RESUMEN
The thiopurine derivatives azathioprine (AZA), mercaptopurine (MP) and tioguanine (TG) remain standard treatment of inflammatory bowel disease (IBD). The immune suppressive effect of thiopurines is primarily based on blocking the Ras-related C3 botulinum toxin substrate 1 (Rac1) causing apoptosis of T lymphocytes by inhibition of the phosphorylated downstream transcription factor Signal Transducer and Activator of Transcription 3 (pSTAT3). A functional pharmacodynamic marker in T lymphocytes may be useful to predict therapeutic outcome of thiopurine therapy. The aim of this study was to explore whether protein levels of Rac1 and pSTAT3 in T lymphocytes may be applied as a specific pharmacodynamic marker for thiopurine therapy in IBD patients. Rac1 and pSTAT3 protein levels in T lymphocytes were explored in 57 IBD patients (median age 51 years, 56% female), subdivided into six groups based on IBD activity and its treatment: patients with active disease without IBD maintenance medication (1) or patients in remission on AZA/MP (2), TG (3), infliximab (IFX) (4), thiopurine and IFX combination-treatment (5) or without IBD medication (6). Reference values were obtained from healthy subjects. Rac1 and pSTAT3 protein levels in T lymphocytes from patients on thiopurine monotherapy (group 2 and 3) were compared to the other groups, and to healthy subjects. Absolute Rac1 and pSTAT3 protein levels showed no differences between the thiopurine monotherapy groups when compared to patients with active disease. However, the ratio of Rac1 and pSTAT3 protein levels was lower in thiopurine patients groups compared to patients with active disease. Rac1-corrected pSTAT3 protein levels may serve as a pharmacodynamic marker of thiopurine monotherapy and may be a potential tool to predict therapeutic effectiveness in IBD patients.
Asunto(s)
Enfermedades Inflamatorias del Intestino , Mercaptopurina , Azatioprina/uso terapéutico , Femenino , Humanos , Inmunosupresores/uso terapéutico , Enfermedades Inflamatorias del Intestino/metabolismo , Infliximab/metabolismo , Infliximab/farmacología , Infliximab/uso terapéutico , Masculino , Mercaptopurina/uso terapéutico , Persona de Mediana Edad , Factor de Transcripción STAT3/metabolismo , Linfocitos T/metabolismo , Tioguanina , Proteína de Unión al GTP rac1/metabolismoRESUMEN
Thiopurine derivatives, such as azathioprine and mercaptopurine, are standard conventional treatment options in inflammatory bowel disease (IBD). Unfortunately, approximately half of patients discontinue thiopurine therapy within 2 years. To improve the prediction of clinical effectiveness, thiopurine therapy is currently optimized using therapeutic drug monitoring. Ras-related C3 botulinum toxin substrate 1 (Rac1) has been suggested as a potential pharmacodynamic marker of the thiopurine effect in lymphocytes. The active thiopurine metabolite 6-thioguanine triphosphate (6-Thio-GTP) causes T cell apoptosis via Rac1 and the downstream transcription factor signal transducer and activator of transcription 3 (STAT3). The aim of this study was to develop and validate a functional pharmacodynamic multiparameter flow cytometric assay to determine Rac1/pSTAT3 expression in the various leukocyte subpopulations in peripheral blood in order to predict therapeutic response in IBD patients in the future. Peripheral blood samples of healthy subjects (no fever or clinical complaints of active disease, C-reactive protein < 10 mg/L) were used for immunocytochemical labeling, applying an optimized fixation and permeabilization strategy. A gating procedure was performed to separate all leukocyte subpopulations. Quantitative data were obtained by measuring presence and median fluorescent intensity. In vitro, Rac1 presence and expression were detectable in all leukocyte subpopulations. After IL-6 stimulation, used as proxy for inflammation, a distinct pSTAT3 signal could be detected in T lymphocytes of healthy subjects. In vivo, an upregulated pSTAT3 signal was detected in nearly all IBD patients with active disease and differed substantially from the signal found in IBD patients in remission on thiopurines and healthy subjects. We developed and validated a functional flow cytometric assay to assess Rac1 and pSTAT3 presence and expression. This opens a venue for a pharmacodynamic assay to predict thiopurine effectiveness in IBD patients.
Asunto(s)
Enfermedades Inflamatorias del Intestino , Mercaptopurina , Azatioprina/farmacología , Azatioprina/uso terapéutico , Biomarcadores , Humanos , Inmunosupresores/uso terapéutico , Enfermedades Inflamatorias del Intestino/tratamiento farmacológico , Enfermedades Inflamatorias del Intestino/metabolismo , Mercaptopurina/farmacología , Mercaptopurina/uso terapéutico , Linfocitos T/metabolismo , Proteína de Unión al GTP rac1/metabolismoRESUMEN
OBJECTIVES: COVID-19 is an ongoing global pandemic. There is an urgent need for identification and understanding of clinical and laboratory parameters related to progression towards a severe and fatal form of this illness, often preceded by a so-called cytokine-storm syndrome (CSS). Therefore, we explored the hemocytometric characteristics of COVID-19 patients in relation to the deteriorating clinical condition CSS, using the Sysmex XN-10 hematology analyzer. METHODS: From March 1st till May 16th, 2020, all patients admitted to our hospital with respiratory complaints and suspected for COVID-19 were included (n=1,140 of whom n=533 COVID-19 positive). The hemocytometric parameters of immunocompetent cells in peripheral blood (neutrophils [NE], lymphocytes [LY] and monocytes [MO]) obtained upon admission to the emergency department (ED) of COVID-19 positive patients were compared with those of the COVID-19 negative ones. Moreover, patients with CSS (n=169) were compared with COVID-19 positive patients without CSS, as well as with COVID-19 negative ones. RESULTS: In addition to a significant reduction in leukocytes, thrombocytes and absolute neutrophils, it appeared that lymphocytes-forward scatter (LY-FSC), and reactive lymphocytes (RE-LYMPHO)/leukocytes were higher in COVID-19-positive than negative patients. At the moment of presentation, COVID-19 positive patients with CSS had different neutrophils-side fluorescence (NE-SFL), neutrophils-forward scatter (NE-FSC), LY-FSC, RE-LYMPHO/lymphocytes, antibody-synthesizing (AS)-LYMPHOs, high fluorescence lymphocytes (HFLC), MO-SSC, MO-SFL, and Reactive (RE)-MONOs. Finally, absolute eosinophils, basophils, lymphocytes, monocytes and MO-FSC were lower in patients with CSS. CONCLUSIONS: Hemocytometric parameters indicative of changes in immunocompetent peripheral blood cells and measured at admission to the ED were associated with COVID-19 with and without CSS.
Asunto(s)
COVID-19/sangre , Síndrome de Liberación de Citoquinas/sangre , Anciano , Anciano de 80 o más Años , Células Sanguíneas/metabolismo , COVID-19/diagnóstico , COVID-19/metabolismo , Síndrome de Liberación de Citoquinas/diagnóstico , Síndrome de Liberación de Citoquinas/metabolismo , Progresión de la Enfermedad , Recuento de Eritrocitos/instrumentación , Femenino , Humanos , Recuento de Linfocitos/instrumentación , Masculino , Persona de Mediana Edad , Pronóstico , SARS-CoV-2RESUMEN
STAT3 mediates signalling downstream of cytokine and growth factor receptors where it acts as a transcription factor for its target genes, including oncogenes and cell survival regulating genes. STAT3 has been found to be persistently activated in many types of cancers, primarily through its tyrosine phosphorylation (Y705). Here, we show that constitutive STAT3 activation protects cells from cytotoxic drug responses of several drug classes. To find novel and potentially targetable STAT3 regulators we performed a kinase and phosphatase siRNA screen with cells expressing either a hyperactive STAT3 mutant or IL6-induced wild type STAT3. The screen identified cell division cycle 7-related protein kinase (CDC7), casein kinase 2, alpha 1 (CSNK2), discoidin domain-containing receptor 2 (DDR2), cyclin-dependent kinase 8 (CDK8), phosphatidylinositol 4-kinase 2-alpha (PI4KII), C-terminal Src kinase (CSK) and receptor-type tyrosine-protein phosphatase H (PTPRH) as potential STAT3 regulators. Using small molecule inhibitors targeting these proteins, we confirmed dose and time dependent inhibition of STAT3-mediated transcription, suggesting that inhibition of these kinases may provide strategies for dampening STAT3 activity in cancers.
Asunto(s)
Antineoplásicos/farmacología , Biología Computacional , Factor de Transcripción STAT3/metabolismo , Relación Dosis-Respuesta a Droga , Células HEK293 , Humanos , Monoéster Fosfórico Hidrolasas/metabolismo , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Quinasas/metabolismo , ARN Interferente Pequeño/genética , Factor de Transcripción STAT3/deficiencia , Factor de Transcripción STAT3/genética , Factores de TiempoRESUMEN
Post-translational modification (PTM) of proteins plays essential regulatory roles in a variety of pathological conditions. Reliable and practical assays are required to accelerate the discovery of inhibitors and activators for PTM related diseases. Today, methodologies are based on specific or group-specific PTM recognition of e.g. phosphate for kinase activity without extending to other type of PTMs. Here we have established a universal time-resolved luminescence assay on a peptide-break platform for the direct detection of wide variety of PTMs. The developed assay is based on the leucine zipper concept wherein a europium-chelate labeled detection peptide and a non-labeled peptide substrate form a highly luminescent dimer. As an active PTM enzyme at sub or low nanomolar concentration modifies the substrate peptide, the luminescent signal of the detached detection peptide is quenched in the presence of soluble quenchers. The functionality of this universal assay technique has been demonstrated for the monitoring of phosphorylation, dephosphorylation, deacetylation, and citrullination with high applicability also to other PTMs in a high throughput format.
RESUMEN
Knowledge of the full target space of bioactive substances, approved and investigational drugs as well as chemical probes, provides important insights into therapeutic potential and possible adverse effects. The existing compound-target bioactivity data resources are often incomparable due to non-standardized and heterogeneous assay types and variability in endpoint measurements. To extract higher value from the existing and future compound target-profiling data, we implemented an open-data web platform, named Drug Target Commons (DTC), which features tools for crowd-sourced compound-target bioactivity data annotation, standardization, curation, and intra-resource integration. We demonstrate the unique value of DTC with several examples related to both drug discovery and drug repurposing applications and invite researchers to join this community effort to increase the reuse and extension of compound bioactivity data.
Asunto(s)
Consenso , Bases del Conocimiento , Descubrimiento de Drogas , Interacciones Farmacológicas , Reposicionamiento de Medicamentos , Humanos , Preparaciones FarmacéuticasRESUMEN
Constitutive JAK/STAT3 signaling contributes to disease progression in many lymphoproliferative disorders. Recent genetic analyses have revealed gain-of-function STAT3 mutations in lymphoid cancers leading to hyperactivation of STAT3, which may represent a potential therapeutic target. Using a functional reporter assay, we screened 306 compounds with selective activity against various target molecules to identify drugs capable of inhibiting the cellular activity of STAT3. Top hits were further validated with additional models including STAT3-mutated natural killer (NK)-cell leukemia/lymphoma cell lines and primary large granular lymphocytic (LGL) leukemia cells to assess their ability to inhibit STAT3 phosphorylation and STAT3 dependent cell viability. We identified JAK, mTOR, Hsp90 and CDK inhibitors as potent inhibitors of both WT and mutant STAT3 activity. The Hsp90 inhibitor luminespib was highly effective at reducing the viability of mutant STAT3 NK cell lines and LGL leukemia patient samples. Luminespib decreased the phosphorylation of mutant STAT3 at Y705, whereas JAK1/JAK2 inhibitor ruxolitinib had reduced efficacy on mutant STAT3 phosphorylation. Additionally, combinations involving Hsp90, JAK and mTOR inhibitors were more effective at reducing cell viability than single agents. Our findings show alternative approaches to inhibit STAT3 activity and suggest Hsp90 as a therapeutic target in lymphoproliferative disorders with constitutively active STAT3.
RESUMEN
Ras GTPases act as "molecular switches", alternating between inactive GDP-bound and active GTP-bound conformation. Ras-oncogenes were discovered over three decades ago, but there are still no effective therapies for Ras-driven cancers. So far, drug discovery strategies have been unsuccessful, because of a lack of suitable screening methodologies and well-defined binding pockets on the Ras proteins. Here, we addressed the former by introducing a homogeneous quenching resonance energy transfer (QRET) technique-based screening strategy for Ras interfacial and competitive inhibitors. We demonstrate that using a unique GTP-specific antibody fragment to monitor GTPase cycling in the presence of a guanine nucleotide exchange factor (GEF) and a GTPase activating protein (GAP) is an efficient method for Ras inhibitor high-throughput screening. When compared to a conventional GEF-stimulated nucleotide exchange assay in a proof-of-concept screen, we identified an overlapping set of potential inhibitor compounds but also compounds found exclusively with the new GTP hydrolysis monitoring-based GTPase cycling assay.
Asunto(s)
Proteínas Activadoras de GTPasa/metabolismo , Factores de Intercambio de Guanina Nucleótido/metabolismo , Proteínas ras/metabolismo , Sitios de Unión , Línea Celular Tumoral , Complejos de Coordinación/química , Europio/química , Transferencia Resonante de Energía de Fluorescencia , Proteínas Activadoras de GTPasa/química , Factores de Intercambio de Guanina Nucleótido/química , Guanosina Trifosfato/química , Guanosina Trifosfato/metabolismo , Ensayos Analíticos de Alto Rendimiento , Humanos , Simulación del Acoplamiento Molecular , Unión Proteica , Proteínas ras/antagonistas & inhibidoresRESUMEN
Male germ cell Rac GTPase-activating protein (MgcRacGAP) is a core regulator of cytokinesis. Furthermore, it appears to be involved in human oncogenesis through cytokinesis-independent mechanisms and has been reported to be essential for nuclear translocation of signal transducer and activator of transcription (STAT) proteins, including the oncoprotein STAT3. Here we utilized MgcRacGAP inhibitor compound 1 (MINC1), a small molecule inhibitor of MgcRacGAP, to further investigate how MgcRacGAP regulates STAT3. Surprisingly, both MINC1 treatment and small interference RNA (siRNA)-mediated gene silencing of MgcRacGAP resulted in increased STAT3 phosphorylation and STAT3-driven transcriptional activity in our experimental systems. Finally, we demonstrated that MINC1-induced STAT3 activation likely is due to increased STAT3 phosphorylation caused by a Rac1-PAR3-IL6-IL6R-JAK2 mediated autocrine/paracrine mechanism.
Asunto(s)
Proteínas Activadoras de GTPasa/antagonistas & inhibidores , Quinasas Janus/metabolismo , Factor de Transcripción STAT3/metabolismo , Relación Dosis-Respuesta a Droga , Proteínas Activadoras de GTPasa/deficiencia , Proteínas Activadoras de GTPasa/genética , Silenciador del Gen , Células HEK293 , Células HeLa , Humanos , Janus Quinasa 2/metabolismo , Fosforilación/efectos de los fármacos , ARN Interferente Pequeño/genética , Receptores de Interleucina-6/metabolismo , Factor de Transcripción STAT3/química , Factor de Transcripción STAT3/deficiencia , Factor de Transcripción STAT3/genética , Transducción de Señal/efectos de los fármacos , Transcripción Genética/efectos de los fármacos , Tirosina/metabolismo , Proteína de Unión al GTP rac1/metabolismoRESUMEN
The signal transducer and activator of transcription (STAT) family of transcription factors orchestrate hematopoietic cell differentiation. Recently, mutations in STAT1, STAT5B, and STAT3 have been linked to development of immunodysregulation polyendocrinopathy enteropathy X-linked-like syndrome. Here, we immunologically characterized 3 patients with de novo activating mutations in the DNA binding or dimerization domains of STAT3 (p.K392R, p.M394T, and p.K658N, respectively). The patients displayed multiorgan autoimmunity, lymphoproliferation, and delayed-onset mycobacterial disease. Immunologically, we noted hypogammaglobulinemia with terminal B-cell maturation arrest, dendritic cell deficiency, peripheral eosinopenia, increased double-negative (CD4(-)CD8(-)) T cells, and decreased natural killer, T helper 17, and regulatory T-cell numbers. Notably, the patient harboring the K392R mutation developed T-cell large granular lymphocytic leukemia at age 14 years. Our results broaden the spectrum of phenotypes caused by activating STAT3 mutations, highlight the role of STAT3 in the development and differentiation of multiple immune cell lineages, and strengthen the link between the STAT family of transcription factors and autoimmunity.
Asunto(s)
Agammaglobulinemia , Enfermedades Autoinmunes , Enfermedades Genéticas Congénitas , Leucemia Linfocítica Granular Grande , Mutación Missense , Infecciones por Mycobacterium , Factor de Transcripción STAT3 , Adolescente , Adulto , Agammaglobulinemia/genética , Agammaglobulinemia/inmunología , Agammaglobulinemia/patología , Sustitución de Aminoácidos , Enfermedades Autoinmunes/genética , Enfermedades Autoinmunes/inmunología , Enfermedades Autoinmunes/patología , Linfocitos B/inmunología , Linfocitos B/patología , Diferenciación Celular/genética , Diferenciación Celular/inmunología , Células Dendríticas/inmunología , Células Dendríticas/patología , Femenino , Enfermedades Genéticas Congénitas/genética , Enfermedades Genéticas Congénitas/inmunología , Enfermedades Genéticas Congénitas/patología , Humanos , Células Asesinas Naturales/inmunología , Células Asesinas Naturales/patología , Leucemia Linfocítica Granular Grande/genética , Leucemia Linfocítica Granular Grande/inmunología , Leucemia Linfocítica Granular Grande/patología , Infecciones por Mycobacterium/genética , Infecciones por Mycobacterium/inmunología , Infecciones por Mycobacterium/patología , Estructura Terciaria de Proteína , Factor de Transcripción STAT3/genética , Factor de Transcripción STAT3/inmunología , Linfocitos T Reguladores/inmunología , Linfocitos T Reguladores/patología , Células Th17/inmunología , Células Th17/patologíaRESUMEN
The Rho family of Ras superfamily small GTPases regulates a broad range of biological processes such as migration, differentiation, cell growth and cell survival. Therefore, the availability of small molecule modulators as tool compounds could greatly enhance research on these proteins and their biological function. To this end, we designed a biochemical, high throughput screening assay with complementary follow-up assays to identify small molecule compounds inhibiting MgcRacGAP, a Rho family GTPase activating protein involved in cytokinesis and transcriptionally upregulated in many cancers. We first performed an in-house screen of 20,480 compounds, and later we tested the assay against 342,046 compounds from the NIH Molecular Libraries Small Molecule Repository. Primary screening hit rates were about 1% with the majority of those affecting the primary readout, an enzyme-coupled GDP detection assay. After orthogonal and counter screens, we identified two hits with high selectivity towards MgcRacGAP, compared with other RhoGAPs, and potencies in the low micromolar range. The most promising hit, termed MINC1, was then examined with cell-based testing where it was observed to induce an increased rate of cytokinetic failure and multinucleation in addition to other cell division defects, suggesting that it may act as an MgcRacGAP inhibitor also in cells.
Asunto(s)
Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/farmacología , Proteínas Activadoras de GTPasa/antagonistas & inhibidores , Bibliotecas de Moléculas Pequeñas/química , Bibliotecas de Moléculas Pequeñas/farmacología , Animales , Ciclo Celular/efectos de los fármacos , Línea Celular , Movimiento Celular/efectos de los fármacos , Descubrimiento de Drogas , Proteínas Activadoras de GTPasa/metabolismo , Células HeLa , Ensayos Analíticos de Alto Rendimiento , HumanosRESUMEN
A quenching resonance energy transfer (QRET) assay for small GTPase nucleotide exchange kinetic monitoring is demonstrated using nanomolar protein concentrations. Small GTPases are central signaling proteins in all eukaryotic cells acting as a "molecular switches" that are active in the GTP-state and inactive in the GDP-state. GTP-loading is highly regulated by guanine nucleotide exchange factors (GEFs). In several diseases, most prominently cancer, this process in misregulated. The kinetics of the nucleotide exchange reaction reports on the enzymatic activity of the GEF reaction system and is, therefore, of special interest. We determined the nucleotide exchange kinetics using europium-labeled GTP (Eu-GTP) in the QRET assay for small GTPases. After GEF catalyzed GTP-loading of a GTPase, a high time-resolved luminescence signal was found to be associated with GTPase bound Eu-GTP, whereas the non-bound Eu-GTP fraction was quenched by soluble quencher. The association kinetics of the Eu-GTP was measured after GEF addition, whereas the dissociation kinetics could be determined after addition of unlabeled GTP. The resulting association and dissociation rates were in agreement with previously published values for H-Ras(Wt), H-Ras(Q61G), and K-Ras(Wt), respectively. The broader applicability of the QRET assay for small GTPases was demonstrated by determining the kinetics of the Ect2 catalyzed RhoA(Wt) GTP-loading. The QRET assay allows the use of nanomolar protein concentrations, as more than 3-fold signal-to-background ratio was achieved with 50 nM GTPase and GEF proteins. Thus, small GTPase exchange kinetics can be efficiently determined in a HTS compatible 384-well plate format.
Asunto(s)
Transferencia de Energía por Resonancia de Bioluminiscencia/métodos , GTP Fosfohidrolasas/química , Nucleótidos/química , Animales , Transferencia de Energía , HumanosRESUMEN
Large granular lymphocytic (LGL) leukemia is characterized by clonal expansion of cytotoxic T cells or natural killer cells. Recently, somatic mutations in the signal transducer and activator of transcription 3 (STAT3) gene were discovered in 28% to 40% of LGL leukemia patients. By exome and transcriptome sequencing of 2 STAT3 mutation-negative LGL leukemia patients, we identified a recurrent, somatic missense mutation (Y665F) in the Src-like homology 2 domain of the STAT5b gene. Targeted amplicon sequencing of 211 LGL leukemia patients revealed 2 additional patients with STAT5b mutations (N642H), resulting in a total frequency of 2% (4 of 211) of STAT5b mutations across all patients. The Y665F and N642H mutant constructs increased the transcriptional activity of STAT5 and tyrosine (Y694) phosphorylation, which was also observed in patient samples. The clinical course of the disease in patients with the N642H mutation was aggressive and fatal, clearly different from typical LGL leukemia with a relatively favorable outcome. This is the first time somatic STAT5 mutations are discovered in human cancer and further emphasizes the role of STAT family genes in the pathogenesis of LGL leukemia.
Asunto(s)
Leucemia Linfocítica Granular Grande/genética , Factor de Transcripción STAT5/genética , Dominios Homologos src/genética , Anciano , Estudios de Cohortes , Dimerización , Exoma/genética , Femenino , Pruebas Genéticas , Células HeLa , Humanos , Masculino , Persona de Mediana Edad , Mutagénesis , Mutación , Fosforilación/genética , Estructura Terciaria de Proteína , Factor de Transcripción STAT5/química , Factor de Transcripción STAT5/metabolismo , Transcripción Genética/genética , Proteínas Supresoras de Tumor/genéticaRESUMEN
BACKGROUND: T-cell large granular lymphocytic leukemia is a rare lymphoproliferative disorder characterized by the expansion of clonal CD3+CD8+ cytotoxic T lymphocytes (CTLs) and often associated with autoimmune disorders and immune-mediated cytopenias. METHODS: We used next-generation exome sequencing to identify somatic mutations in CTLs from an index patient with large granular lymphocytic leukemia. Targeted resequencing was performed in a well-characterized cohort of 76 patients with this disorder, characterized by clonal T-cell-receptor rearrangements and increased numbers of large granular lymphocytes. RESULTS: Mutations in the signal transducer and activator of transcription 3 gene (STAT3) were found in 31 of 77 patients (40%) with large granular lymphocytic leukemia. Among these 31 patients, recurrent mutational hot spots included Y640F in 13 (17%), D661V in 7 (9%), D661Y in 7 (9%), and N647I in 3 (4%). All mutations were located in exon 21, encoding the Src homology 2 (SH2) domain, which mediates the dimerization and activation of STAT protein. The amino acid changes resulted in a more hydrophobic protein surface and were associated with phosphorylation of STAT3 and its localization in the nucleus. In vitro functional studies showed that the Y640F and D661V mutations increased the transcriptional activity of STAT3. In the affected patients, downstream target genes of the STAT3 pathway (IFNGR2, BCL2L1, and JAK2) were up-regulated. Patients with STAT3 mutations presented more often with neutropenia and rheumatoid arthritis than did patients without these mutations. CONCLUSIONS: The SH2 dimerization and activation domain of STAT3 is frequently mutated in patients with large granular lymphocytic leukemia; these findings suggest that aberrant STAT3 signaling underlies the pathogenesis of this disease. (Funded by the Academy of Finland and others.).
Asunto(s)
Leucemia Linfocítica Granular Grande/genética , Factor de Transcripción STAT3/genética , Anciano , Exoma , Expresión Génica , Humanos , Masculino , Mutación , Receptores de Antígenos de Linfocitos T , Análisis de Secuencia de ARN , Transcripción Genética , Regulación hacia ArribaRESUMEN
MicroRNAs (miRNAs) are pivotal for regulation of hematopoiesis but their critical targets remain largely unknown. Here, we show that ectopic expression of miR-17, -20,-93 and -106, all AAAGUGC seed-containing miRNAs, increases proliferation, colony outgrowth and replating capacity of myeloid progenitors and results in enhanced P-ERK levels. We found that these miRNAs are endogenously and abundantly expressed in myeloid progenitors and down-regulated in mature neutrophils. Quantitative proteomics identified sequestosome 1 (SQSTM1), an ubiquitin-binding protein and regulator of autophagy-mediated protein degradation, as a major target for these miRNAs in myeloid progenitors. In addition, we found increased expression of Sqstm1 transcripts during CSF3-induced neutrophil differentiation of 32D-CSF3R cells and an inverse correlation of SQSTM1 protein levels and miR-106 expression in AML samples. ShRNA-mediated silencing of Sqstm1 phenocopied the effects of ectopic miR-17/20/93/106 expression in hematopoietic progenitors in vitro and in mice. Further, SQSTM1 binds to the ligand-activated colony-stimulating factor 3 receptor (CSF3R) mainly in the late endosomal compartment, but not in LC3 positive autophagosomes. SQSTM1 regulates CSF3R stability and ligand-induced mitogen-activated protein kinase signaling. We demonstrate that AAAGUGC seed-containing miRNAs promote cell expansion, replating capacity and signaling in hematopoietic cells by interference with SQSTM1-regulated pathways.