RESUMEN
Recently we have shown that capsaicin attenuates the strength of LTP in the lateral amygdala (LA) and demonstrated that this effect is mediated by the transient receptor potential (TRP) channel TRPV1. Here we further show that capsaicin, which is thought to act primarily through TRPV1, modifies long term depression (LTD) in the LA. Yet the application of various TRPV1 antagonists does not reverse this effect and it remains in TRPV1-deficient mice. In addition, voltage gated calcium channels, nitric oxide and CB1 receptors are not involved. Using pharmacology and TRPM1-/- mice, our electrophysiological data indicate that capsaicin-induced activation of TRPM1 channels contribute to the induction of LA-LTD. Whereas LA-LTD in general depends on the acitvation of NMDA receptors- and group II metabotropic glutamate receptors (mGluR), the modifying effect of capsaicin on LA-LTD via TRPM1 appears to be specifically mediated by group I mGluRs and in interaction with another member of the TRP family, TRPC5. Additionally, intact GABAergic transmission is required for the capsaicin-effect to take place. This is the first documentation that beside their function in the retina TRPM1 proteins are expressed in the brain and have a functional relevance in modifying synaptic plasticity.
Asunto(s)
Complejo Nuclear Basolateral/efectos de los fármacos , Capsaicina/farmacología , Depresión Sináptica a Largo Plazo/efectos de los fármacos , Fármacos del Sistema Sensorial/farmacología , Canales Catiónicos TRPC/efectos de los fármacos , Canales Catiónicos TRPM/efectos de los fármacos , Animales , Inmunohistoquímica , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Canales Catiónicos TRPM/deficienciaRESUMEN
Adult T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive hematologic tumor associated with poor outcome. In this study, we analyzed the prognostic relevance of genetic alterations, immunophenotypic markers, and microarray gene expression signatures in a panel of 53 adult T-ALL patients treated in the Eastern Cooperative Oncology Group E2993 clinical trial. An early immature gene expression signature, the absence of bi-allelic TCRG deletion, CD13 surface expression, heterozygous deletions of the short arm of chromosome 17, and mutations in IDH1/IDH2 and DNMT3A genes are associated with poor prognosis in this series. In contrast, expression of CD8 or CD62L, homozygous deletion of CDKN2A/CDKN2B, NOTCH1 and/or FBXW7 mutations, and mutations or deletions in the BCL11B tumor suppressor gene were associated with improved overall survival. Importantly, the prognostic relevance of CD13 expression and homozygous CDKN2A/CDKN2B deletions was restricted to cortical and mature T-ALLs. Conversely, mutations in IDH1/IDH2 and DNMT3A were specifically associated with poor outcome in early immature adult T-ALLs. This trial was registered at www.clinicaltrials.gov as #NCT00002514.
Asunto(s)
Regulación Leucémica de la Expresión Génica , Genómica , Leucemia-Linfoma Linfoblástico de Células T Precursoras/genética , Leucemia-Linfoma Linfoblástico de Células T Precursoras/mortalidad , Adulto , Femenino , Dosificación de Gen/genética , Humanos , Inmunofenotipificación , Masculino , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Análisis por Micromatrices , Leucemia-Linfoma Linfoblástico de Células T Precursoras/metabolismo , Pronóstico , Factores de Riesgo , Transcripción Genética/genéticaRESUMEN
The SH2B adaptor protein 3 (SH2B3) gene encodes a negative regulator of cytokine signaling with a critical role in the homeostasis of hematopoietic stem cells and lymphoid progenitors. Here, we report the identification of germline homozygous SH2B3 mutations in 2 siblings affected with developmental delay and autoimmunity, one in whom B-precursor acute lymphoblastic leukemia (ALL) developed. Mechanistically, loss of SH2B3 increases Janus kinase-signal transducer and activator of transcription signaling, promotes lymphoid cell proliferation, and accelerates leukemia development in a mouse model of NOTCH1-induced ALL. Moreover, extended mutation analysis showed homozygous somatic mutations in SH2B3 in 2 of 167 ALLs analyzed. Overall, these results demonstrate a Knudson tumor suppressor role for SH2B3 in the pathogenesis of ALL and highlight a possible link between genetic predisposition factors in the pathogenesis of autoimmunity and leukemogenesis.
Asunto(s)
Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , Proteínas/genética , Proteínas Adaptadoras Transductoras de Señales , Animales , Enfermedades Autoinmunes/genética , Secuencia de Bases , Western Blotting , Niño , Preescolar , Análisis Mutacional de ADN , Discapacidades del Desarrollo/genética , Femenino , Genotipo , Mutación de Línea Germinal , Humanos , Lactante , Recién Nacido , Péptidos y Proteínas de Señalización Intracelular , Masculino , Ratones , Ratones Endogámicos C57BL , Datos de Secuencia Molecular , Análisis de Secuencia por Matrices de Oligonucleótidos , Linaje , HermanosRESUMEN
Alzheimer's disease (AD) and schizophrenia (SCZ) represent two major neuropathological conditions with a high disease burden. Despite their distinct etiologies, patients suffering from AD or SCZ share a common burden of disrupted memory functions unattended by current therapies. Recent preclinical analyses highlight cell-type-specific contributions of parvalbumin interneurons (PVIs), particularly the plasticity of their cellular excitability, towards intact neuronal network function (cell-to-network plasticity) and memory performance. Here we argue that deficits of PVI cell-to-network plasticity may underlie memory deficits in AD and SCZ, and we explore two therapeutic avenues: the targeting of PVI-specific neuromodulation, including by neuropeptides, and the recruitment of network synchrony in the gamma frequency range (40 Hz) by external stimulation. We finally propose that these approaches be merged under consideration of recent insights into human brain physiology.
Asunto(s)
Interneuronas , Plasticidad Neuronal , Parvalbúminas , Esquizofrenia , Humanos , Interneuronas/fisiología , Parvalbúminas/metabolismo , Plasticidad Neuronal/fisiología , Animales , Esquizofrenia/fisiopatología , Esquizofrenia/terapia , Enfermedad de Alzheimer/fisiopatología , Enfermedad de Alzheimer/terapiaRESUMEN
Understanding the plasticity of neuronal networks is an emerging field of (patho-) physiological research, yet the underlying cellular mechanisms remain poorly understood. Gamma oscillations (30 to 80 hertz), a biomarker of cognitive performance, require and potentiate glutamatergic transmission onto parvalbumin-positive interneurons (PVIs), suggesting an interface for cell-to-network plasticity. In ex vivo local field potential recordings, we demonstrate long-term potentiation of hippocampal gamma power. Gamma potentiation obeys established rules of PVI plasticity, requiring calcium-permeable AMPA receptors (CP-AMPARs) and metabotropic glutamate receptors (mGluRs). A microcircuit computational model of CA3 gamma oscillations predicts CP-AMPAR plasticity onto PVIs critically outperforms pyramidal cell plasticity in increasing gamma power and completely accounts for gamma potentiation. We reaffirm this ex vivo in three PVI-targeting animal models, demonstrating that gamma potentiation requires PVI-specific signaling via a Gq/PKC pathway comprising mGluR5 and a Gi-sensitive, PKA-dependent pathway. Gamma activity-dependent, metabotropically mediated CP-AMPAR plasticity on PVIs may serve as a guiding principle in understanding network plasticity in health and disease.
Asunto(s)
Hipocampo , Parvalbúminas , Animales , Parvalbúminas/metabolismo , Hipocampo/metabolismo , Potenciación a Largo Plazo/fisiología , Transducción de Señal , Interneuronas/fisiología , Plasticidad Neuronal/fisiologíaAsunto(s)
Antineoplásicos/farmacología , Duplicación de Gen , Leucemia/genética , Metiltransferasas/antagonistas & inhibidores , Proteína de la Leucemia Mieloide-Linfoide/genética , Secuencias Repetidas en Tándem , Apoptosis/efectos de los fármacos , Apoptosis/genética , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Supervivencia Celular/genética , Resistencia a Antineoplásicos/genética , Inhibidores Enzimáticos/farmacología , N-Metiltransferasa de Histona-Lisina , Histonas/metabolismo , Humanos , Leucemia/metabolismo , Leucemia/patologíaRESUMEN
BACKGROUND: Glioblastoma (GBM) is the most common and aggressive primary brain tumor. Prognosis remains poor despite multimodal therapy. Developing alternative treatments is essential. Drugs targeting kinases within the phosphoinositide 3-kinase (PI3K) and mitogen-activated protein kinase (MAPK) effectors of receptor tyrosine kinase (RTK) signaling represent promising candidates. METHODS: We previously developed a non-germline genetically engineered mouse model of GBM in which PI3K and MAPK are activated via Pten deletion and KrasG12D in immortalized astrocytes. Using this model, we examined the influence of drug potency on target inhibition, alternate pathway activation, efficacy, and synergism of single agent and combination therapy with inhibitors of these 2 pathways. Efficacy was then examined in GBM patient-derived xenografts (PDX) in vitro and in vivo. RESULTS: PI3K and mitogen-activated protein kinase kinase (MEK) inhibitor potency was directly associated with target inhibition, alternate RTK effector activation, and efficacy in mutant murine astrocytes in vitro. The kinomes of GBM PDX and tumor samples were heterogeneous, with a subset of the latter harboring MAPK hyperactivation. Dual PI3K/MEK inhibitor treatment overcame alternate effector activation, was synergistic in vitro, and was more effective than single agent therapy in subcutaneous murine allografts. However, efficacy in orthotopic allografts was minimal. This was likely due to dose-limiting toxicity and incomplete target inhibition. CONCLUSION: Drug potency influences PI3K/MEK inhibitor-induced target inhibition, adaptive kinome reprogramming, efficacy, and synergy. Our findings suggest that combination therapies with highly potent, brain-penetrant kinase inhibitors will be required to improve patient outcomes.
Asunto(s)
Neoplasias Encefálicas/tratamiento farmacológico , Resistencia a Antineoplásicos , Glioblastoma/tratamiento farmacológico , Proteínas Quinasas Activadas por Mitógenos/antagonistas & inhibidores , Inhibidores de las Quinasa Fosfoinosítidos-3 , Inhibidores de Proteínas Quinasas/farmacología , Animales , Apoptosis/efectos de los fármacos , Neoplasias Encefálicas/metabolismo , Neoplasias Encefálicas/patología , Proliferación Celular/efectos de los fármacos , Sinergismo Farmacológico , Glioblastoma/metabolismo , Glioblastoma/patología , Humanos , Ratones , Fosforilación , Transducción de Señal/efectos de los fármacos , Células Tumorales Cultivadas , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
The DOT1L lysine methyltransferase has emerged as a validated therapeutic target in MLL-rearranged (MLLr) acute leukemias. Although S-adenosylmethionine competitive inhibitors have demonstrated pharmacological proof-of-principle in MLLr-leukemia, these compounds require further optimization to improve cellular potency and pharmacokinetic stability. Limiting DOT1L inhibitor discovery and ligand optimization have been complex biochemical methods often using radionucleotides and cellular methods requiring prolonged culture. We therefore developed a new suite of assay technologies that allows comparative assessment of chemical tools for DOT1L in a miniaturized format. Coupling these assays with structural information, we developed new insights into DOT1L ligand binding and identified several functionalized probes with increased cellular potency (IC50 values â¼10 nM) and excellent selectivity for DOT1L. Together these assay technologies define a platform capability for discovery and optimization of small-molecule DOT1L inhibitors.
Asunto(s)
Antineoplásicos/síntesis química , Inhibidores Enzimáticos/síntesis química , Regulación Neoplásica de la Expresión Génica , Ensayos Analíticos de Alto Rendimiento , Histonas/antagonistas & inhibidores , Metiltransferasas/antagonistas & inhibidores , Adenosina/análogos & derivados , Adenosina/síntesis química , Adenosina/farmacología , Antineoplásicos/farmacología , Línea Celular Tumoral , Inhibidores Enzimáticos/farmacología , Células Epiteliales/efectos de los fármacos , Células Epiteliales/enzimología , Células Epiteliales/patología , N-Metiltransferasa de Histona-Lisina , Histonas/genética , Histonas/metabolismo , Humanos , Ligandos , Metilación , Metiltransferasas/química , Metiltransferasas/genética , Metiltransferasas/metabolismo , Modelos Moleculares , Compuestos de Fenilurea/síntesis química , Compuestos de Fenilurea/farmacología , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , S-Adenosilmetionina/química , S-Adenosilmetionina/metabolismo , Bibliotecas de Moléculas Pequeñas/síntesis química , Bibliotecas de Moléculas Pequeñas/farmacología , Relación Estructura-ActividadRESUMEN
Tissue-specific alternative splicing is critical for the emergence of tissue identity during development, yet the role of this process in malignant transformation is undefined. Tissue-specific splicing involves evolutionarily conserved, alternative exons that represent only a minority of the total alternative exons identified. Many of these conserved exons have functional features that influence signaling pathways to profound biological effect. Here, we determined that lineage-specific splicing of a brain-enriched cassette exon in the membrane-binding tumor suppressor annexin A7 (ANXA7) diminishes endosomal targeting of the EGFR oncoprotein, consequently enhancing EGFR signaling during brain tumor progression. ANXA7 exon splicing was mediated by the ribonucleoprotein PTBP1, which is normally repressed during neuronal development. PTBP1 was highly expressed in glioblastomas due to loss of a brain-enriched microRNA (miR-124) and to PTBP1 amplification. The alternative ANXA7 splicing trait was present in precursor cells, suggesting that glioblastoma cells inherit the trait from a potential tumor-initiating ancestor and that these cells exploit this trait through accumulation of mutations that enhance EGFR signaling. Our data illustrate that lineage-specific splicing of a tissue-regulated alternative exon in a constituent of an oncogenic pathway eliminates tumor suppressor functions and promotes glioblastoma progression. This paradigm may offer a general model as to how tissue-specific regulatory mechanisms can reprogram normal developmental processes into oncogenic ones.
Asunto(s)
Empalme Alternativo , Anexina A7/genética , Neoplasias Encefálicas/genética , Glioblastoma/genética , Neoplasias Encefálicas/metabolismo , Neoplasias Encefálicas/patología , Linaje de la Célula/genética , Transformación Celular Neoplásica/genética , Progresión de la Enfermedad , Receptores ErbB/genética , Receptores ErbB/metabolismo , Exones , Técnicas de Silenciamiento del Gen , Glioblastoma/metabolismo , Glioblastoma/patología , Ribonucleoproteínas Nucleares Heterogéneas/antagonistas & inhibidores , Ribonucleoproteínas Nucleares Heterogéneas/genética , Ribonucleoproteínas Nucleares Heterogéneas/metabolismo , Humanos , MicroARNs/genética , MicroARNs/metabolismo , Células Madre Neoplásicas/metabolismo , Células Madre Neoplásicas/patología , Neovascularización Patológica/genética , Proteína de Unión al Tracto de Polipirimidina/antagonistas & inhibidores , Proteína de Unión al Tracto de Polipirimidina/genética , Proteína de Unión al Tracto de Polipirimidina/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , ARN Neoplásico/genética , ARN Neoplásico/metabolismo , Transducción de Señal/genética , Células Tumorales CultivadasRESUMEN
T-cell acute lymphoblastic leukemias (T-ALL) and lymphomas are aggressive hematologic cancers frequently associated with activating mutations in NOTCH1. Early studies identified NOTCH1 as an attractive therapeutic target for the treatment of T-ALL through the use of γ-secretase inhibitors (GSI). Here, we characterized the interaction between PF-03084014, a clinically relevant GSI, and dexamethasone in preclinical models of glucocorticoid-resistant T-ALL. Combination treatment of the GSI PF-03084014 with glucocorticoids induced a synergistic antileukemic effect in human T-ALL cell lines and primary human T-ALL patient samples. Mechanistically PF-03084014 plus glucocorticoid treatment induced increased transcriptional upregulation of the glucocorticoid receptor and glucocorticoid target genes. Treatment with PF-03084014 and glucocorticoids in combination was highly efficacious in vivo, with enhanced reduction of tumor burden in a xenograft model of T-ALL. Finally, glucocorticoid treatment effectively reversed PF-03084014-induced gastrointestinal toxicity via inhibition of goblet cell metaplasia. These results warrant the analysis of PF-03084014 and glucocorticoids in combination for the treatment of glucocorticoid-resistant T-ALL.
Asunto(s)
Secretasas de la Proteína Precursora del Amiloide/antagonistas & inhibidores , Antineoplásicos/farmacología , Glucocorticoides/farmacología , Leucemia-Linfoma Linfoblástico de Células T Precursoras/metabolismo , Tetrahidronaftalenos/farmacología , Valina/análogos & derivados , Secretasas de la Proteína Precursora del Amiloide/metabolismo , Animales , Línea Celular Tumoral , Análisis por Conglomerados , Dexametasona/administración & dosificación , Resistencia a Antineoplásicos/efectos de los fármacos , Femenino , Perfilación de la Expresión Génica , Regulación Leucémica de la Expresión Génica/efectos de los fármacos , Humanos , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos NOD , Ratones SCID , Leucemia-Linfoma Linfoblástico de Células T Precursoras/tratamiento farmacológico , Receptor Notch1/antagonistas & inhibidores , Receptor Notch1/metabolismo , Tetrahidronaftalenos/química , Tetrahidronaftalenos/toxicidad , Valina/química , Valina/farmacología , Valina/toxicidadRESUMEN
T cell acute lymphoblastic leukemia (T-ALL) is an immature hematopoietic malignancy driven mainly by oncogenic activation of NOTCH1 signaling. In this study we report the presence of loss-of-function mutations and deletions of the EZH2 and SUZ12 genes, which encode crucial components of the Polycomb repressive complex 2 (PRC2), in 25% of T-ALLs. To further study the role of PRC2 in T-ALL, we used NOTCH1-dependent mouse models of the disease, as well as human T-ALL samples, and combined locus-specific and global analysis of NOTCH1-driven epigenetic changes. These studies demonstrated that activation of NOTCH1 specifically induces loss of the repressive mark Lys27 trimethylation of histone 3 (H3K27me3) by antagonizing the activity of PRC2. These studies suggest a tumor suppressor role for PRC2 in human leukemia and suggest a hitherto unrecognized dynamic interplay between oncogenic NOTCH1 and PRC2 function for the regulation of gene expression and cell transformation.
Asunto(s)
Leucemia-Linfoma Linfoblástico de Células T Precursoras/genética , Proteínas Represoras/metabolismo , Animales , Proteínas Portadoras/genética , Proteínas Portadoras/metabolismo , Línea Celular Tumoral , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Proteína Potenciadora del Homólogo Zeste 2 , Epigénesis Genética/genética , Regulación Neoplásica de la Expresión Génica/genética , Silenciador del Gen/fisiología , N-Metiltransferasa de Histona-Lisina/genética , N-Metiltransferasa de Histona-Lisina/metabolismo , Humanos , Ratones , Proteínas de Neoplasias , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Análisis de Secuencia por Matrices de Oligonucleótidos , Complejo Represivo Polycomb 2 , Proteínas del Grupo Polycomb , Leucemia-Linfoma Linfoblástico de Células T Precursoras/metabolismo , Receptor Notch1/genética , Receptor Notch1/metabolismo , Proteínas Represoras/genética , Factores de Transcripción/genética , Factores de Transcripción/metabolismoRESUMEN
Early immature T cell acute lymphoblastic leukemias (T-ALLs) account for ~5-10% of pediatric T-ALLs and are associated with poor prognosis. However, the genetic defects that drive the biology of these tumors remain largely unknown. In this study, analysis of microarray gene expression signatures in adult T-ALL demonstrated a high prevalence of early immature leukemias and revealed a close relationship between these tumors and myeloid leukemias. Many adult immature T-ALLs harbored mutations in myeloid-specific oncogenes and tumor suppressors including IDH1, IDH2, DNMT3A, FLT3, and NRAS. Moreover, we identified ETV6 mutations as a novel genetic lesion uniquely present in immature adult T-ALL. Our results demonstrate that early immature adult T-ALL represents a heterogeneous category of leukemias characterized by the presence of overlapping myeloid and T-ALL characteristics, and highlight the potential role of ETV6 mutations in these tumors.
Asunto(s)
Mutación , Leucemia-Linfoma Linfoblástico de Células T Precursoras/genética , Leucemia-Linfoma Linfoblástico de Células T Precursoras/metabolismo , Proteínas Proto-Oncogénicas c-ets/genética , Proteínas Proto-Oncogénicas c-ets/metabolismo , Proteínas Represoras/genética , Proteínas Represoras/metabolismo , Adulto , Línea Celular Tumoral , Femenino , Perfilación de la Expresión Génica , Regulación Leucémica de la Expresión Génica/genética , Genes Supresores de Tumor , Humanos , Lactante , Masculino , Análisis de Secuencia por Matrices de Oligonucleótidos , Proteína ETS de Variante de Translocación 6RESUMEN
The TLX1 oncogene (encoding the transcription factor T cell leukemia homeobox protein-1) has a major role in the pathogenesis of T cell acute lymphoblastic leukemia (T-ALL). However, the specific mechanisms of T cell transformation downstream of TLX1 remain to be elucidated. Here we show that transgenic expression of human TLX1 in mice induces T-ALL with frequent deletions and mutations in Bcl11b (encoding B cell leukemia/lymphoma-11B) and identify the presence of recurrent mutations and deletions in BCL11B in 16% of human T-ALLs. Most notably, mouse TLX1 tumors were typically aneuploid and showed a marked defect in the activation of the mitotic checkpoint. Mechanistically, TLX1 directly downregulates the expression of CHEK1 (encoding CHK1 checkpoint homolog) and additional mitotic control genes and induces loss of the mitotic checkpoint in nontransformed preleukemic thymocytes. These results identify a previously unrecognized mechanism contributing to chromosomal missegregation and aneuploidy active at the earliest stages of tumor development in the pathogenesis of cancer.
Asunto(s)
Aneuploidia , Transformación Celular Neoplásica/genética , Proteínas de Homeodominio/genética , Proteínas Proto-Oncogénicas/genética , Linfocitos T/patología , Animales , Secuencia de Bases , Aberraciones Cromosómicas , Hibridación Genómica Comparativa , Análisis Mutacional de ADN , Eliminación de Gen , Perfilación de la Expresión Génica , Regulación Leucémica de la Expresión Génica , Proteínas de Homeodominio/metabolismo , Ratones , Ratones Transgénicos , Datos de Secuencia Molecular , Tamaño de los Órganos , Leucemia-Linfoma Linfoblástico de Células T Precursoras/genética , Leucemia-Linfoma Linfoblástico de Células T Precursoras/patología , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Represoras/genética , Cariotipificación Espectral , Timo/crecimiento & desarrollo , Timo/patología , Trisomía/genética , Proteínas Supresoras de Tumor/genéticaRESUMEN
The establishment and maintenance of centromeric heterochromatin in fission yeast require the RITS complex. Comprised of centromeric siRNAs, the chromodomain protein Chp1, Argonaute (Ago1), and Tas3, RITS couples the cellular RNAi pathway with assembly of constitutive heterochromatin. However, the mechanisms governing RITS-dependent establishment versus maintenance of centromeric heterochromatin remain unresolved. Here, we report that a mutant Tas3 protein that cannot bind Ago1 supports the maintenance of centromeric heterochromatin but cannot mediate efficient de novo establishment from cells transiently depleted for the histone H3 lysine 9 methyltransferase Clr4. In contrast, centromeric heterochromatin efficiently assembles in mutant cells transiently depleted for dicer. This mutant therefore allows ordering of the events leading to establishment of centromeric heterochromatin and places lysine 9 methylation of histone H3 upstream of dicer function.