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1.
Pediatr Blood Cancer ; : e28748, 2020 Oct 06.
Artigo em Inglês | MEDLINE | ID: mdl-33025707

RESUMO

BACKGROUND: Diamond-Blackfan anemia (DBA) is an inherited bone marrow failure syndrome characterized by anemia, short stature, congenital anomalies, and cancer predisposition. Most cases are due to mutations in genes encoding ribosomal proteins (RP) leading to RP haploinsufficiency. Effective treatments for the anemia of DBA include chronic red cell transfusions, long-term corticosteroid therapy, or hematopoietic stem cell transplantation. In a small patient series and in animal models, there have been hematologic responses to L-leucine with amelioration of anemia. The study objectives of this clinical trial were to determine feasibility, safety, and efficacy of L-leucine in transfusion-dependent patients with DBA. PROCEDURE: Patients ≥2 years of age received L-leucine 700 mg/m2 orally three times daily for nine months to determine a hematologic response and any improvement in growth (NCT01362595). RESULTS: This multicenter, phase I/II study enrolled 55 subjects; 43 were evaluable. There were 21 males; the median age at enrollment was 10.4 years (range, 2.5-46.1 years). No significant adverse events were attributable to L-leucine. Two subjects had a complete erythroid response and five had a partial response. Nine of 25, and 11 of 25, subjects experienced a positive weight and height percentile change, respectively, at the end of therapy. CONCLUSIONS: L-leucine is safe, resulted in an erythroid response in 16% of subjects with DBA, and led to an increase in weight and linear growth velocity in 36% and 44% of evaluable subjects, respectively. Further studies will be critical to understand the role of L-leucine in the management of patients with DBA.

2.
Haematologica ; 105(9): 2229-2239, 2020 Jun 05.
Artigo em Inglês | MEDLINE | ID: mdl-33054048

RESUMO

Pyruvate kinase deficiency (PKD) is the most common cause of chronic hereditary non-spherocytic hemolytic anemia and results in a broad spectrum of disease. The diagnosis of PKD requires a high index of suspicion and judicious use of laboratory tests that may not always be informative, including pyruvate kinase enzyme assay and genetic analysis of the PKLR gene. A significant minority of patients with PKD have occult mutations in non-coding regions of PKLR which are missed on standard genetic tests. The biochemical consequences of PKD result in hemolytic anemia due to red cell pyruvate and ATP deficiency while simultaneously causing increased red cell 2,3-diphosphoglycerate, which facilitates oxygen unloading. This phenomenon, in addition to numerous other factors such as genetic background and differences in splenic function result in a poor correlation between symptoms and degree of anemia from patient to patient. Red cell transfusions should, therefore, be symptom-directed and not based on a hemoglobin threshold. Patients may experience specific complications, such as paravertebral extramedullary hematopoiesis and chronic debilitating icterus, which require personalized treatment. The decision to perform splenectomy or hematopoietic stem cell transplantation is nuanced and depends on disease burden and long-term outlook given that targeted therapeutics are in development. In recognition of the complicated nature of the disease and its management and the limitations of the PKD literature, an international working group of ten PKD experts convened to better define the disease burden and manifestations. This article summarizes the conclusions of this working group and is a guide for clinicians and investigators caring for patients with PKD.

4.
Br J Haematol ; 2020 May 28.
Artigo em Inglês | MEDLINE | ID: mdl-32463523

RESUMO

Diagnosis of pyruvate kinase deficiency (PKD), the most common cause of hereditary non-spherocytic haemolytic anaemia, remains challenging in routine practice and no biomarkers for clinical severity have been characterised. This prospective study enrolled 41 patients with molecularly confirmed PKD from nine North American centres to evaluate the diagnostic sensitivity of pyruvate kinase (PK) enzyme activity and PK:hexokinase (HK) enzyme activity ratio, and evaluate the erythrocyte PK (PK-R) protein level and erythrocyte metabolites as biomarkers for clinical severity. In this population not transfused for ≥90 days before sampling, the diagnostic sensitivity of the PK enzyme assay was 90% [95% confidence interval (CI) 77-97%], whereas the PK:HK ratio sensitivity was 98% (95% CI 87-100%). There was no correlation between PK enzyme activity and clinical severity. Transfusion requirements correlated with normalised erythrocyte ATP levels (r = 0·527, P = 0·0016) and PK-R protein levels (r = -0·527, P = 0·0028). PK-R protein levels were significantly higher in the never transfused [median (range) 40·1 (9·8-73·9)%] versus ever transfused [median (range) 7·7 (0·4-15·1)%] patients (P = 0·0014). The PK:HK ratio had excellent sensitivity for PK diagnosis, superior to PKLR exon sequencing. Given that the number of PKLR variants and genotype combinations limits prognostication based on molecular findings, PK-R protein level may be a useful prognostic biomarker of disease severity and merits further study.

5.
Haematologica ; 2020 Mar 12.
Artigo em Inglês | MEDLINE | ID: mdl-32165483

RESUMO

Pyruvate kinase deficiency (PKD) is the most common cause of chronic hereditary nonspherocytic hemolytic anemia and results in a broad spectrum of disease. Diagnosis of PKD requires a high index of suspicion and judicious use of laboratory tests that may not always be informative, including PK enzyme assay and genetic analysis of the PKLR gene. A significant minority of patients with PKD have occult mutations in non-coding regions of PKLR that are missed on standard genetic tests. The biochemical consequences of PKD result in hemolytic anemia due to red cell pyruvate and ATP deficiency while simultaneously causing increased red cell 2,3-diphosphoglycerate, which facilitates oxygen unloading. This phenomenon, in addition to numerous other factors such as genetic background and differences in splenic function result in a poor correlation between symptoms and degree of anemia from patient to patient. Therefore, red cell transfusions should be symptom-directed and not based on a hemoglobin threshold. Patients may experience specific complications, such as paravertebral extramedullary hematopoiesis and chronic debilitating icterus, that require personalized treatment. The decision to perform splenectomy or hematopoietic stem cell transplantation is nuanced and depends on disease burden and long-term outlook given that targeted therapeutics are in development. In recognition of the complicated nature of the disease and its management and the limitations of the PKD literature, an international working group of 10 PKD experts convened to better define the burden and manifestations of disease. This manuscript summarizes the conclusions of this working group and is a guide for clinicians and investigators caring for patients with PKD.

6.
Am J Hematol ; 95(5): 472-482, 2020 05.
Artigo em Inglês | MEDLINE | ID: mdl-32043619

RESUMO

Pyruvate kinase (PK) deficiency is a rare recessive congenital hemolytic anemia caused by mutations in the PKLR gene. This study reports the molecular features of 257 patients enrolled in the PKD Natural History Study. Of the 127 different pathogenic variants detected, 84 were missense and 43 non-missense, including 20 stop-gain, 11 affecting splicing, five large deletions, four in-frame indels, and three promoter variants. Within the 177 unrelated patients, 35 were homozygous and 142 compound heterozygous (77 for two missense, 48 for one missense and one non-missense, and 17 for two non-missense variants); the two most frequent mutations were p.R510Q in 23% and p.R486W in 9% of mutated alleles. Fifty-five (21%) patients were found to have at least one previously unreported variant with 45 newly described mutations. Patients with two non-missense mutations had lower hemoglobin levels, higher numbers of lifetime transfusions, and higher rates of complications including iron overload, extramedullary hematopoiesis, and pulmonary hypertension. Rare severe complications, including lower extremity ulcerations and hepatic failure, were seen more frequently in patients with non-missense mutations or with missense mutations characterized by severe protein instability. The PKLR genotype did not correlate with the frequency of complications in utero or in the newborn period. With ICCs ranging from 0.4 to 0.61, about the same degree of clinical similarity exists within siblings as it does between siblings, in terms of hemoglobin, total bilirubin, splenectomy status, and cholecystectomy status. Pregnancy outcomes were similar across genotypes in PK deficient women. This report confirms the wide genetic heterogeneity of PK deficiency.


Assuntos
Anemia Hemolítica Congênita não Esferocítica/genética , Estudos de Associação Genética/métodos , Piruvato Quinase/deficiência , Erros Inatos do Metabolismo dos Piruvatos/genética , Adolescente , Adulto , Criança , Pré-Escolar , Feminino , Humanos , Lactente , Recém-Nascido , Masculino , Pessoa de Meia-Idade , Piruvato Quinase/genética , Adulto Jovem
7.
N Engl J Med ; 381(10): 933-944, 2019 09 05.
Artigo em Inglês | MEDLINE | ID: mdl-31483964

RESUMO

BACKGROUND: Pyruvate kinase deficiency is caused by mutations in PKLR and leads to congenital hemolytic anemia. Mitapivat is an oral, small-molecule allosteric activator of pyruvate kinase in red cells. METHODS: In this uncontrolled, phase 2 study, we evaluated the safety and efficacy of mitapivat in 52 adults with pyruvate kinase deficiency who were not receiving red-cell transfusions. The patients were randomly assigned to receive either 50 mg or 300 mg of mitapivat twice daily for a 24-week core period; eligible patients could continue treatment in an ongoing extension phase. RESULTS: Common adverse events, including headache and insomnia, occurred at the time of drug initiation and were transient; 92% of the episodes of headache and 47% of the episodes of insomnia resolved within 7 days. The most common serious adverse events, hemolytic anemia and pharyngitis, each occurred in 2 patients (4%). A total of 26 patients (50%) had an increase of more than 1.0 g per deciliter in the hemoglobin level. Among these patients, the mean maximum increase was 3.4 g per deciliter (range, 1.1 to 5.8), and the median time until the first increase of more than 1.0 g per deciliter was 10 days (range, 7 to 187); 20 patients (77%) had an increase of more than 1.0 g per deciliter in the hemoglobin level at more than 50% of visits during the core study period, with improvement in markers of hemolysis. The response was sustained in all 19 patients remaining in the extension phase, with a median follow-up of 29 months (range, 22 to 35). Hemoglobin responses were observed only in patients who had at least one missense PKLR mutation and were associated with the red-cell pyruvate kinase protein level at baseline. CONCLUSIONS: The administration of mitapivat was associated with a rapid increase in the hemoglobin level in 50% of adults with pyruvate kinase deficiency, with a sustained response during a median follow-up of 29 months during the extension phase. Adverse effects were mainly low-grade and transient. (Funded by Agios Pharmaceuticals; ClinicalTrials.gov number, NCT02476916.).


Assuntos
Anemia Hemolítica Congênita não Esferocítica/tratamento farmacológico , Hemoglobinas/metabolismo , Piperazinas/administração & dosagem , Piruvato Quinase/deficiência , Erros Inatos do Metabolismo dos Piruvatos/tratamento farmacológico , Quinolinas/administração & dosagem , Administração Oral , Adolescente , Adulto , Anemia Hemolítica Congênita não Esferocítica/sangue , Anemia Hemolítica Congênita não Esferocítica/genética , Catecóis , Esquema de Medicação , Feminino , Seguimentos , Cefaleia/induzido quimicamente , Humanos , Masculino , Mutação , Piperazinas/efeitos adversos , Piruvato Quinase/sangue , Piruvato Quinase/genética , Erros Inatos do Metabolismo dos Piruvatos/sangue , Erros Inatos do Metabolismo dos Piruvatos/genética , Quinolinas/efeitos adversos , Distúrbios do Início e da Manutenção do Sono/induzido quimicamente , Tirfostinas , Adulto Jovem
8.
Blood Adv ; 3(18): 2751-2763, 2019 09 24.
Artigo em Inglês | MEDLINE | ID: mdl-31540902

RESUMO

The del(5q) myelodysplastic syndrome (MDS) is a distinct subtype of MDS, associated with deletion of the ribosomal protein S14 (RPS14) gene that results in macrocytic anemia. This study sought to identify novel targets for the treatment of patients with del(5q) MDS by performing an in vivo drug screen using an rps14-deficient zebrafish model. From this, we identified the secreted gelatinase matrix metalloproteinase 9 (MMP9). MMP9 inhibitors significantly improved the erythroid defect in rps14-deficient zebrafish. Similarly, treatment with MMP9 inhibitors increased the number of colony forming unit-erythroid colonies and the CD71+ erythroid population from RPS14 knockdown human BMCD34+ cells. Importantly, we found that MMP9 expression is upregulated in RPS14-deficient cells by monocyte chemoattractant protein 1. Double knockdown of MMP9 and RPS14 increased the CD71+ population compared with RPS14 single knockdown, suggesting that increased expression of MMP9 contributes to the erythroid defect observed in RPS14-deficient cells. In addition, transforming growth factor ß (TGF-ß) signaling is activated in RPS14 knockdown cells, and treatment with SB431542, a TGF-ß inhibitor, improved the defective erythroid development of RPS14-deficient models. We found that recombinant MMP9 treatment decreases the CD71+ population through increased SMAD2/3 phosphorylation, suggesting that MMP9 directly activates TGF-ß signaling in RPS14-deficient cells. Finally, we confirmed that MMP9 inhibitors reduce SMAD2/3 phosphorylation in RPS14-deficient cells to rescue the erythroid defect. In summary, these study results support a novel role for MMP9 in the pathogenesis of del(5q) MDS and the potential for the clinical use of MMP9 inhibitors in the treatment of patients with del(5q) MDS.

9.
Haematologica ; 104(10): 1974-1983, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-30948484

RESUMO

Quality of response to immunosuppressive therapy and long-term outcomes for pediatric severe aplastic anemia remain incompletely characterized. Contemporary evidence to inform treatment of relapsed or refractory severe aplastic anemia for pediatric patients is also limited. The clinical features and outcomes for 314 children treated from 2002 to 2014 with immunosuppressive therapy for acquired severe aplastic anemia were analyzed retrospectively from 25 institutions in the North American Pediatric Aplastic Anemia Consortium. The majority of subjects (n=264) received horse anti-thymocyte globulin (hATG) plus cyclosporine (CyA) with a median 61 months follow up. Following hATG/CyA, 71.2% (95%CI: 65.3,76.6) achieved an objective response. In contrast to adult studies, the quality of response achieved in pediatric patients was high, with 59.8% (95%CI: 53.7,65.8) complete response and 68.2% (95%CI: 62.2,73.8) achieving at least a very good partial response with a platelet count ≥50×109L. At five years post-hATG/CyA, overall survival was 93% (95%CI: 89,96), but event-free survival without subsequent treatment was only 64% (95%CI: 57,69) without a plateau. Twelve of 171 evaluable patients (7%) acquired clonal abnormalities after diagnosis after a median 25.2 months (range: 4.3-71 months) post treatment. Myelodysplastic syndrome or leukemia developed in 6 of 314 (1.9%). For relapsed/refractory disease, treatment with a hematopoietic stem cell transplant had a superior event-free survival compared to second immunosuppressive therapy treatment in a multivariate analysis (HR=0.19, 95%CI: 0.08,0.47; P=0.0003). This study highlights the need for improved therapies to achieve sustained high-quality remission for children with severe aplastic anemia.

12.
Am J Hematol ; 94(1): 149-161, 2019 01.
Artigo em Inglês | MEDLINE | ID: mdl-30358897

RESUMO

Pyruvate kinase deficiency (PKD) is the most common enzyme defect of glycolysis and an important cause of hereditary, nonspherocytic hemolytic anemia. The disease has a worldwide geographical distribution but there are no verified data regarding its frequency. Difficulties in the diagnostic workflow and interpretation of PK enzyme assay likely play a role. By the creation of a global PKD International Working Group in 2016, involving 24 experts from 20 Centers of Expertise we studied the current gaps in the diagnosis of PKD in order to establish diagnostic guidelines. By means of a detailed survey and subsequent discussions, multiple aspects of the diagnosis of PKD were evaluated and discussed by members of Expert Centers from Europe, USA, and Asia directly involved in diagnosis. Broad consensus was reached among the Centers on many clinical and technical aspects of the diagnosis of PKD. The results of this study are here presented as recommendations for the diagnosis of PKD and used to prepare a diagnostic algorithm. This information might be helpful for other Centers to deliver timely and appropriate diagnosis and to increase awareness in PKD.


Assuntos
Anemia Hemolítica Congênita não Esferocítica/diagnóstico , Piruvato Quinase/deficiência , Erros Inatos do Metabolismo dos Piruvatos/diagnóstico , Anemia Hemolítica Congênita não Esferocítica/sangue , Anemia Hemolítica Congênita não Esferocítica/genética , Anemia Hemolítica Congênita não Esferocítica/fisiopatologia , Artefatos , Contagem de Células Sanguíneas , Preservação de Sangue , Análise Mutacional de DNA , Eritrócitos/enzimologia , Reações Falso-Negativas , Reações Falso-Positivas , Humanos , Piruvato Quinase/sangue , Piruvato Quinase/genética , Erros Inatos do Metabolismo dos Piruvatos/sangue , Erros Inatos do Metabolismo dos Piruvatos/genética , Erros Inatos do Metabolismo dos Piruvatos/fisiopatologia , Valores de Referência , Reticulócitos , Sensibilidade e Especificidade , Análise de Sequência de DNA , Espectrofotometria , Fatores de Tempo
13.
Am J Hum Genet ; 103(6): 930-947, 2018 12 06.
Artigo em Inglês | MEDLINE | ID: mdl-30503522

RESUMO

Diamond-Blackfan anemia (DBA) is a rare bone marrow failure disorder that affects 7 out of 1,000,000 live births and has been associated with mutations in components of the ribosome. In order to characterize the genetic landscape of this heterogeneous disorder, we recruited a cohort of 472 individuals with a clinical diagnosis of DBA and performed whole-exome sequencing (WES). We identified relevant rare and predicted damaging mutations for 78% of individuals. The majority of mutations were singletons, absent from population databases, predicted to cause loss of function, and located in 1 of 19 previously reported ribosomal protein (RP)-encoding genes. Using exon coverage estimates, we identified and validated 31 deletions in RP genes. We also observed an enrichment for extended splice site mutations and validated their diverse effects using RNA sequencing in cell lines obtained from individuals with DBA. Leveraging the size of our cohort, we observed robust genotype-phenotype associations with congenital abnormalities and treatment outcomes. We further identified rare mutations in seven previously unreported RP genes that may cause DBA, as well as several distinct disorders that appear to phenocopy DBA, including nine individuals with biallelic CECR1 mutations that result in deficiency of ADA2. However, no new genes were identified at exome-wide significance, suggesting that there are no unidentified genes containing mutations readily identified by WES that explain >5% of DBA-affected case subjects. Overall, this report should inform not only clinical practice for DBA-affected individuals, but also the design and analysis of rare variant studies for heterogeneous Mendelian disorders.


Assuntos
Anemia de Diamond-Blackfan/genética , Adolescente , Criança , Pré-Escolar , Estudos de Coortes , Exoma/genética , Éxons/genética , Feminino , Deleção de Genes , Estudos de Associação Genética/métodos , Humanos , Peptídeos e Proteínas de Sinalização Intercelular/genética , Masculino , Mutação/genética , Fenótipo , Proteínas Ribossômicas/genética , Ribossomos/genética , Análise de Sequência de RNA/métodos , Sequenciamento Completo do Exoma/métodos
14.
Pediatr Clin North Am ; 65(3): 579-595, 2018 06.
Artigo em Inglês | MEDLINE | ID: mdl-29803284

RESUMO

Mature red blood cells are reliant on the glycolytic pathway for energy production and the hexose monophosphate shunt for cell protection from oxidative insults. The most common red blood cell enzyme disorders are characterized by hemolysis but with wide clinical variability. Glucose-6-phosphate dehydrogenase deficiency is the most common red cell enzyme disorder worldwide. Frequent clinical presentations include neonatal jaundice and episodic hemolysis after exposure to oxidative stress. Symptoms of pyruvate kinase deficiency and other glycolytic enzyme disorders include neonatal jaundice, chronic hemolytic anemia, gallstones, and transfusion-related and transfusion-independent iron overload. Diagnosis is critical for appropriate supportive care, monitoring, and treatment.


Assuntos
Anemia Hemolítica Congênita não Esferocítica/diagnóstico , Anemia Hemolítica Congênita não Esferocítica/etiologia , Deficiência de Glucosefosfato Desidrogenase/diagnóstico , Deficiência de Glucosefosfato Desidrogenase/etiologia , Piruvato Quinase/deficiência , Erros Inatos do Metabolismo dos Piruvatos/diagnóstico , Erros Inatos do Metabolismo dos Piruvatos/etiologia , Deficiência de Glucosefosfato Desidrogenase/terapia , Humanos , Piruvato Quinase/uso terapêutico
16.
Pediatr Blood Cancer ; 65(9): e27220, 2018 09.
Artigo em Inglês | MEDLINE | ID: mdl-29749692

RESUMO

Alpha thalassemia due to nondeletional mutations usually leads to more severe disease than that caused by deletional mutations. Devastating outcomes such as hydrops fetalis can occur with two nondeletional mutations, therefore warranting DNA-based workup for suspected carriers with subtle hematological abnormalities for family counseling purposes. We describe three cases with hemoglobin (Hb) Adana, a nondeletional alpha chain mutation, compounded with an alpha globin gene deletion resulting in thalassemia intermedia. We review the literature, draw genotype-phenotype correlations from published cases of Hb Adana, and propose that this correlation can be used by clinicians to help direct diagnostic studies and urge hematologists to thoroughly workup high-risk patients.


Assuntos
Hemoglobinas Anormais/genética , alfa-Globinas/genética , Talassemia alfa/genética , Pré-Escolar , Códon/genética , Feminino , Deleção de Genes , Estudos de Associação Genética , Aconselhamento Genético , Humanos , Hidropisia Fetal/etiologia , Hidropisia Fetal/genética , Masculino , Mutação Puntual , Deleção de Sequência
17.
Blood ; 131(20): 2183-2192, 2018 05 17.
Artigo em Inglês | MEDLINE | ID: mdl-29549173

RESUMO

An international, multicenter registry was established to collect retrospective and prospective clinical data on patients with pyruvate kinase (PK) deficiency, the most common glycolytic defect causing congenital nonspherocytic hemolytic anemia. Medical history and laboratory and radiologic data were retrospectively collected at enrollment for 254 patients with molecularly confirmed PK deficiency. Perinatal complications were common, including anemia that required transfusions, hyperbilirubinemia, hydrops, and prematurity. Nearly all newborns were treated with phototherapy (93%), and many were treated with exchange transfusions (46%). Children age 5 years and younger were often transfused until splenectomy. Splenectomy (150 [59%] of 254 patients) was associated with a median increase in hemoglobin of 1.6 g/dL and a decreased transfusion burden in 90% of patients. Predictors of a response to splenectomy included higher presplenectomy hemoglobin (P = .007), lower indirect bilirubin (P = .005), and missense PKLR mutations (P = .0017). Postsplenectomy thrombosis was reported in 11% of patients. The most frequent complications included iron overload (48%) and gallstones (45%), but other complications such as aplastic crises, osteopenia/bone fragility, extramedullary hematopoiesis, postsplenectomy sepsis, pulmonary hypertension, and leg ulcers were not uncommon. Overall, 87 (34%) of 254 patients had both a splenectomy and cholecystectomy. In those who had a splenectomy without simultaneous cholecystectomy, 48% later required a cholecystectomy. Although the risk of complications increases with severity of anemia and a genotype-phenotype relationship was observed, complications were common in all patients with PK deficiency. Diagnostic testing for PK deficiency should be considered in patients with apparent congenital hemolytic anemia and close monitoring for iron overload, gallstones, and other complications is needed regardless of baseline hemoglobin. This trial was registered at www.clinicaltrials.gov as #NCT02053480.


Assuntos
Anemia Hemolítica Congênita não Esferocítica/diagnóstico , Estudos de Associação Genética , Piruvato Quinase/deficiência , Erros Inatos do Metabolismo dos Piruvatos/diagnóstico , Adolescente , Adulto , Anemia Hemolítica Congênita não Esferocítica/etiologia , Anemia Hemolítica Congênita não Esferocítica/metabolismo , Anemia Hemolítica Congênita não Esferocítica/terapia , Transfusão de Sangue , Criança , Pré-Escolar , Colecistectomia/efeitos adversos , Colecistectomia/métodos , Terapia Combinada , Ativação Enzimática , Feminino , Genótipo , Humanos , Lactente , Recém-Nascido , Masculino , Pessoa de Meia-Idade , Mutação , Fenótipo , Piruvato Quinase/metabolismo , Erros Inatos do Metabolismo dos Piruvatos/etiologia , Erros Inatos do Metabolismo dos Piruvatos/metabolismo , Erros Inatos do Metabolismo dos Piruvatos/terapia , Esplenectomia/efeitos adversos , Esplenectomia/métodos , Avaliação de Sintomas , Resultado do Tratamento , Adulto Jovem
18.
Haematologica ; 102(5): 826-834, 2017 05.
Artigo em Inglês | MEDLINE | ID: mdl-28154085

RESUMO

Forkhead box M1 (FOXM1) belongs to the forkhead/winged-helix family of transcription factors and regulates a network of proliferation-associated genes. Its abnormal upregulation has been shown to be a key driver of cancer progression and an initiating factor in oncogenesis. FOXM1 is also highly expressed in stem/progenitor cells and inhibits their differentiation, suggesting that FOXM1 plays a role in the maintenance of multipotency. However, the exact molecular mechanisms by which FOXM1 regulates human stem/progenitor cells are still uncharacterized. To understand the role of FOXM1 in normal hematopoiesis, human cord blood CD34+ cells were transduced with FOXM1 short hairpin ribonucleic acid (shRNA) lentivirus. Knockdown of FOXM1 resulted in a 2-fold increase in erythroid cells compared to myeloid cells. Additionally, knockdown of FOXM1 increased bromodeoxyuridine (BrdU) incorporation in erythroid cells, suggesting greater proliferation of erythroid progenitors. We also observed that the defective phosphorylation of FOXM1 by checkpoint kinase 2 (CHK2) or cyclin-dependent kinases 1/2 (CDK1/2) increased the erythroid population in a manner similar to knockdown of FOXM1. Finally, we found that an inhibitor of FOXM1, forkhead domain inhibitor-6 (FDI-6), increased red blood cell numbers through increased proliferation of erythroid precursors. Overall, our data suggest a novel function of FOXM1 in normal human hematopoiesis.


Assuntos
Proliferação de Células/genética , Células Precursoras Eritroides/metabolismo , Eritropoese/genética , Proteína Forkhead Box M1/genética , Antígenos CD34/sangue , Diferenciação Celular/genética , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Quinase do Ponto de Checagem 2/metabolismo , Células Eritroides/metabolismo , Sangue Fetal/citologia , Sangue Fetal/metabolismo , Proteína Forkhead Box M1/metabolismo , Perfilação da Expressão Gênica , Células HEK293 , Humanos , Células K562 , Fosforilação , Piridinas/farmacologia , Interferência de RNA , Tiofenos/farmacologia
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