RESUMO
The transcription factor RUNX1 is mutated in familial platelet disorder with associated myeloid malignancy (FPDMM) and in sporadic myelodysplastic syndrome and leukemia. RUNX1 was shown to regulate inflammation in multiple cell types. Here we show that RUNX1 is required in granulocyte-monocyte progenitors (GMPs) to epigenetically repress two inflammatory signaling pathways in neutrophils: Toll-like receptor 4 (TLR4) and type I interferon (IFN) signaling. RUNX1 loss in GMPs augments neutrophils' inflammatory response to the TLR4 ligand lipopolysaccharide through increased expression of the TLR4 coreceptor CD14. RUNX1 binds Cd14 and other genes encoding proteins in the TLR4 and type I IFN signaling pathways whose chromatin accessibility increases when RUNX1 is deleted. Transcription factor footprints for the effectors of type I IFN signaling-the signal transducer and activator of transcription (STAT1::STAT2) and interferon regulatory factors (IRFs)-were enriched in chromatin that gained accessibility in both GMPs and neutrophils when RUNX1 was lost. STAT1::STAT2 and IRF motifs were also enriched in the chromatin of retrotransposons that were derepressed in RUNX1-deficient GMPs and neutrophils. We conclude that a major direct effect of RUNX1 loss in GMPs is the derepression of type I IFN and TLR4 signaling, resulting in a state of fixed maladaptive innate immunity.
Assuntos
Neutrófilos , Receptor 4 Toll-Like , Receptor 4 Toll-Like/metabolismo , Monócitos/metabolismo , Subunidade alfa 2 de Fator de Ligação ao Core/genética , Subunidade alfa 2 de Fator de Ligação ao Core/metabolismo , Citocinas/metabolismo , Cromatina/metabolismo , Fator de Transcrição STAT1/metabolismoRESUMO
The transcription factor GATA2 has a pivotal role in haematopoiesis. Heterozygous germline GATA2 mutations result in a syndrome characterized by immunodeficiency, bone marrow failure and predispositions to myelodysplastic syndrome (MDS) and acute myeloid leukaemia. Clinical symptoms in these patients are diverse and mechanisms driving GATA2-related phenotypes are largely unknown. To explore the impact of GATA2 haploinsufficiency on haematopoiesis, we generated a zebrafish model carrying a heterozygous mutation of gata2b (gata2b+/-), an orthologue of GATA2. Morphological analysis revealed myeloid and erythroid dysplasia in gata2b+/- kidney marrow. Because Gata2b could affect both transcription and chromatin accessibility during lineage differentiation, this was assessed by single-cell (sc) RNA-seq and single-nucleus (sn) ATAC-seq. Sn-ATAC-seq showed that the co-accessibility between the transcription start site (TSS) and a -3.5-4.1 kb putative enhancer was more robust in gata2b+/- zebrafish HSPCs compared to wild type, increasing gata2b expression and resulting in higher genome-wide Gata2b motif use in HSPCs. As a result of increased accessibility of the gata2b locus, gata2b+/- chromatin was also more accessible during lineage differentiation. scRNA-seq data revealed myeloid differentiation defects, that is, impaired cell cycle progression, reduced expression of cebpa and cebpb and increased signatures of ribosome biogenesis. These data also revealed a differentiation delay in erythroid progenitors, aberrant proliferative signatures and down-regulation of Gata1a, a master regulator of erythropoiesis, which worsened with age. These findings suggest that cell-intrinsic compensatory mechanisms, needed to obtain normal levels of Gata2b in heterozygous HSPCs to maintain their integrity, result in aberrant lineage differentiation, thereby representing a critical step in the predisposition to MDS.
Assuntos
Epigênese Genética , Fator de Transcrição GATA2 , Heterozigoto , Peixe-Zebra , Animais , Fator de Transcrição GATA2/genética , Proteínas de Peixe-Zebra/genética , Células Eritroides/metabolismo , Células Eritroides/patologia , Células Mieloides/metabolismo , Células Mieloides/patologia , Eritropoese/genética , Síndromes Mielodisplásicas/genética , Síndromes Mielodisplásicas/patologia , Síndromes Mielodisplásicas/metabolismoRESUMO
Severe congenital neutropenia (SCN) patients are prone to develop myelodysplastic syndrome (MDS) or acute myeloid leukaemia (AML). Leukaemic progression of SCN is associated with the early acquisition of CSF3R mutations in haematopoietic progenitor cells (HPCs), which truncate the colony-stimulating factor 3 receptor (CSF3R). These mutant clones may arise years before MDS/AML becomes overt. Introduction and activation of CSF3R truncation mutants in normal HPCs causes a clonally dominant myeloproliferative state in mice treated with CSF3. Paradoxically, in SCN patients receiving CSF3 therapy, clonal dominance of CSF3R mutant clones usually occurs only after the acquisition of additional mutations shortly before frank MDS or AML is diagnosed. To seek an explanation for this discrepancy, we introduced a patient-derived CSF3R-truncating mutation in ELANE-SCN and HAX1-SCN derived and control induced pluripotent stem cells and compared the CSF3 responses of HPCs generated from these lines. In contrast to CSF3R-mutant control HPCs, CSF3R-mutant HPCs from SCN patients do not show increased proliferation but display elevated levels of inflammatory signalling. Thus, activation of the truncated CSF3R in SCN-HPCs does not evoke clonal outgrowth but causes a sustained pro-inflammatory state, which has ramifications for how these CSF3R mutants contribute to the leukaemic transformation of SCN.
Assuntos
Leucemia Mieloide Aguda , Síndromes Mielodisplásicas , Camundongos , Animais , Síndrome Congênita de Insuficiência da Medula Óssea/genética , Leucemia Mieloide Aguda/diagnóstico , Mutação , Síndromes Mielodisplásicas/genética , Síndromes Mielodisplásicas/complicaçõesRESUMO
We describe a patient with congenital neutropenia (CN) with a homozygous germline mutation in the colony-stimulating factor 3 receptor gene (CSF3R). The patient's bone marrow shows lagging neutrophil development with subtle left shift and unresponsiveness to CSF3 in in vitro colony assays. This patient illustrates that the di-proline hinge motif in the extracellular cytokine receptor homology domain of CSF3R is critical for adequate neutrophil production, but dispensable for in vivo terminal neutrophil maturation. This report underscores that CN patients with inherited CSF3R mutations should be marked as a separate clinical entity, characterized by a failure to respond to CSF3.
Assuntos
Neutropenia , Receptores de Fator Estimulador de Colônias , Humanos , Receptores de Fator Estimulador de Colônias/genética , Mutação , Receptores de Citocinas/genética , Fator Estimulador de Colônias de Granulócitos , Neutropenia/genéticaRESUMO
PURPOSE OF REVIEW: Myeloid diseases are often characterized by a disturbed regulation of myeloid cell proliferation, survival, and maturation. This may either result in a severe paucity of functional neutrophils (neutropenia), an excess production of mature cells (myeloproliferative disorders) or in clonal expansions of dysplastic or immature myeloid cells (myelodysplasia and acute myeloid leukemia). Although these conditions can be regarded as separate entities, caused by the accumulation of distinct sets of somatic gene mutations, it becomes increasingly clear that they may also evolve as the prime consequence of a congenital defect resulting in severe neutropenia. Prominent examples of such conditions include the genetically heterogeneous forms of severe congenital neutropenia (SCN) and Shwachman-Diamond Syndrome. CSF3 treatment is a successful therapy to alleviate neutropenia in the majority of these patients but does not cure the disease nor does it prevent malignant transformation. Allogeneic stem cell transplantation is currently the only therapeutic option to cure SCN, but is relatively cumbersome, e.g., hampered by treatment-related mortality and donor availability. Hence, there is a need for new therapeutic approaches. RECENT FINDINGS: Developments in disease modeling, amongst others based on induced pluripotent stem cell and CRISPR/Cas9 based gene-editing technologies, have created new insights in disease biology and possibilities for treatment. In addition, they are fueling expectations for advanced disease monitoring to prevent malignant transformation. SUMMARY: This review highlights the recent progress made in SCN disease modeling and discusses the challenges that are still ahead of us to gain a better understanding of the biological heterogeneity of the disease and its consequences for patient care.
Assuntos
Síndromes Mielodisplásicas , Neutropenia , Síndrome Congênita de Insuficiência da Medula Óssea , Humanos , Mutação , Neutropenia/congênito , Neutropenia/genética , Neutropenia/terapiaRESUMO
Neutrophilic differentiation is dependent on CCAAT enhancer-binding protein α (C/EBPα), a transcription factor expressed in multiple organs including the bone marrow. Using functional genomic technologies in combination with clustered regularly-interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein 9 genome editing and in vivo mouse modeling, we show that CEBPA is located in a 170-kb topological-associated domain that contains 14 potential enhancers. Of these, 1 enhancer located +42 kb from CEBPA is active and engages with the CEBPA promoter in myeloid cells only. Germ line deletion of the homologous enhancer in mice in vivo reduces Cebpa levels exclusively in hematopoietic stem cells (HSCs) and myeloid-primed progenitor cells leading to severe defects in the granulocytic lineage, without affecting any other Cebpa-expressing organ studied. The enhancer-deleted progenitor cells lose their myeloid transcription program and are blocked in differentiation. Deletion of the enhancer also causes loss of HSC maintenance. We conclude that a single +42-kb enhancer is essential for CEBPA expression in myeloid cells only.
Assuntos
Proteína alfa Estimuladora de Ligação a CCAAT/metabolismo , Diferenciação Celular/genética , Linhagem da Célula/genética , Elementos Facilitadores Genéticos , Células Mieloides/fisiologia , Mielopoese/genética , Neutrófilos/fisiologia , Animais , Proteína alfa Estimuladora de Ligação a CCAAT/genética , Linhagem Celular Tumoral , Regulação da Expressão Gênica no Desenvolvimento , Células HEK293 , Células HL-60 , Células HeLa , Células Hep G2 , Humanos , Células Jurkat , Células K562 , Camundongos , Camundongos Knockout , Células U937RESUMO
Interstrand crosslinks (ICLs) are toxic DNA lesions that cause severe genomic damage during replication, especially in Fanconi anemia pathway-deficient cells. This results in progressive bone marrow failure and predisposes to acute myeloid leukemia (AML). The molecular mechanisms responsible for these defects are largely unknown. Using Ercc1-deficient mice, we show that Trp53 is responsible for ICL-induced bone marrow failure and that loss of Trp53 is leukemogenic in this model. In addition, Ercc1-deficient myeloid progenitors gain elevated levels of miR-139-3p and miR-199a-3p with age. These microRNAs exert opposite effects on hematopoiesis. Ectopic expression of miR-139-3p strongly inhibited proliferation of myeloid progenitors, whereas inhibition of miR-139-3p activity restored defective proliferation of Ercc1-deficient progenitors. Conversely, the inhibition of miR-199a-3p functions aggravated the myeloid proliferation defect in the Ercc1-deficient model, whereas its enforced expression enhanced proliferation of progenitors. Importantly, miR-199a-3p caused AML in a pre-leukemic mouse model, supporting its role as an onco-microRNA. Target genes include HuR for miR-139-3p and Prdx6, Runx1, and Suz12 for miR-199a-3p. The latter genes have previously been implicated as tumor suppressors in de novo and secondary AML. These findings show that, in addition to TRP53-controlled mechanisms, miR-139-3p and miR-199a-3p are involved in the defective hematopoietic function of ICL-repair deficient myeloid progenitors.
Assuntos
Transformação Celular Neoplásica/genética , Células-Tronco Hematopoéticas/patologia , Leucemia/genética , MicroRNAs/genética , Animais , Transformação Celular Neoplásica/metabolismo , Transformação Celular Neoplásica/patologia , Reparo do DNA/genética , Proteínas de Ligação a DNA/deficiência , Modelos Animais de Doenças , Endonucleases/deficiência , Células-Tronco Hematopoéticas/metabolismo , Leucemia/metabolismo , Leucemia/patologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismoRESUMO
Serpinb1 is an inhibitor of neutrophil granule serine proteases cathepsin G, proteinase-3 and elastase. One of its core physiological functions is to protect neutrophils from granule protease-mediated cell death. Mice lacking Serpinb1a (Sb1a-/-), its mouse ortholog, have reduced bone marrow neutrophil numbers due to cell death mediated by cathepsin G and the mice show increased susceptibility to lung infections. Here, we show that conditional deletion of Serpinb1a using the Lyz2-cre and Cebpa-cre knock-in mice effectively leads to recombination-mediated deletion in neutrophils but protein-null neutrophils were only obtained using the latter recombinase-expressing strain. Absence of Serpinb1a protein in neutrophils caused neutropenia and increased granule permeabilization-induced cell death. We then generated transgenic mice expressing human Serpinb1 in neutrophils under the human MRP8 (S100A8) promoter. Serpinb1a expression levels in founder lines correlated positively with increased neutrophil survival when crossed with Sb1a-/- mice, which had their defective neutrophil phenotype rescued in the higher expressing transgenic line. Using new conditional and transgenic mouse models, our study demonstrates the presence of a relatively low Serpinb1a protein threshold in neutrophils that is required for sustained survival. These models will also be helpful in delineating recently described functions of Serpinb1 in metabolism and cancer.
Assuntos
Deleção de Genes , Células Mieloides/metabolismo , Neutrófilos/citologia , Serpinas/deficiência , Serpinas/genética , Alelos , Animais , Sobrevivência Celular , Técnicas de Introdução de Genes , Humanos , Camundongos Transgênicos , Recombinação GenéticaRESUMO
Severe congenital neutropenia (SCN) is characterized by low numbers of peripheral neutrophil granulocytes and a predisposition to life-threatening bacterial infections. We describe a novel genetic SCN type in 2 unrelated families associated with recessively inherited loss-of-function mutations in CSF3R, encoding the granulocyte colony-stimulating factor (G-CSF) receptor. Family A, with 3 affected children, carried a homozygous missense mutation (NM_000760.3:c.922C>T, NP_000751.1:p.Arg308Cys), which resulted in perturbed N-glycosylation and aberrant localization to the cell surface. Family B, with 1 affected infant, carried compound heterozygous deletions provoking frameshifts and premature stop codons (NM_000760.3:c.948_963del, NP_000751.1:p.Gly316fsTer322 and NM_000760.3:c.1245del, NP_000751.1:p.Gly415fsTer432). Despite peripheral SCN, all patients had morphologic evidence of full myeloid cell maturation in bone marrow. None of the patients responded to treatment with recombinant human G-CSF. Our study highlights the genetic and morphologic SCN variability and provides evidence both for functional importance and redundancy of G-CSF receptor-mediated signaling in human granulopoiesis.
Assuntos
Mutação de Sentido Incorreto , Neutropenia/congênito , Receptores de Fator Estimulador de Colônias/genética , Sequência de Bases , Criança , Pré-Escolar , Síndrome Congênita de Insuficiência da Medula Óssea , Feminino , Células HeLa , Homozigoto , Humanos , Lactente , Recém-Nascido , Masculino , Modelos Moleculares , Neutropenia/genética , Linhagem , Receptores de Fator Estimulador de Colônias/químicaRESUMO
Severe congenital neutropenia (CN) is a preleukemic bone marrow failure syndrome with a 20% risk of evolving into leukemia or myelodysplastic syndrome (MDS). Patterns of acquisition of leukemia-associated mutations were investigated using next-generation deep-sequencing in 31 CN patients who developed leukemia or MDS. Twenty (64.5%) of the 31 patients had mutations in RUNX1. A majority of patients with RUNX1 mutations (80.5%) also had acquired CSF3R mutations. In contrast to their high frequency in CN patients who developed leukemia or MDS, RUNX1 mutations were found in only 9 of 307 (2.9%) patients with de novo pediatric acute myeloid leukemia. A sequential analysis at stages prior to overt leukemia revealed RUNX1 mutations to be late events in leukemic transformation. Single-cell analyses in 2 patients showed that RUNX1 and CSF3R mutations were present in the same malignant clone. Functional studies demonstrated elevated granulocyte colony-stimulating factor (G-CSF)-induced proliferation with diminished myeloid differentiation of hematopoietic CD34(+) cells coexpressing mutated forms of RUNX1 and CSF3R. The high frequency of cooperating RUNX1 and CSF3R mutations in CN patients suggests a novel molecular pathway of leukemogenesis: mutations in the hematopoietic cytokine receptor (G-CSFR) in combination with the second mutations in the downstream hematopoietic transcription fator (RUNX1). The detection of both RUNX1 and CSF3R mutations could be used as a marker for identifying CN patients with a high risk of progressing to leukemia or MDS.
Assuntos
Transformação Celular Neoplásica/genética , Subunidade alfa 2 de Fator de Ligação ao Core/genética , Leucemia Mieloide/genética , Mutação , Neutropenia/congênito , Receptores de Fator Estimulador de Colônias/genética , Adolescente , Adulto , Criança , Pré-Escolar , Síndrome Congênita de Insuficiência da Medula Óssea , Análise Citogenética , Feminino , Humanos , Masculino , Neutropenia/genética , Neutropenia/patologia , Transdução de Sinais/genética , Adulto JovemRESUMO
JAK2 tyrosine kinase regulates many cellular functions. Its activity is controlled by the pseudokinase (JH2) domain by still poorly understood mechanisms. The V617F mutation in the pseudokinase domain activates JAK2 and causes myeloproliferative neoplasms. We conducted a detailed kinetic analysis of recombinant JAK2 tyrosine kinase domain (JH1) and wild-type and V617F tandem kinase (JH1JH2) domains using peptide microarrays to define the functions of the kinase domains. The results show that i) JAK2 follows a random Bi-Bi reaction mechanism ii) JH2 domain restrains the activity of the JH1 domain by reducing the affinity for ATP and ATP competitive inhibitors iii) V617F decreases affinity for ATP but increases catalytic activity compared to wild-type and iv) the SH2-JH2 linker region participates in controlling activity by reducing the affinity for ATP.
RESUMO
Shwachman-Diamond syndrome is a congenital bone marrow failure disorder characterized by debilitating neutropenia. The disease is associated with loss-of-function mutations in the SBDS gene, implicated in ribosome biogenesis, but the cellular and molecular events driving cell specific phenotypes in ribosomopathies remain poorly defined. Here, we established what is to our knowledge the first mammalian model of neutropenia in Shwachman-Diamond syndrome through targeted downregulation of Sbds in hematopoietic stem and progenitor cells expressing the myeloid transcription factor CCAAT/enhancer binding protein α (Cebpa). Sbds deficiency in the myeloid lineage specifically affected myelocytes and their downstream progeny while, unexpectedly, it was well tolerated by rapidly cycling hematopoietic progenitor cells. Molecular insights provided by massive parallel sequencing supported cellular observations of impaired cell cycle exit and formation of secondary granules associated with the defect of myeloid lineage progression in myelocytes. Mechanistically, Sbds deficiency activated the p53 tumor suppressor pathway and induced apoptosis in these cells. Collectively, the data reveal a previously unanticipated, selective dependency of myelocytes and downstream progeny, but not rapidly cycling progenitors, on this ubiquitous ribosome biogenesis protein, thus providing a cellular basis for the understanding of myeloid lineage biased defects in Shwachman-Diamond syndrome.
Assuntos
Diferenciação Celular/genética , Linhagem da Célula/genética , Células-Tronco Hematopoéticas/metabolismo , Células Mieloides/citologia , Células Mieloides/metabolismo , Neutropenia/genética , Proteínas/genética , Animais , Apoptose/genética , Doenças da Medula Óssea/genética , Proteínas Estimuladoras de Ligação a CCAAT/genética , Proteínas Estimuladoras de Ligação a CCAAT/metabolismo , Ciclo Celular/genética , Modelos Animais de Doenças , Insuficiência Pancreática Exócrina/genética , Deleção de Genes , Hematopoese/genética , Transplante de Células-Tronco Hematopoéticas , Células-Tronco Hematopoéticas/citologia , Lipomatose/genética , Camundongos , Camundongos Knockout , Síndrome de Shwachman-Diamond , Transdução de Sinais , Proteína Supressora de Tumor p53/metabolismoRESUMO
FcγRIIb is the sole inhibitory FcR for IgG in humans and mice, where it is involved in the negative regulation of Ab production and cellular activation. FcγRIIb-deficient mice show exacerbated disease following the induction of nephrotoxic nephritis (NTN). In this study, we determined the cellular origin of the FcγRIIb-knockout phenotype by inducing NTN in mice with a deficiency of FcγRIIb on either B cells alone (FcγRIIB(fl/fl)/CD19Cre(+)) or myeloid cells (FcγRIIB(fl/fl)/CEBPαCre(+)). Deletion of FcγRIIb from B cells did not increase susceptibility to NTN, compared with wild-type (WT) mice, despite higher Ab titers in the FcγRIIB(fl/fl)/CD19Cre(+) mice compared with the WT littermate controls. In contrast, mice lacking FcγRIIb on myeloid cells had exacerbated disease as measured by increased glomerular thrombosis, glomerular crescents, albuminuria, serum urea, and glomerular neutrophil infiltration when compared with WT littermate controls. The role for FcγRIIb expression on radioresistant intrinsic renal cells in the protection from NTN was then investigated using bone marrow chimeric mice. FcγRIIb(-/-) mice transplanted with FcγRIIb(-/-) bone marrow were more susceptible to NTN than WT mice transplanted with FcγRIIb(-/-) bone marrow, indicating that the presence of WT intrinsic renal cells protects from NTN. These results demonstrate that FcγRIIb on myeloid cells plays a major role in protection from NTN, and therefore, augmentation of FcγRIIb on these cells could be a therapeutic target in human Ab-mediated glomerulonephritis. Where there was a lack of FcγRIIb on circulating myeloid cells, expression of FcγRIIb on intrinsic renal cells provided an additional level of protection from Ab-mediated glomerulonephritis.
Assuntos
Subpopulações de Linfócitos B/imunologia , Glomerulonefrite/imunologia , Glomerulonefrite/prevenção & controle , Rim/imunologia , Células Mieloides/imunologia , Receptores de IgG/fisiologia , Animais , Subpopulações de Linfócitos B/citologia , Subpopulações de Linfócitos B/metabolismo , Células Cultivadas , Predisposição Genética para Doença , Glomerulonefrite/patologia , Rim/metabolismo , Rim/patologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Células Mieloides/citologia , Células Mieloides/metabolismo , Infiltração de Neutrófilos/genética , Infiltração de Neutrófilos/imunologia , Quimera por Radiação/genética , Quimera por Radiação/imunologia , Receptores de IgG/deficiência , Receptores de IgG/genéticaRESUMO
Steady-state hematopoiesis is altered on infection, but the cellular and molecular mechanisms driving these changes are largely unknown. Modulation of hematopoiesis is essential to increase the output of the appropriate type of effector cell required to combat the invading pathogen. In the present study, we demonstrate that the pro-inflammatory cytokine IFNγ is involved in orchestrating inflammation-induced myelopoiesis. Using both mouse models and in vitro assays, we show that IFNγ induces the differentiation of monocytes over neutrophils at the level of myeloid progenitors. Infection with lymphocytic choriomeningitis virus induces monopoiesis in wild-type mice, but causes increased neutrophil production in IFNγ(-/-) mice. We demonstrate that IFNγ enhances the expression of the monopoiesis-inducing transcription factors IRF8 and PU.1 in myeloid progenitor cells, whereas it reduces G-CSF-driven neutrophil differentiation via a SOCS3-dependent inhibition of STAT3 phosphorylation. These results establish a critical role for IFNγ in directing monocyte versus neutrophil development during immune activation.
Assuntos
Mediadores da Inflamação/fisiologia , Inflamação/metabolismo , Interferon gama/fisiologia , Neutrófilos/metabolismo , Animais , Células da Medula Óssea/imunologia , Células da Medula Óssea/metabolismo , Ligante CD27/genética , Ligante CD27/metabolismo , Diferenciação Celular/efeitos dos fármacos , Células Cultivadas , Fator Estimulador de Colônias de Granulócitos/farmacologia , Inflamação/genética , Mediadores da Inflamação/metabolismo , Mediadores da Inflamação/farmacologia , Fatores Reguladores de Interferon/metabolismo , Interferon gama/genética , Interferon gama/farmacologia , Coriomeningite Linfocítica/genética , Coriomeningite Linfocítica/imunologia , Coriomeningite Linfocítica/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Monócitos/citologia , Monócitos/metabolismo , Células Progenitoras Mieloides/metabolismo , Mielopoese/efeitos dos fármacos , Neutrófilos/citologia , Fosforilação , Proteínas Proto-Oncogênicas/metabolismo , Fator de Transcrição STAT3/metabolismo , Proteína 3 Supressora da Sinalização de Citocinas , Proteínas Supressoras da Sinalização de Citocina/metabolismo , Linfócitos T/imunologia , Linfócitos T/metabolismo , Transativadores/metabolismoRESUMO
Diamond-Blackfan anemia (DBA) is associated with developmental defects and profound anemia. Mutations in genes encoding a ribosomal protein of the small (e.g., RPS19) or large (e.g., RPL11) ribosomal subunit are found in more than half of these patients. The mutations cause ribosomal haploinsufficiency, which reduces overall translation efficiency of cellular mRNAs. We reduced the expression of Rps19 or Rpl11 in mouse erythroblasts and investigated mRNA polyribosome association, which revealed deregulated translation initiation of specific transcripts. Among these were Bag1, encoding a Hsp70 cochaperone, and Csde1, encoding an RNA-binding protein, and both were expressed at increased levels in erythroblasts. Their translation initiation is cap independent and starts from an internal ribosomal entry site, which appeared sensitive to knockdown of Rps19 or Rpl11. Mouse embryos lacking Bag1 die at embryonic day 13.5, with reduced erythroid colony forming cells in the fetal liver, and low Bag1 expression impairs erythroid differentiation in vitro. Reduced expression of Csde1 impairs the proliferation and differentiation of erythroid blasts. Protein but not mRNA expression of BAG1 and CSDE1 was reduced in erythroblasts cultured from DBA patients. Our data suggest that impaired internal ribosomal entry site-mediated translation of mRNAs expressed at increased levels in erythroblasts contributes to the erythroid phenotype of DBA.
Assuntos
Anemia de Diamond-Blackfan/genética , Anemia de Diamond-Blackfan/patologia , Biomarcadores/metabolismo , Diferenciação Celular , Eritroblastos/citologia , Polirribossomos/patologia , Biossíntese de Proteínas , Animais , Western Blotting , Proliferação de Células , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Proteínas de Ligação a DNA/fisiologia , Embrião de Mamíferos/citologia , Embrião de Mamíferos/metabolismo , Eritroblastos/metabolismo , Feminino , Citometria de Fluxo , Perfilação da Expressão Gênica , Humanos , Camundongos , Camundongos Knockout , Mutação/genética , Análise de Sequência com Séries de Oligonucleotídeos , Fenótipo , Polirribossomos/genética , Polirribossomos/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA Interferente Pequeno/genética , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Reação em Cadeia da Polimerase em Tempo Real , Proteínas Ribossômicas/antagonistas & inibidores , Proteínas Ribossômicas/genética , Proteínas Ribossômicas/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Fatores de Transcrição/fisiologiaRESUMO
MicroRNAs (miRNAs) have the potential to regulate cellular differentiation programs; however, miRNA deficiency in primary hematopoietic stem cells (HSCs) results in HSC depletion in mice, leaving the question of whether miRNAs play a role in early-lineage decisions un-answered. To address this issue, we deleted Dicer1, which encodes an essential RNase III enzyme for miRNA biogenesis, in murine CCAAT/enhancer-binding protein α (C/EBPA)-positive myeloid-committed progenitors in vivo. In contrast to the results in HSCs, we found that miRNA depletion affected neither the number of myeloid progenitors nor the percentage of C/EBPA-positive progenitor cells. Analysis of gene-expression profiles from wild-type and Dicer1-deficient granulocyte-macrophage progenitors (GMPs) revealed that 20 miRNA families were active in GMPs. Of the derepressed miRNA targets in Dicer1-null GMPs, 27% are normally exclusively expressed in HSCs or are specific for multipotent progenitors and erythropoiesis, indicating an altered gene-expression landscape. Dicer1-deficient GMPs were defective in myeloid development in vitro and exhibited an increased replating capacity, indicating the regained self-renewal potential of these cells. In mice, Dicer1 deletion blocked monocytic differentiation, depleted macrophages, and caused myeloid dysplasia with morphologic features of Pelger-Huët anomaly. These results provide evidence for a miRNA-controlled switch for a cellular program of self-renewal and expansion toward myeloid differentiation in GMPs.
Assuntos
Diferenciação Celular/genética , RNA Helicases DEAD-box/genética , Células Dendríticas/fisiologia , Macrófagos/fisiologia , Células Progenitoras Mieloides/fisiologia , Neutrófilos/patologia , Ribonuclease III/genética , Animais , Proteínas Estimuladoras de Ligação a CCAAT/genética , Proteínas Estimuladoras de Ligação a CCAAT/metabolismo , Proteínas Estimuladoras de Ligação a CCAAT/fisiologia , Células Cultivadas , RNA Helicases DEAD-box/metabolismo , RNA Helicases DEAD-box/fisiologia , Células Dendríticas/citologia , Células Dendríticas/metabolismo , Embrião de Mamíferos , Deleção de Genes , Contagem de Leucócitos , Macrófagos/citologia , Macrófagos/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Células Progenitoras Mieloides/citologia , Células Progenitoras Mieloides/metabolismo , Neutrófilos/citologia , Neutrófilos/metabolismo , Neutrófilos/fisiologia , Anomalia de Pelger-Huët/genética , Anomalia de Pelger-Huët/patologia , Ribonuclease III/metabolismo , Ribonuclease III/fisiologiaRESUMO
Severe congenital neutropenia (SCN) is a BM failure syndrome with a high risk of progression to acute myeloid leukemia (AML). The underlying genetic changes involved in SCN evolution to AML are largely unknown. We obtained serial hematopoietic samples from an SCN patient who developed AML 17 years after the initiation of G-CSF treatment. Next- generation sequencing was performed to identify mutations during disease progression. In the AML phase, we found 12 acquired nonsynonymous mutations. Three of these, in CSF3R, LLGL2, and ZC3H18, co-occurred in a subpopulation of progenitor cells already in the early SCN phase. This population expanded over time, whereas clones harboring only CSF3R mutations disappeared from the BM. The other 9 mutations were only apparent in the AML cells and affected known AML-associated genes (RUNX1 and ASXL1) and chromatin remodelers (SUZ12 and EP300). In addition, a novel CSF3R mutation that conferred autonomous proliferation to myeloid progenitors was found. We conclude that progression from SCN to AML is a multistep process, with distinct mutations arising early during the SCN phase and others later in AML development. The sequential gain of 2 CSF3R mutations implicates abnormal G-CSF signaling as a driver of leukemic transformation in this case of SCN.
Assuntos
Transformação Celular Neoplásica/genética , Leucemia Mieloide Aguda/genética , Mutação , Proteínas de Neoplasias/genética , Neutropenia/genética , Adulto , Medula Óssea/metabolismo , Transformação Celular Neoplásica/metabolismo , Fator Estimulador de Colônias de Granulócitos/uso terapêutico , Humanos , Leucemia Mieloide Aguda/complicações , Leucemia Mieloide Aguda/tratamento farmacológico , Leucemia Mieloide Aguda/metabolismo , Masculino , Proteínas de Neoplasias/metabolismo , Neutropenia/complicações , Neutropenia/congênito , Neutropenia/tratamento farmacológico , Neutropenia/metabolismo , Células-Tronco/metabolismoRESUMO
Reactive oxygen species (ROS) regulate growth factor receptor signalling at least in part by inhibiting oxidation-sensitive phosphatases. An emerging concept is that ROS act locally to affect signal transduction in different subcellular compartments and that ROS levels are regulated by antioxidant proteins at the same local level. Here, we show that the ER-resident antioxidant peroxiredoxin 4 (Prdx4) interacts with the cytoplasmic domain of the granulocyte colony-stimulating factor receptor (G-CSFR). This interaction occurs when the activated G-CSFR resides in early endosomes. Prdx4 inhibits G-CSF-induced signalling and proliferation in myeloid progenitors, depending on its redox-active cysteine core. Protein tyrosine phosphatase 1b (Ptp1b) appears to be a major downstream effector controlling these responses. Conversely, Ptp1b might keep Prdx4 active by reducing its phosphorylation. These findings unveil a new signal transduction regulatory circuitry involving redox-controlled processes in the ER and activated cytokine receptors in endosomes.