RESUMO
The biology of haematopoietic stem cells (HSCs) has predominantly been studied under transplantation conditions1,2. It has been particularly challenging to study dynamic HSC behaviour, given that the visualization of HSCs in the native niche in live animals has not, to our knowledge, been achieved. Here we describe a dual genetic strategy in mice that restricts reporter labelling to a subset of the most quiescent long-term HSCs (LT-HSCs) and that is compatible with current intravital imaging approaches in the calvarial bone marrow3-5. We show that this subset of LT-HSCs resides close to both sinusoidal blood vessels and the endosteal surface. By contrast, multipotent progenitor cells (MPPs) show greater variation in distance from the endosteum and are more likely to be associated with transition zone vessels. LT-HSCs are not found in bone marrow niches with the deepest hypoxia and instead are found in hypoxic environments similar to those of MPPs. In vivo time-lapse imaging revealed that LT-HSCs at steady-state show limited motility. Activated LT-HSCs show heterogeneous responses, with some cells becoming highly motile and a fraction of HSCs expanding clonally within spatially restricted domains. These domains have defined characteristics, as HSC expansion is found almost exclusively in a subset of bone marrow cavities with bone-remodelling activity. By contrast, cavities with low bone-resorbing activity do not harbour expanding HSCs. These findings point to previously unknown heterogeneity within the bone marrow microenvironment, imposed by the stages of bone turnover. Our approach enables the direct visualization of HSC behaviours and dissection of heterogeneity in HSC niches.
Assuntos
Células-Tronco Hematopoéticas/metabolismo , Imagem Molecular , Animais , Remodelação Óssea , Movimento Celular , Proliferação de Células , Sobrevivência Celular , Feminino , Genes Reporter , Hipóxia/metabolismo , Proteína do Locus do Complexo MDS1 e EVI1/genética , Proteína do Locus do Complexo MDS1 e EVI1/metabolismo , Masculino , Camundongos , Oxigênio/metabolismo , Crânio/citologia , Tirosina Quinase 3 Semelhante a fms/genética , Tirosina Quinase 3 Semelhante a fms/metabolismoRESUMO
One mechanism by which oncoproteins work is through perturbation of cellular maturation; understanding the mechanisms by which this occurs can lead to the development of targeted therapies. EVI1 is a zinc finger oncoprotein involved in the development of acute myeloid leukemia; previous work has shown it to interfere with the maturation of granulocytes from immature precursors. Here we investigate the mechanism by which that occurs, using an immortalized hematopoietic progenitor cell line, EML-C1, as a model system. We document that overexpression of EVI1 abrogates retinoic acid-induced maturation of EML cells into committed myeloid cells, a process that can be documented by the down-regulation of stem cell antigen-1 and acquisition of responsiveness to granulocyte-macrophage colony-stimulating factor. We show that this requires DNA binding capacity of EVI1, suggesting that downstream target genes are involved. We identify the myeloid regulator Cebpa as a target gene and identify two EVI1 binding regions within evolutionarily conserved enhancer elements at +35 and +37 kb relative to the gene. EVI1 can strongly suppress Cebpa transcription, and add-back of Cebpa into EVI1-expressing EML cells partially corrects the block in maturation. We identify the DNA sequences to which EVI1 binds at +35 and +37 kb and show that mutation of one of these releases Cebpa from EVI1-induced suppression. We observe a more complex picture in primary bone marrow cells, where EVI1 suppresses Cebpa in stem cells but not in more committed progenitors. Our data thus identify a regulatory node by which EVI1 contributes to leukemia, and this represents a possible therapeutic target for treatment of EVI1-expressing leukemia.
Assuntos
Proteínas Estimuladoras de Ligação a CCAAT/metabolismo , Diferenciação Celular , Proteínas de Ligação a DNA/metabolismo , Células-Tronco Hematopoéticas/metabolismo , Elementos de Resposta , Fatores de Transcrição/metabolismo , Transcrição Gênica , Animais , Antígenos Ly/genética , Antígenos Ly/metabolismo , Células da Medula Óssea/metabolismo , Proteínas Estimuladoras de Ligação a CCAAT/genética , Linhagem Celular , Cricetinae , Proteínas de Ligação a DNA/genética , Leucemia/genética , Leucemia/metabolismo , Proteína do Locus do Complexo MDS1 e EVI1 , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Camundongos , Proto-Oncogenes/genética , Fatores de Transcrição/genéticaRESUMO
A subgroup of leukemogenic mixed-lineage leukemia (MLL) fusion proteins (MFPs) including MLL-AF9 activates the Mecom locus and exhibits extremely poor clinical prognosis. Mecom encodes EVI1 and MDS1-EVI1 (ME) proteins via alternative transcription start sites; these differ by the presence of a PRDI-BF1-RIZ1 (PR) domain with histone methyltransferase activity in the ME isoform. Using an ME-deficient mouse, we show that ME is required for MLL-AF9-induced transformation both in vitro and in vivo. And, although Nup98-HOXA9, MEIS1-HOXA9, and E2A-Hlf could transform ME-deficient cells, both MLL-AF9 and MLL-ENL were ineffective, indicating that the ME requirement is specific to MLL fusion leukemia. Further, we show that the PR domain is essential for MFP-induced transformation. These studies clearly indicate an essential role of PR-domain protein ME in MFP leukemia, suggesting that ME may be a novel target for therapeutic intervention for this group of leukemias.
Assuntos
Regulação Leucêmica da Expressão Gênica , Leucemia Aguda Bifenotípica/metabolismo , Proteína de Leucina Linfoide-Mieloide/genética , Proteína de Leucina Linfoide-Mieloide/metabolismo , Proteínas de Fusão Oncogênica/genética , Proteínas de Fusão Oncogênica/metabolismo , Alelos , Animais , Medula Óssea/patologia , Linhagem da Célula , Transformação Celular Neoplásica , Éxons , Humanos , Camundongos , Camundongos Knockout , Fenótipo , Isoformas de ProteínasRESUMO
The EVI1 oncogene at human chr 3q26 is rearranged and/or overexpressed in a subset of acute myeloid leukemias and myelodysplasias. The EVI1 protein is a 135 kDa transcriptional regulator with DNA-binding zinc finger domains. Here we provide a critical review of the current state of research into the molecular mechanisms by which this gene plays a role in myeloid malignancies. The major pertinent cellular effects are blocking myeloid differentiation and preventing cellular apoptosis, and several potential mechanisms for these phenomena have been identified. Evidence supports a role for EVI1 in inducing cellular quiescence, and this may contribute to the resistance to chemotherapy seen in patients with neoplasms that overexpress EVI1. Another isoform, MDS1-EVI1 (or PRDM3), encoded by the same locus as EVI1, harbors an N-terminal histone methyltransferase(HMT) domain; experimental findings indicate that this protein and its HMT activity are critical for the progression of a subset of AMLs, and this provides a potential target for therapeutic intervention.
Assuntos
Proteínas de Ligação a DNA/genética , Leucemia Mieloide/genética , Proto-Oncogenes/genética , Fatores de Transcrição/genética , Animais , Aberrações Cromossômicas , Proteínas de Ligação a DNA/metabolismo , Modelos Animais de Doenças , Epigênese Genética , Regulação Leucêmica da Expressão Gênica , Loci Gênicos , Humanos , Leucemia Mieloide/metabolismo , Leucemia Mieloide/patologia , Proteína do Locus do Complexo MDS1 e EVI1 , Camundongos , Transdução de Sinais , Fatores de Transcrição/metabolismoAssuntos
Linfoma Difuso de Grandes Células B/diagnóstico por imagem , Neoplasias Esplênicas/diagnóstico por imagem , Humanos , Imunofenotipagem , Leucemia , Linfoma Difuso de Grandes Células B/diagnóstico , Linfoma Difuso de Grandes Células B/patologia , Masculino , Pessoa de Meia-Idade , Neoplasias Esplênicas/diagnósticoRESUMO
Numerous examples exist of how disrupting the actions of physiological regulators of blood cell development yields hematologic malignancies. The master regulator of hematopoietic stem/progenitor cells GATA-2 was cloned almost 20 years ago, and elegant genetic analyses demonstrated its essential function to promote hematopoiesis. While certain GATA-2 target genes are implicated in leukemogenesis, only recently have definitive insights emerged linking GATA-2 to human hematologic pathophysiologies. These pathophysiologies include myelodysplastic syndrome, acute myeloid leukemia and an immunodeficiency syndrome with complex phenotypes including leukemia. As GATA-2 has a pivotal role in the etiology of human cancer, it is instructive to consider mechanisms underlying normal GATA factor function/regulation and how dissecting such mechanisms may reveal unique opportunities for thwarting GATA-2-dependent processes in a therapeutic context. This article highlights GATA factor mechanistic principles, with a heavy emphasis on GATA-1 and GATA-2 functions in the hematopoietic system, and new links between GATA-2 dysregulation and human pathophysiologies.
Assuntos
Fatores de Transcrição GATA/metabolismo , Neoplasias Hematológicas/genética , Fator de Transcrição GATA2/metabolismo , Neoplasias Hematológicas/metabolismo , Humanos , Processamento de Proteína Pós-TraducionalRESUMO
Human colorectal cancer (CRC) cells are resistant to the anti-proliferative effect of transforming growth factor-ß (TGF-ß), suggesting that disruption of TGF-ß signaling plays an important role in colorectal carcinogenesis. Ecotropic virus integration site-1 (Evi-1) oncoprotein represses TGF-ß signaling by interacting with Smads, but its role in CRC has not been established. The purpose of this study is to determine whether Evi-1 plays role(s) in CRCs and to characterize Evi-1 transcript(s) in CRCs. Evi-1 was overexpressed in 53% of human CRC samples, 100% of colon adenoma samples, and 100% of human colon cancer cell lines tested. Using 5' RACE, we cloned a novel Evi-1 transcript (Evi-1e) from a human CRC tissue and found that this novel transcript was expressed at a higher level in CRC tissues than in normal tissues and was the major Evi-1 transcript in CRCs. Transient Evi-1 transfection inhibited TGF-ß-induced transcriptional activity and reversed the growth inhibitory effect of TGF-ß in MC-26 mouse colon cancer cells. In conclusion, we have identified overexpression of Evi-1 oncoprotein as a novel mechanism by which a subset of human CRCs may escape TGF-ß regulation. We have also identified a novel Evi-1 transcript, Evi-1e, as the major Evi-1 transcript expressed in human CRCs.
Assuntos
Neoplasias Colorretais/genética , Neoplasias Colorretais/metabolismo , Proteínas de Ligação a DNA/genética , Proto-Oncogenes/genética , Fatores de Transcrição/genética , Fator de Crescimento Transformador beta/metabolismo , Animais , Sequência de Bases , Linhagem Celular Tumoral , Colo/metabolismo , Colo/patologia , Neoplasias Colorretais/patologia , Proteínas de Ligação a DNA/metabolismo , Éxons , Regulação Neoplásica da Expressão Gênica , Humanos , Proteína do Locus do Complexo MDS1 e EVI1 , Camundongos , Regiões Promotoras Genéticas , Reto/metabolismo , Reto/patologia , Transdução de Sinais , Fatores de Transcrição/metabolismo , Regulação para CimaRESUMO
The Mds1 and Evi1 complex locus (Mecom) gives rise to several alternative transcripts implicated in leukemogenesis. However, the contribution that Mecom-derived gene products make to normal hematopoiesis remains largely unexplored. To investigate the role of the upstream transcription start site of Mecom in adult hematopoiesis, we created a mouse model with a lacZ knock-in at this site, termed ME(m1), which eliminates Mds1-Evi1 (ME), the longer, PR-domain-containing isoform produced by the gene (also known as PRDM3). ß-galactosidase-marking studies revealed that, within hematopoietic cells, ME is exclusively expressed in the stem cell compartment. ME deficiency leads to a reduction in the number of HSCs and a complete loss of long-term repopulation capacity, whereas the stem cell compartment is shifted from quiescence to active cycling. Genetic exploration of the relative roles of endogenous ME and EVI1 isoforms revealed that ME preferentially rescues long-term HSC defects. RNA-seq analysis in Lin(-)Sca-1(+)c-Kit(+) cells (LSKs) of ME(m1) documents near complete silencing of Cdkn1c, encoding negative cell-cycle regulator p57-Kip2. Reintroduction of ME into ME(m1) LSKs leads to normalization of both p57-Kip2 expression and growth control. Our results clearly demonstrate a critical role of PR-domain-containing ME in linking p57-kip2 regulation to long-term HSC function.
Assuntos
Hematopoese , Células-Tronco Hematopoéticas/citologia , Animais , Inibidor de Quinase Dependente de Ciclina p57/genética , Éxons , Regulação da Expressão Gênica no Desenvolvimento , Técnicas de Introdução de Genes , Técnicas de Inativação de Genes , Células-Tronco Hematopoéticas/metabolismo , Óperon Lac , Leucemia/genética , Leucocitose/genética , Camundongos , Camundongos Endogâmicos C57BL , Proteínas de Fusão Oncogênica/genética , Proteínas de Fusão Oncogênica/metabolismo , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Estrutura Terciária de Proteína , Trombocitopenia/genéticaRESUMO
A cis-regulatory genetic element which targets gene expression to stem cells, termed stem cell enhancer, serves as a molecular handle for stem cell-specific genetic engineering. Here we show the generation and characterization of a tamoxifen-inducible CreERT2 transgenic (Tg) mouse employing previously identified hematopoietic stem cell (HSC) enhancer for Runx1, eR1 (+24 m). Kinetic analysis of labeled cells after tamoxifen injection and transplantation assays revealed that eR1-driven CreERT2 activity marks dormant adult HSCs which slowly but steadily contribute to unperturbed hematopoiesis. Fetal and child HSCs that are uniformly or intermediately active were also efficiently targeted. Notably, a gene ablation at distinct developmental stages, enabled by this system, resulted in different phenotypes. Similarly, an oncogenic Kras induction at distinct ages caused different spectrums of malignant diseases. These results demonstrate that the eR1-CreERT2 Tg mouse serves as a powerful resource for the analyses of both normal and malignant HSCs at all developmental stages.
Assuntos
Células-Tronco Adultas , Células-Tronco Hematopoéticas , Animais , Camundongos , Cinética , Feto , Engenharia Genética , Camundongos Transgênicos , Subunidade alfa 2 de Fator de Ligação ao Core/genéticaRESUMO
The zinc finger protein EVI1 is causally associated with acute myeloid leukemogenesis, and inhibition of its function with a small molecule therapeutic may provide effective therapy for EVI1-expressing leukemias. In this paper we describe the development of a pyrrole-imidazole polyamide to specifically block EVI1 binding to DNA. We first identify essential domains for leukemogenesis through structure-function studies on both EVI1 and the t(3;21)(q26;q22)-derived RUNX1-MDS1-EVI1 (RME) protein, which revealed that DNA binding to the cognate motif GACAAGATA via the first of two zinc finger domains (ZF1, encompassing fingers 1-7) is essential transforming activity. To inhibit DNA binding via ZF1, we synthesized a pyrrole-imidazole polyamide 1, designed to bind to a subsite within the GACAAGATA motif and thereby block EVI1 binding. DNase I footprinting and electromobility shift assays revealed a specific and high affinity interaction between polyamide 1 and the GACAAGATA motif. In an in vivo CAT reporter assay using NIH-3T3-derived cell line with a chromosome-embedded tet-inducible EVI1-VP16 as well as an EVI1-responsive reporter, polyamide 1 completely blocked EVI1-responsive reporter activity. Growth of a leukemic cell line bearing overexpressed EVI1 was also inhibited by treatment with polyamide 1, while a control cell line lacking EVI1 was not. Finally, colony formation by RME was attenuated by polyamide 1 in a serial replating assay. These studies provide evidence that a cell permeable small molecule may effectively block the activity of a leukemogenic transcription factor and provide a valuable tool to dissect critical functions of EVI1 in leukemogenesis.
Assuntos
Proteínas de Ligação a DNA/antagonistas & inibidores , Proteínas de Ligação a DNA/química , Inibidores do Crescimento/farmacologia , Imidazóis/farmacologia , Nylons/farmacologia , Pirróis/farmacologia , Fatores de Transcrição/antagonistas & inibidores , Fatores de Transcrição/química , Sequência de Aminoácidos , Animais , Sequência de Bases , Linhagem Celular Transformada , Linhagem Celular Tumoral , Proteínas de Ligação a DNA/genética , Sistemas de Liberação de Medicamentos/métodos , Inibidores do Crescimento/química , Inibidores do Crescimento/metabolismo , Humanos , Imidazóis/química , Imidazóis/metabolismo , Proteína do Locus do Complexo MDS1 e EVI1 , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Células Mieloides/efeitos dos fármacos , Células Mieloides/metabolismo , Células Mieloides/patologia , Nylons/química , Nylons/metabolismo , Ligação Proteica/genética , Proto-Oncogenes/genética , Pirróis/química , Pirróis/metabolismo , Ratos , Retroviridae/genética , Fatores de Transcrição/genéticaRESUMO
Hematopoietic ontogeny consists of two broad programs: an initial hematopoietic stem cell (HSC)-independent program followed by HSC-dependent hematopoiesis that sequentially seed the fetal liver and generate blood cells. However, the transition from HSC-independent to HSC-derived hematopoiesis remains poorly characterized. To help resolve this question, we developed Mds1CreERT2 mice, which inducibly express Cre-recombinase in emerging HSCs in the aorta and label long-term adult HSCs, but not HSC-independent yolk-sac-derived primitive or definitive erythromyeloid (EMP) hematopoiesis. Our lineage-tracing studies indicate that HSC-derived erythroid, myeloid, and lymphoid progeny significantly expand in the liver and blood stream between E14.5 and E16.5. Additionally, we find that HSCs contribute the majority of F4/80+ macrophages in adult spleen and marrow, in contrast to their limited contribution to macrophage populations in brain, liver, and lungs. The Mds1CreERT2 mouse model will be useful to deconvolute the complexity of hematopoiesis as it unfolds in the embryo and functions postnatally.
Assuntos
Envelhecimento/metabolismo , Alelos , Células-Tronco Hematopoéticas/metabolismo , Integrases/metabolismo , Animais , Linhagem da Célula/efeitos dos fármacos , Embrião de Mamíferos/metabolismo , Feto/citologia , Hemangioblastos/metabolismo , Hematopoese/efeitos dos fármacos , Fígado/embriologia , Proteína do Locus do Complexo MDS1 e EVI1 , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Tamoxifeno/farmacologiaRESUMO
Mds1-Evi1 (also known as Prdm3) and Prdm16 are two highly related zinc finger transcription factors that, within the hematopoietic system, are both expressed primarily in hematopoietic stem cells (HSCs). Our laboratory previously found that constitutive Mds1-Evi1 knockout mice are viable, but their HSCs are unable to withstand myeloablative chemotherapy or effectively transplant irradiated recipient mice. A similar phenotype has been observed for Prdm16, except that the Prdm16 constitutive knockout is lethal. Here, we created a novel double-knockout model of Mds1-Evi1 and Prdm16 in the bone marrow, in which double knockout occurs only in cells that endogenously express Mds1-Evi1 and only upon induction with tamoxifen. We show that combined Mds1-Evi1/Prdm16 deficiency causes bone marrow failure within 15 days, with rapid loss in all progenitor compartments, while the peripheral blood exhibits progressive reductions in peripheral monocytes and granulocytes. We found that surviving hematopoietic stem cells and granulocytic progenitors had elevated apoptosis and cell division, and were unable to form colonies in vitro; adding back wild-type Mds1-Evi1 or Prdm16 to double-knockout bone marrow restores colony formation, and for MDS1-EVI1, this activity depends on a functional PR domain. All of these phenotypic effects were exhibited at milder levels in Mds1-Evi1 and Prdm16 single-knockout controls. Overall, these results illustrate that Mds1-Evi1 and Prdm16 play additive roles in maintaining normal hematopoietic stem cell survival.
Assuntos
Apoptose/fisiologia , Proteínas de Ligação a DNA/metabolismo , Células Precursoras de Granulócitos/metabolismo , Hematopoese/fisiologia , Proteína do Locus do Complexo MDS1 e EVI1/metabolismo , Modelos Biológicos , Fatores de Transcrição/metabolismo , Animais , Linhagem Celular , Sobrevivência Celular/genética , Proteínas de Ligação a DNA/genética , Células Precursoras de Granulócitos/citologia , Proteína do Locus do Complexo MDS1 e EVI1/genética , Camundongos , Camundongos Knockout , Fatores de Transcrição/genéticaRESUMO
The bone marrow microenvironment (BMME) contributes to the regulation of hematopoietic stem cell (HSC) function, though its role in age-associated lineage skewing is poorly understood. Here we show that dysfunction of aged marrow macrophages (Mφs) directs HSC platelet-bias. Mφs from the marrow of aged mice and humans exhibited an activated phenotype, with increased expression of inflammatory signals. Aged marrow Mφs also displayed decreased phagocytic function. Senescent neutrophils, typically cleared by marrow Mφs, were markedly increased in aged mice, consistent with functional defects in Mφ phagocytosis and efferocytosis. In aged mice, Interleukin 1B (IL1B) was elevated in the bone marrow and caspase 1 activity, which can process pro-IL1B, was increased in marrow Mφs and neutrophils. Mechanistically, IL1B signaling was necessary and sufficient to induce a platelet bias in HSCs. In young mice, depletion of phagocytic cell populations or loss of the efferocytic receptor Axl expanded platelet-biased HSCs. Our data support a model wherein increased inflammatory signals and decreased phagocytic function of aged marrow Mφs induce the acquisition of platelet bias in aged HSCs. This work highlights the instructive role of Mφs and IL1B in the age-associated lineage-skewing of HSCs, and reveals the therapeutic potential of their manipulation as antigeronic targets.
Assuntos
Envelhecimento/fisiologia , Plaquetas/metabolismo , Medula Óssea/metabolismo , Células-Tronco Hematopoéticas/metabolismo , Interleucina-1beta/metabolismo , Macrófagos/metabolismo , Animais , Medula Óssea/patologia , Caspase 1/metabolismo , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Neutrófilos , Fagocitose , Fenótipo , Proteínas Proto-Oncogênicas , Receptores Proteína Tirosina Quinases , Receptor Tirosina Quinase AxlRESUMO
Inv(3q26) and t(3:3)(q21;q26) are specific to poor-prognosis myeloid malignancies, and result in marked overexpression of EVI1, a zinc-finger transcription factor and myeloid-specific oncoprotein. Despite extensive study, the mechanism by which EVI1 contributes to myeloid malignancy remains unclear. Here we describe a new mouse model that mimics the transcriptional effects of 3q26 rearrangement. We show that EVI1 overexpression causes global distortion of hematopoiesis, with suppression of erythropoiesis and lymphopoiesis, and marked premalignant expansion of myelopoiesis that eventually results in leukemic transformation. We show that myeloid skewing is dependent on DNA binding by EVI1, which upregulates Spi1, encoding master myeloid regulator PU.1. We show that EVI1 binds to the -14 kb upstream regulatory element (-14kbURE) at Spi1; knockdown of Spi1 dampens the myeloid skewing. Furthermore, deletion of the -14kbURE at Spi1 abrogates the effects of EVI1 on hematopoietic stem cells. These findings support a novel mechanism of leukemogenesis through EVI1 overexpression.
Assuntos
Proteína do Locus do Complexo MDS1 e EVI1/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Transativadores/metabolismo , Alelos , Animais , Apoptose/genética , Apoptose/fisiologia , Linhagem Celular , Proliferação de Células/genética , Proliferação de Células/fisiologia , Citometria de Fluxo , Hematopoese/genética , Hematopoese/fisiologia , Proteína do Locus do Complexo MDS1 e EVI1/genética , Camundongos , Proteínas Proto-Oncogênicas/genética , Transativadores/genéticaRESUMO
Expression of the MECOM (also known as EVI1) proto-oncogene is deregulated by chromosomal translocations in some cases of acute myeloid leukemia (AML) and is associated with poor clinical outcome. Here, through transcriptomic and metabolomic profiling of hematopoietic cells, we reveal that EVI1 overexpression alters cellular metabolism. A screen using pooled short hairpin RNAs (shRNAs) identified the ATP-buffering, mitochondrial creatine kinase CKMT1 as necessary for survival of EVI1-expressing cells in subjects with EVI1-positive AML. EVI1 promotes CKMT1 expression by repressing the myeloid differentiation regulator RUNX1. Suppression of arginine-creatine metabolism by CKMT1-directed shRNAs or by the small molecule cyclocreatine selectively decreased the viability, promoted the cell cycle arrest and apoptosis of human EVI1-positive cell lines, and prolonged survival in both orthotopic xenograft models and mouse models of primary AML. CKMT1 inhibition altered mitochondrial respiration and ATP production, an effect that was abrogated by phosphocreatine-mediated reactivation of the arginine-creatine pathway. Targeting CKMT1 is thus a promising therapeutic strategy for this EVI1-driven AML subtype that is highly resistant to current treatment regimens.
Assuntos
Subunidade alfa 2 de Fator de Ligação ao Core/genética , Creatina Quinase/genética , Proteínas de Ligação a DNA/genética , Regulação Neoplásica da Expressão Gênica/genética , Leucemia Mieloide Aguda/genética , Proto-Oncogenes/genética , Fatores de Transcrição/genética , Adulto , Idoso , Idoso de 80 Anos ou mais , Western Blotting , Simulação por Computador , Subunidade alfa 2 de Fator de Ligação ao Core/metabolismo , Creatina Quinase/metabolismo , Feminino , Citometria de Fluxo , Perfilação da Expressão Gênica , Estudo de Associação Genômica Ampla , Humanos , Leucemia Mieloide Aguda/metabolismo , Proteína do Locus do Complexo MDS1 e EVI1 , Masculino , Redes e Vias Metabólicas , Metabolômica , Pessoa de Meia-Idade , Mitocôndrias , Proto-Oncogene Mas , RNA Interferente PequenoRESUMO
Overexpression of ErbB2 and ErbB4 receptors in breast cancers may be accompanied by contrasting clinical outcomes. To investigate the molecular mechanisms contributing to these differences, we undertook a comparative study of gene expression regulated by the two receptors. Agonistic antibodies were employed to activate ErbB2 and ErbB4 in isolation from the other ErbBs in breast cancer cells. Gene expression profiling using a 16 755-gene oligonucleotide array was performed to identify transcriptional targets of receptor activation. Our results indicate that, in the same cell line, ErbB2 and ErbB4 activation influence gene transcription differentially. Although there are genes that are regulated by signaling from both receptors, there are also receptor-specific targets that are preferentially regulated by each receptor. We further show that two ligands acting via the same receptor homodimer may activate different subsets of genes. Many of the induced genes are hitherto unidentified targets of ErbB signaling. These include ErbB4 targets EPS15R, GATA4, and RAB2 and ErbB2-activated HRY/HES1 and PPAP2A. Targets of ErbB2 homodimer signaling may be especially important as markers in breast cancer, where ErbB2 homodimerization mediated by overexpression and ligand-independent activation is common.
Assuntos
Receptores ErbB/metabolismo , Regulação Neoplásica da Expressão Gênica/fisiologia , Receptor ErbB-2/genética , Transcrição Gênica/fisiologia , Neoplasias da Mama/metabolismo , Feminino , Perfilação da Expressão Gênica , Humanos , Ligantes , Análise de Sequência com Séries de Oligonucleotídeos , Receptor ErbB-2/biossíntese , Receptor ErbB-4 , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Células Tumorais Cultivadas , Regulação para CimaRESUMO
Several lines of evidence suggest that production of parathyroid hormone-related protein (PTHrP) by breast cancer cells contributes to the formation of bone metastases. However, it is not clear if PTHrP promotes access of cancer cells to the skeleton or if it simply promotes bone resorption around cells already within bone. To study the effects of PTHrP on the development of bone metastases, we treated mice overexpressing PTHrP in their mammary glands (K14-PTHrP transgenic mice) with 9,10-dimethyl-1,2-benz-anthracene (DMBA), a known mammary carcinogen. After DMBA treatment, K14-PTHrP mice showed a higher incidence of tumor formation and a shorter latency to tumor formation than wild-type littermates. Transgenic tumors expressed the K14-PTHrP transgene and secreted excess amounts of PTHrP. In response, tumor-bearing transgenic mice became hypercalcemic and had elevated circulating levels of PTHrP. Despite the development of visceral metastases, neither transgenic mice nor wild-type controls developed bone metastases. This was true even if tumor cells were introduced into the arterial circulation of immunodeficient mice. Our results are consistent with the emerging notion that the ability of breast cancer cells to produce PTHrP in response to cues from the bone microenvironment may be more important to the development of skeletal metastases than the production of PTHrP by cells within the primary breast cancer.
Assuntos
Neoplasias Ósseas/secundário , Expressão Gênica , Hipercalcemia/etiologia , Neoplasias Mamárias Experimentais/fisiopatologia , Hormônios Peptídicos/genética , Animais , Modelos Animais de Doenças , Feminino , Masculino , Neoplasias Mamárias Experimentais/sangue , Camundongos , Camundongos Transgênicos , Metástase Neoplásica , Proteína Relacionada ao Hormônio Paratireóideo , Células Tumorais CultivadasRESUMO
Recent studies have indicated a role for a MECOM allele in susceptibility to osteoporotic fractures in humans. We have generated a mutation in Mecom in mouse (termed ME(m1)) via lacZ knock-in into the upstream transcription start site for the gene, resulting in disruption of Mds1 and Mds1-Evi1 transcripts, but not of Evi1 transcripts. We demonstrate that ME(m1/m1) mice have severe kyphoscoliosis that is reminiscent of human congenital or primary kyphoscoliosis. ME(m1/m1) mice appear normal at birth, but by 2weeks, they exhibit a slight lumbar lordosis and narrowed intervertebral space. This progresses to severe lordosis with disc collapse and synostosis, together with kyphoscoliosis. Bone formation and strength testing show that ME(m1/m1) mice have normal bone formation and composition but are osteopenic. While endochondral bone development is normal, it is markedly dysplastic in its organization. Electron micrographs of the 1week postnatal intervertebral discs reveals marked disarray of collagen fibers, consistent with an inherent weakness in the non-osseous connective tissue associated with the spine. These findings indicate that lack of ME leads to a complex defect in both osseous and non-osseous musculoskeletal tissues, including a marked vertebral osteopenia, degeneration of the IVD, and disarray of connective tissues, which is likely due to an inherent inability to establish and/or maintain components of these tissues.
Assuntos
Doenças Ósseas Metabólicas/complicações , Doenças Ósseas Metabólicas/patologia , Proteínas de Ligação a DNA/metabolismo , Deleção de Genes , Coluna Vertebral/anormalidades , Fatores de Transcrição/metabolismo , Animais , Fenômenos Biomecânicos , Doenças Ósseas Metabólicas/diagnóstico por imagem , Doenças Ósseas Metabólicas/genética , Colágeno/genética , Colágeno/ultraestrutura , Feminino , Marcação de Genes , Loci Gênicos/genética , Proteínas Hedgehog/genética , Humanos , Disco Intervertebral/diagnóstico por imagem , Disco Intervertebral/patologia , Cifose/congênito , Cifose/diagnóstico por imagem , Cifose/genética , Cifose/patologia , Lordose/congênito , Lordose/diagnóstico por imagem , Lordose/genética , Lordose/patologia , Vértebras Lombares/diagnóstico por imagem , Vértebras Lombares/patologia , Proteína do Locus do Complexo MDS1 e EVI1 , Masculino , Camundongos , Mutação/genética , Osteogênese , Proto-Oncogenes , Receptor Tipo 1 de Hormônio Paratireóideo/genética , Coluna Vertebral/diagnóstico por imagem , Coluna Vertebral/patologia , Tendões/diagnóstico por imagem , Tendões/patologia , Tendões/ultraestrutura , Vértebras Torácicas/diagnóstico por imagem , Vértebras Torácicas/patologia , Microtomografia por Raio-XRESUMO
The ecotropic viral integration site 1 (Evi1) oncogenic transcription factor is one of a number of alternative transcripts encoded by the Mds1 and Evi1 complex locus (Mecom). Overexpression of Evi1 has been observed in a number of myeloid disorders and is associated with poor patient survival. It is also amplified and/or overexpressed in many epithelial cancers including nasopharyngeal carcinoma, ovarian carcinoma, ependymomas, and lung and colorectal cancers. Two murine knockout models have also demonstrated Evi1's critical role in the maintenance of hematopoietic stem cell renewal with its absence resulting in the death of mutant embryos due to hematopoietic failure. Here we characterize a novel mouse model (designated Evi1(fl3)) in which Evi1 exon 3, which carries the ATG start, is flanked by loxP sites. Unexpectedly, we found that germline deletion of exon3 produces a hypomorphic allele due to the use of an alternative ATG start site located in exon 4, resulting in a minor Evi1 N-terminal truncation and a block in expression of the Mds1-Evi1 fusion transcript. Evi1(δex3/δex3) mutant embryos showed only a mild non-lethal hematopoietic phenotype and bone marrow failure was only observed in adult Vav-iCre/+, Evi1(fl3/fl3) mice in which exon 3 was specifically deleted in the hematopoietic system. Evi1(δex3/δex3) knockout pups are born in normal numbers but die during the perinatal period from congenital heart defects. Database searches identified 143 genes with similar mutant heart phenotypes as those observed in Evi1(δex3/δex3) mutant pups. Interestingly, 42 of these congenital heart defect genes contain known Evi1-binding sites, and expression of 18 of these genes are also effected by Evi1 siRNA knockdown. These results show a potential functional involvement of Evi1 target genes in heart development and indicate that Evi1 is part of a transcriptional program that regulates cardiac development in addition to the development of blood.
Assuntos
Alelos , Proteínas de Ligação a DNA/genética , Estudos de Associação Genética , Cardiopatias Congênitas/genética , Proto-Oncogenes/genética , Fatores de Transcrição/genética , Animais , Animais Recém-Nascidos , Sequência de Bases , Medula Óssea/patologia , Proteínas de Ligação a DNA/química , Modelos Animais de Doenças , Éxons , Deleção de Genes , Regulação da Expressão Gênica no Desenvolvimento , Genes Letais , Cardiopatias Congênitas/mortalidade , Cardiopatias Congênitas/patologia , Cardiopatias Congênitas/fisiopatologia , Hematopoese , Células-Tronco Hematopoéticas/metabolismo , Imunofenotipagem , Proteína do Locus do Complexo MDS1 e EVI1 , Camundongos , Camundongos Knockout , Dados de Sequência Molecular , Mutação , Fenótipo , Alinhamento de Sequência , Índice de Gravidade de Doença , Fatores de Transcrição/químicaRESUMO
This review will begin with a detail of the revision of the WHO classification, and pathological definitions of Burkitt lymphoma. Over the past several years, molecular understanding of Burkitt lymphoma has improved significantly. Using gene expression profiling, a genomic "signature" of Burkitt lymphoma may be identified, that has fidelity beyond c-myc expression, and the presence of the classical t(8;14). Then, evaluation and therapy of the adult patient with Burkitt lymphoma will be reviewed. Relatively few data exist on optimal therapy of the adult patient with Burkitt lymphoma. Principles of therapy should include high doses of alkylating agents, frequent administration of chemotherapy, and attention to central nervous system (CNS) prophylaxis with high doses of systemic chemotherapy, intrathecal therapy, or both. The outcome of adult patients with Burkitt lymphoma, particularly those over 40 years of age, is inferior to the outcome of younger patients, but may be improving over the past few years. Results from an international collaborative effort, which are helpful in evaluating results of Burkitt lymphoma therapy in adults, will be presented. HIV-associated Burkitt lymphoma, and elderly patients with Burkitt lymphoma, comprise special clinical situations that will be also covered in this review.