDK7 inhibition with YKL-5-124 did not lead to significant cell death, but resulted in aberrant cell cycle progression especially in MYCN-amplified cells. Unlike THZ1, YKL-5-124 had minimal effects on Pol II C-terminal domain phosphorylation, but significantly inhibited that of the CDK1 and CDK2 cell cycle kinases. Combining YKL-5-124 with the BRD4 inhibitor JQ1 resulted in synergistic cytotoxicity. A distinct MYCN-gene expression signature associated with resistance to BRD4 inhibition was suppressed with the combination. The synergy between YKL-5-124 and JQ1 translated into significant tumor regression in cell line and patient-derived xenograft mouse models of neuroblastoma. CONCLUSIONS: The combination of CDK7 and BRD4 inhibition provides a therapeutic option for neuroblastoma and suggests that the addition of YKL-5-124 could improve the therapeutic efficacy of JQ1 and delay resistance to BRD4 inhibition.
RESUMEN
Only one class of targeted agents (anti-GD2 antibodies) has been incorporated into front-line therapy for neuroblastoma since the 1980s. The Neuroblastoma New Drug Development Strategy (NDDS) initiative commenced in 2012 to accelerate the development of new drugs for neuroblastoma. Advances have occurred, with eight of nine high-priority targets being evaluated in paediatric trials including anaplastic lymphoma kinase inhibitors being investigated in front-line, but significant challenges remain. This article reports the conclusions of the second NDDS forum, which expanded across the Atlantic to further develop the initiative. Pre-clinical and clinical data for 40 genetic targets and mechanisms of action were prioritised and drugs were identified for early-phase trials. Strategies to develop drugs targeting TERT, telomere maintenance, ATRX, alternative lengthening of telomeres (ALT), BRIP1 and RRM2 as well as direct targeting of MYCN are high priority and should be championed for drug discovery. Promising pre-clinical data suggest that targeting of ALT by ATM or PARP inhibition may be potential strategies. Drugs targeting CDK2/9, CDK7, ATR and telomere maintenance should enter paediatric clinical development rapidly. Optimising the response to anti-GD2 by combinations with chemotherapy, targeted agents and other immunological targets are crucial. Delivering this strategy in the face of small patient cohorts, genomically defined subpopulations and a large number of permutations of combination trials, demands even greater international collaboration. In conclusion, the NDDS provides an internationally agreed, biologically driven selection of prioritised genetic targets and drugs. Improvements in the strategy for conducting trials in neuroblastoma will accelerate bringing these new drugs more rapidly to front-line therapy.
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Neoplasias Encefálicas/tratamiento farmacológico , Desarrollo de Medicamentos , Neuroblastoma/tratamiento farmacológico , Antineoplásicos/aislamiento & purificación , Antineoplásicos/uso terapéutico , Neoplasias Encefálicas/patología , Niño , Congresos como Asunto , Desarrollo de Medicamentos/métodos , Desarrollo de Medicamentos/organización & administración , Desarrollo de Medicamentos/tendencias , Descubrimiento de Drogas/métodos , Descubrimiento de Drogas/organización & administración , Descubrimiento de Drogas/tendencias , Europa (Continente) , Humanos , Oncología Médica/métodos , Oncología Médica/organización & administración , Oncología Médica/tendencias , Terapia Molecular Dirigida/métodos , Terapia Molecular Dirigida/tendencias , Neuroblastoma/patología , Pediatría/métodos , Pediatría/organización & administración , Pediatría/tendencias , Inhibidores de Proteínas Quinasas/aislamiento & purificación , Inhibidores de Proteínas Quinasas/uso terapéutico , Terapias en Investigación/métodos , Terapias en Investigación/tendenciasRESUMEN
Neuroblastoma (NB), derived from the neural crest (NC), is the most common pediatric extracranial solid tumor. Here, we establish a platform that allows the study of human NBs in mouse-human NC chimeras. Chimeric mice were produced by injecting human NC cells carrying NB relevant oncogenes in utero into gastrulating mouse embryos. The mice developed tumors composed of a heterogenous cell population that resembled that seen in primary NBs of patients but were significantly different from homogeneous tumors formed in xenotransplantation models. The human tumors emerged in immunocompetent hosts and were extensively infiltrated by mouse cytotoxic T cells, reflecting a vigorous host anti-tumor immune response. However, the tumors blunted the immune response by inducing infiltration of regulatory T cells and expression of immune-suppressive molecules similar to escape mechanisms seen in human cancer patients. Thus, this experimental platform allows the study of human tumor initiation, progression, manifestation, and tumor-immune-system interactions in an animal model system.
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Cresta Neural , Neuroblastoma , Animales , Niño , Quimera , Modelos Animales de Enfermedad , Humanos , RatonesRESUMEN
Aggressive cancers often have activating mutations in growth-controlling oncogenes and inactivating mutations in tumor-suppressor genes. In neuroblastoma, amplification of the MYCN oncogene and inactivation of the ATRX tumor-suppressor gene correlate with high-risk disease and poor prognosis. Here we show that ATRX mutations and MYCN amplification are mutually exclusive across all ages and stages in neuroblastoma. Using human cell lines and mouse models, we found that elevated MYCN expression and ATRX mutations are incompatible. Elevated MYCN levels promote metabolic reprogramming, mitochondrial dysfunction, reactive-oxygen species generation, and DNA-replicative stress. The combination of replicative stress caused by defects in the ATRX-histone chaperone complex, and that induced by MYCN-mediated metabolic reprogramming, leads to synthetic lethality. Therefore, ATRX and MYCN represent an unusual example, where inactivation of a tumor-suppressor gene and activation of an oncogene are incompatible. This synthetic lethality may eventually be exploited to improve outcomes for patients with high-risk neuroblastoma.
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Proteína Proto-Oncogénica N-Myc/genética , Neuroblastoma/metabolismo , Proteína Nuclear Ligada al Cromosoma X/genética , Animales , Preescolar , Estudios de Cohortes , Femenino , Amplificación de Genes , Humanos , Lactante , Masculino , Ratones , Mitocondrias/genética , Mitocondrias/metabolismo , Mutación , Proteína Proto-Oncogénica N-Myc/metabolismo , Neuroblastoma/genética , Especies Reactivas de Oxígeno/metabolismo , Proteína Nuclear Ligada al Cromosoma X/metabolismoRESUMEN
The lack of model systems has limited the preclinical discovery and testing of therapies for Wilms tumor (WT) patients who have poor outcomes. Herein, we establish 45 heterotopic WT patient-derived xenografts (WTPDX) in CB17 scid-/- mice that capture the biological heterogeneity of Wilms tumor (WT). Among these 45 total WTPDX, 6 from patients with diffuse anaplastic tumors, 9 from patients who experienced disease relapse, and 13 from patients with bilateral disease are included. Early passage WTPDX show evidence of clonal selection, clonal evolution and enrichment of blastemal gene expression. Favorable histology WTPDX are sensitive, whereas unfavorable histology WTPDX are resistant to conventional chemotherapy with vincristine, actinomycin-D, and doxorubicin given singly or in combination. This WTPDX library is a unique scientific resource that retains the spectrum of biological heterogeneity present in WT and provides an essential tool to test targeted therapies for WT patient groups with poor outcomes.
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Protocolos de Quimioterapia Combinada Antineoplásica/farmacología , Evolución Clonal , Resistencia a Antineoplásicos/genética , Neoplasias Renales/genética , Tumor de Wilms/genética , Animales , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Humanos , Neoplasias Renales/tratamiento farmacológico , Neoplasias Renales/patología , Masculino , Ratones , Ratones SCID , Secuenciación del Exoma , Tumor de Wilms/tratamiento farmacológico , Tumor de Wilms/patología , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Despite progress in intensification of therapy, outcomes for patients with metastatic osteosarcoma (OS) have not improved in thirty years. We developed a system that enabled preclinical screening of compounds against metastatic OS cells in the context of the native lung microenvironment. Using this strategy to screen a library of epigenetically targeted compounds, we identified inhibitors of CDK12 to be most effective, reducing OS cell outgrowth in the lung by more than 90% at submicromolar doses. We found that knockout of CDK12 in an in vivo model of lung metastasis significantly decreased the ability of OS to colonize the lung. CDK12 inhibition led to defects in transcription elongation in a gene length- and expression-dependent manner. These effects were accompanied by defects in RNA processing and altered the expression of genes involved in transcription regulation and the DNA damage response. We further identified OS models that differ in their sensitivity to CDK12 inhibition in the lung and provided evidence that upregulated MYC levels may mediate these differences. Our studies provided a framework for rapid preclinical testing of compounds with antimetastatic activity and highlighted CDK12 as a potential therapeutic target in OS.
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Quinasas Ciclina-Dependientes/antagonistas & inhibidores , Quinasas Ciclina-Dependientes/metabolismo , Osteosarcoma/enzimología , Osteosarcoma/secundario , Animales , Línea Celular Tumoral , Quinasas Ciclina-Dependientes/genética , Ensayos de Selección de Medicamentos Antitumorales , Femenino , Técnicas de Inactivación de Genes , Humanos , Neoplasias Pulmonares/enzimología , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/secundario , Ratones , Ratones SCID , Osteosarcoma/genética , Inhibidores de Proteínas Quinasas/farmacología , Microambiente Tumoral/efectos de los fármacos , Microambiente Tumoral/genética , Microambiente Tumoral/fisiologíaRESUMEN
The CCCTC-binding factor (CTCF), which anchors DNA loops that organize the genome into structural domains, has a central role in gene control by facilitating or constraining interactions between genes and their regulatory elements1,2. In cancer cells, the disruption of CTCF binding at specific loci by somatic mutation3,4 or DNA hypermethylation5 results in the loss of loop anchors and consequent activation of oncogenes. By contrast, the germ-cell-specific paralogue of CTCF, BORIS (brother of the regulator of imprinted sites, also known as CTCFL)6, is overexpressed in several cancers7-9, but its contributions to the malignant phenotype remain unclear. Here we show that aberrant upregulation of BORIS promotes chromatin interactions in ALK-mutated, MYCN-amplified neuroblastoma10 cells that develop resistance to ALK inhibition. These cells are reprogrammed to a distinct phenotypic state during the acquisition of resistance, a process defined by the initial loss of MYCN expression followed by subsequent overexpression of BORIS and a concomitant switch in cellular dependence from MYCN to BORIS. The resultant BORIS-regulated alterations in chromatin looping lead to the formation of super-enhancers that drive the ectopic expression of a subset of proneural transcription factors that ultimately define the resistance phenotype. These results identify a previously unrecognized role of BORIS-to promote regulatory chromatin interactions that support specific cancer phenotypes.
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Cromatina/genética , Cromatina/metabolismo , Proteínas de Unión al ADN/metabolismo , Resistencia a Antineoplásicos/efectos de los fármacos , Resistencia a Antineoplásicos/genética , Neuroblastoma/tratamiento farmacológico , Neuroblastoma/patología , Quinasa de Linfoma Anaplásico/antagonistas & inhibidores , Quinasa de Linfoma Anaplásico/genética , Animales , Factor de Unión a CCCTC/metabolismo , Línea Celular Tumoral , Proteínas de Unión al ADN/genética , Femenino , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Regulación Neoplásica de la Expresión Génica/genética , Células HEK293 , Humanos , Ratones , Terapia Molecular Dirigida , Proteína Proto-Oncogénica N-Myc/genética , Neuroblastoma/enzimología , Neuroblastoma/genética , Fenotipo , Unión ProteicaRESUMEN
Chromosome 17q21-ter is commonly gained in neuroblastoma, but it is unclear which gene in the region is important for tumorigenesis. The JMJD6 gene at 17q21-ter activates gene transcription. Here we show that JMJD6 forms protein complexes with N-Myc and BRD4, and is important for E2F2, N-Myc and c-Myc transcription. Knocking down JMJD6 reduces neuroblastoma cell proliferation and survival in vitro and tumor progression in mice, and high levels of JMJD6 expression in human neuroblastoma tissues independently predict poor patient prognosis. In addition, JMJD6 gene is associated with transcriptional super-enhancers. Combination therapy with the CDK7/super-enhancer inhibitor THZ1 and the histone deacetylase inhibitor panobinostat synergistically reduces JMJD6, E2F2, N-Myc, c-Myc expression, induces apoptosis in vitro and leads to neuroblastoma tumor regression in mice, which are significantly reversed by forced JMJD6 over-expression. Our findings therefore identify JMJD6 as a neuroblastoma tumorigenesis factor, and the combination therapy as a treatment strategy.
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Histona Demetilasas con Dominio de Jumonji/metabolismo , Neuroblastoma/tratamiento farmacológico , Receptores de Superficie Celular/metabolismo , Animales , Apoptosis/efectos de los fármacos , Carcinogénesis , Proliferación Celular/efectos de los fármacos , Factor de Transcripción E2F2/genética , Factor de Transcripción E2F2/metabolismo , Femenino , Regulación Neoplásica de la Expresión Génica , Inhibidores de Histona Desacetilasas/administración & dosificación , Humanos , Histona Demetilasas con Dominio de Jumonji/antagonistas & inhibidores , Histona Demetilasas con Dominio de Jumonji/genética , Masculino , Ratones , Ratones Endogámicos BALB C , Neuroblastoma/genética , Neuroblastoma/metabolismo , Neuroblastoma/fisiopatología , Unión Proteica , Proteínas Proto-Oncogénicas c-myc/genética , Proteínas Proto-Oncogénicas c-myc/metabolismo , Receptores de Superficie Celular/antagonistas & inhibidores , Receptores de Superficie Celular/genéticaRESUMEN
Cyclin-dependent kinase 12 (CDK12) modulates transcription elongation by phosphorylating the carboxy-terminal domain of RNA polymerase II and selectively affects the expression of genes involved in the DNA damage response (DDR) and mRNA processing. Yet, the mechanisms underlying such selectivity remain unclear. Here we show that CDK12 inhibition in cancer cells lacking CDK12 mutations results in gene length-dependent elongation defects, inducing premature cleavage and polyadenylation (PCPA) and loss of expression of long (>45 kb) genes, a substantial proportion of which participate in the DDR. This early termination phenotype correlates with an increased number of intronic polyadenylation sites, a feature especially prominent among DDR genes. Phosphoproteomic analysis indicated that CDK12 directly phosphorylates pre-mRNA processing factors, including those regulating PCPA. These results support a model in which DDR genes are uniquely susceptible to CDK12 inhibition primarily due to their relatively longer lengths and lower ratios of U1 snRNP binding to intronic polyadenylation sites.
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Quinasas Ciclina-Dependientes/genética , Daño del ADN , Reparación del ADN/genética , Línea Celular Tumoral , Cromatografía Líquida de Alta Presión , Cromatografía Liquida , Humanos , Modelos Moleculares , Fosforilación , Poliadenilación , Procesamiento Postranscripcional del ARN , Espectrometría de Masas en TándemRESUMEN
N6-methyladenosine (m6A) modification of mRNA is emerging as an important regulator of gene expression that affects different developmental and biological processes, and altered m6A homeostasis is linked to cancer1-5. m6A modification is catalysed by METTL3 and enriched in the 3' untranslated region of a large subset of mRNAs at sites close to the stop codon5. METTL3 can promote translation but the mechanism and relevance of this process remain unknown1. Here we show that METTL3 enhances translation only when tethered to reporter mRNA at sites close to the stop codon, supporting a mechanism of mRNA looping for ribosome recycling and translational control. Electron microscopy reveals the topology of individual polyribosomes with single METTL3 foci in close proximity to 5' cap-binding proteins. We identify a direct physical and functional interaction between METTL3 and the eukaryotic translation initiation factor 3 subunit h (eIF3h). METTL3 promotes translation of a large subset of oncogenic mRNAs-including bromodomain-containing protein 4-that is also m6A-modified in human primary lung tumours. The METTL3-eIF3h interaction is required for enhanced translation, formation of densely packed polyribosomes and oncogenic transformation. METTL3 depletion inhibits tumorigenicity and sensitizes lung cancer cells to BRD4 inhibition. These findings uncover a mechanism of translation control that is based on mRNA looping and identify METTL3-eIF3h as a potential therapeutic target for patients with cancer.
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Carcinogénesis , Factor 3 de Iniciación Eucariótica/metabolismo , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patología , Metiltransferasas/metabolismo , Biosíntesis de Proteínas , ARN Mensajero/química , ARN Mensajero/metabolismo , Animales , Línea Celular Tumoral , Ciclización , Femenino , Humanos , Neoplasias Pulmonares/metabolismo , Ratones , Ratones Desnudos , Conformación de Ácido Nucleico , Polirribosomas/química , Polirribosomas/metabolismo , Unión Proteica , ARN Mensajero/genéticaRESUMEN
We present the development of the first small molecule degraders that can induce anaplastic lymphoma kinase (ALK) degradation, including in non-small-cell lung cancer (NSCLC), anaplastic large-cell lymphoma (ALCL), and neuroblastoma (NB) cell lines. These degraders were developed through conjugation of known pyrimidine-based ALK inhibitors, TAE684 or LDK378, and the cereblon ligand pomalidomide. We demonstrate that in some cell types degrader potency is compromised by expression of drug transporter ABCB1. In addition, proteomic profiling demonstrated that these compounds also promote the degradation of additional kinases including PTK2 (FAK), Aurora A, FER, and RPS6KA1 (RSK1).
Asunto(s)
Quinasa de Linfoma Anaplásico/metabolismo , Inhibidores de Proteínas Quinasas/farmacología , Proteolisis/efectos de los fármacos , Quinasa de Linfoma Anaplásico/antagonistas & inhibidores , Carcinoma de Pulmón de Células no Pequeñas/patología , Línea Celular Tumoral , Humanos , Neoplasias Pulmonares/patología , Pirimidinas/farmacología , Sulfonas/farmacologíaRESUMEN
Irreversible inhibition of transcriptional cyclin-dependent kinases (CDKs) provides a therapeutic strategy for cancers that rely on aberrant transcription; however, lack of understanding of resistance mechanisms to these agents will likely impede their clinical evolution. Here, we demonstrate upregulation of multidrug transporters ABCB1 and ABCG2 as a major mode of resistance to THZ1, a covalent inhibitor of CDKs 7, 12, and 13 in neuroblastoma and lung cancer. To counter this obstacle, we developed a CDK inhibitor, E9, that is not a substrate for ABC transporters, and by selecting for resistance, determined that it exerts its cytotoxic effects through covalent modification of cysteine 1039 of CDK12. These results highlight the importance of considering this common mode of resistance in the development of clinical analogs of THZ1, identify a covalent CDK12 inhibitor that is not susceptible to ABC transporter-mediated drug efflux, and demonstrate that target deconvolution can be accomplished through selection for resistance.