RESUMEN
Double knockout of the two miR-15/16 loci in mouse resulted in the development of acute myeloid leukemia (AML). This result suggested that, at least, a fraction of human AMLs could be due to a similar mechanism. We analyzed the role of the two miR-15/16 clusters in 93 myelodysplastic syndrome (MDS) patients divided in three subgroups: patients with MDS, patients with MDS before transforming into AML (MDS-T), and patients with AML evolving from MDS (MDS-AML). Then, we tested 139 AML cases and 14 different AML cell lines by assessing microRNA (miRNA) expression, target protein expression, genetic loss, and silencing. MDS-T and MDS-AML patients show a reduction of the expression of miR-15a/-15b/-16 compared to MDS patients. Each miRNA can significantly predict MDS and MDS-T groups. Then, 79% of primary AMLs show a reduced expression of miR-15a and/or miR-15b. The expression of miR-15a/-15b/-16 significantly stratified AML patients in two prognostic classes. Furthermore, 40% of AML cell lines showed a combined loss of the expression of miR-15a/-15b and overexpression of their direct/indirect targets. As potential mechanisms involved in the silencing of the two miR-15/16 loci, we identified a genetic loss of miR-15a and miR-15b and silencing of these two loci by methylation. We identified a potential driver oncogenic role in the loss of expression of both miR-15/16 clusters in the progression of MDS into AML and in AML pathogenesis. The stratification of AML patients, based on miR-15/16 expression, can lead to targeted and combination therapies for the treatment of this incurable disease.
Asunto(s)
Leucemia Mieloide Aguda/genética , MicroARNs/genética , Anciano , Anciano de 80 o más Años , Estudios de Cohortes , Progresión de la Enfermedad , Femenino , Regulación Leucémica de la Expresión Génica , Humanos , Leucemia Mieloide Aguda/metabolismo , Leucemia Mieloide Aguda/patología , Masculino , MicroARNs/metabolismo , Persona de Mediana EdadRESUMEN
Unexplained blood cytopenias, in particular anemia, are often found in older persons. The relationship between these cytopenias and myeloid neoplasms like myelodysplastic syndromes is currently poorly defined. We studied a prospective cohort of patients with unexplained cytopenia with the aim to estimate the predictive value of somatic mutations for identifying subjects with, or at risk of, developing a myeloid neoplasm. The study included a learning cohort of 683 consecutive patients investigated for unexplained cytopenia, and a validation cohort of 190 patients referred for suspected myeloid neoplasm. Using granulocyte DNA, we looked for somatic mutations in 40 genes that are recurrently mutated in myeloid malignancies. Overall, 435/683 patients carried a somatic mutation in at least 1 of these genes. Carrying a somatic mutation with a variant allele frequency ≥0.10, or carrying 2 or more mutations, had a positive predictive value for diagnosis of myeloid neoplasm equal to 0.86 and 0.88, respectively. Spliceosome gene mutations and comutation patterns involving TET2, DNMT3A, or ASXL1 had positive predictive values for myeloid neoplasm ranging from 0.86 to 1.0. Within subjects with inconclusive diagnostic findings, carrying 1 or more somatic mutations was associated with a high probability of developing a myeloid neoplasm during follow-up (hazard ratio = 13.9, P < .001). The predictive values of mutation analysis were confirmed in the independent validation cohort. The findings of this study indicate that mutation analysis on peripheral blood granulocytes may significantly improve the current diagnostic approach to unexplained cytopenia and more generally the diagnostic accuracy of myeloid neoplasms.
Asunto(s)
Anemia/genética , Neoplasias Hematológicas/genética , Mutación , Pancitopenia/genética , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Análisis Mutacional de ADN , Femenino , Humanos , Leucemia Mieloide/genética , Masculino , Persona de Mediana Edad , Síndromes Mielodisplásicos/genética , Estudios Prospectivos , Adulto JovenAsunto(s)
Interferón Tipo I , Trastornos Mieloproliferativos , Neoplasias , Autoanticuerpos , HumanosRESUMEN
Chronic myelomonocytic leukemia (CMML) is a myelodysplastic/myeloproliferative neoplasm with variable clinical course. To predict the clinical outcome, we previously developed a CMML-specific prognostic scoring system (CPSS) based on clinical parameters and cytogenetics. In this work, we tested the hypothesis that accounting for gene mutations would further improve risk stratification of CMML patients. We therefore sequenced 38 genes to explore the role of somatic mutations in disease phenotype and clinical outcome. Overall, 199 of 214 (93%) CMML patients carried at least 1 somatic mutation. Stepwise linear regression models showed that these mutations accounted for 15% to 24% of variability of clinical phenotype. Based on multivariable Cox regression analyses, cytogenetic abnormalities and mutations in RUNX1, NRAS, SETBP1, and ASXL1 were independently associated with overall survival (OS). Using these parameters, we defined a genetic score that identified 4 categories with significantly different OS and cumulative incidence of leukemic evolution. In multivariable analyses, genetic score, red blood cell transfusion dependency, white blood cell count, and marrow blasts retained independent prognostic value. These parameters were included into a clinical/molecular CPSS (CPSS-Mol) model that identified 4 risk groups with markedly different median OS (from >144 to 18 months, hazard ratio [HR] = 2.69) and cumulative incidence of leukemic evolution (from 0% to 48% at 4 years, HR = 3.84) (P < .001). The CPSS-Mol fully retained its ability to risk stratify in an independent validation cohort of 260 CMML patients. In conclusion, integrating conventional parameters and gene mutations significantly improves risk stratification of CMML patients, providing a robust basis for clinical decision-making and a reliable tool for clinical trials.
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Biomarcadores de Tumor/genética , Aberraciones Cromosómicas , Leucemia Mielomonocítica Crónica/genética , Mutación/genética , Medición de Riesgo/métodos , Adulto , Anciano , Anciano de 80 o más Años , Toma de Decisiones Clínicas , Estudios de Cohortes , Femenino , Estudios de Seguimiento , Humanos , Leucemia Mielomonocítica Crónica/patología , Masculino , Persona de Mediana Edad , Clasificación del Tumor , Fenotipo , Pronóstico , Factores de Riesgo , Tasa de Supervivencia , Adulto JovenRESUMEN
Hereditary thrombocytosis (HT) is a rare inherited disorder with clinical features resembling those of sporadic essential thrombocythemia. This study included 933 patients with persistent isolated thrombocytosis for whom secondary reactive causes were excluded. Of 933 patients screened, 567 were JAK2-mutated, 255 CALR-mutated, 41 MPL-mutated, 2 double-mutated, and 68 were triple-negative. Two patients carried germline non-canonical mutations in exon 10: MPL W515* and MPL V501A. One triple-negative patient carried another germline non-canonical MPL mutation outside exon 10: MPL R102P. As germline MPL mutations may be underlying causes of HT, we recommend screening patients with triple-negative isolated thrombocytosis for non-canonical MPL mutations. Although clear evidence concerning HT treatment is still lacking, individuals with HT should probably be excluded from cytoreductive treatment. Thus, an accurate diagnosis is pivotal in avoiding unnecessary treatments.
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Receptores de Trombopoyetina , Trombocitosis , Humanos , Receptores de Trombopoyetina/genética , Receptores de Trombopoyetina/metabolismo , Calreticulina/genética , Trombocitosis/genética , Mutación , Janus Quinasa 2/genética , Células Germinativas/metabolismoRESUMEN
Alzheimer Disease (AD) is the most common neurodegenerative disorder worldwide, and account for 60% to 70% of all cases of progressive cognitive impairment in elderly patients. At the microscopic level distinctive features of AD are neurons and synapses degeneration, together with extensive amounts of senile plaques and neurofibrillars tangles. The degenerative process probably starts 20-30 years before the clinical onset of the disease. Senile plaques are composed of a central core of amyloid ß peptide, Aß, derived from the metabolism of the larger amyloid precursor protein, APP, which is expressed not only in the brain, but even in non neuronal tissues. More than 30 years ago, some studies reported that human platelets express APP and all the enzymatic activities necessary to process this protein through the same pathways described in the brain. Since then a large number of evidence has been accumulated to suggest that platelets may be a good peripheral model to study the metabolism of APP, and the pathophysiology of the onset of AD. In this review, we will summarize the current knowledge on the involvement of platelets in Alzheimer Disease. Although platelets are generally accepted as a suitable model for AD, the current scientific interest on this model is very high, because many concepts still remain debated and controversial. At the same time, however, these still unsolved divergences mirror a difficulty to establish constant parameters to better defined the role of platelets in AD.
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Approximately 20% of patients with myeloproliferative neoplasms (MPN) harbor mutations in the gene calreticulin (CALR), with 80% of those mutations classified as either type I or type II. While type II CALR-mutant proteins retain many of the Ca2+ binding sites present in the wild-type protein, type I CALR-mutant proteins lose these residues. The functional consequences of this differential loss of Ca2+ binding sites remain unexplored. Here, we show that the loss of Ca2+ binding residues in the type I mutant CALR protein directly impairs its Ca2+ binding ability, which in turn leads to depleted endoplasmic reticulum (ER) Ca2+ and subsequent activation of the IRE1α/XBP1 pathway of the unfolded protein response. Genetic or pharmacologic inhibition of IRE1α/XBP1 signaling induces cell death in type I mutant but not type II mutant or wild-type CALR-expressing cells, and abrogates type I mutant CALR-driven MPN disease progression in vivo. SIGNIFICANCE: Current targeted therapies for CALR-mutated MPNs are not curative and fail to differentiate between type I- versus type II-driven disease. To improve treatment strategies, it is critical to identify CALR mutation type-specific vulnerabilities. Here we show that IRE1α/XBP1 represents a unique, targetable dependency specific to type I CALR-mutated MPNs. This article is highlighted in the In This Issue feature, p. 265.
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Calreticulina , Trastornos Mieloproliferativos , Neoplasias , Respuesta de Proteína Desplegada , Calcio/metabolismo , Calreticulina/genética , Endorribonucleasas/genética , Humanos , Proteínas Mutantes/química , Mutación , Trastornos Mieloproliferativos/genética , Proteínas Serina-Treonina Quinasas/genética , Proteína 1 de Unión a la X-Box/genéticaRESUMEN
Somatic mutations in splicing factor genes frequently occur in myeloid neoplasms. While SF3B1 mutations are associated with myelodysplastic syndromes (MDS) with ring sideroblasts, SRSF2P95 mutations are found in different disease categories, including MDS, myeloproliferative neoplasms (MPN), myelodysplastic/myeloproliferative neoplasms (MDS/MPN), and acute myeloid leukemia (AML). To identify molecular determinants of this phenotypic heterogeneity, we explored molecular and clinical features of a prospective cohort of 279 SRSF2P95-mutated cases selected from a population of 2663 patients with myeloid neoplasms. Median number of somatic mutations per subject was 3. Multivariate regression analysis showed associations between co-mutated genes and clinical phenotype, including JAK2 or MPL with myelofibrosis (OR = 26.9); TET2 with monocytosis (OR = 5.2); RAS-pathway genes with leukocytosis (OR = 5.1); and STAG2, RUNX1, or IDH1/2 with blast phenotype (MDS or AML) (OR = 3.4, 1.9, and 2.1, respectively). Within patients with SRSF2-JAK2 co-mutation, JAK2 dominance was invariably associated with clinical feature of MPN, whereas SRSF2 mutation was dominant in MDS/MPN. Within patients with SRSF2-TET2 co-mutation, clinical expressivity of monocytosis was positively associated with co-mutated clone size. This study provides evidence that co-mutation pattern, clone size, and hierarchy concur to determine clinical phenotype, tracing relevant genotype-phenotype associations across disease entities and giving insight on unaccountable clinical heterogeneity within current WHO classification categories.
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Células Clonales/patología , Leucemia Mieloide Aguda/patología , Mutación , Síndromes Mielodisplásicos/patología , Enfermedades Mielodisplásicas-Mieloproliferativas/patología , Trastornos Mieloproliferativos/patología , Factores de Empalme Serina-Arginina/genética , Adulto , Anciano , Anciano de 80 o más Años , Biomarcadores de Tumor/genética , Células Clonales/metabolismo , Femenino , Estudios de Seguimiento , Estudios de Asociación Genética , Humanos , Leucemia Mieloide Aguda/genética , Masculino , Persona de Mediana Edad , Síndromes Mielodisplásicos/genética , Enfermedades Mielodisplásicas-Mieloproliferativas/genética , Trastornos Mieloproliferativos/genética , Pronóstico , Estudios Prospectivos , Tasa de SupervivenciaRESUMEN
The Rap family of small GTP-binding proteins is composed by four different members: Rap1A, Rap1B, Rap2A and Rap2B. In this work we report the identification and characterization of a fifth member of this family of small GTPases. This new protein is highly homologous to Rap2A and Rap2B, binds labeled GTP on nitrocellulose, and is recognized by a specific anti-Rap2 antibody, but not by an anti-Rap1 antibody. The protein has thus been named Rap2C. Binding of GTP to recombinant purified Rap2C was Mg(2+)-dependent. However, accurate comparison of the kinetics of nucleotide binding and release revealed that Rap2C bound GTP less efficiently and possessed slower rate of GDP release compared to the highly homologous Rap2B. Moreover, in the presence of Mg(2+), the relative affinity of Rap2C for GTP was only about twofold higher than that for GDP, while, under the same conditions, Rap2B was able to bind GTP with about sevenfold higher affinity than GDP. When expressed in eukaryotic cells, Rap2C localized at the plasma membrane, as dictated by the presence of a CAAX motif at the C-terminus. We found that Rap2C represented the predominant Rap2 protein expressed in circulating mononuclear leukocytes, but was not present in platelets. Importantly, Rap2C was found to be expressed in human megakaryocytes, suggesting that the protein may be down-regulated during platelets generation. This work demonstrates that Rap2C is a new member of the Rap2 subfamily of proteins, able to bind guanine nucleotides with peculiar properties, and differently expressed by various hematopoietic subsets. This new protein may therefore contribute to the still poorly clarified cellular events regulated by this subfamily of GTP-binding proteins.
Asunto(s)
Proteínas de Unión al GTP/genética , Proteínas de Unión al GTP/metabolismo , Proteínas de Unión al GTP rap/genética , Proteínas de Unión al GTP rap/metabolismo , Secuencia de Aminoácidos , Animales , Plaquetas/metabolismo , Etiquetas de Secuencia Expresada , Guanina/metabolismo , Humanos , Células Jurkat , Leucocitos Mononucleares/metabolismo , Megacariocitos/metabolismo , Ratones , Datos de Secuencia Molecular , Células 3T3 NIH , Unión Proteica , ARN Mensajero/metabolismo , Alineación de Secuencia , Factores de Tiempo , Proteínas de Unión al GTP rap1/genética , Proteínas de Unión al GTP rap1/metabolismo , Proteínas rasRESUMEN
PURPOSE: The genetic basis of myelodysplastic syndromes (MDS) is heterogeneous, and various combinations of somatic mutations are associated with different clinical phenotypes and outcomes. Whether the genetic basis of MDS influences the outcome of allogeneic hematopoietic stem-cell transplantation (HSCT) is unclear. PATIENTS AND METHODS: We studied 401 patients with MDS or acute myeloid leukemia (AML) evolving from MDS (MDS/AML). We used massively parallel sequencing to examine tumor samples collected before HSCT for somatic mutations in 34 recurrently mutated genes in myeloid neoplasms. We then analyzed the impact of mutations on the outcome of HSCT. RESULTS: Overall, 87% of patients carried one or more oncogenic mutations. Somatic mutations of ASXL1, RUNX1, and TP53 were independent predictors of relapse and overall survival after HSCT in both patients with MDS and patients with MDS/AML (P values ranging from .003 to .035). In patients with MDS/AML, gene ontology (ie, secondary-type AML carrying mutations in genes of RNA splicing machinery, TP53-mutated AML, or de novo AML) was an independent predictor of posttransplantation outcome (P = .013). The impact of ASXL1, RUNX1, and TP53 mutations on posttransplantation survival was independent of the revised International Prognostic Scoring System (IPSS-R). Combining somatic mutations and IPSS-R risk improved the ability to stratify patients by capturing more prognostic information at an individual level. Accounting for various combinations of IPSS-R risk and somatic mutations, the 5-year probability of survival after HSCT ranged from 0% to 73%. CONCLUSION: Somatic mutation in ASXL1, RUNX1, or TP53 is independently associated with unfavorable outcomes and shorter survival after allogeneic HSCT for patients with MDS and MDS/AML. Accounting for these genetic lesions may improve the prognostication precision in clinical practice and in designing clinical trials.
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Accumulation of amyloidogenic Aß peptides in the brain contributes to the onset of Alzheimer disease. Aß peptide deposits are also present in blood vessel walls, mainly deriving from circulating platelets. However, their effect on platelet function is unclear. We demonstrate that immobilized Aß peptides induce platelet adhesion and spreading through metalloproteinase-sensitive surface receptors. Aß peptides also fasten platelet spreading on collagen, and support the time- and ADP-dependent activation of adherent platelets, leading to stimulation of several signalling proteins. Our results indicate a potential role for peripheral Aß peptides in promoting platelet adhesion and activation in the initiation of thrombus formation.
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Péptidos beta-Amiloides/metabolismo , Activación Plaquetaria , Adhesividad Plaquetaria , Adenosina Difosfato/metabolismo , Enfermedad de Alzheimer/metabolismo , Vasos Sanguíneos/metabolismo , Adhesión Celular , Membrana Celular/metabolismo , Trastornos Cerebrovasculares/sangre , Trastornos Cerebrovasculares/metabolismo , Colágeno/metabolismo , Humanos , Proteínas Inmovilizadas/metabolismo , Metaloproteasas/metabolismo , Fosfatidilinositol 3-Quinasas/metabolismo , Proteína Quinasa C/metabolismo , Trombosis/metabolismoRESUMEN
We have recently shown that ADP-induced activation of protein kinase C (PKC) requires the co-stimulation of both P2Y1 and P2Y12 receptors. In this work, we show that inhibition of ADP-mediated phosphorylation of pleckstrin, the main PKC substrate, caused by antagonists of the P2Y12 receptor can be reversed by stimulation of the α2-adrenergic receptor by epinephrine. However, we also observed that addition of epinephrine alone caused a marked phosphorylation of pleckstrin. This effect occurred in the absence of Gq stimulation, as it was not associated to intracellular Ca2+ release. Epinephrine-induced pleckstrin phosphorylation was time- and dose-dependent, and was inhibited by the α2-adrenergic antagonist yohimbin. Phosphorylation of pleckstrin did not occur when platelet stimulation with epinephrine was performed in the presence of the ADP scavenger apyrase, and was suppressed by antagonists of both P2Y1 and P2Y12 ADP receptors. Importantly, no release of dense granules was measured in epinephrine-treated platelets. Addition of epinephrine to platelets was also able to stimulate Rap1b activation. Similarly to pleckstrin phosphorylation, however, this effect was prevented in the presence of apyrase or upon pharmacologic blockade of either P2Y1 or P2Y12 receptors. These results indicate that sub-threshold amounts of ADP in the medium are essential to allow epinephrine stimulation of α2-adrenergic receptor to elicit platelet responses, and reveal a novel synergism among strong stimulation of Gz and sub-threshold stimulation of both Gq and Gi, able to dissociate PKC activation from intracellular Ca2+ mobilisation.