RESUMO
Cholesterol homeostasis has been proposed as one mechanism contributing to chemoresistance in AML and hence, inclusion of statins in therapeutic regimens as part of clinical trials in AML has shown encouraging results. Chemical screening of primary human AML specimens by our group led to the identification of lipophilic statins as potent inhibitors of AMLs from a wide range of cytogenetic groups. Genetic screening to identify modulators of the statin response uncovered the role of protein geranylgeranylation and of RAB proteins, coordinating various aspect of vesicular trafficking, in mediating the effects of statins on AML cell viability. We further show that statins can inhibit vesicle-mediated transport in primary human specimens, and that statins sensitive samples show expression signatures reminiscent of enhanced vesicular trafficking. Overall, this study sheds light into the mechanism of action of statins in AML and identifies a novel vulnerability for cytogenetically diverse AML.
Assuntos
Inibidores de Hidroximetilglutaril-CoA Redutases , Leucemia Mieloide Aguda , Humanos , Inibidores de Hidroximetilglutaril-CoA Redutases/farmacologia , Inibidores de Hidroximetilglutaril-CoA Redutases/uso terapêutico , Leucemia Mieloide Aguda/tratamento farmacológico , Leucemia Mieloide Aguda/genéticaRESUMO
Acute myeloid leukemias (AML) with mutations in the NPM1 gene (NPM1c+) represent a large AML subgroup with varying response to conventional treatment, highlighting the need to develop targeted therapeutic strategies for this disease. We screened a library of clinical drugs on a cohort of primary human AML specimens and identified the BCL2 inhibitor ABT-199 as a selective agent against NPM1c+ AML. Mutational analysis of ABT-199-sensitive and -resistant specimens identified mutations in NPM1, RAD21, and IDH1/IDH2 as predictors of ABT-199 sensitivity. Comparative transcriptome analysis further uncovered BCL2A1 as a potential mediator of ABT-199 resistance in AML. In line with our observation that RAD21 mutation confers sensitivity to ABT-199, we provide functional evidence that reducing RAD21 levels can sensitize AML cells to BCL2 inhibition. Moreover, we demonstrate that ABT-199 is able to produce selective anti-AML activity in vivo toward AML with mutations associated with compound sensitivity in PDX models. Overall, this study delineates the contribution of several genetic events to the response to ABT-199 and provides a rationale for the development of targeted therapies for NPM1c+ AML.
Assuntos
Antineoplásicos/farmacologia , Compostos Bicíclicos Heterocíclicos com Pontes/farmacologia , Resistencia a Medicamentos Antineoplásicos/genética , Leucemia Mieloide Aguda/genética , Antígenos de Histocompatibilidade Menor/genética , Proteínas Proto-Oncogênicas c-bcl-2/genética , Sulfonamidas/farmacologia , Humanos , Leucemia Mieloide Aguda/tratamento farmacológico , Mutação , Proteínas Nucleares/genética , Nucleofosmina , Células Tumorais CultivadasRESUMO
To identify therapeutic targets in acute myeloid leukemia (AML), we chemically interrogated 200 sequenced primary specimens. Mubritinib, a known ERBB2 inhibitor, elicited strong anti-leukemic effects in vitro and in vivo. In the context of AML, mubritinib functions through ubiquinone-dependent inhibition of electron transport chain (ETC) complex I activity. Resistance to mubritinib characterized normal CD34+ hematopoietic cells and chemotherapy-sensitive AMLs, which displayed transcriptomic hallmarks of hypoxia. Conversely, sensitivity correlated with mitochondrial function-related gene expression levels and characterized a large subset of chemotherapy-resistant AMLs with oxidative phosphorylation (OXPHOS) hyperactivity. Altogether, our work thus identifies an ETC complex I inhibitor and reveals the genetic landscape of OXPHOS dependency in AML.
Assuntos
Antineoplásicos/farmacologia , Complexo I de Transporte de Elétrons/antagonistas & inibidores , Leucemia Mieloide Aguda/metabolismo , Oxazóis/farmacologia , Fosforilação Oxidativa/efeitos dos fármacos , Inibidores de Proteínas Quinases/farmacologia , Triazóis/farmacologia , Animais , Biomarcadores , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Feminino , Hematopoese/efeitos dos fármacos , Humanos , Leucemia Mieloide Aguda/tratamento farmacológico , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/mortalidade , Camundongos , Modelos Biológicos , Receptor ErbB-2/antagonistas & inibidoresRESUMO
Acute promyelocytic leukemia (APL) is a medical emergency because of associated lethal early bleeding, a condition preventable by prompt diagnosis and therapeutic intervention. The mechanisms underlying the hemostatic anomalies of APL are not completely elucidated. RNA-sequencing-based characterization of APL (n = 30) was performed and compared to that of other acute myeloid leukemia (n = 400) samples and normal promyelocytes. Perturbations in the transcriptome of coagulation and fibrinolysis-related genes in APL extend beyond known culprits and now include Thrombin, Factor X and Urokinase Receptor. Most intriguingly, the Podoplanin (PDPN) gene, involved in platelet aggregation, is aberrantly expressed in APL promyelocytes and is the most distinctive transcript for this disease. Using an antibody panel optimized for AML diagnosis by flow cytometry, we also found that PDPN was the most specific surface marker for APL, and that all-trans retinoic acid therapy rapidly decreases its expression. Functional studies showed that engineered overexpression of this gene in human leukemic cells causes aberrant platelet binding, activation and aggregation. PDPN-expressing primary APL cells, but not PDPN-negative primary leukemias, specifically induce platelet binding, activation and aggregation. Finally, PDPN expression on leukemia cells in a xenograft model was associated with thrombocytopenia and prolonged bleeding time in vivo. Together our results suggest that PDPN may contribute to the hemostatic perturbations found in APL.
Assuntos
Hemorragia/etiologia , Leucemia Promielocítica Aguda/complicações , Glicoproteínas de Membrana/fisiologia , Transcriptoma , Adulto , Idoso , Animais , Feminino , Citometria de Fluxo , Humanos , Leucemia Promielocítica Aguda/genética , Masculino , Glicoproteínas de Membrana/genética , Camundongos , Pessoa de Meia-Idade , Agregação Plaquetária , Trombocitopenia/etiologia , Tretinoína/farmacologiaRESUMO
Purpose:RUNX1-mutated (RUNX1mut) acute myeloid leukemia (AML) is associated with adverse outcome, highlighting the urgent need for a better genetic characterization of this AML subgroup and for the design of efficient therapeutic strategies for this disease. Toward this goal, we further dissected the mutational spectrum and gene expression profile of RUNX1mut AML and correlated these results to drug sensitivity to identify novel compounds targeting this AML subgroup.Experimental Design: RNA-sequencing of 47 RUNX1mut primary AML specimens was performed and sequencing results were compared to those of RUNX1 wild-type samples. Chemical screens were also conducted using RUNX1mut specimens to identify compounds selectively affecting the viability of RUNX1mut AML.Results: We show that samples with no remaining RUNX1 wild-type allele are clinically and genetically distinct and display a more homogeneous gene expression profile. Chemical screening revealed that most RUNX1mut specimens are sensitive to glucocorticoids (GCs) and we confirmed that GCs inhibit AML cell proliferation through their interaction with the glucocorticoid receptor (GR). We observed that specimens harboring RUNX1 mutations expected to result in low residual RUNX1 activity are most sensitive to GCs, and that coassociating mutations as well as GR levels contribute to GC sensitivity. Accordingly, acquired glucocorticoid sensitivity was achieved by negatively regulating RUNX1 expression in human AML cells.Conclusions: Our findings show the profound impact of RUNX1 allele dosage on gene expression profile and glucocorticoid sensitivity in AML, thereby opening opportunities for preclinical testing which may lead to drug repurposing and improved disease characterization. Clin Cancer Res; 23(22); 6969-81. ©2017 AACR.
Assuntos
Alelos , Subunidade alfa 2 de Fator de Ligação ao Core/genética , Resistencia a Medicamentos Antineoplásicos/genética , Dosagem de Genes , Glucocorticoides/farmacologia , Leucemia Mieloide Aguda/genética , Mutação , Adulto , Idoso , Idoso de 80 Anos ou mais , Linhagem Celular Tumoral , Feminino , Regulação Leucêmica da Expressão Gênica , Inativação Gênica , Humanos , Leucemia Mieloide Aguda/diagnóstico , Leucemia Mieloide Aguda/tratamento farmacológico , Leucemia Mieloide Aguda/mortalidade , Masculino , Pessoa de Meia-IdadeRESUMO
Current chemotherapies for T cell acute lymphoblastic leukemia (T-ALL) efficiently reduce tumor mass. Nonetheless, disease relapse attributed to survival of preleukemic stem cells (pre-LSCs) is associated with poor prognosis. Herein, we provide direct evidence that pre-LSCs are much less chemosensitive to existing chemotherapy drugs than leukemic blasts because of a distinctive lower proliferative state. Improving therapies for T-ALL requires the development of strategies to target pre-LSCs that are absolutely dependent on their microenvironment. Therefore, we designed a robust protocol for high-throughput screening of compounds that target primary pre-LSCs maintained in a niche-like environment, on stromal cells that were engineered for optimal NOTCH1 activation. The multiparametric readout takes into account the intrinsic complexity of primary cells in order to specifically monitor pre-LSCs, which were induced here by the SCL/TAL1 and LMO1 oncogenes. We screened a targeted library of compounds and determined that the estrogen derivative 2-methoxyestradiol (2-ME2) disrupted both cell-autonomous and non-cell-autonomous pathways. Specifically, 2-ME2 abrogated pre-LSC viability and self-renewal activity in vivo by inhibiting translation of MYC, a downstream effector of NOTCH1, and preventing SCL/TAL1 activity. In contrast, normal hematopoietic stem/progenitor cells remained functional. These results illustrate how recapitulating tissue-like properties of primary cells in high-throughput screening is a promising avenue for innovation in cancer chemotherapy.
Assuntos
Estradiol/análogos & derivados , Células-Tronco Neoplásicas/metabolismo , Leucemia-Linfoma Linfoblástico de Células Precursoras/tratamento farmacológico , Leucemia-Linfoma Linfoblástico de Células Precursoras/metabolismo , Microambiente Tumoral/efeitos dos fármacos , 2-Metoxiestradiol , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Estradiol/farmacologia , Humanos , Células Jurkat , Proteínas com Domínio LIM/genética , Proteínas com Domínio LIM/metabolismo , Camundongos , Células-Tronco Neoplásicas/patologia , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras/patologia , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas/metabolismo , Receptor Notch1/genética , Receptor Notch1/metabolismo , Proteína 1 de Leucemia Linfocítica Aguda de Células T , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Microambiente Tumoral/genética , Ensaio Tumoral de Célula-Tronco , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
In this study, we analyzed RNA-sequencing data of 14 samples characterized by biallelic CEBPA (CEBPA(bi)) mutations included in the Leucegene collection of 415 primary acute myeloid leukemia (AML) specimens, and describe for the first time high frequency recurrent mutations in the granulocyte colony-stimulating factor receptor gene CSF3R, which signals through JAK-STAT proteins. Chemical interrogation of these primary human specimens revealed a uniform and specific sensitivity to all JAK inhibitors tested irrespective of their CSF3R mutation status, indicating a general sensitization of JAK-STAT signaling in this leukemia subset. Altogether, these results identified the co-occurrence of mutations in CSF3R and CEBPA in a well-defined AML subset, which uniformly responds to JAK inhibitors and paves the way to personalized clinical trials for this disease.
Assuntos
Proteínas Estimuladoras de Ligação a CCAAT/genética , Resistencia a Medicamentos Antineoplásicos/genética , Janus Quinases/antagonistas & inibidores , Leucemia Mieloide Aguda/tratamento farmacológico , Leucemia Mieloide Aguda/genética , Inibidores de Proteínas Quinases/uso terapêutico , Receptores de Fator Estimulador de Colônias/genética , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Estudos de Coortes , Análise Mutacional de DNA/métodos , Ensaios de Seleção de Medicamentos Antitumorais , Feminino , Perfilação da Expressão Gênica , Frequência do Gene , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Leucemia Mieloide Aguda/classificação , Leucemia Mieloide Aguda/patologia , Masculino , Pessoa de Meia-Idade , Mutação , Medicina de Precisão , Transcriptoma , Células Tumorais Cultivadas , Adulto JovemRESUMO
Acute myeloid leukemia (AML) is a genetically heterogeneous hematologic malignancy, which is initiated and driven by a rare fraction of leukemia stem cells (LSCs). Despite the difficulties of identifying a common LSC phenotype, there is increasing evidence that high expression of stem cell gene signatures is associated with poor clinical outcome. Identification of functionally distinct subpopulations in this disease is therefore crucial to dissecting the molecular machinery underlying LSC self-renewal. Here, we combined next-generation sequencing technology with in vivo assessment of LSC frequencies and identified the adhesion G protein-coupled receptor 56 (GPR56) as a novel and stable marker for human LSCs for the majority of AML samples. High GPR56 expression was significantly associated with high-risk genetic subgroups and poor outcome. Analysis of GPR56 in combination with CD34 expression revealed engraftment potential of GPR56(+)cells in both the CD34(-)and CD34(+)fractions, thus defining a novel LSC compartment independent of the CD34(+)CD38(-)LSC phenotype.
Assuntos
Biomarcadores Tumorais/metabolismo , Proliferação de Células , Leucemia Mieloide Aguda/patologia , Células-Tronco Neoplásicas/patologia , Receptores Acoplados a Proteínas G/metabolismo , Adulto , Animais , Separação Celular , Células Cultivadas , Células HEK293 , Humanos , Leucemia Mieloide Aguda/metabolismo , Leucemia Mieloide Aguda/mortalidade , Camundongos , Camundongos Endogâmicos NOD , Camundongos Transgênicos , Células-Tronco Neoplásicas/fisiologia , Receptores Acoplados a Proteínas G/fisiologia , Análise de SobrevidaRESUMO
Using next-generation sequencing of primary acute myeloid leukemia (AML) specimens, we identified to our knowledge the first unifying genetic network common to the two subgroups of KMT2A (MLL)-rearranged leukemia, namely having MLL fusions or partial tandem duplications. Within this network, we experimentally confirmed upregulation of the gene with the most subtype-specific increase in expression, LOC100289656, and identified cryptic MLL fusions, including a new MLL-ENAH fusion. We also identified a subset of MLL fusion specimens carrying mutations in SPI1 accompanied by inactivation of its transcriptional network, as well as frequent RAS pathway mutations, which sensitized the leukemias to synthetic lethal interactions between MEK and receptor tyrosine kinase inhibitors. This transcriptomics-based characterization and chemical interrogation of human MLL-rearranged AML was a valuable approach for identifying complementary features that define this disease.
Assuntos
Regulação Leucêmica da Expressão Gênica , Histona-Lisina N-Metiltransferase/genética , Leucemia Mieloide Aguda/genética , Proteína de Leucina Linfoide-Mieloide/genética , Transcriptoma , Animais , Antineoplásicos/farmacologia , Estudos de Casos e Controles , Resistencia a Medicamentos Antineoplásicos , Redes Reguladoras de Genes , Humanos , Leucemia Mieloide Aguda/metabolismo , Camundongos Endogâmicos NOD , Camundongos SCID , Mutação , Transplante de Neoplasias , Proteínas de Fusão Oncogênica/genética , Translocação Genética , Proteínas ras/genéticaRESUMO
Leukemic stem cells (LSCs) are considered a major cause of relapse in acute myeloid leukemia (AML). Defining pathways that control LSC self-renewal is crucial for a better understanding of underlying mechanisms and for the development of targeted therapies. However, currently available culture conditions do not prevent spontaneous differentiation of LSCs, which greatly limits the feasibility of cell-based assays. To overcome these constraints we conducted a high-throughput chemical screen and identified small molecules that inhibit differentiation and support LSC activity in vitro. Similar to reports with cord blood stem cells, several of these compounds suppressed the aryl-hydrocarbon receptor (AhR) pathway, which we show to be inactive in vivo and rapidly activated ex vivo in AML cells. We also identified a compound, UM729, that collaborates with AhR suppressors in preventing AML cell differentiation. Together, these findings provide newly defined culture conditions for improved ex vivo culture of primary human AML cells.
Assuntos
Adenina/análogos & derivados , Técnicas de Cultura de Células/métodos , Indóis/farmacologia , Leucemia/metabolismo , Células-Tronco Neoplásicas/fisiologia , Pirimidinas/farmacologia , Adenina/farmacologia , Meios de Cultura Livres de Soro , Ensaios de Seleção de Medicamentos Antitumorais , Humanos , Leucemia Mieloide Aguda , Estrutura MolecularRESUMO
Chromatin modulators are emerging as attractive drug targets, given their widespread implication in human cancers and susceptibility to pharmacological inhibition. Here we establish the histone methyltransferase G9a/EHMT2 as a selective regulator of fast proliferating myeloid progenitors with no discernible function in hematopoietic stem cells (HSCs). In mouse models of acute myeloid leukemia (AML), loss of G9a significantly delays disease progression and reduces leukemia stem cell (LSC) frequency. We connect this function of G9a to its methyltransferase activity and its interaction with the leukemogenic transcription factor HoxA9 and provide evidence that primary human AML cells are sensitive to G9A inhibition. Our results highlight a clinical potential of G9A inhibition as a means to counteract the proliferation and self-renewal of AML cells by attenuating HoxA9-dependent transcription.
Assuntos
Regulação Neoplásica da Expressão Gênica , Histona-Lisina N-Metiltransferase/metabolismo , Proteínas de Homeodomínio/metabolismo , Leucemia Mieloide Aguda/enzimologia , Leucemia Mieloide Aguda/genética , Animais , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Inibidores Enzimáticos/farmacologia , Células HEK293 , Células-Tronco Hematopoéticas/enzimologia , Histona-Lisina N-Metiltransferase/genética , Humanos , Leucemia Mieloide Aguda/patologia , Camundongos Endogâmicos C57BL , Quinazolinas/farmacologiaRESUMO
Histone methylation is a dynamic and reversible process proposed to directly impact on stem cell fate. The Jumonji (JmjC) domain-containing family of demethylases comprises 27 members that target mono-, di-, and trimethylated lysine residues of histone (or nonhistone) proteins. To evaluate their role in regulation of hematopoietic stem cell (HSC) behavior, we performed an in vivo RNAi-based functional screen and demonstrated that Jarid1b and Jhdm1f play opposing roles in regulation of HSC activity. Decrease in Jarid1b levels correlated with an in vitro expansion of HSCs with preserved long-term in vivo lymphomyeloid differentiation potential. Through RNA sequencing analysis, Jarid1b knockdown was associated with increased expression levels of several HSC regulators (Hoxa7, Hoxa9, Hoxa10, Hes1, Gata2) and reduced levels of differentiation-associated genes. shRNA against Jhdmlf, in contrast, impaired hematopoietic reconstitution of bone marrow cells. Together, our studies identified Jarid1b as a negative regulator of HSC activity and Jhdmlf as a positive regulator of HSC activity.
Assuntos
Proteínas de Ligação a DNA/fisiologia , Hematopoese/genética , Células-Tronco Hematopoéticas/fisiologia , Ensaios de Triagem em Larga Escala/métodos , Histona Desmetilases com o Domínio Jumonji/fisiologia , Interferência de RNA/fisiologia , Animais , Células Cultivadas , Proteínas de Ligação a DNA/genética , Regulação da Expressão Gênica/efeitos dos fármacos , Técnicas de Silenciamento de Genes , Hematopoese/efeitos dos fármacos , Células-Tronco Hematopoéticas/metabolismo , Histona Desmetilases/genética , Histona Desmetilases/fisiologia , Histona Desmetilases com o Domínio Jumonji/genética , Camundongos , Camundongos Congênicos , Camundongos Endogâmicos C57BL , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/farmacologia , Fatores de Transcrição/genética , Fatores de Transcrição/fisiologia , Estudos de Validação como AssuntoRESUMO
The incidence of refractory acute myeloid leukemia (AML) is on the increase due in part to an aging population that fails to respond to traditional therapies. High throughput genomic analysis promises better diagnosis, prognosis, and therapeutic intervention based on improved patient stratification. Relevant preclinical models are urgently required to advance drug development in this area. The collaborating oncogenes, HOXA9 and MEIS1, are frequently co-overexpressed in cytogenetically normal AML (CN-AML), and a conditional transplantation mouse model was developed that demonstrated oncogene dependency and expression levels comparable to CN-AML patients. Integration of gene signatures obtained from the mouse model and a cohort of CN-AML patients using statistically significant connectivity map analysis identified Entinostat as a drug with the potential to alter the leukemic condition toward the normal state. Ex vivo treatment of leukemic cells, but not age-matched normal bone marrow controls, with Entinostat validated the gene signature and resulted in reduced viability in liquid culture, impaired colony formation, and loss of the leukemia initiating cell. Furthermore, in vivo treatment with Entinostat resulted in prolonged survival of leukemic mice. This study demonstrates that the HDAC inhibitor Entinostat inhibits disease maintenance and prolongs survival in a clinically relevant murine model of cytogenetically normal AML.
Assuntos
Benzamidas/farmacologia , Inibidores de Histona Desacetilases/farmacologia , Leucemia Mieloide Aguda/tratamento farmacológico , Piridinas/farmacologia , Animais , Perfilação da Expressão Gênica , Regulação Leucêmica da Expressão Gênica , Imunofenotipagem , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/patologia , Camundongos , Camundongos Endogâmicos C57BLRESUMO
In this study, we show the high frequency of spontaneous γδ T-cell leukemia (T-ALL) occurrence in mice with biallelic deletion of enhancer of zeste homolog 2 (Ezh2). Tumor cells show little residual H3K27 trimethylation marks compared with controls. EZH2 is a component of the PRC2 Polycomb group protein complex, which is associated with DNA methyltransferases. Using next-generation sequencing, we identify alteration in gene expression levels of EZH2 and acquired mutations in PRC2-associated genes (DNMT3A and JARID2) in human adult T-ALL. Together, these studies document that deregulation of EZH2 and associated genes leads to the development of mouse, and likely human, T-ALL.
Assuntos
Proteínas de Ligação a DNA/metabolismo , Histona-Lisina N-Metiltransferase/metabolismo , Leucemia-Linfoma de Células T do Adulto/metabolismo , Fatores de Transcrição/metabolismo , Doença Aguda , Animais , Proteínas de Ligação a DNA/genética , Proteína Potenciadora do Homólogo 2 de Zeste , Histona-Lisina N-Metiltransferase/genética , Humanos , Leucemia-Linfoma de Células T do Adulto/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Complexo Repressor Polycomb 2 , Proteínas do Grupo Polycomb , Ligação Proteica , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Fatores de Transcrição/genéticaRESUMO
The three-amino-acid loop extension (TALE) class homeodomain proteins MEIS1 and PKNOX1 (PREP1) share the ability to interact with PBX and HOX family members and bind similar DNA sequences but appear to play opposing roles in tumor development. Elevated levels of MEIS1 accelerate development of HOX- and MLL-induced leukemias, and this pro-tumorigenic property has been associated with transcriptional activity of MEIS1. In contrast, reduction of PKNOX1 levels has been linked with cancer development despite the absence of an identifiable transactivating domain. In this report, we show that a chimeric protein generated by fusion of the MEIS1 C-terminal region encompassing the transactivating domain with the full-length PKNOX1 (PKNOX1-MC) acquired the ability to accelerate the onset of Hoxa9-induced leukemia in the mouse bone marrow transduction/transplantation model. Gene expression profiling of primary bone marrow cells transduced with Hoxa9 plus Meis1, or Hoxa9 plus Pknox1-MC revealed perturbations in overlapping functional gene subsets implicated in DNA packaging, chromosome organization, and in cell cycle regulation. Together, results presented in this report suggest that the C-terminal domain of MEIS1 confers to PKNOX1 an ectopic transactivating function that promotes leukemogenesis by regulating expression of genes involved in chromatin accessibility and cell cycle progression.
Assuntos
Proteínas de Homeodomínio/química , Proteínas de Homeodomínio/metabolismo , Proteínas de Neoplasias/química , Domínios e Motivos de Interação entre Proteínas/fisiologia , Animais , Ciclo Celular/genética , Transformação Celular Neoplásica/genética , Transformação Celular Neoplásica/metabolismo , Transformação Celular Neoplásica/patologia , Células Cultivadas , Cromatina/metabolismo , Proteínas de Homeodomínio/genética , Leucemia/genética , Leucemia/metabolismo , Leucemia/mortalidade , Leucemia/patologia , Camundongos , Camundongos Endogâmicos C3H , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Proteína Meis1 , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Proteínas Oncogênicas/metabolismo , Proteínas Oncogênicas/fisiologia , Ligação Proteica/genética , Ligação Proteica/fisiologia , Domínios e Motivos de Interação entre Proteínas/genética , TransfecçãoRESUMO
BACKGROUND: While the role of canonical (ß-catenin-mediated) Wnt signaling in hematolymphopoiesis has been studied extensively, little is known of the potential importance of non-canonical Wnt signals in hematopoietic cells. Wnt4 is one of the Wnt proteins that can elicit non-canonical pathways. We have previously shown that retroviral overexpression of Wnt4 by hematopoietic cells increased thymic cellularity as well as the frequency of early thymic progenitors and bone marrow hematopoietic progenitor cells (HPCs). However, the molecular pathways responsible for its effect in HPCs are not known. METHODOLOGY/PRINCIPAL FINDINGS: Here we report that Wnt4 stimulation resulted in the activation of the small GTPase Rac1 as well as Jnk kinases in an HPC cell line. Jnk activity was necessary, while ß-catenin was dispensable, for the Wnt4-mediated expansion of primary fetal liver HPCs in culture. Furthermore, Jnk2-deficient and Wnt4 hemizygous mice presented lower numbers of HPCs in their bone marrow, and Jnk2-deficient HPCs showed increased rates of apoptosis. Wnt4 also improved HPC activity in a competitive reconstitution model in a cell-autonomous, Jnk2-dependent manner. Lastly, we identified Fz6 as a receptor for Wnt4 in immature HPCs and showed that the absence of Wnt4 led to a decreased expression of four polarity complex genes. CONCLUSIONS/SIGNIFICANCE: Our results establish a functional role for non-canonical Wnt signaling in hematopoiesis through a pathway involving Wnt4, Fz6, Rac1 and Jnk kinases.
Assuntos
Polaridade Celular , Células-Tronco Hematopoéticas/citologia , Células-Tronco Hematopoéticas/metabolismo , Transdução de Sinais , Proteínas Wnt/metabolismo , Animais , Células da Medula Óssea/citologia , Cálcio/metabolismo , Linhagem da Célula , Proliferação de Células , Células Cultivadas , Ativação Enzimática , Receptores Frizzled/metabolismo , Hematopoese , Células-Tronco Hematopoéticas/enzimologia , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Células NIH 3T3 , Receptores Acoplados a Proteínas G/metabolismo , Proteína Wnt4RESUMO
Understanding the function of important DNA elements in mammalian stem cell genomes would be enhanced by the availability of deletion collections in which segmental haploidies are precisely characterized. Using a modified Cre-loxP-based system, we now report the creation and characterization of a collection of â¼1,300 independent embryonic stem cell (ESC) clones enriched for nested chromosomal deletions. Mapping experiments indicate that this collection spans over 25% of the mouse genome with good representative coverage of protein-coding genes, regulatory RNAs, and other non-coding sequences. This collection of clones was screened for in vitro defects in differentiation of ESC into embryoid bodies (EB). Several putative novel haploinsufficient regions, critical for EB development, were identified. Functional characterization of one of these regions, through BAC complementation, identified the ribosomal gene Rps14 as a novel haploinsufficient determinant of embryoid body formation. This new library of chromosomal deletions in ESC (DelES: http://bioinfo.iric.ca/deles) will serve as a unique resource for elucidation of novel protein-coding and non-coding regulators of ESC activity.
Assuntos
Diferenciação Celular , Deleção Cromossômica , Células-Tronco Embrionárias/citologia , Genoma , Mamíferos/genética , Animais , Linhagem Celular , Mapeamento Cromossômico , Feminino , Humanos , Masculino , CamundongosRESUMO
It is believed that hemopoietic stem cells (HSC), which colonize the fetal liver (FL) rapidly, expand to establish a supply of HSCs adequate for maintenance of hemopoiesis throughout life. Accordingly, FL HSCs are actively cycling as opposed to their predominantly quiescent bone marrow counterparts, suggesting that the FL microenvironment provides unique signals that support HSC proliferation and self-renewal. We now report the generation and characterization of mice with a mutant allele of Baf250a lacking exons 2 and 3. Baf250a(E2E3/E2E3) mice are viable until E19.5, but do not survive beyond birth. Most interestingly, FL HSC numbers are markedly higher in these mice than in control littermates, thus raising the possibility that Baf250a determines the HSC pool size in vivo. Limit dilution experiments indicate that the activity of Baf250a(E2E3/E2E3) HSC is equivalent to that of the wild-type counterparts. The Baf250a(E2E3/E2E3) FL-derived stroma, in contrast, exhibits a hemopoiesis-supporting potential superior to the developmentally matched controls. To our knowledge, this demonstration is the first that a mechanism operating in a cell nonautonomous manner canexpand the pool size of the fetal HSC populations.
Assuntos
Proteínas de Ligação a DNA/genética , Células-Tronco Hematopoéticas/metabolismo , Fígado/metabolismo , Mutação , Proteínas Nucleares/genética , Alelos , Animais , Western Blotting , Contagem de Células , Proliferação de Células , Ensaio de Unidades Formadoras de Colônias , Proteínas de Ligação a DNA/metabolismo , Feminino , Citometria de Fluxo , Perfilação da Expressão Gênica , Hematopoese , Transplante de Células-Tronco Hematopoéticas , Células-Tronco Hematopoéticas/citologia , Fígado/embriologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos , Proteínas Nucleares/metabolismo , Análise de Sequência com Séries de Oligonucleotídeos , Fatores de Tempo , Fatores de TranscriçãoRESUMO
Factors that trigger and sustain self-renewal divisions in tissue stem cells remain poorly characterized. By modulating the levels of Hoxb4 and its co-factor Pbxl in primary hematopoietic cells (Hoxb4hiPbxl(10) cells), we report an in vitro expansion of mouse hematopoietic stem cells (HSCs) by 105-fold over 2 weeks, with subsequent preservation of HSC properties. Clonal analyses of the hematopoietic system in recipients of expanded HSCs indicate that up to 70% of Hoxb4hiPbxl(10) stem cells present at initiation of culture underwent self-renewal in vitro. In this setting, Hoxb4 and its co-factor did not promote an increase in DNA synthesis, or a decrease in doubling time of Scal+Lin- cells when compared to controls. Q-PCR analyses further revealed a downregulation of Cdknlb (p27Kipl) and Mxdl (MadI) transcript levels in Hoxb4hiPbxl(l0) primitive cells, accompanied by a more subtle increase in c-myc and reduction in Ccnd3 (Cyclin D3). We thus put forward this strategy as an efficient in vitro HSC expansion tool, enabling a further step into the avenue of self-renewal molecular effectors.
Assuntos
Células-Tronco Hematopoéticas/fisiologia , Proteínas de Homeodomínio/fisiologia , Fatores de Transcrição/fisiologia , Animais , Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/genética , Divisão Celular/fisiologia , Células Cultivadas , Ciclina D3 , Inibidor de Quinase Dependente de Ciclina p27/genética , Ciclinas/genética , Regulação para Baixo/genética , Perfilação da Expressão Gênica , Células-Tronco Hematopoéticas/citologia , Camundongos , Camundongos Endogâmicos C3H , Camundongos Endogâmicos C57BL , Fator de Transcrição 1 de Leucemia de Células Pré-B , Proteínas Proto-Oncogênicas c-myc/genética , Proteínas Repressoras/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Transcrição Gênica/genéticaRESUMO
The Hoxa9 and Meis1 genes represent important oncogenic collaborators activated in a significant proportion of human leukemias with genetic alterations in the MLL gene. In this study, we show that the transforming property of Meis1 is modulated by 3 conserved domains, namely the Pbx interaction motif (PIM), the homeodomain, and the C-terminal region recently described to possess transactivating properties. Meis1 and Pbx1 interaction domain-swapping mutants are dysfunctional separately, but restore the full oncogenic activity of Meis1 when cotransduced in primary cells engineered to overexpress Hoxa9, thus implying a modular nature for PIM in Meis1-accelerated transformation. Moreover, we show that the transactivating domain of VP16 can restore, and even enhance, the oncogenic potential of the Meis1 mutant lacking the C-terminal 49 amino acids. In contrast to Meis1, the fusion VP16-Meis1 is spontaneously oncogenic, and all leukemias harbor genetic activation of endogenous Hoxa9 and/or Hoxa7, suggesting that Hoxa gene activation represents a key event required for the oncogenic activity of VP16-Meis1.