RESUMEN
Sonic hedgehog (Shh) signaling is essential to the patterning of the embryonic neural tube, but its presence and function in the postmitotic differentiated neurons in the brain remain largely uncharacterized. We recently showed that Shh and its signaling components, Patched and Smoothened, are expressed in postnatal and adult hippocampal neurons. We have now examined whether Shh signaling has a function in these neurons. Using cultured hippocampal neurons as a model system, we found that presynaptic terminals become significantly larger in response to the application of Shh. Ultrastructural examination confirmed the enlarged presynaptic profiles and also revealed variable increases in the size of synaptic vesicles, with a resulting loss of uniformity. Furthermore, electrophysiological analyses showed significant increases in the frequency, but not the amplitude, of spontaneous miniature excitatory postsynaptic currents (mEPSCs) in response to Shh, providing functional evidence of the selective role of Shh in presynaptic terminals. Thus, we conclude that Shh signaling regulates the structure and functional properties of presynaptic terminals of hippocampal neurons.
Asunto(s)
Proteínas Hedgehog/metabolismo , Hipocampo/citología , Terminales Presinápticos/fisiología , Terminales Presinápticos/ultraestructura , Animales , Células HEK293 , Humanos , Neurotransmisores/metabolismo , Tamaño de los Órganos , Terminales Presinápticos/metabolismo , Ratas , Transducción de Señal , Vesículas Sinápticas/metabolismo , Vesículas Sinápticas/ultraestructuraRESUMEN
BACKGROUND: CAR T cell therapy is a promising approach to improve outcomes and decrease toxicities for patients with cancer. While extraordinary success has been achieved using CAR T cells to treat patients with CD19-positive malignancies, multiple obstacles have so far limited the benefit of CAR T cell therapy for patients with solid tumors. Novel manufacturing and engineering approaches show great promise to enhance CAR T cell function against solid tumors. However, similar to single agent chemotherapy approaches, CAR T cell monotherapy may be unable to achieve high cure rates for patients with difficult to treat solid tumors. Thus, combinatorial drug plus CAR T cell approaches are likely required to achieve widespread clinical success. METHODS: We developed a novel, confocal microscopy based, high-content screen to evaluate 1114 FDA approved drugs for the potential to increase expression of the solid tumor antigen B7-H3 on the surface of osteosarcoma cells. Western blot, RT-qPCR, siRNA knockdown and flow cytometry assays were used to validate screening results and identify mechanisms of drug-induced B7-H3 upregulation. Cytokine and cytotoxicity assays were used to determine if drug pre-treatment enhanced B7-H3-CAR T cell effector function. RESULTS: Fifty-five drugs were identified to increase B7-H3 expression on the surface of LM7 osteosarcoma cells using a novel high-content, high-throughput screen. One drug, ingenol-3-angelate (I3A), increased B7-H3 expression by up to 100%, and was evaluated in downstream experiments. Validation assays confirmed I3A increased B7-H3 expression in a biphasic dose response and cell dependent fashion. Mechanistic studies demonstrated that I3A increased B7-H3 (CD276) mRNA, total protein, and cell surface expression via protein kinase C alpha activation. Functionally, I3A induced B7-H3 expression enhanced B7-H3-CAR T cell function in cytokine production and cytotoxicity assays. CONCLUSIONS: This study demonstrates a novel high-content and high-throughput screen can identify drugs to enhance CAR T cell activity. This and other high-content technologies will pave the way to develop clinical trials implementing rational drug plus CAR T cell combinatorial therapies. Importantly, the technique could also be repurposed for an array of basic and translational research applications where drugs are needed to modulate cell surface protein expression.
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Neoplasias Óseas , Diterpenos , Osteosarcoma , Humanos , Proteína Quinasa C-alfa/metabolismo , Antígenos B7/genética , Antígenos B7/metabolismo , Osteosarcoma/metabolismo , Neoplasias Óseas/patología , Linfocitos T , Citocinas/metabolismo , Línea Celular TumoralRESUMEN
Pediatric high-grade glioma (pHGG) is a major contributor to cancer-related death in children. In vitro and in vivo disease models reflecting the intimate connection between developmental context and pathogenesis of pHGG are essential to advance understanding and identify therapeutic vulnerabilities. Here we report establishment of 21 patient-derived pHGG orthotopic xenograft (PDOX) models and eight matched cell lines from diverse groups of pHGG. These models recapitulate histopathology, DNA methylation signatures, mutations and gene expression patterns of the patient tumors from which they were derived, and include rare subgroups not well-represented by existing models. We deploy 16 new and existing cell lines for high-throughput screening (HTS). In vitro HTS results predict variable in vivo response to PI3K/mTOR and MEK pathway inhibitors. These unique new models and an online interactive data portal for exploration of associated detailed molecular characterization and HTS chemical sensitivity data provide a rich resource for pediatric brain tumor research.
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Heterogeneidad Genética/efectos de los fármacos , Glioma/tratamiento farmacológico , Glioma/genética , Animales , Neoplasias Encefálicas , Línea Celular Tumoral , Proliferación Celular , Niño , Modelos Animales de Enfermedad , Regulación Neoplásica de la Expresión Génica , Glioma/patología , Ensayos Analíticos de Alto Rendimiento , Humanos , Ratones , Mutación , Inhibidores de Proteínas Quinasas/uso terapéutico , Serina-Treonina Quinasas TOR , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
BACKGROUND: Meiotic crossovers are the major mechanism by which haplotypes are shuffled to generate genetic diversity. Previously available methods for the genome-wide, high-resolution identification of meiotic crossover sites are limited by the laborious nature of the assay (as in sperm typing). METHODS: Several methods have been introduced to identify crossovers using high density single nucleotide polymorphism (SNP) array technologies, although programs are not widely available to implement such analyses. RESULTS: Here we present a two-generation "reverse pedigree analysis" method (analyzing the genotypes of two children relative to each parent) and a web-accessible tool to determine and visualize inheritance differences among siblings and crossover locations on each parental gamete. This approach is complementary to existing methods and uses informative markers which provide high resolution for locating meiotic crossover sites. We introduce a segmentation algorithm to identify crossover sites, and used a synthetic data set to determine that the segmentation algorithm specificity was 92% and sensitivity was 89%. The use of reverse pedigrees allows the inference of crossover locations on the X chromosome in a maternal gamete through analysis of two sons and their father. We further analyzed genotypes from eight multiplex autism families, observing a 1.462 maternal to paternal recombination ratio and no significant differences between affected and unaffected children. Meiotic recombination results from pediSNP can also be used to identify haplotypes that are shared by probands within a pedigree, as we demonstrated with a multiplex autism family. CONCLUSION: Using "reverse pedigrees" and defining unique sets of genotype markers within pedigree data, we introduce a method that identifies inherited allelic differences and meiotic crossovers. We implemented the method in the pediSNP software program, and we applied it to several data sets. This approach uses data from two generations to identify crossover sites, facilitating studies of recombination in disease. pediSNP is available online at http://pevsnerlab.kennedykrieger.org/pediSNP.
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Intercambio Genético , Patrón de Herencia , Meiosis , Modelos Genéticos , Linaje , Adulto , Niño , Marcadores Genéticos , Genotipo , Humanos , Polimorfismo de Nucleótido SimpleRESUMEN
BACKGROUND: Medulloblastoma is the most frequently occurring malignant brain tumor in children. Current treatment strategies for medulloblastoma include aggressive surgery, cranio-spinal irradiation and adjuvant chemotherapy. Because current treatments can cause severe long-term side effects and are not curative, successful treatment remains a challenge. METHODS: In this study, we employed a high-throughput cell viability assay to screen 12,800 compounds and to identify drug candidates with anti-proliferative properties for medulloblastoma cells. We also tested these compounds for attenuating medulloblastoma tumor development using mouse xenografts. RESULTS: We identified two histone deacetylase inhibitors (dacinostat and quisinostat) with anti-proliferative properties for medulloblastoma cells. We showed that both compounds induce cytotoxicity, trigger cell apoptosis, and block cell cycle progression at the G2/M phase. In addition, dacinostat and quisinostat attenuated xenograft medulloblastoma growth in mice. CONCLUSIONS: Our findings suggest that histone deacetylase inhibitors are potent therapeutic agents against medulloblastoma.
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There are currently no FDA-approved therapies to prevent the hearing loss associated with the usage of cisplatin in chemotherapeutic regimens. We recently demonstrated that the pharmacologic inhibition with kenpaullone or genetic deletion of CDK2 preserved hearing function in animal models treated with cisplatin, which suggests that CDK2 is a promising therapeutic target to prevent cisplatin-induced ototoxicity. In this study, we identified two lead compounds, AT7519 and AZD5438, from a focused library screen of 187 CDK2 inhibitors, performed in an immortalized cell line derived from neonatal mouse cochleae treated with cisplatin. Moreover, we screened 36 analogues of AT7519 and identified analogue 7, which exhibited an improved therapeutic index. When delivered locally, analogue 7 and AZD5438 both provided significant protection against cisplatin-induced ototoxicity in mice. Thus, we have identified two additional compounds that prevent cisplatin-induced ototoxicity in vivo and provided further evidence that CDK2 is a druggable target for treating cisplatin-induced ototoxicity.
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Cisplatino/efectos adversos , Quinasa 2 Dependiente de la Ciclina/antagonistas & inhibidores , Pérdida Auditiva/inducido químicamente , Inhibidores de Proteínas Quinasas/farmacología , Animales , Antineoplásicos/efectos adversos , Cóclea/efectos de los fármacos , Evaluación Preclínica de Medicamentos/métodos , Pérdida Auditiva/prevención & control , Humanos , Imidazoles/química , Imidazoles/farmacología , Ratones Endogámicos , Técnicas de Cultivo de Órganos , Sustancias Protectoras/química , Sustancias Protectoras/farmacología , Inhibidores de Proteínas Quinasas/química , Pirimidinas/química , Pirimidinas/farmacología , Relación Estructura-ActividadRESUMEN
Human pregnane X receptor (hPXR) is a nuclear receptor that regulates the expression of phase I and phase II drug-metabolism enzymes, as well as that of drug transporters. hPXR is a "xenobiotics sensor" and can be activated by structurally diverse compounds. The activation of hPXR by its agonists increases the clearance of xenobiotics by increasing the expression of drug-metabolism enzymes and drug transporters, possibly leading to drug toxicity, drug resistance, and other adverse drug reactions. Therefore, hPXR antagonists might attenuate agonist-mediated activation of hPXR and reduce the risk of adverse drug reactions. Several hPXR antagonists have been reported, but none of them is specific for hPXR. In this study, we present the first large-scale, unbiased, cell-based high-throughput screen to identify specific hPXR antagonists. Among the 132,975 compounds screened, we identified the 1,4,5-substituted 1,2,3-triazole analogs as potent and specific hPXR antagonists by sequentially performing primary screening, retesting, and dose-response analysis using cell-based hPXR gene reporter and receptor binding assays, as well as receptor and promoter specificity assays. The compound SJ000076745-1 is the most potent and specific hPXR antagonist in the 1,4,5-substituted 1,2,3-triazole chemical class, having a cell-based hPXR antagonist 50% inhibitory concentration (IC50) value of 377 ± 16 nM and an hPXR binding inhibitory IC50 value of 563 ± 40 nM.
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Ensayos Analíticos de Alto Rendimiento , Receptores de Esteroides/antagonistas & inhibidores , Triazoles/análisis , Triazoles/farmacología , Relación Dosis-Respuesta a Droga , Células Hep G2 , Humanos , Estructura Molecular , Receptor X de Pregnano , Relación Estructura-Actividad , Células Tumorales CultivadasRESUMEN
Phenotypic screening through high-content automated microscopy is a powerful tool for evaluating the mechanism of action of candidate therapeutics. Despite more than a decade of development, however, high content assays have yielded mixed results, identifying robust phenotypes in only a small subset of compound classes. This has led to a combinatorial explosion of assay techniques, analyzing cellular phenotypes across dozens of assays with hundreds of measurements. Here, using a minimalist three-stain assay and only 23 basic cellular measurements, we developed an analytical approach that leverages informative dimensions extracted by linear discriminant analysis to evaluate similarity between the phenotypic trajectories of different compounds in response to a range of doses. This method enabled us to visualize biologically-interpretable phenotypic tracks populated by compounds of similar mechanism of action, cluster compounds according to phenotypic similarity, and classify novel compounds by comparing them to phenotypically active exemplars. Hierarchical clustering applied to 154 compounds from over a dozen different mechanistic classes demonstrated tight agreement with published compound mechanism classification. Using 11 phenotypically active mechanism classes, classification was performed on all 154 compounds: 78% were correctly identified as belonging to one of the 11 exemplar classes or to a different unspecified class, with accuracy increasing to 89% when less phenotypically active compounds were excluded. Importantly, several apparent clustering and classification failures, including rigosertib and 5-fluoro-2'-deoxycytidine, instead revealed more complex mechanisms or off-target effects verified by more recent publications. These results show that a simple, easily replicated, minimalist high-content assay can reveal subtle variations in the cellular phenotype induced by compounds and can correctly predict mechanism of action, as long as the appropriate analytical tools are used.
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Microscopía/métodos , Bibliotecas de Moléculas Pequeñas/análisis , Bibliotecas de Moléculas Pequeñas/farmacología , Análisis por Conglomerados , Daño del ADN , Análisis Discriminante , Células HeLa , Histonas/metabolismo , Humanos , Fenotipo , Análisis de Componente PrincipalRESUMEN
The hedgehog (HH) family of growth factors is involved in many aspects of growth and development, from the establishment of left-right axes at gastrulation to the patterning and formation of multiple structures in essentially every tissue, to the maintenance and regulation of stem cell populations in adults. Sonic hedgehog (Shh) in particular acts as a mitogen, regulating proliferation of target cells, a growth factor that triggers differentiation in target populations, and a morphogen causing cells to respond differently based on their positions along a spatial and temporal concentration gradient. Given its very broad range of effects in development, it is not surprising that many of the structures affected by a disruption in Shh signaling are also affected in Down syndrome (DS). However, recent studies have shown that trisomic cerebellar granule cell precursors have a deficit, compared to their euploid counterparts, in their response to the mitogenic effects of Shh. This deficit substantially contributes to the hypocellular cerebellum in mouse models that parallels the human DS phenotype and can be corrected in early development by a single exposure to a small-molecule agonist of the Shh pathway. Here, we consider how an attenuated Shh response might affect several aspects of development to produce multiple phenotypic outcomes observed in DS.
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Síndrome de Down/patología , Proteínas Hedgehog/deficiencia , Transducción de Señal/fisiología , Trisomía/patología , Animales , Sistema Cardiovascular/fisiopatología , Modelos Animales de Enfermedad , Huesos Faciales/patología , Proteínas Hedgehog/genética , Humanos , Ratones , Mutación/genética , Cráneo/patologíaRESUMEN
BACKGROUND: Chromosomal translocations generating oncogenic transcription factors are the hallmark of a variety of tumors, including many sarcomas. Ewing sarcoma family of tumors (ESFTs) are characterized by the t(11;22)(q24;q12) translocation that generates the Ewing sarcoma breakpoint region 1 and Friend leukemia virus integration 1 (EWS-FLI1) fusion transcription factor responsible for the highly malignant phenotype of this tumor. Although continued expression of EWS-FLI1 is believed to be critical for ESFT cell survival, a clinically effective small-molecule inhibitor remains elusive likely because EWS-FLI1 is a transcription factor and therefore widely felt to be "undruggable." METHODS: We developed a high-throughput screen to evaluate more than 50 000 compounds for inhibition of EWS-FLI1 activity in TC32 ESFT cells. We used a TC32 cell-based luciferase reporter screen using the EWS-FLI1 downstream target NR0B1 promoter and a gene signature secondary screen to sort and prioritize the compounds. We characterized the lead compound, mithramycin, based on its ability to inhibit EWS-FLI1 activity in vitro using microarray expression profiling, quantitative reverse transcription-polymerase chain reaction, and immunoblot analysis, and in vivo using immunohistochemistry. We studied the impact of this inhibition on cell viability in vitro and on tumor growth in ESFT xenograft models in vivo (n = 15-20 mice per group). All statistical tests were two-sided. RESULTS: Mithramycin inhibited expression of EWS-FLI1 downstream targets at the mRNA and protein levels and decreased the growth of ESFT cells at half maximal inhibitory concentrations between 10 (95% confidence interval [CI] = 8 to 13 nM) and 15 nM (95% CI = 13 to 19 nM). Mithramycin suppressed the growth of two different ESFT xenograft tumors and prolonged the survival of ESFT xenograft-bearing mice by causing a decrease in mean tumor volume. For example, in the TC32 xenograft model, on day 15 of treatment, the mean tumor volume for the mithramycin-treated mice was approximately 3% of the tumor volume observed in the control mice (mithramycin vs control: 69 vs 2388 mm(3), difference = 2319 mm(3), 95% CI = 1766 to 2872 mm(3), P < .001). CONCLUSION: Mithramycin inhibits EWS-FLI1 activity and demonstrates ESFT antitumor activity both in vitro and in vivo.