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1.
Br J Haematol ; 199(4): 482-495, 2022 11.
Artigo em Inglês | MEDLINE | ID: mdl-35753998

RESUMO

The importance of predisposition to leukaemia in clinical practice is being increasingly recognized. This is emphasized by the establishment of a novel WHO disease category in 2016 called "myeloid neoplasms with germline predisposition". A major syndrome within this group is GATA2 deficiency, a heterogeneous immunodeficiency syndrome with a very high lifetime risk to develop myelodysplastic syndrome (MDS) and acute myeloid leukaemia (AML). GATA2 deficiency has been identified as the most common hereditary cause of MDS in adolescents with monosomy 7. Allogenic haematopoietic stem cell transplantation is the only curative option; however, chances of survival decrease with progression of immunodeficiency and MDS evolution. Penetrance and expressivity within families carrying GATA2 mutations is often variable, suggesting that co-operating extrinsic events are required to trigger the disease. Predictive tools are lacking, and intrafamilial heterogeneity is poorly understood; hence there is a clear unmet medical need. On behalf of the ERAPerMed GATA2 HuMo consortium, in this review we describe the genetic, clinical, and biological aspects of familial GATA2-related MDS, highlighting the importance of developing robust disease preclinical models to improve early detection and clinical decision-making of GATA2 carriers.


Assuntos
Deficiência de GATA2 , Transplante de Células-Tronco Hematopoéticas , Síndromes de Imunodeficiência , Leucemia Mieloide Aguda , Síndromes Mielodisplásicas , Transtornos Mieloproliferativos , Humanos , Suscetibilidade a Doenças , Deficiência de GATA2/genética , Deficiência de GATA2/terapia , Fator de Transcrição GATA2/genética , Síndromes de Imunodeficiência/genética , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/terapia , Síndromes Mielodisplásicas/genética , Síndromes Mielodisplásicas/terapia , Transtornos Mieloproliferativos/complicações
2.
Stem Cells ; 35(11): 2253-2266, 2017 11.
Artigo em Inglês | MEDLINE | ID: mdl-28869683

RESUMO

Runt-related transcription factor 1 (Runx1) is a master hematopoietic transcription factor essential for hematopoietic stem cell (HSC) emergence. Runx1-deficient mice die during early embryogenesis due to the inability to establish definitive hematopoiesis. Here, we have used human pluripotent stem cells (hPSCs) as model to study the role of RUNX1 in human embryonic hematopoiesis. Although the three RUNX1 isoforms a, b, and c were induced in CD45+ hematopoietic cells, RUNX1c was the only isoform induced in hematoendothelial progenitors (HEPs)/hemogenic endothelium. Constitutive expression of RUNX1c in human embryonic stem cells enhanced the appearance of HEPs, including hemogenic (CD43+) HEPs and promoted subsequent differentiation into blood cells. Conversely, specific deletion of RUNX1c dramatically reduced the generation of hematopoietic cells from HEPs, indicating that RUNX1c is a master regulator of human hematopoietic development. Gene expression profiling of HEPs revealed a RUNX1c-induced proinflammatory molecular signature, supporting previous studies demonstrating proinflammatory signaling as a regulator of HSC emergence. Collectively, RUNX1c orchestrates hematopoietic specification of hPSCs, possibly in cooperation with proinflammatory signaling. Stem Cells 2017;35:2253-2266.


Assuntos
Subunidade alfa 2 de Fator de Ligação ao Core/genética , Perfilação da Expressão Gênica/métodos , Células-Tronco Pluripotentes/metabolismo , Animais , Diferenciação Celular , Subunidade alfa 2 de Fator de Ligação ao Core/metabolismo , Humanos , Camundongos , Transdução de Sinais
3.
Stem Cells ; 35(7): 1687-1703, 2017 07.
Artigo em Inglês | MEDLINE | ID: mdl-28472853

RESUMO

Coenzyme Q10 (CoQ10 ) plays a crucial role in mitochondria as an electron carrier within the mitochondrial respiratory chain (MRC) and is an essential antioxidant. Mutations in genes responsible for CoQ10 biosynthesis (COQ genes) cause primary CoQ10 deficiency, a rare and heterogeneous mitochondrial disorder with no clear genotype-phenotype association, mainly affecting tissues with high-energy demand including brain and skeletal muscle (SkM). Here, we report a four-year-old girl diagnosed with minor mental retardation and lethal rhabdomyolysis harboring a heterozygous mutation (c.483G > C (E161D)) in COQ4. The patient's fibroblasts showed a decrease in [CoQ10 ], CoQ10 biosynthesis, MRC activity affecting complexes I/II + III, and respiration defects. Bona fide induced pluripotent stem cell (iPSCs) lines carrying the COQ4 mutation (CQ4-iPSCs) were generated, characterized and genetically edited using the CRISPR-Cas9 system (CQ4ed -iPSCs). Extensive differentiation and metabolic assays of control-iPSCs, CQ4-iPSCs and CQ4ed -iPSCs demonstrated a genotype association, reproducing the disease phenotype. The COQ4 mutation in iPSC was associated with CoQ10 deficiency, metabolic dysfunction, and respiration defects. iPSC differentiation into SkM was compromised, and the resulting SkM also displayed respiration defects. Remarkably, iPSC differentiation in dopaminergic or motor neurons was unaffected. This study offers an unprecedented iPSC model recapitulating CoQ10 deficiency-associated functional and metabolic phenotypes caused by COQ4 mutation. Stem Cells 2017;35:1687-1703.


Assuntos
Ataxia/genética , Deficiência Intelectual/genética , Mitocôndrias/genética , Doenças Mitocondriais/genética , Proteínas Mitocondriais/genética , Debilidade Muscular/genética , Rabdomiólise/genética , Ubiquinona/análogos & derivados , Ubiquinona/deficiência , Ataxia/enzimologia , Ataxia/patologia , Sistemas CRISPR-Cas , Diferenciação Celular , Pré-Escolar , Neurônios Dopaminérgicos/citologia , Neurônios Dopaminérgicos/metabolismo , Complexo de Proteínas da Cadeia de Transporte de Elétrons/genética , Complexo de Proteínas da Cadeia de Transporte de Elétrons/metabolismo , Evolução Fatal , Feminino , Fibroblastos/metabolismo , Fibroblastos/patologia , Edição de Genes/métodos , Expressão Gênica , Genes Letais , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismo , Células-Tronco Pluripotentes Induzidas/patologia , Deficiência Intelectual/enzimologia , Deficiência Intelectual/patologia , Mitocôndrias/enzimologia , Mitocôndrias/patologia , Doenças Mitocondriais/enzimologia , Doenças Mitocondriais/patologia , Proteínas Mitocondriais/deficiência , Neurônios Motores/citologia , Neurônios Motores/metabolismo , Debilidade Muscular/enzimologia , Debilidade Muscular/patologia , Cultura Primária de Células , Rabdomiólise/enzimologia , Rabdomiólise/patologia , Ubiquinona/genética
5.
Stem Cells ; 34(3): 581-7, 2016 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-26850912

RESUMO

Although B cells have been shown to be refractory to reprogramming into pluripotency, induced pluripotent stem cells (iPSCs) have been very recently generated, at very low efficiency, from human cord blood (CB)- and peripheral blood (PB)-derived CD19+CD20 + B cells using nonintegrative tetracistronic OSKM-expressing Sendai Virus (SeV). Here, we addressed whether cell ontogeny and hierarchy influence the reprogramming efficiency of the B-cell compartment. We demonstrate that human fetal liver (FL)-derived CD19 + B cells are 110-fold easier to reprogram into iPSCs than those from CB/PB. Similarly, FL-derived CD34+CD19 + B progenitors are reprogrammed much easier than mature B cells (0.46% vs. 0.11%). All FL B-cell iPSCs carry complete VDJH rearrangements while 55% and 45% of the FL B-progenitor iPSCs carry incomplete and complete VDJH rearrangements, respectively, reflecting the reprogramming of developmentally different B progenitors (pro-B vs. pre-B) within a continuous differentiation process. Finally, our data suggest that successful B-cell reprogramming relies on active cell proliferation, and it is MYC-dependent as identical nonintegrative polycistronic SeV lacking MYC (OSKL or OSKLN) fail to reprogram B cells. The ability to efficiently reprogram human fetal primary B cells and B precursors offers an unprecedented opportunity for studying developmental B-lymphopoiesis and modeling B-cell malignances.


Assuntos
Linfócitos B/citologia , Diferenciação Celular/genética , Reprogramação Celular/genética , Células-Tronco Pluripotentes Induzidas , Animais , Técnicas de Cultura de Células , Proliferação de Células/genética , Sangue Fetal/citologia , Vetores Genéticos , Histona-Lisina N-Metiltransferase/genética , Humanos , Fator 4 Semelhante a Kruppel , Fatores de Transcrição Kruppel-Like/genética , Camundongos , Fator 3 de Transcrição de Octâmero/genética , Proteínas Proto-Oncogênicas c-myc/genética , Fatores de Transcrição SOXB1/genética
6.
Blood ; 121(19): 3867-78, S1-3, 2013 May 09.
Artigo em Inglês | MEDLINE | ID: mdl-23479570

RESUMO

Mixed-lineage leukemia (MLL)-AF4 fusion arises prenatally in high-risk infant acute pro-B-lymphoblastic leukemia (pro-B-ALL). In human embryonic stem cells (hESCs), MLL-AF4 skewed hematoendothelial specification but was insufficient for transformation, suggesting that additional oncogenic insults seem required for MLL-AF4-mediated transformation. MLL-AF4+ pro-B-ALL expresses enormous levels of FLT3, occasionally because of activating mutations, thus representing a candidate cooperating event in MLL-AF4+ pro-B-ALL. Here, we explored the developmental impact of FLT3 activation alone, or together with MLL-AF4, in the hematopoietic fate of hESCs. FLT3 activation does not affect specification of hemogenic precursors but significantly enhances the formation of CD45(+) blood cells, and CD45(+)CD34(+) blood progenitors with clonogenic potential. However, overexpression of FLT3 mutations or wild-type FLT3 (FLT3-WT) completely abrogates hematopoietic differentiation from MLL-AF4-expressing hESCs, indicating that FLT3 activation cooperates with MLL-AF4 to inhibit human embryonic hematopoiesis. Cell cycle/apoptosis analyses suggest that FLT3 activation directly affects hESC specification rather than proliferation or survival of hESC-emerging hematopoietic derivatives. Transcriptional profiling of hESC-derived CD45(+) cells supports the FLT3-mediated inhibition of hematopoiesis in MLL-AF4-expressing hESCs, which is associated with large transcriptional changes and downregulation of genes involved in hematopoietic system development and function. Importantly, FLT3 activation does not cooperate with MLL-AF4 to immortalize/transform hESC-derived hematopoietic cells, suggesting the need of alternative (epi)-genetic cooperating hits.


Assuntos
Diferenciação Celular/genética , Células-Tronco Embrionárias/fisiologia , Hematopoese/genética , Proteína de Leucina Linfoide-Mieloide/fisiologia , Proteínas de Fusão Oncogênica/fisiologia , Tirosina Quinase 3 Semelhante a fms/fisiologia , Animais , Linhagem da Célula/genética , Células Cultivadas , Células-Tronco Embrionárias/metabolismo , Ativação Enzimática/fisiologia , Perfilação da Expressão Gênica , Células-Tronco Hematopoéticas/metabolismo , Células-Tronco Hematopoéticas/fisiologia , Humanos , Camundongos , Camundongos SCID , Análise em Microsséries , Proteína de Leucina Linfoide-Mieloide/genética , Proteína de Leucina Linfoide-Mieloide/metabolismo , Proteínas de Fusão Oncogênica/genética , Proteínas de Fusão Oncogênica/metabolismo , Tirosina Quinase 3 Semelhante a fms/genética , Tirosina Quinase 3 Semelhante a fms/metabolismo
7.
Stem Cells ; 32(11): 2811-7, 2014 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-24989459

RESUMO

Access to healthy or diseased human neural tissue is a daunting task and represents a barrier for advancing our understanding about the cellular, genetic, and molecular mechanisms underlying neurogenesis and neurodegeneration. Reprogramming of somatic cells to pluripotency by transient expression of transcription factors was achieved a few years ago. Induced pluripotent stem cells (iPSC) from both healthy individuals and patients suffering from debilitating, life-threatening neurological diseases have been differentiated into several specific neuronal subtypes. An alternative emerging approach is the direct conversion of somatic cells (i.e., fibroblasts, blood cells, or glial cells) into neuron-like cells. However, to what extent neuronal direct conversion of diseased somatic cells can be achieved remains an open question. Optimization of current expansion and differentiation approaches is highly demanded to increase the differentiation efficiency of specific phenotypes of functional neurons from iPSCs or through somatic cell direct conversion. The realization of the full potential of iPSCs relies on the ability to precisely modify specific genome sequences. Genome editing technologies including zinc finger nucleases, transcription activator-like effector nucleases, and clustered regularly interspaced short palindromic repeat/CAS9 RNA-guided nucleases have progressed very fast over the last years. The combination of genome-editing strategies and patient-specific iPSC biology will offer a unique platform for in vitro generation of diseased and corrected neural derivatives for personalized therapies, disease modeling and drug screening.


Assuntos
Diferenciação Celular/fisiologia , Reprogramação Celular/fisiologia , Engenharia Genética , Células-Tronco Pluripotentes Induzidas/citologia , Neurônios/citologia , Animais , Diferenciação Celular/genética , Fibroblastos/citologia , Engenharia Genética/métodos , Humanos , Neurônios/metabolismo
8.
Haematologica ; 99(7): 1168-75, 2014 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-24727813

RESUMO

Aplastic anemia is a life-threatening bone marrow failure disorder characterized by peripheral pancytopenia and marrow hypoplasia. The majority of cases of aplastic anemia remain idiopathic, although hematopoietic stem cell deficiency and impaired immune responses are hallmarks underlying the bone marrow failure in this condition. Mesenchymal stem/stromal cells constitute an essential component of the bone marrow hematopoietic microenvironment because of their immunomodulatory properties and their ability to support hematopoiesis, and they have been involved in the pathogenesis of several hematologic malignancies. We investigated whether bone marrow mesenchymal stem cells contribute, directly or indirectly, to the pathogenesis of aplastic anemia. We found that mesenchymal stem cell cultures can be established from the bone marrow of aplastic anemia patients and display the same phenotype and differentiation potential as their counterparts from normal bone marrow. Mesenchymal stem cells from aplastic anemia patients support the in vitro homeostasis and the in vivo repopulating function of CD34(+) cells, and maintain their immunosuppressive and anti-inflammatory properties. These data demonstrate that bone marrow mesenchymal stem cells from patients with aplastic anemia do not have impaired functional and immunological properties, suggesting that they do not contribute to the pathogenesis of the disease.


Assuntos
Anemia Aplástica/imunologia , Anemia Aplástica/metabolismo , Células-Tronco Mesenquimais/imunologia , Células-Tronco Mesenquimais/metabolismo , Adolescente , Adulto , Idoso , Anemia Aplástica/diagnóstico , Anemia Aplástica/etiologia , Antígenos CD34/metabolismo , Estudos de Casos e Controles , Diferenciação Celular , Células Cultivadas , Criança , Técnicas de Cocultura , Feminino , Sangue Fetal/citologia , Sobrevivência de Enxerto , Transplante de Células-Tronco Hematopoéticas , Células-Tronco Hematopoéticas/citologia , Células-Tronco Hematopoéticas/metabolismo , Humanos , Imunomodulação , Imunofenotipagem , Masculino , Células-Tronco Mesenquimais/citologia , Pessoa de Meia-Idade , Fenótipo , Adulto Jovem
9.
Haematologica ; 98(7): 1022-9, 2013 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-23349299

RESUMO

The homeostasis of the hematopoietic stem/progenitor cell pool relies on a fine-tuned balance between self-renewal, differentiation and proliferation. Recent studies have proposed that mitochondria regulate these processes. Although recent work has contributed to understanding the role of mitochondria during stem cell differentiation, it remains unclear whether the mitochondrial content/function affects human hematopoietic stem versus progenitor function. We found that mitochondrial mass correlates strongly with mitochondrial membrane potential in CD34(+) hematopoietic stem/progenitor cells. We, therefore, sorted cord blood CD34(+) cells on the basis of their mitochondrial mass and analyzed the in vitro homeostasis and clonogenic potential as well as the in vivo repopulating potential of CD34(+) cells with high (CD34(+) Mito(High)) versus low (CD34(+) Mito(Low)) mitochondrial mass. The CD34(+) Mito(Low) fraction contained 6-fold more CD34(+)CD38(-) primitive cells and was enriched in hematopoietic stem cell function, as demonstrated by its significantly greater hematopoietic reconstitution potential in immuno-deficient mice. In contrast, the CD34(+) Mito(High) fraction was more enriched in hematopoietic progenitor function with higher in vitro clonogenic capacity. In vitro differentiation of CD34(+) Mito(Low) cells was significantly delayed as compared to that of CD34(+) Mito(High) cells. The eventual complete differentiation of CD34(+) Mito(Low) cells, which coincided with a robust expansion of the CD34(-) differentiated progeny, was accompanied by mitochondrial adaptation, as shown by significant increases in ATP production and expression of the mitochondrial genes ND1 and COX2. In conclusion, cord blood CD34(+) cells with low levels of mitochondrial mass are enriched in hematopoietic repopulating stem cell function whereas high levels of mitochondrial mass identify hematopoietic progenitors. A mitochondrial response underlies hematopoietic stem/progenitor cell differentiation and proliferation of lineage-committed CD34(-) cells.


Assuntos
Antígenos CD34/biossíntese , Diferenciação Celular/fisiologia , Proliferação de Células , Sangue Fetal/citologia , Sangue Fetal/fisiologia , Células-Tronco Hematopoéticas/citologia , Células-Tronco Hematopoéticas/fisiologia , Tamanho Mitocondrial/fisiologia , Animais , Antígenos CD34/sangue , Células Cultivadas , Humanos , Recém-Nascido , Potencial da Membrana Mitocondrial/fisiologia , Camundongos , Camundongos SCID
10.
Stem Cell Res ; 64: 102906, 2022 10.
Artigo em Inglês | MEDLINE | ID: mdl-36087523

RESUMO

Germline SAMD9 mutations are one of the most common alterations that predispose to pediatric myelodysplastic syndrome (MDS), a clonal disorder characterized by ineffective hematopoiesis, increasing the risk of developing acute myeloid leukemia (AML). Up to date, a disease model to study the role of SAMD9 mutation in MDS is still lacking. Here, we have generated a human induced pluripotent stem cell (hiPSC) line carrying SAMD9mut (p.I1567M), taking advantage of CRISPR/Cas9 system. As a result, the genetic engineered hiPSC line represent a new in vitro disease model to understand the impact of SAMD9 mutation at molecular and cellular level during hematopoiesis.


Assuntos
Células-Tronco Pluripotentes Induzidas , Leucemia Mieloide Aguda , Síndromes Mielodisplásicas , Humanos , Criança , Células-Tronco Pluripotentes Induzidas/metabolismo , Sistemas CRISPR-Cas/genética , Síndromes Mielodisplásicas/genética , Síndromes Mielodisplásicas/metabolismo , Heterozigoto , Mutação/genética , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/genética
11.
Methods Mol Biol ; 2185: 281-298, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33165855

RESUMO

Umbilical Cord Blood (CB) is a rich source of hematopoietic stem/progenitor cells (HSPCs) with high proliferative capacity and a naïve immune status. These characteristics, among others, make CB a good source of HSPCs not only for transplantation, but also for biomedical research purposes. Here we describe the methods for human CB-HSPCs isolation, as well as their culture and cryopreservation, viral transduction and sorting, and in vivo and in vitro assays in order to study leukemic processes.


Assuntos
Técnicas de Cultura de Células , Criopreservação , Sangue Fetal , Citometria de Fluxo , Células-Tronco Hematopoéticas , Sangue Fetal/citologia , Sangue Fetal/metabolismo , Células-Tronco Hematopoéticas/citologia , Células-Tronco Hematopoéticas/metabolismo , Humanos
12.
Stem Cell Res ; 55: 102445, 2021 08.
Artigo em Inglês | MEDLINE | ID: mdl-34284273

RESUMO

Germline heterozygous GATA2 mutations underlie a complex disorder characterized by bone marrow failure, immunodeficiency and high risk to develop myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML). Our understanding about GATA2 deficiency is limited due to the lack of relevant disease models. Here we generated high quality human induced pluripotent stem cell (iPSC) lines carrying two of the most recurrent germline GATA2 mutations (R389W and R396Q) associated with MDS, using CRISPR/Cas9. These hiPSCs represent an in vitro model to study the molecular and cellular mechanisms underlying GATA2 deficiency, when differentiated into blood progenitors.


Assuntos
Deficiência de GATA2 , Células-Tronco Pluripotentes Induzidas , Síndromes Mielodisplásicas , Sistemas CRISPR-Cas/genética , Fator de Transcrição GATA2/genética , Heterozigoto , Humanos , Síndromes Mielodisplásicas/genética
13.
STAR Protoc ; 1(1)2020 06 19.
Artigo em Inglês | MEDLINE | ID: mdl-32984851

RESUMO

Here, we provide a detailed protocol for synthetic lethality screens in a Jurkat T cell leukemia line using cell death as the readout measuring the combinatorial effect of a pan-PI3K inhibitor (GDC0941) with specific gene depletion by shRNA. We describe the use of an ultra-complex shRNA library, coverage considerations, time frames, protocol details, and bottlenecks with images to facilitate similar approaches. We discuss how this protocol resource can be readily adapted by investigators. For complete details on the use and execution of this protocol, please refer to (Mues et al., 2019).


Assuntos
RNA Interferente Pequeno , Testes de Toxicidade/métodos , Biblioteca Gênica , Humanos , Indazóis , Células Jurkat , Inibidores de Fosfoinositídeo-3 Quinase , Sulfonamidas
14.
STAR Protoc ; 1(2): 100067, 2020 09 18.
Artigo em Inglês | MEDLINE | ID: mdl-33111105

RESUMO

Stimulation of naive T lymphocytes via the T cell receptor (TCR) induces distinct phosphorylation patterns that can be used to explore various signaling pathways within the cell. This protocol can be used to characterize different perturbations to the signaling pathways and the variations in time of stimulation. Here, we provide a method of barcoding and consolidating a maximum of 24 different sample conditions using two florescent dyes. This single sample for phospho-staining and flow cytometry saves time and reagents. For complete details on the use and execution of this protocol, please refer to Krutzik and Nolan (2006), Krutzik et al. (2012), Vercoulen et al. (2017), Ksionda et al. (2018), and Myers et al. (2019).


Assuntos
Citometria de Fluxo/métodos , Corantes Fluorescentes , Técnicas de Sonda Molecular , Linfócitos T , Animais , Células Cultivadas , Corantes Fluorescentes/química , Corantes Fluorescentes/metabolismo , Camundongos , Fosforilação , Receptores de Antígenos de Linfócitos T/química , Receptores de Antígenos de Linfócitos T/metabolismo , Transdução de Sinais , Linfócitos T/química , Linfócitos T/citologia , Linfócitos T/metabolismo , Fatores de Tempo
15.
Oncogene ; 39(45): 6920-6934, 2020 11.
Artigo em Inglês | MEDLINE | ID: mdl-32989257

RESUMO

Oncogenic mutations in RAS genes, like KRASG12D or NRASG12D, trap Ras in the active state and cause myeloproliferative disorder and T cell leukemia (T-ALL) when induced in the bone marrow via Mx1CRE. The RAS exchange factor RASGRP1 is frequently overexpressed in T-ALL patients. In T-ALL cell lines overexpression of RASGRP1 increases flux through the RASGTP/RasGDP cycle. Here we expanded RASGRP1 expression surveys in pediatric T-ALL and generated a RoLoRiG mouse model crossed to Mx1CRE to determine the consequences of induced RASGRP1 overexpression in primary hematopoietic cells. RASGRP1-overexpressing, GFP-positive cells outcompeted wild type cells and dominated the peripheral blood compartment over time. RASGRP1 overexpression bestows gain-of-function colony formation properties to bone marrow progenitors in medium containing limited growth factors. RASGRP1 overexpression enhances baseline mTOR-S6 signaling in the bone marrow, but not in vitro cytokine-induced signals. In agreement with these mechanistic findings, hRASGRP1-ires-EGFP enhances fitness of stem- and progenitor- cells, but only in the context of native hematopoiesis. RASGRP1 overexpression is distinct from KRASG12D or NRASG12D, does not cause acute leukemia on its own, and leukemia virus insertion frequencies predict that RASGRP1 overexpression can effectively cooperate with lesions in many other genes to cause acute T-ALL.


Assuntos
Proteínas de Ligação a DNA/metabolismo , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Hematopoese/fisiologia , Células-Tronco Hematopoéticas/patologia , Leucemia-Linfoma Linfoblástico de Células T Precursoras/patologia , Animais , Medula Óssea/patologia , Células Cultivadas , Criança , Ensaio de Unidades Formadoras de Colônias , Proteínas de Ligação a DNA/genética , Modelos Animais de Doenças , Feminino , Regulação Leucêmica da Expressão Gênica , Fatores de Troca do Nucleotídeo Guanina/genética , Transplante de Células-Tronco Hematopoéticas , Humanos , Masculino , Camundongos , Camundongos Transgênicos , Leucemia-Linfoma Linfoblástico de Células T Precursoras/genética , Cultura Primária de Células , Proteínas Quinases S6 Ribossômicas/metabolismo , Transdução de Sinais/genética , Serina-Treonina Quinases TOR/metabolismo , Quimeras de Transplante
16.
Cell Rep ; 27(2): 631-647.e5, 2019 04 09.
Artigo em Inglês | MEDLINE | ID: mdl-30970263

RESUMO

Deregulated signal transduction is a cancer hallmark, and its complexity and interconnectivity imply that combination therapy should be considered, but large data volumes that cover the complexity are required in user-friendly ways. Here, we present a searchable database resource of synthetic lethality with a PI3 kinase signal transduction inhibitor by performing a saturation screen with an ultra-complex shRNA library containing 30 independent shRNAs per gene target. We focus on Ras-PI3 kinase signaling with T cell leukemia as a screening platform for multiple clinical and experimental reasons. Our resource predicts multiple combination-based therapies with high fidelity, ten of which we confirmed with small molecule inhibitors. Included are biochemical assays, as well as the IPI145 (duvelisib) inhibitor. We uncover the mechanism of synergy between the PI3 kinase inhibitor GDC0941 (pictilisib) and the tubulin inhibitor vincristine and demonstrate broad synergy in 28 cell lines of 5 cancer types and efficacy in preclinical leukemia mouse trials.


Assuntos
Neoplasias/tratamento farmacológico , Inibidores de Proteínas Quinases/uso terapêutico , RNA Interferente Pequeno/genética , Mutações Sintéticas Letais/genética , Animais , Modelos Animais de Doenças , Humanos , Camundongos , Inibidores de Proteínas Quinases/farmacologia , Transdução de Sinais
17.
Oncoimmunology ; 7(9): e1477460, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30228947

RESUMO

Treatment for acute myeloid leukemia (AML) remains suboptimal and many patients remain refractory or relapse upon standard chemotherapy based on nucleoside analogs plus anthracyclines. The crosstalk between AML cells and the BM stroma is a major mechanism underlying therapy resistance in AML. Lenalidomide and pomalidomide, a new generation immunomodulatory drugs (IMiDs), possess pleiotropic anti-leukemic properties including potent immune-modulating effects and are commonly used in hematological malignances associated with intrinsic dysfunctional BM such as myelodysplastic syndromes and multiple myeloma. Whether IMiDs may improve the efficacy of current standard treatment in AML remains understudied. Here, we have exploited in vitro and in vivo preclinical AML models to analyze whether IMiDs potentiate the efficacy of AraC/Idarubicin-based standard AML chemotherapy by interfering with the BM stroma-mediated chemoresistance. We report that IMiDs do not exert cytotoxic effects on either non-del5q/5q- AML cells nor BM-MSCs, but they enhance the immunomodulatory properties of BM-MSCs. When combined with AraC/Idarubicin, IMiDs fail to circumvent BM stroma-mediated resistance of non-del5q/5q- AML cells in vitro and in vivo but induce robust extramedullary mobilization of AML cells. When administered as a single agent, lenalidomide specifically mobilizes non-del5q/5q- AML cells, but not healthy CD34+ cells, to peripheral blood (PB) through specific downregulation of CXCR4 in AML blasts. Global gene expression profiling supports a migratory/mobilization gene signature in lenalidomide-treated non-del5q/5q- AML blasts but not in CD34+ cells. Collectively, IMiDs mobilize non-del5q/5q- AML blasts to PB through CXCR4 downregulation, but fail to potentiate AraC/Idarubicin activity in preclinical models of non-del5q/5q- AML.

19.
Stem Cell Res ; 24: 144-147, 2017 10.
Artigo em Inglês | MEDLINE | ID: mdl-28465093

RESUMO

We report the generation, CRISPR/Cas9-edition and characterization of induced pluripotent stem cell (iPSC) lines from a patient with coenzyme Q10 deficiency harboring the heterozygous mutation c.483G>C in the COQ4 gene. iPSCs were generated using non-integrative Sendai Viruses containing the reprogramming factors OCT4, SOX2, KLF4 and C-MYC. The iPSC lines carried the c.483G>C COQ4 mutation, silenced the OKSM expression and were mycoplasma-free. They were bona fide pluripotent cells as characterized by morphology, immunophenotype/gene expression for pluripotent-associated markers/genes, NANOG and OCT4 promoter demethylation, karyotype and teratoma formation. The COQ4 mutation was CRISPR/Cas9 edited resulting in isogenic, diploid and off-target free COQ4-corrected iPSCs.


Assuntos
Genômica/métodos , Células-Tronco Pluripotentes Induzidas/metabolismo , Proteínas Mitocondriais/genética , Ubiquinona/análogos & derivados , Diferenciação Celular , Pré-Escolar , Feminino , Heterozigoto , Humanos , Fator 4 Semelhante a Kruppel , Proteínas Mitocondriais/metabolismo , Mutação , Ubiquinona/deficiência
20.
Stem Cells Dev ; 25(3): 259-65, 2016 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-26603126

RESUMO

Hematopoietic stem cell (HSC) engraftment has been achieved using single-cell transplantation of prospectively highly purified adult HSC populations. However, bulk transplants are still performed when assessing the HSC potential of early embryonic hematopoietic tissues such as the aorta-gonad mesonephros (AGM) due to very low HSC activity content early in development. Intra-bone marrow transplantation (IBMT) has emerged as a superior administration route over intravenous (IV) transplantation for assessing the reconstituting ability of human HSCs in the xenotransplant setting since it bypasses the requirement for homing to the BM. In this study, we compared the ability of IBMT and IV administration of embryonic day 11.5 AGM-derived cells to reconstitute the hematopoietic system of myeloablated recipients. IBMT resulted in higher levels of AGM HSC long-term multilineage engraftment in the peripheral blood, BM, spleen, and thymus of primary and secondary recipients, and in limiting dilution experiments. The administration route did not skew the multilineage contribution pattern, but IBMT conferred higher Lineage(-)Sca-1(+)c-kit(+) long-term engraftment, in line with the superior IBMT reconstitution. Therefore, IBMT represents a superior administration route to detect HSC activity from developmentally early sources with limited HSC activity content, such as the AGM.


Assuntos
Transplante de Medula Óssea/métodos , Linhagem da Célula , Células-Tronco Embrionárias/citologia , Transplante de Células-Tronco Hematopoéticas/métodos , Mesonefro/citologia , Animais , Aorta/citologia , Aorta/embriologia , Células da Medula Óssea/citologia , Células Cultivadas , Células-Tronco Embrionárias/transplante , Gônadas/citologia , Gônadas/embriologia , Injeções Intravenosas , Camundongos , Camundongos Endogâmicos C57BL , Baço/citologia , Timo/citologia
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