RESUMEN
Somatic mutations in UBA1 cause vacuoles, E1 ubiquitin-activating enzyme, X-linked, autoinflammatory somatic (VEXAS) syndrome, an adult-onset inflammatory disease with an overlap of hematologic manifestations. VEXAS syndrome is characterized by a high mortality rate and significant clinical heterogeneity. We sought to determine independent predictors of survival in VEXAS and to understand the mechanistic basis for these factors. We analyzed 83 patients with somatic pathogenic variants in UBA1 at p.Met41 (p.Met41Leu/Thr/Val), the start codon for translation of the cytoplasmic isoform of UBA1 (UBA1b). Patients with the p.Met41Val genotype were most likely to have an undifferentiated inflammatory syndrome. Multivariate analysis showed ear chondritis was associated with increased survival, whereas transfusion dependence and the p.Met41Val variant were independently associated with decreased survival. Using in vitro models and patient-derived cells, we demonstrate that p.Met41Val variant supports less UBA1b translation than either p.Met41Leu or p.Met41Thr, providing a molecular rationale for decreased survival. In addition, we show that these 3 canonical VEXAS variants produce more UBA1b than any of the 6 other possible single-nucleotide variants within this codon. Finally, we report a patient, clinically diagnosed with VEXAS syndrome, with 2 novel mutations in UBA1 occurring in cis on the same allele. One mutation (c.121 A>T; p.Met41Leu) caused severely reduced translation of UBA1b in a reporter assay, but coexpression with the second mutation (c.119 G>C; p.Gly40Ala) rescued UBA1b levels to those of canonical mutations. We conclude that regulation of residual UBA1b translation is fundamental to the pathogenesis of VEXAS syndrome and contributes to disease prognosis.
Asunto(s)
Nucleótidos , Enzimas Activadoras de Ubiquitina , Codón Iniciador , Humanos , Mutación , Enzimas Activadoras de Ubiquitina/genética , UbiquitinaciónAsunto(s)
Proteína Antagonista del Receptor de Interleucina 1 , Linfohistiocitosis Hemofagocítica , Humanos , Linfohistiocitosis Hemofagocítica/tratamiento farmacológico , Linfohistiocitosis Hemofagocítica/etiología , Linfohistiocitosis Hemofagocítica/diagnóstico , Linfohistiocitosis Hemofagocítica/complicaciones , Proteína Antagonista del Receptor de Interleucina 1/uso terapéutico , Proteína Antagonista del Receptor de Interleucina 1/efectos adversos , Masculino , Femenino , Resultado del Tratamiento , Persona de Mediana Edad , Adulto , Anciano , Leucemia/tratamiento farmacológico , Leucemia/complicacionesAsunto(s)
Síndromes Mielodisplásicos/terapia , Adulto , Anemia/complicaciones , Anemia/terapia , Antineoplásicos/uso terapéutico , Azacitidina/uso terapéutico , Transfusión Sanguínea/métodos , Decitabina/uso terapéutico , Manejo de la Enfermedad , Hematínicos/uso terapéutico , Trasplante de Células Madre Hematopoyéticas/métodos , Hemorragia/complicaciones , Hemorragia/terapia , Humanos , Quelantes del Hierro/uso terapéutico , Síndromes Mielodisplásicos/complicaciones , Neutropenia/complicaciones , Neutropenia/terapia , Trombocitopenia/complicaciones , Trombocitopenia/terapiaAsunto(s)
Antineoplásicos/uso terapéutico , Linfoma/tratamiento farmacológico , Mieloma Múltiple/tratamiento farmacológico , Adulto , Anciano , Anciano de 80 o más Años , Antineoplásicos/efectos adversos , Femenino , Humanos , Estimación de Kaplan-Meier , Londres/epidemiología , Linfoma/mortalidad , Masculino , Persona de Mediana Edad , Mieloma Múltiple/mortalidad , Pronóstico , Resultado del Tratamiento , Privación de Tratamiento/estadística & datos numéricosRESUMEN
Haploinsufficiency of ribosomal proteins (RPs) and upregulation of the tumour suppressor TP53 have been shown to be the common basis for the anaemia observed in Diamond Blackfan anaemia and 5q- myelodysplastic syndrome. We previously demonstrated that treatment with L-Leucine resulted in a marked improvement in anaemia in disease models. To determine if the L-Leucine effect was Tp53-dependent, we used antisense MOs to rps19 and rps14 in zebrafish; expression of tp53 and its downstream target cdkn1a remained elevated following L-leucine treatment. We confirmed this observation in human CD34+ cells. L-Leucine thus alleviates anaemia in RP-deficient cells in a TP53-independent manner.
Asunto(s)
Anemia de Diamond-Blackfan/tratamiento farmacológico , Anemia Macrocítica/tratamiento farmacológico , Proteína p53 Supresora de Tumor/metabolismo , Proteínas de Pez Cebra/metabolismo , Anemia de Diamond-Blackfan/genética , Anemia de Diamond-Blackfan/metabolismo , Anemia de Diamond-Blackfan/patología , Anemia Macrocítica/genética , Anemia Macrocítica/metabolismo , Anemia Macrocítica/patología , Animales , Deleción Cromosómica , Cromosomas Humanos Par 5/genética , Cromosomas Humanos Par 5/metabolismo , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/genética , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/metabolismo , Modelos Animales de Enfermedad , Humanos , Leucina , Proteínas Ribosómicas/genética , Proteínas Ribosómicas/metabolismo , Proteína p53 Supresora de Tumor/genética , Proteínas de Pez Cebra/genéticaRESUMEN
Haploinsufficiency of ribosomal proteins (RPs) has been proposed to be the common basis for the anemia observed in Diamond-Blackfan anemia (DBA) and myelodysplastic syndrome with loss of chromosome 5q [del(5q) MDS]. We have modeled DBA and del(5q) MDS in zebrafish using antisense morpholinos to rps19 and rps14, respectively, and have demonstrated that, as in humans, haploinsufficient levels of these proteins lead to a profound anemia. To address the hypothesis that RP loss results in impaired mRNA translation, we treated Rps19 and Rps14-deficient embryos with the amino acid L-leucine, a known activator of mRNA translation. This resulted in a striking improvement of the anemia associated with RP loss. We confirmed our findings in primary human CD34⺠cells, after shRNA knockdown of RPS19 and RPS14. Furthermore, we showed that loss of Rps19 or Rps14 activates the mTOR pathway, and this is accentuated by L-leucine in both Rps19 and Rps14 morphants. This effect could be abrogated by rapamycin suggesting that mTOR signaling may be responsible for the improvement in anemia associated with L-leucine. Our studies support the rationale for ongoing clinical trials of L-leucine as a therapeutic agent for DBA, and potentially for patients with del(5q) MDS.
Asunto(s)
Anemia de Diamond-Blackfan/tratamiento farmacológico , Desarrollo Embrionario/efectos de los fármacos , Leucina/uso terapéutico , Síndromes Mielodisplásicos/tratamiento farmacológico , Transducción de Señal/efectos de los fármacos , Serina-Treonina Quinasas TOR/metabolismo , Regulación hacia Arriba/efectos de los fármacos , Anemia de Diamond-Blackfan/sangre , Anemia de Diamond-Blackfan/embriología , Anemia de Diamond-Blackfan/metabolismo , Anemia Macrocítica/tratamiento farmacológico , Anemia Macrocítica/metabolismo , Animales , Animales Modificados Genéticamente , Células Cultivadas , Deleción Cromosómica , Cromosomas Humanos Par 5/metabolismo , Modelos Animales de Enfermedad , Embrión no Mamífero/efectos de los fármacos , Hematínicos/farmacología , Hematínicos/uso terapéutico , Hematopoyesis/efectos de los fármacos , Células Madre Hematopoyéticas/efectos de los fármacos , Células Madre Hematopoyéticas/inmunología , Células Madre Hematopoyéticas/metabolismo , Células Madre Hematopoyéticas/patología , Humanos , Leucina/farmacología , Síndromes Mielodisplásicos/embriología , Síndromes Mielodisplásicos/genética , Síndromes Mielodisplásicos/metabolismo , ARN Interferente Pequeño , Proteínas Ribosómicas/antagonistas & inhibidores , Serina-Treonina Quinasas TOR/antagonistas & inhibidores , Pez Cebra , Proteínas de Pez Cebra/antagonistas & inhibidoresRESUMEN
A comprehensive understanding of the genes and pathways regulating hematopoiesis is needed to identify genes causally related to bone marrow failure syndromes, myelodysplastic syndromes, and hematopoietic neoplasms. To identify novel genes involved in hematopoiesis, we performed an ethyl-nitrosourea mutagenesis screen in zebrafish (Danio rerio) to search for mutants with defective definitive hematopoiesis. We report the recovery and analysis of the grechetto mutant, which harbors an inactivating mutation in cleavage and polyadenylation specificity factor 1 (cpsf1), a gene ubiquitously expressed and required for 3' untranslated region processing of a subset of pre-mRNAs. grechetto mutants undergo normal primitive hematopoiesis and specify appropriate numbers of definitive HSCs at 36 hours postfertilization. However, when HSCs migrate to the caudal hematopoietic tissue at 3 days postfertilization, their numbers start decreasing as a result of apoptotic cell death. Consistent with Cpsf1 function, c-myb:EGFP(+) cells in grechetto mutants also show defective polyadenylation of snrnp70, a gene required for HSC development. By 5 days postfertilization, definitive hematopoiesis is compromised and severely decreased blood cell numbers are observed across the myeloid, erythroid, and lymphoid cell lineages. These studies show that cpsf1 is essential for HSC survival and differentiation in caudal hematopoietic tissue.
Asunto(s)
Factor de Especificidad de Desdoblamiento y Poliadenilación/genética , Hematopoyesis/genética , Células Madre Hematopoyéticas/citología , Células Madre Hematopoyéticas/fisiología , Animales , Diferenciación Celular/fisiología , Supervivencia Celular/fisiología , Regulación del Desarrollo de la Expresión Génica/fisiología , Masculino , Mutagénesis/fisiología , Fenotipo , Pez CebraRESUMEN
The regulation of blood vessel formation is of fundamental importance to many physiological processes, and angiogenesis is a major area for novel therapeutic approaches to diseases from ischemia to cancer. A poorly understood clinical manifestation of pathological angiogenesis is angiodysplasia, vascular malformations that cause severe gastrointestinal bleeding. Angiodysplasia can be associated with von Willebrand disease (VWD), the most common bleeding disorder in man. VWD is caused by a defect or deficiency in von Willebrand factor (VWF), a glycoprotein essential for normal hemostasis that is involved in inflammation. We hypothesized that VWF regulates angiogenesis. Inhibition of VWF expression by short interfering RNA (siRNA) in endothelial cells (ECs) caused increased in vitro angiogenesis and increased vascular endothelial growth factor (VEGF) receptor-2 (VEGFR-2)-dependent proliferation and migration, coupled to decreased integrin αvß3 levels and increased angiopoietin (Ang)-2 release. ECs expanded from blood-derived endothelial progenitor cells of VWD patients confirmed these results. Finally, 2 different approaches, in situ and in vivo, showed increased vascularization in VWF-deficient mice. We therefore identify a new function of VWF in ECs, which confirms VWF as a protein with multiple vascular roles and defines a novel link between hemostasis and angiogenesis. These results may have important consequences for the management of VWD, with potential therapeutic implications for vascular diseases.
Asunto(s)
Células Endoteliales/metabolismo , Neovascularización Fisiológica , Factor de von Willebrand/metabolismo , Adulto , Anciano de 80 o más Años , Angiopoyetina 2/genética , Angiopoyetina 2/metabolismo , Animales , Línea Celular , Movimiento Celular , Proliferación Celular , Células Endoteliales/citología , Femenino , Hemostasis , Humanos , Immunoblotting , Integrina alfaVbeta3/genética , Integrina alfaVbeta3/metabolismo , Masculino , Ratones , Ratones Noqueados , Persona de Mediana Edad , Neovascularización Patológica , Interferencia de ARN , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Receptor 2 de Factores de Crecimiento Endotelial Vascular/genética , Receptor 2 de Factores de Crecimiento Endotelial Vascular/metabolismo , Enfermedades de von Willebrand/genética , Enfermedades de von Willebrand/metabolismo , Enfermedades de von Willebrand/patología , Factor de von Willebrand/genéticaRESUMEN
In a zebrafish mutagenesis screen to identify genes essential for myelopoiesis, we identified an insertional allele hi1727, which disrupts the gene encoding RNA helicase dead-box 18 (Ddx18). Homozygous Ddx18 mutant embryos exhibit a profound loss of myeloid and erythroid cells along with cardiovascular abnormalities and reduced size. These mutants also display prominent apoptosis and a G1 cell-cycle arrest. Loss of p53, but not Bcl-xl overexpression, rescues myeloid cells to normal levels, suggesting that the hematopoietic defect is because of p53-dependent G1 cell-cycle arrest. We then sequenced primary samples from 262 patients with myeloid malignancies because genes essential for myelopoiesis are often mutated in human leukemias. We identified 4 nonsynonymous sequence variants (NSVs) of DDX18 in acute myeloid leukemia (AML) patient samples. RNA encoding wild-type DDX18 and 3 NSVs rescued the hematopoietic defect, indicating normal DDX18 activity. RNA encoding one mutation, DDX18-E76del, was unable to rescue hematopoiesis, and resulted in reduced myeloid cell numbers in ddx18(hi1727/+) embryos, indicating this NSV likely functions as a dominant-negative allele. These studies demonstrate the use of the zebrafish as a robust in vivo system for assessing the function of genes mutated in AML, which will become increasingly important as more sequence variants are identified by next-generation resequencing technologies.
Asunto(s)
Ciclo Celular/genética , ARN Helicasas DEAD-box/genética , ARN Helicasas DEAD-box/metabolismo , Hematopoyesis/genética , Células Madre Hematopoyéticas/metabolismo , Leucemia Mieloide Aguda/genética , Proteínas de Pez Cebra/metabolismo , Pez Cebra/metabolismo , Alelos , Animales , Western Blotting , Separación Celular , Embrión no Mamífero , Citometría de Flujo , Células Madre Hematopoyéticas/citología , Humanos , Hibridación in Situ , Mutagénesis Sitio-Dirigida , Mutación , Células Mieloides/citología , Células Mieloides/metabolismo , Reacción en Cadena de la Polimerasa , Proteínas de Pez Cebra/genéticaRESUMEN
Mutations in the human nucleophosmin (NPM1) gene are the most frequent genetic alteration in adult acute myeloid leukemias (AMLs) and result in aberrant cytoplasmic translocation of this nucleolar phosphoprotein (NPMc+). However, underlying mechanisms leading to leukemogenesis remain unknown. To address this issue, we took advantage of the zebrafish model organism, which expresses 2 genes orthologous to human NPM1, referred to as npm1a and npm1b. Both genes are ubiquitously expressed, and their knockdown produces a reduction in myeloid cell numbers that is specifically rescued by NPM1 expression. In zebrafish, wild-type human NPM1 is nucleolar while NPMc+ is cytoplasmic, as in human AML, and both interact with endogenous zebrafish Npm1a and Npm1b. Forced NPMc+ expression in zebrafish causes an increase in pu.1(+) primitive early myeloid cells. A more marked perturbation of myelopoiesis occurs in p53(m/m) embryos expressing NPMc+, where mpx(+) and csf1r(+) cell numbers are also expanded. Importantly, NPMc+ expression results in increased numbers of definitive hematopoietic cells, including erythromyeloid progenitors in the posterior blood island and c-myb/cd41(+) cells in the ventral wall of the aorta. These results are likely to be relevant to human NPMc+ AML, where the observed NPMc+ multilineage expression pattern implies transformation of a multipotent stem or progenitor cell.
Asunto(s)
Hematopoyesis/genética , Leucemia Mieloide Aguda/genética , Células Mieloides/fisiología , Proteínas Nucleares/genética , Animales , Apoptosis/genética , Secuencia de Bases , Western Blotting , Separación Celular , Citoplasma/metabolismo , Embrión no Mamífero , Citometría de Flujo , Técnica del Anticuerpo Fluorescente , Células Madre Hematopoyéticas/fisiología , Humanos , Inmunoprecipitación , Leucemia Mieloide Aguda/metabolismo , Datos de Secuencia Molecular , Mutación , Proteínas Nucleares/metabolismo , Nucleofosmina , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Homología de Secuencia de Ácido Nucleico , Pez CebraRESUMEN
Fundamental life science and pharmaceutical research are continually striving to provide physiologically relevant context for their biological studies. Zebrafish present an opportunity for high-content screening (HCS) to bring a true in vivo model system to screening studies. Zebrafish embryos and young larvae are an economical, human-relevant model organism that are amenable to both genetic engineering and modification, and direct inspection via microscopy. The use of these organisms entails unique challenges that new technologies are overcoming, including artificial intelligence (AI). In this perspective article, we describe the state-of-the-art in terms of automated sample handling, imaging, and data analysis with zebrafish during early developmental stages. We highlight advances in orienting the embryos, including the use of robots, microfluidics, and creative multi-well plate solutions. Analyzing the micrographs in a fast, reliable fashion that maintains the anatomical context of the fluorescently labeled cells is a crucial step. Existing software solutions range from AI-driven commercial solutions to bespoke analysis algorithms. Deep learning appears to be a critical tool that researchers are only beginning to apply, but already facilitates many automated steps in the experimental workflow. Currently, such work has permitted the cellular quantification of multiple cell types in vivo, including stem cell responses to stress and drugs, neuronal myelination and macrophage behavior during inflammation and infection. We evaluate pro and cons of proprietary versus open-source methodologies for combining technologies into fully automated workflows of zebrafish studies. Zebrafish are poised to charge into HCS with ever-greater presence, bringing a new level of physiological context.
Asunto(s)
Inteligencia Artificial , Pez Cebra , Algoritmos , Animales , Programas Informáticos , Flujo de Trabajo , Pez Cebra/fisiologíaRESUMEN
Germline loss or mutation of one copy of the transcription factor GATA2 in humans leads to a range of clinical phenotypes affecting hematopoietic, lymphatic and vascular systems. GATA2 heterozygous mice show only a limited repertoire of the features observed in humans. Zebrafish have two copies of the Gata2 gene as a result of an additional round of ancestral whole genome duplication. These genes, Gata2a and Gata2b, show distinct but overlapping expression patterns, and between them, highlight a significantly broader range of the phenotypes observed in GATA2 deficient syndromes, than each one alone. In this manuscript, we use mutants for Gata2a and Gata2b to interrogate the effects on hematopoiesis of these two ohnologs, alone and in combination, during development in order to further define the role of GATA2 in developmental hematopoiesis. We define unique roles for each ohnolog at different stages of developmental myelopoiesis and for the emergence of hematopoietic stem and progenitor cells. These effects are not additive in the haploinsufficient state suggesting a redundancy between these two genes in hematopoietic stem and progenitor cells. Rescue studies additionally support that Gata2b can compensate for the effects of Gata2a loss. Finally we show that adults with loss of combined heterozygosity show defects in the myeloid compartment consistent with GATA2 loss in humans. These results build on existing knowledge from other models of GATA2 deficiency and refine our understanding of the early developmental effects of GATA2. In addition, these studies shed light on the complexity and potential structure-function relationships as well as sub-functionalization of Gata2 genes in the zebrafish model.
RESUMEN
Myelodysplastic syndrome (MDS) is a hematological malignancy characterized by blood cytopenias and predisposition to acute myeloid leukemia (AML). Therapies for MDS are lacking, particularly those that have an impact in the early stages of disease. We developed a model of MDS in zebrafish with knockout of Rps14, the primary mediator of the anemia associated with del(5q) MDS. These mutant animals display dose- and age-dependent abnormalities in hematopoiesis, culminating in bone marrow failure with dysplastic features. We used Rps14 knockdown to undertake an in vivo small-molecule screening, to identify compounds that ameliorate the MDS phenotype, and we identified imiquimod, an agonist of Toll-like receptor-7 (TLR7) and TLR8. Imiquimod alleviates anemia by promoting hematopoietic stem and progenitor cell expansion and erythroid differentiation, the mechanism of which is dependent on TLR7 ligation and Myd88. TLR7 activation in this setting paradoxically promoted an anti-inflammatory gene signature, indicating cross talk via TLR7 between proinflammatory pathways endogenous to Rps14 loss and the NF-κB pathway. Finally, in highly purified human bone marrow samples from anemic patients, imiquimod led to an increase in erythroid output from myeloerythroid progenitors and common myeloid progenitors. Our findings have both specific implications for the development of targeted therapeutics for del(5q) MDS and wider significance identifying a potential role for TLR7 ligation in modifying anemia.
Asunto(s)
Síndromes Mielodisplásicos , Pez Cebra , Animales , Hematopoyesis , Humanos , Síndromes Mielodisplásicos/genética , Transducción de Señal , Receptor Toll-Like 7/genéticaRESUMEN
The inclusion of familial myeloid malignancies as a separate disease entity in the revised WHO classification has renewed efforts to improve the recognition and management of this group of at risk individuals. Here we report a cohort of 86 acute myeloid leukemia (AML) and myelodysplastic syndrome (MDS) families with 49 harboring germline variants in 16 previously defined loci (57%). Whole exome sequencing in a further 37 uncharacterized families (43%) allowed us to rationalize 65 new candidate loci, including genes mutated in rare hematological syndromes (ADA, GP6, IL17RA, PRF1 and SEC23B), reported in prior MDS/AML or inherited bone marrow failure series (DNAH9, NAPRT1 and SH2B3) or variants at novel loci (DHX34) that appear specific to inherited forms of myeloid malignancies. Altogether, our series of MDS/AML families offer novel insights into the etiology of myeloid malignancies and provide a framework to prioritize variants for inclusion into routine diagnostics and patient management.
Asunto(s)
Mutación de Línea Germinal , Leucemia Mieloide Aguda/genética , Síndromes Mielodisplásicos/genética , Proteínas Adaptadoras Transductoras de Señales/genética , Adenosina Desaminasa/genética , Dineínas Axonemales/genética , Estudios de Cohortes , Humanos , Degradación de ARNm Mediada por Codón sin Sentido , Linaje , Perforina/genética , Glicoproteínas de Membrana Plaquetaria/genética , ARN Helicasas/genética , Receptores de Interleucina-17/genética , Proteínas de Transporte Vesicular/genética , Secuenciación del ExomaRESUMEN
Recombinant activated factor VII (rFVIIa /Novoseven) has been used in a wide variety of circumstances as a treatment for uncontrolled bleeding. We present a retrospective report of the use of rFVIIa in 40 consecutive patients without inherited bleeding disorders in a single centre. Twenty-one (68%) of the 31 patients whose response to rFVIIa was documented showed a reduction or cessation in bleeding; in nine patients (29%) bleeding was unchanged and in one patient (3%) bleeding increased despite rFVIIa. One person suffered a thrombotic stroke after rFVIIa treatment. There were no other adverse events directly attributable to rFVIIa. Twenty-four patients (60%) died during the hospital admission in which the rFVIIa was administered. Twelve patients (30%) who received rFVIIa had bleeding secondary to haematological malignancy and 21 patients (53%) had bleeding complicating a surgical procedure. There were 11 deaths (92%) in the haematological malignancy group and 10 deaths (48%) in the surgical group. Patients with haematological malignancy received a significantly greater median number of doses of rFVIIa than patients with surgical bleeding complications (three versus one dose, P = 0.04). We conclude that rFVIIa can be used safely in uncontrolled haemorrhage and the majority of patients show a response.