RESUMO
Prognostic gene expression signatures have been proposed as clinical tools to clarify therapeutic options in acute myeloid leukemia (AML). However, these signatures rely on measuring large numbers of genes and often perform poorly when applied to independent cohorts or those with older patients. Long intergenic non-coding RNAs (lincRNAs) are emerging as important regulators of cell identity and oncogenesis, but knowledge of their utility as prognostic markers in AML is limited. Here we analyze transcriptomic data from multiple cohorts of clinically annotated AML patients and report that (i) microarrays designed for coding gene expression can be repurposed to yield robust lincRNA expression data, (ii) some lincRNA genes are located in close proximity to hematopoietic coding genes and show strong expression correlations in AML, (iii) lincRNA gene expression patterns distinguish cytogenetic and molecular subtypes of AML, (iv) lincRNA signatures composed of three or four genes are independent predictors of clinical outcome and further dichotomize survival in European Leukemia Net (ELN) risk groups and (v) an analytical tool based on logistic regression analysis of quantitative PCR measurement of four lincRNA genes (LINC4) can be used to determine risk in AML.
Assuntos
Leucemia Mieloide Aguda/genética , RNA Longo não Codificante/genética , Transcriptoma/genética , Adolescente , Adulto , Feminino , Perfilação da Expressão Gênica/métodos , Humanos , Leucemia Mieloide Aguda/patologia , Masculino , Pessoa de Meia-Idade , Prognóstico , Medição de Risco , Fatores de Risco , Adulto JovemRESUMO
MicroRNAs (miRNAs) are crucial components of homeostatic and developmental gene regulation. In turn, dysregulation of miRNA expression is a common feature of different types of cancer, which can be harnessed therapeutically. Here we identify miR-139-5p suppression across several cytogenetically defined acute myeloid leukemia (AML) subgroups. The promoter of mir-139 was transcriptionally silenced and could be reactivated by histone deacetylase inhibitors in a dose-dependent manner. Restoration of mir-139 expression in cell lines representing the major AML subgroups (t[8;21], inv[16], mixed lineage leukemia-rearranged and complex karyotype AML) caused cell cycle arrest and apoptosis in vitro and in xenograft mouse models in vivo. During normal hematopoiesis, mir-139 is exclusively expressed in terminally differentiated neutrophils and macrophages. Ectopic expression of mir-139 repressed proliferation of normal CD34(+)-hematopoietic stem and progenitor cells and perturbed myelomonocytic in vitro differentiation. Mechanistically, mir-139 exerts its effects by repressing the translation initiation factor EIF4G2, thereby reducing overall protein synthesis while specifically inducing the translation of cell cycle inhibitor p27(Kip1). Knockdown of EIF4G2 recapitulated the effects of mir-139, whereas restoring EIF4G2 expression rescued the mir-139 phenotype. Moreover, elevated miR-139-5p expression is associated with a favorable outcome in a cohort of 165 pediatric patients with AML. Thus, mir-139 acts as a global tumor suppressor-miR in AML by controlling protein translation. As AML cells are dependent on high protein synthesis rates controlling the expression of mir-139 constitutes a novel path for the treatment of AML.
Assuntos
Fator de Iniciação Eucariótico 4G/genética , Leucemia Mieloide/genética , MicroRNAs/biossíntese , Biossíntese de Proteínas , Animais , Diferenciação Celular/genética , Linhagem Celular Tumoral , Proliferação de Células/genética , Fator de Iniciação Eucariótico 4G/biossíntese , Feminino , Regulação Leucêmica da Expressão Gênica , Técnicas de Silenciamento de Genes , Humanos , Leucemia Mieloide/patologia , Masculino , Camundongos , MicroRNAs/genética , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Histone deacetylase (HDAC) inhibitors (HDACis) are well-characterized anti-cancer agents with promising results in clinical trials. However, mechanistically little is known regarding their selectivity in killing malignant cells while sparing normal cells. Gene expression-based chemical genomics identified HDACis as being particularly potent against Down syndrome-associated myeloid leukemia (DS-AMKL) blasts. Investigating the antileukemic function of HDACis revealed their transcriptional and post-translational regulation of key autophagic proteins, including ATG7. This leads to suppression of autophagy, a lysosomal degradation process that can protect cells against damaged or unnecessary organelles and protein aggregates. DS-AMKL cells exhibit low baseline autophagy due to mammalian target of rapamycin (mTOR) activation. Consequently, HDAC inhibition repressed autophagy below a critical threshold, which resulted in accumulation of mitochondria, production of reactive oxygen species, DNA damage and apoptosis. Those HDACi-mediated effects could be reverted upon autophagy activation or aggravated upon further pharmacological or genetic inhibition. Our findings were further extended to other major acute myeloid leukemia subgroups with low basal level autophagy. The constitutive suppression of autophagy due to mTOR activation represents an inherent difference between cancer and normal cells. Thus, via autophagy suppression, HDACis deprive cells of an essential pro-survival mechanism, which translates into an attractive strategy to specifically target cancer cells.
Assuntos
Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Autofagia/efeitos dos fármacos , Inibidores de Histona Desacetilases/farmacologia , Leucemia Mieloide/patologia , Animais , Humanos , Leucemia Mieloide/imunologia , Leucemia Mieloide/metabolismo , Camundongos , Espécies Reativas de Oxigênio/metabolismo , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Transient leukemia (TL) is evident in 5-10% of all neonates with Down syndrome (DS) and associated with N-terminal truncating GATA1 mutations (GATA1s). Here we report that TL-cell clones generate abundant eosinophils in a substantial fraction of patients. Sorted eosinophils from patients with TL and eosinophilia carried the same GATA1s mutations as sorted TL blasts, consistent with their clonal origin. TL blasts exhibited a genetic program characteristic of eosinophils and differentiated along the eosinophil lineage in vitro. Similarly, ectopic expression of Gata1s, but not Gata1, in wild-type CD34(+)-hematopoietic stem and progenitor cells induced hyperproliferation of eosinophil promyelocytes in vitro. Although GATA1s retained the function of GATA1 to induce eosinophil genes by occupying their promoter regions, GATA1s was impaired in its ability to repress oncogenic MYC and the pro-proliferative E2F transcription network. Chromatin Immunoprecipitation Sequencing (ChIP-seq) indicated reduced GATA1s occupancy at the MYC promoter. Knockdown of MYC, or the obligate E2F-cooperation partner DP1, rescued the GATA1s-induced hyperproliferative phenotype. In agreement, terminal eosinophil maturation was blocked in Gata1(Δe2) knockin mice, exclusively expressing Gata1s, leading to accumulation of eosinophil precursors in blood and bone marrow. These data suggest a direct relationship between the N-terminal truncating mutations of GATA1 and clonal eosinophilia in DS patients.
Assuntos
Proliferação de Células , Síndrome de Down/patologia , Eosinofilia/patologia , Fator de Transcrição GATA1/genética , Leucemia Mieloide Aguda/patologia , Mutação/genética , Animais , Apoptose , Diferenciação Celular , Síndrome de Down/complicações , Síndrome de Down/genética , Eosinofilia/etiologia , Células-Tronco Hematopoéticas/metabolismo , Células-Tronco Hematopoéticas/patologia , Humanos , Lactente , Recém-Nascido , Leucemia Mieloide Aguda/complicações , Leucemia Mieloide Aguda/genética , Camundongos , RNA Mensageiro/genética , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Células Tumorais CultivadasRESUMO
MicroRNAs (miRNAs) play a pivotal role in the regulation of hematopoiesis and development of leukemia. Great interest emerged in modulating miRNA expression for therapeutic purposes. In order to identify miRNAs, which specifically suppress leukemic growth of acute myeloid leukemia (AML) with t(8;21), inv(16) or mixed lineage leukemia (MLL) rearrangement by inducing differentiation, we conducted a miRNA expression profiling in a cohort of 90 cytogenetically characterized, de novo pediatric AML cases. Four miRNAs, specifically downregulated in MLL-rearranged, t(8;21) or inv(16) AMLs, were characterized by their tumor-suppressive properties in cell lines representing those respective cytogenetic groups. Among those, forced expression of miR-9 reduced leukemic growth and induced monocytic differentiation of t(8;21) AML cell lines in vitro and in vivo. The tumor-suppressive functions of miR-9 were specifically restricted to AML cell lines and primary leukemic blasts with t(8;21). On the other hand, these functions were not evident in AML blasts from patients with MLL rearrangements. We showed that miR-9 exerts its effects through the cooperation with let-7 to repress the oncogenic LIN28B/HMGA2 axis. Thus, miR-9 is a tumor suppressor-miR which acts in a stringent cell context-dependent manner.
Assuntos
Cromossomos Humanos Par 21 , Cromossomos Humanos Par 8 , Genes Supressores de Tumor , Leucemia Mieloide Aguda/genética , MicroRNAs/genética , Translocação Genética , Animais , Divisão Celular , Criança , Feminino , Citometria de Fluxo , Xenoenxertos , Humanos , Leucemia Mieloide Aguda/patologia , Masculino , Camundongos , Camundongos NusAssuntos
Transformação Celular Neoplásica/genética , Regulação Leucêmica da Expressão Gênica/genética , Hematopoese/genética , Leucemia/genética , RNA Longo não Codificante/genética , Montagem e Desmontagem da Cromatina/genética , Pesquisa em Genética , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , RNA Longo não Codificante/classificaçãoRESUMO
Reprogramming of somatic cells into patient-specific pluripotent analogues of human embryonic stem cells (ESCs) emerges as a prospective therapeutic angle in molecular medicine and a tool for basic stem cell biology. However, the combination of relative inefficiency and high variability of non-defined culture conditions precluded the use of this technique in a clinical setting and impeded comparability between laboratories. To overcome these obstacles, we sequentially devised a reprogramming protocol using one lentiviral-based polycistronic reprogramming construct, optimized for high co-expression of OCT4, SOX2, KLF4 and MYC in conjunction with small molecule inhibitors of non-permissive signaling cascades, such as transforming growth factor ß (SB431542), MEK/ERK (PD0325901) and Rho-kinase signaling (Thiazovivin), in a defined extracellular environment. Based on human fetal liver fibroblasts we could efficiently derive induced pluripotent stem cells (iPSCs) within 14 days. We attained efficiencies of up to 10.97±1.71% resulting in 79.5- fold increase compared to non-defined reprogramming using four singular vectors. We show that the overall increase of efficiency and temporal kinetics is a combinatorial effect of improved lentiviral vector design, signaling inhibition and definition of extracellular matrix (Matrigel®) and culture medium (mTESR®1). Using this protocol, we could derive iPSCs from patient fibroblasts, which were impermissive to classical reprogramming efforts, and from a patient suffering from familial platelet disorder. Thus, our defined protocol for highly efficient reprogramming to generate patient-specific iPSCs, reflects a big step towards therapeutic and broad scientific application of iPSCs, even in previously unfeasible settings.
Assuntos
Colágeno/química , Células-Tronco Pluripotentes Induzidas/fisiologia , Laminina/química , Proteoglicanas/química , Animais , Benzamidas/farmacologia , Transtornos Plaquetários/genética , Transtornos Plaquetários/patologia , Proliferação de Células , Células Cultivadas , Reprogramação Celular , Subunidade alfa 2 de Fator de Ligação ao Core/genética , Meios de Cultura/química , Dioxóis/farmacologia , Difenilamina/análogos & derivados , Difenilamina/farmacologia , Combinação de Medicamentos , Células-Tronco Embrionárias/fisiologia , Humanos , Células-Tronco Pluripotentes Induzidas/efeitos dos fármacos , Recém-Nascido , Fator 4 Semelhante a Kruppel , Camundongos , Receptores de Fatores de Crescimento Transformadores beta/antagonistas & inibidores , Fatores de Transcrição/biossíntese , Fatores de Transcrição/genéticaRESUMO
Mutations of the hematopoietic transcription factor GATA1 (GATA1s) are pathognomonic in newborn with transient leukemia and children with Down syndrome and myeloid leukemia (ML-DS). Both TL and ML-DS can also occur in children with trisomy 21 mosaic.Between 2002 and 2011, 15 newborns and infants were diagnosed with DS mosaic. 9 of them presented with TL and 8 children suffered from ML-DS; 2 of them with a history of TL. In children without stigmata the special morphology and immunophenotype of blasts triggered the screening for GATA1 mutation and trisomy 21 mosaic.All newborns with TL achieved complete remission (CR). Due to clinical symptoms caused by the leukemic blasts, in 3 children low-dose cytarabine was applied. 1 patient died due to cardiac defect. In all patients GATA 1 s was confirmed. 6 children with ML-DS were initially treated according the AML-BFM protocol. After ML-DS was confirmed, therapy was continued with the intensity reduced schedule according to the ML-DS 2006 protocol. All children are still in CR (follow-up 1.8-7 years, median 2.7 yrs). 2 children with unknown trisomy 21 mosaic were diagnosed as acute megakaryoblastic leukemia (AMKL) and treated according the high risk arm of the AML-BFM 2004 including allogeneic stem cell transplantation in one child). GATA1 mutation was identified retrospectively. Both children are alive in CR.GATA1s associated leukemia has to be excluded in all young children with AMKL (<5 years old) to prevent overtreatment. Treatment with reduced intensity seems sufficient in children trisomy 21 mosaic and ML-DS.
Assuntos
Síndrome de Down/genética , Fator de Transcrição GATA1/genética , Leucemia Megacarioblástica Aguda/genética , Leucemia Mieloide Aguda/genética , Mosaicismo , Mutação , Mielopoese/genética , Transtornos Mieloproliferativos/genética , Antimetabólitos Antineoplásicos/uso terapêutico , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Criança , Pré-Escolar , Terapia Combinada , Citarabina/uso terapêutico , Síndrome de Down/diagnóstico , Feminino , Seguimentos , Transplante de Células-Tronco Hematopoéticas , Humanos , Lactente , Recém-Nascido , Leucemia Megacarioblástica Aguda/diagnóstico , Leucemia Megacarioblástica Aguda/tratamento farmacológico , Leucemia Mieloide Aguda/diagnóstico , Leucemia Mieloide Aguda/tratamento farmacológico , Masculino , Transtornos Mieloproliferativos/diagnóstico , Transtornos Mieloproliferativos/tratamento farmacológicoRESUMO
Mutations in the NADP(+)-dependent isocitrate dehydrogenase genes 1 and 2 (IDH1 and IDH2) have recently been found in adult acute myeloid leukemia (AML) patients with a prevalence rising up to 33%. To investigate the frequency of IDH1/2 mutations in pediatric AML, we characterized the mutational hotspot (exon 4) of these genes in diagnostic samples from 460 pediatric AML patients. Our analysis identified somatic IDH1/2 mutations in 4% of cases (IDH1 R132 n=8; IDH2 R140 n=10) and the minor allele of single-nucleotide polymorphism (SNP) rs11554137 in 47 children (10.2%). IDH mutations were associated with an intermediate age (P=0.008), FAB M1/M2 (P=0.013) and nucleophosmin1 mutations (P=0.001). In univariate analysis, IDH(mutated) compared with IDH(wildtype) patients showed a significantly improved overall survival (OS; P=0.032) but not event-free survival (EFS; P=0.14). However, multivariate analysis did not show independent prognostic significance. Children with at least one minor allele of IDH1 SNP rs11554137 had similar EFS (P=0.27) and OS (P=0.62) compared with major allele patients. Gene expression profiles of 12 IDH(mutated) were compared with 201 IDH(wildtype) patients to identify differentially expressed genes and pathways. Although only a small number of discriminating genes were identified, analysis revealed a deregulated tryptophan metabolism, and a significant downregulation of KYNU expression in IDH(mutated) cases.