RESUMO
The role of epigenetic mechanisms in the regulation of microRNAs (miRNAs) with a tumour-suppressor function in human neoplasms has recently been established. Several miRNAs have been found to be inappropriately regulated by DNA methylation in patients with acute lymphoblastic leukaemia (ALL). We analysed the methylation status of the three members of the MIR9 family (MIR9-1, MIR9-2 and MIR9-3) in a uniformly treated cohort of 200 newly diagnosed ALLs. MIR9 was methylated in 54% of the patients and was associated with downregulation of MIR9 (P < 0·01). Hypermethylation of MIR9 was an independent prognostic factor for disease-free survival, overall survival and event-free survival in a multivariate analysis (P < 0·01). Epigenetic downregulation of MIR9 induced upregulation of its targets, FGFR1 and CDK6, while treatment of ALL cells with FGFR1 (PD-173074) and CDK6 (PD-0332991) inhibitors induced a decrease in cell proliferation and an increase in apoptosis of ALL cells. Our results indicate that the MIR9 family is involved in the pathogenesis and clinical behaviour of ALL and provide the basis for new therapeutic strategies in the treatment of ALL, targeting the epigenetic regulation of miRNAs and/or the FGFR1 or CDK6-RB pathway directly.
Assuntos
Quinase 6 Dependente de Ciclina/genética , Epigênese Genética , MicroRNAs/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , Receptor Tipo 1 de Fator de Crescimento de Fibroblastos/genética , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Criança , Pré-Escolar , Aberrações Cromossômicas , Ilhas de CpG/genética , Quinase 6 Dependente de Ciclina/antagonistas & inibidores , Quinase 6 Dependente de Ciclina/biossíntese , Metilação de DNA , DNA de Neoplasias/genética , Regulação para Baixo , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Lactente , Masculino , MicroRNAs/fisiologia , Pessoa de Meia-Idade , Piperazinas/farmacologia , Leucemia-Linfoma Linfoblástico de Células Precursoras/metabolismo , Leucemia-Linfoma Linfoblástico de Células Precursoras/patologia , Prognóstico , Piridinas/farmacologia , Pirimidinas/farmacologia , RNA Neoplásico/genética , RNA Neoplásico/fisiologia , Receptor Tipo 1 de Fator de Crescimento de Fibroblastos/antagonistas & inibidores , Receptor Tipo 1 de Fator de Crescimento de Fibroblastos/biossíntese , Transdução de Sinais/efeitos dos fármacos , Células Tumorais Cultivadas , Adulto JovemRESUMO
Recent studies have shown aberrant expression of SOX11 in various types of aggressive B-cell neoplasms. To elucidate the molecular mechanisms leading to such deregulation, we performed a comprehensive SOX11 gene expression and epigenetic study in stem cells, normal hematopoietic cells and different lymphoid neoplasms. We observed that SOX11 expression is associated with unmethylated DNA and presence of activating histone marks (H3K9/14Ac and H3K4me3) in embryonic stem cells and some aggressive B-cell neoplasms. In contrast, adult stem cells, normal hematopoietic cells and other lymphoid neoplasms do not express SOX11. Such repression was associated with silencing histone marks H3K9me2 and H3K27me3. The SOX11 promoter of non-malignant cells was consistently unmethylated whereas lymphoid neoplasms with silenced SOX11 tended to acquire DNA hypermethylation. SOX11 silencing in cell lines was reversed by the histone deacetylase inhibitor SAHA but not by the DNA methyltransferase inhibitor AZA. These data indicate that, although DNA hypermethylation of SOX11 is frequent in lymphoid neoplasms, it seems to be functionally inert, as SOX11 is already silenced in the hematopoietic system. In contrast, the pathogenic role of SOX11 is associated with its de novo expression in some aggressive lymphoid malignancies, which is mediated by a shift from inactivating to activating histone modifications.
Assuntos
Epigênese Genética/genética , Histonas/metabolismo , Linfoma/genética , Linfoma/metabolismo , Fatores de Transcrição SOXC/genética , Western Blotting , Linhagem Celular Tumoral , Imunoprecipitação da Cromatina , Metilação de DNA/efeitos dos fármacos , Metilação de DNA/genética , Metilação de DNA/fisiologia , Epigênese Genética/efeitos dos fármacos , Inibidores de Histona Desacetilases/farmacologia , Humanos , Técnicas In Vitro , Análise de Sequência com Séries de Oligonucleotídeos , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
Transcription factors are common targets of epigenetic inactivation in human cancer. Promoter hypermethylation and subsequent silencing of transcription factors can lead to further deregulation of their targets. In this study, we explored the potential epigenetic deregulation in cancer of Ikaros family genes, which code for essential transcription factors in cell differentiation and exhibit genetic defects in hematologic neoplasias. Unexpectedly, our analysis revealed that Ikaros undergoes very specific promoter hypermethylation in colorectal cancer, including in all the cell lines studied and around 64% of primary colorectal adenocarcinomas, with increasing proportions in advanced Duke's stages. Ikaros hypermethylation occurred in the context of a novel long-range epigenetic silencing (LRES) region. Reintroduction of Ikaros in colorectal cancer cells, ChIP-chip analysis, and validation in primary samples led us to identify a number of direct targets that are possibly related with colorectal cancer progression. Our results not only provide the first evidence that LRES can have functional specific effects in cancer but also identify several deregulated Ikaros targets that may contribute to progression in colorectal adenocarcinoma.
Assuntos
Adenocarcinoma/genética , Adenocarcinoma/metabolismo , Neoplasias Colorretais/genética , Neoplasias Colorretais/metabolismo , Inativação Gênica , Fator de Transcrição Ikaros/genética , Fator de Transcrição Ikaros/metabolismo , Diferenciação Celular , Ilhas de CpG , Metilação de DNA/genética , Progressão da Doença , Regulação Neoplásica da Expressão Gênica , Células HCT116 , Humanos , Estadiamento de Neoplasias , Regiões Promotoras GenéticasAssuntos
Antineoplásicos/uso terapêutico , Leucemia Mielogênica Crônica BCR-ABL Positiva/genética , Leucemia Mielogênica Crônica BCR-ABL Positiva/terapia , Piperazinas/uso terapêutico , Pirimidinas/uso terapêutico , Transplante de Células-Tronco , Adolescente , Adulto , Benzamidas , Criança , Terapia Combinada , Feminino , Genes abl , Humanos , Mesilato de Imatinib , Transfusão de Linfócitos , Masculino , Pessoa de Meia-Idade , Recidiva , Fatores de Tempo , Quimeras de Transplante , Transplante Homólogo , Adulto JovemRESUMO
BACKGROUND: Fms-like tyrosine kinase-3 (FLT3) gene mutations are frequent in acute promyelocytic leukemia but their prognostic value is not well established. DESIGN AND METHODS: We evaluated FLT3-internal tandem duplication and FLT3-D835 mutations in patients treated with all-trans retinoic acid and anthracycline-based chemotherapy enrolled in two subsequent trials of the Programa de Estudio y Tratamiento de las Hemopatías Malignas (PETHEMA) and Hemato-Oncologie voor Volwassenen Nederland (HOVON) groups between 1996 and 2005. RESULTS: FLT3-internal tandem duplication and FLT3-D835 mutation status was available for 306 (41%) and 213 (29%) patients, respectively. Sixty-eight (22%) and 20 (9%) patients had internal tandem duplication and D835 mutations, respectively. Internal tandem duplication was correlated with higher white blood cell and blast counts, lactate dehydrogenase, relapse-risk score, fever, hemorrhage, coagulopathy, BCR3 isoform, M3 variant subtype, and expression of CD2, CD34, human leukocyte antigen-DR, and CD11b surface antigens. The FLT3-D835 mutation was not significantly associated with any clinical or biological characteristic. Univariate analysis showed higher relapse and lower survival rates in patients with a FLT3-internal tandem duplication, while no impact was observed in relation to FLT3-D835. The prognostic value of the FLT3-internal tandem duplication was not retained in the multivariate analysis. CONCLUSIONS: FLT3-internal tandem duplication mutations are associated with several hematologic features in acute promyelocytic leukemia, in particular with high white blood cell counts, but we were unable to demonstrate an independent prognostic value in patients with acute promyelocytic leukemia treated with all-trans retinoic acid and anthracycline-based regimens.
Assuntos
Antraciclinas/uso terapêutico , Antineoplásicos/uso terapêutico , Leucemia Promielocítica Aguda/tratamento farmacológico , Mutação , Tretinoína/uso terapêutico , Tirosina Quinase 3 Semelhante a fms/genética , Adolescente , Adulto , Idoso , Criança , Feminino , Humanos , Leucemia Promielocítica Aguda/genética , Leucemia Promielocítica Aguda/mortalidade , Masculino , Pessoa de Meia-Idade , Prognóstico , Indução de Remissão , Análise de Sobrevida , Resultado do Tratamento , Adulto JovemRESUMO
BACKGROUND: LMO2 is highly expressed at the most immature stages of lymphopoiesis. In T-lymphocytes, aberrant LMO2 expression beyond those stages leads to T-cell acute lymphoblastic leukemia, while in B cells LMO2 is also expressed in germinal center lymphocytes and diffuse large B-cell lymphomas, where it predicts better clinical outcome. The implication of LMO2 in B-cell acute lymphoblastic leukemia must still be explored. DESIGN AND METHODS: We measured LMO2 expression by real time RT-PCR in 247 acute lymphoblastic leukemia patient samples with cytogenetic data (144 of them also with survival and immunophenotypical data) and in normal hematopoietic and lymphoid cells. RESULTS: B-cell acute lymphoblastic leukemia cases expressed variable levels of LMO2 depending on immunophenotypical and cytogenetic features. Thus, the most immature subtype, pro-B cells, displayed three-fold higher LMO2 expression than pre-B cells, common-CD10+ or mature subtypes. Additionally, cases with TEL-AML1 or MLL rearrangements exhibited two-fold higher LMO2 expression compared to cases with BCR-ABL rearrangements or hyperdyploid karyotype. Clinically, high LMO2 expression correlated with better overall survival in adult patients (5-year survival rate 64.8% (42.5%-87.1%) vs. 25.8% (10.9%-40.7%), P= 0.001) and constituted a favorable independent prognostic factor in B-ALL with normal karyotype: 5-year survival rate 80.3% (66.4%-94.2%) vs. 63.0% (46.1%-79.9%) (P= 0.043). CONCLUSIONS: Our data indicate that LMO2 expression depends on the molecular features and the differentiation stage of B-cell acute lymphoblastic leukemia cells. Furthermore, assessment of LMO2 expression in adult patients with a normal karyotype, a group which lacks molecular prognostic factors, could be of clinical relevance.
Assuntos
Proteínas de Ligação a DNA/genética , Regulação Leucêmica da Expressão Gênica , Metaloproteínas/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras B/diagnóstico , Leucemia-Linfoma Linfoblástico de Células Precursoras B/genética , Proteínas Adaptadoras de Transdução de Sinal , Adolescente , Adulto , Fatores Etários , Idoso , Idoso de 80 Anos ou mais , Subpopulações de Linfócitos B/metabolismo , Diferenciação Celular/genética , Linhagem Celular Tumoral , Criança , Pré-Escolar , Humanos , Imunofenotipagem , Lactente , Cariotipagem , Proteínas com Domínio LIM , Pessoa de Meia-Idade , Leucemia-Linfoma Linfoblástico de Células Precursoras B/metabolismo , Leucemia-Linfoma Linfoblástico de Células Precursoras B/mortalidade , Leucemia-Linfoma Linfoblástico de Células T Precursoras/genética , Prognóstico , Proteínas Proto-Oncogênicas , Análise de Sobrevida , Resultado do Tratamento , Adulto JovemRESUMO
Aberrant DNA methylation is one of the most frequent alterations in patients with Acute Lymphoblastic Leukemia (ALL). Using methylation bead arrays we analyzed the methylation status of 807 genes implicated in cancer in a group of ALL samples at diagnosis (nâ=â48). We found that 154 genes were methylated in more than 10% of ALL samples. Interestingly, the expression of 13 genes implicated in the TP53 pathway was downregulated by hypermethylation. Direct or indirect activation of TP53 pathway with 5-aza-2'-deoxycitidine, Curcumin or Nutlin-3 induced an increase in apoptosis of ALL cells. The results obtained with the initial group of 48 patients was validated retrospectively in a second cohort of 200 newly diagnosed ALL patients. Methylation of at least 1 of the 13 genes implicated in the TP53 pathway was observed in 78% of the patients, which significantly correlated with a higher relapse (pâ=â0.001) and mortality (p<0.001) rate being an independent prognostic factor for disease-free survival (DFS) (pâ=â0.006) and overall survival (OS) (pâ=â0.005) in the multivariate analysis. All these findings indicate that TP53 pathway is altered by epigenetic mechanisms in the majority of ALL patients and correlates with prognosis. Treatments with compounds that may reverse the epigenetic abnormalities or activate directly the p53 pathway represent a new therapeutic alternative for patients with ALL.
Assuntos
Epigênese Genética/fisiologia , Inativação Gênica/fisiologia , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismo , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Linhagem Celular Tumoral , Criança , Pré-Escolar , Estudos de Coortes , Feminino , Regulação Leucêmica da Expressão Gênica , Frequência do Gene , Humanos , Lactente , Masculino , Pessoa de Meia-Idade , Leucemia-Linfoma Linfoblástico de Células Precursoras/diagnóstico , Leucemia-Linfoma Linfoblástico de Células Precursoras/mortalidade , Prognóstico , Estudos Retrospectivos , Transdução de Sinais/genética , Adulto JovemRESUMO
BACKGROUND: Dendritic cell (DC)-based immunotherapeutic protocols are being developed to treat acute myeloid leukemia (AML). So far, DCs for clinical use are obtained from leukemic blasts or from monocytes, after 6 to 10 days of ex vivo culture. However, DC precursors are easily driven to DCs in short-term culture. We tested if DC precursors contained in peripheral blood stem cell (PBSC) products obtained from AML patients can be used to induce antileukemia responses. STUDY DESIGN AND METHODS: PBSCs obtained from 30 consecutive AML patients were tested. Myeloid DCs (MDCs) were purified by immunomagnetic selection and screened for cytogenetic and/or molecular abnormalities by fluorescence in situ hybridization (FISH) or polymerase chain reaction (PCR) assays. MDCs were matured and pulsed with autologous blast lysates and tested for stimulatory capability against AML cells. RESULTS: A median of 0.62 × 10(6) MDCs (range, 0.04-3.25)/mL were quantified in PBSC products. Isolated MDC expressed Class I and II HLA but CD86, CD54, and CCR5 partially. By FISH or PCR assay, these MDCs lacked cytogenetic or molecular abnormalities detected in leukemia cells at diagnosis. MDCs achieved a maturated stage (mature-MDCs) after 24-hour ex vivo culture with tumor necrosis factor-α and autologous blast lysates. These mature-MDCs were capable of stimulating autologous peripheral blood effectors to exert cytotoxicity against autologous leukemia cells and HL-60 cell line. CONCLUSION: We conclude that PBSCs obtained for autologous stem cell transplantation can constitute a novel source of MDCs to design feasible vaccination trials.
Assuntos
Células Dendríticas/imunologia , Células Dendríticas/transplante , Efeito Enxerto vs Leucemia/imunologia , Leucemia Mieloide Aguda/terapia , Técnicas de Cultura de Células , Estudos de Viabilidade , Humanos , Separação Imunomagnética , Imunoterapia , Leucemia Mieloide Aguda/imunologia , Transplante de Células-Tronco de Sangue Periférico , Fatores de Tempo , Transplante Autólogo , Células Tumorais CultivadasAssuntos
Biomarcadores Tumorais/análise , Biomarcadores Tumorais/genética , Quinase 6 Dependente de Ciclina/metabolismo , MicroRNAs/análise , MicroRNAs/genética , Adulto , Biomarcadores Tumorais/metabolismo , Células da Medula Óssea/citologia , Células da Medula Óssea/metabolismo , Criança , Quinase 6 Dependente de Ciclina/genética , Intervalo Livre de Doença , Epigênese Genética , Feminino , Expressão Gênica , Humanos , Masculino , Metilação , MicroRNAs/metabolismo , Procedimentos Analíticos em Microchip , Leucemia-Linfoma Linfoblástico de Células Precursoras/complicações , Leucemia-Linfoma Linfoblástico de Células Precursoras/mortalidade , Leucemia-Linfoma Linfoblástico de Células Precursoras/fisiopatologia , Leucemia-Linfoma Linfoblástico de Células Precursoras/terapia , Valor Preditivo dos Testes , Fatores de Risco , Espanha , Transplante de Células-Tronco/mortalidade , Regulação para CimaAssuntos
Proteínas de Ligação a DNA/metabolismo , Regulação Leucêmica da Expressão Gênica , Inativação Gênica , Leucemia Mieloide Aguda/metabolismo , MicroRNAs/metabolismo , Fatores de Transcrição/metabolismo , Animais , Linhagem Celular Tumoral , Proteínas de Ligação a DNA/genética , Humanos , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/patologia , Proteína do Locus do Complexo MDS1 e EVI1 , Camundongos , MicroRNAs/genética , Proto-Oncogenes/genética , Fatores de Transcrição/genéticaRESUMO
BACKGROUND: Aberrant promoter DNA methylation has been shown to play a role in acute myeloid leukemia (AML) pathophysiology. However, further studies to discuss the prognostic value and the relationship of the epigenetic signatures with defined genomic rearrangements in acute myeloid leukemia are required. METHODOLOGY/PRINCIPAL FINDINGS: We carried out high-throughput methylation profiling on 116 de novo AML cases and we validated the significant biomarkers in an independent cohort of 244 AML cases. Methylation signatures were associated with the presence of a specific cytogenetic status. In normal karyotype cases, aberrant methylation of the promoter of DBC1 was validated as a predictor of the disease-free and overall survival. Furthermore, DBC1 expression was significantly silenced in the aberrantly methylated samples. Patients with chromosome rearrangements showed distinct methylation signatures. To establish the role of fusion proteins in the epigenetic profiles, 20 additional samples of human hematopoietic stem/progenitor cells (HSPC) transduced with common fusion genes were studied and compared with patient samples carrying the same rearrangements. The presence of MLL rearrangements in HSPC induced the methylation profile observed in the MLL-positive primary samples. In contrast, fusion genes such as AML1/ETO or CBFB/MYH11 failed to reproduce the epigenetic signature observed in the patients. CONCLUSIONS/SIGNIFICANCE: Our study provides a comprehensive epigenetic profiling of AML, identifies new clinical markers for cases with a normal karyotype, and reveals relevant biological information related to the role of fusion proteins on the methylation signature.
Assuntos
Metilação de DNA , Leucemia Mieloide Aguda/genética , Adulto , Cromossomos Humanos/genética , Epigênese Genética/genética , Feminino , Variação Genética , Humanos , Cariotipagem , Leucemia Mieloide Aguda/diagnóstico , Masculino , Pessoa de Meia-Idade , Proteínas de Fusão Oncogênica/metabolismo , Prognóstico , Proteínas Supressoras de Tumor/genéticaRESUMO
In Burkitt lymphoma/leukemia (BL), achievement of complete remission with first-line chemotherapy remains a challenging issue, as most patients who respond remain disease-free, whereas those refractory have few options of being rescued with salvage therapies. The mechanisms underlying BL chemoresistance and how it can be circumvented remain undetermined. We previously reported the frequent inactivation of the proapoptotic BIM gene in B-cell lymphomas. Here we show that BIM epigenetic silencing by concurrent promoter hypermethylation and deacetylation occurs frequently in primary BL samples and BL-derived cell lines. Remarkably, patients with BL with hypermethylated BIM presented lower complete remission rate (24% vs 79%; P = .002) and shorter overall survival (P = .007) than those with BIM-expressing lymphomas, indicating that BIM transcriptional repression may mediate tumor chemoresistance. Accordingly, by combining in vitro and in vivo studies of human BL-xenografts grown in immunodeficient RAG2(-/-)γc(-/-) mice and of murine B220(+)IgM(+) B-cell lymphomas generated in Eµ-MYC and Eµ-MYC-BIM(+/-) transgenes, we demonstrate that lymphoma chemoresistance is dictated by BIM gene dosage and is reversible on BIM reactivation by genetic manipulation or after treatment with histone-deacetylase inhibitors. We suggest that the combination of histone-deacetylase inhibitors and high-dose chemotherapy may overcome chemoresistance, achieve durable remission, and improve survival of patients with BL.
Assuntos
Proteínas Reguladoras de Apoptose/genética , Linfoma de Burkitt/tratamento farmacológico , Linfoma de Burkitt/genética , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Inativação Gênica/efeitos dos fármacos , Inibidores de Histona Desacetilases/uso terapêutico , Proteínas de Membrana/genética , Proteínas Proto-Oncogênicas/genética , Animais , Antibióticos Antineoplásicos/farmacologia , Proteína 11 Semelhante a Bcl-2 , Linhagem Celular Tumoral , Doxorrubicina/farmacologia , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Inibidores de Histona Desacetilases/farmacologia , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Regiões Promotoras Genéticas , Proteínas Proto-Oncogênicas c-myc/genética , Células Tumorais CultivadasRESUMO
BACKGROUND: Despite the favorable results of imatinib front line in chronic-phase chronic myeloid leukemia there is room for improvement. DESIGN AND METHODS: Early intervention during imatinib therapy was undertaken in 210 adults with chronic-phase chronic myeloid leukemia less than three months from diagnosis (Sokal high risk: 16%). Patients received imatinib 400 mg/day. At three months, dose was increased if complete hematologic response was not achieved. At six months, patients in complete cytogenetic response were kept on 400 mg and the remainder randomized to higher imatinib dose or 400 mg plus interferon-alfa. At 18 months, randomized patients were switched to a 2(nd) generation tyrosine kinase inhibitor if not in complete cytogenetic response and imatinib dose increased in non-randomized patients not in major molecular response. RESULTS: Seventy-two percent of patients started imatinib within one month from diagnosis. Median follow-up is 50.5 (range: 1.2-78) months. At three months 4 patients did not have complete hematologic response; at six months 73.8% were in complete cytogenetic response; among the remainder, 9 could not be randomized (toxicity or consent withdrawal), 17 were assigned to high imatinib dose, and 15 to 400 mg + interferon-alpha. The low number of randomized patients precluded comparison between the two arms. Cumulative response at three years was: complete hematologic response 98.6%, complete cytogenetic response 90% and major molecular response 82%. On an intention-to-treat basis, complete cytogenetic response was 78.8% at 18 months. At five years, survival was 97.5%, survival free from accelerated/blastic phase 94.3%, failure free survival 82.5%, and event free survival (including permanent imatinib discontinuation) 71.5%. CONCLUSIONS: These results indicate the benefit of early intervention during imatinib therapy (ClinicalTrials.gov Identifier: NCT00390897).
Assuntos
Leucemia Mieloide de Fase Crônica/tratamento farmacológico , Piperazinas/uso terapêutico , Pirimidinas/uso terapêutico , Adolescente , Adulto , Idoso , Anemia/induzido quimicamente , Antineoplásicos/efeitos adversos , Antineoplásicos/uso terapêutico , Benzamidas , Esquema de Medicação , Feminino , Seguimentos , Humanos , Mesilato de Imatinib , Leucemia Mieloide de Fase Crônica/patologia , Masculino , Pessoa de Meia-Idade , Neutropenia/induzido quimicamente , Piperazinas/efeitos adversos , Pirimidinas/efeitos adversos , Espanha , Trombocitopenia/induzido quimicamente , Fatores de Tempo , Resultado do Tratamento , Adulto JovemRESUMO
Wnt5a is a member of the Wnt family of proteins that signals through the non-canonical Wnt/Ca(2+)pathway to suppress cyclin D1. Deregulation of this pathway has been found in animal models suggesting that it acts as tumour suppressor in acute myeloid leukemia (AML). Although DNA methylation is the main mechanism of regulation of the canonical Wnt pathway in AML, the role of WNT5A abnormalities has never been evaluated in this clinical setting. The methylation status of WNT5A promoter-exon 1 was analyzed by methylation-specific PCR and sequencing in eleven AML-derived cell lines and 252 AML patients. We observed WNT5A hypermethylation in seven cell lines and in 43% (107/252) of AML patients. WNT5A methylation was associated with decreased WNT5A expression (P < 0.001) that was restored after exposure to 5-Aza-2'-deoxycytidine. Moreover, WNT5A hypermethylation correlated with upregulation of CYCLIN D1 expression (P < 0.001). Relapse (15%vs 37%, P < 0.001) and mortality (61%vs 79%, P = 0.004) rates were lower for patients in the non-methylated group. Disease-free survival and overall survival at 6 and 7 years, respectively, were 60% and 27% for unmethylated patients and 20% and 0% for hypermethylated patients (P = 0.0001 and P = 0.04, respectively). Interestingly, significant differences were also observed when the analysis was carried out according to cytogenetic risk groups. We demonstrate that WNT5A, a putative tumor suppressor gene in AML, is silenced by methylation in this disease and that this epigenetic event is associated with upregulation of CYCLIN D1 expression and confers poor prognosis in patients with AML.
Assuntos
Epigênese Genética , Leucemia Mieloide Aguda/genética , Proteínas Proto-Oncogênicas/genética , Transdução de Sinais/fisiologia , Proteínas Wnt/genética , Adulto , Idoso , Ciclina D1/genética , Metilação de DNA , Feminino , Humanos , Leucemia Mieloide Aguda/metabolismo , Leucemia Mieloide Aguda/mortalidade , Masculino , Pessoa de Meia-Idade , Regiões Promotoras Genéticas , Proteínas Proto-Oncogênicas/fisiologia , Proteínas Wnt/fisiologia , Proteína Wnt-5aRESUMO
BACKGROUND: The therapeutic use of multipotent stem cells depends on their differentiation potential, which has been shown to be variable for different populations. These differences are likely to be the result of key changes in their epigenetic profiles. METHODOLOGY/PRINCIPAL FINDINGS: to address this issue, we have investigated the levels of epigenetic regulation in well characterized populations of pluripotent embryonic stem cells (ESC) and multipotent adult stem cells (ASC) at the trancriptome, methylome, histone modification and microRNA levels. Differences in gene expression profiles allowed classification of stem cells into three separate populations including ESC, multipotent adult progenitor cells (MAPC) and mesenchymal stromal cells (MSC). The analysis of the PcG repressive marks, histone modifications and gene promoter methylation of differentiation and pluripotency genes demonstrated that stem cell populations with a wider differentiation potential (ESC and MAPC) showed stronger representation of epigenetic repressive marks in differentiation genes and that this epigenetic signature was progressively lost with restriction of stem cell potential. Our analysis of microRNA established specific microRNA signatures suggesting specific microRNAs involved in regulation of pluripotent and differentiation genes. CONCLUSIONS/SIGNIFICANCE: Our study leads us to propose a model where the level of epigenetic regulation, as a combination of DNA methylation and histone modification marks, at differentiation genes defines degrees of differentiation potential from progenitor and multipotent stem cells to pluripotent stem cells.
Assuntos
Epigênese Genética , Células-Tronco/citologia , Tecido Adiposo/metabolismo , Adulto , Diferenciação Celular , Metilação de DNA , Perfilação da Expressão Gênica , Histonas/metabolismo , Humanos , Células-Tronco Mesenquimais/citologia , MicroRNAs/metabolismo , Pessoa de Meia-Idade , Análise de Sequência com Séries de Oligonucleotídeos , Regiões Promotoras Genéticas , Transcrição GênicaRESUMO
BACKGROUND: Alterations in the DNA methylation pattern are a hallmark of leukemias and lymphomas. However, most epigenetic studies in hematologic neoplasms (HNs) have focused either on the analysis of few candidate genes or many genes and few HN entities, and comprehensive studies are required. METHODOLOGY/PRINCIPAL FINDINGS: Here, we report for the first time a microarray-based DNA methylation study of 767 genes in 367 HNs diagnosed with 16 of the most representative B-cell (n = 203), T-cell (n = 30), and myeloid (n = 134) neoplasias, as well as 37 samples from different cell types of the hematopoietic system. Using appropriate controls of B-, T-, or myeloid cellular origin, we identified a total of 220 genes hypermethylated in at least one HN entity. In general, promoter hypermethylation was more frequent in lymphoid malignancies than in myeloid malignancies, being germinal center mature B-cell lymphomas as well as B and T precursor lymphoid neoplasias those entities with highest frequency of gene-associated DNA hypermethylation. We also observed a significant correlation between the number of hypermethylated and hypomethylated genes in several mature B-cell neoplasias, but not in precursor B- and T-cell leukemias. Most of the genes becoming hypermethylated contained promoters with high CpG content, and a significant fraction of them are targets of the polycomb repressor complex. Interestingly, T-cell prolymphocytic leukemias show low levels of DNA hypermethylation and a comparatively large number of hypomethylated genes, many of them showing an increased gene expression. CONCLUSIONS/SIGNIFICANCE: We have characterized the DNA methylation profile of a wide range of different HNs entities. As well as identifying genes showing aberrant DNA methylation in certain HN subtypes, we also detected six genes--DBC1, DIO3, FZD9, HS3ST2, MOS, and MYOD1--that were significantly hypermethylated in B-cell, T-cell, and myeloid malignancies. These might therefore play an important role in the development of different HNs.
Assuntos
Metilação de DNA , Neoplasias Hematológicas/genética , Neoplasias Hematológicas/metabolismo , Análise de Sequência com Séries de Oligonucleotídeos/métodos , Linfócitos B/metabolismo , Análise por Conglomerados , Ilhas de CpG , Perfilação da Expressão Gênica , Humanos , Regiões Promotoras Genéticas , Linfócitos T/metabolismoRESUMO
The development of Imatinib Mesylate (IM), the first specific inhibitor of BCR-ABL1, has had a major impact in patients with Chronic Myeloid Leukemia (CML), establishing IM as the standard therapy for CML. Despite the clinical success obtained with the use of IM, primary resistance to IM and molecular evidence of persistent disease has been observed in 20-25% of IM treated patients. The existence of second generation TK inhibitors, which are effective in patients with IM resistance, makes identification of predictors of resistance to IM an important goal in CML. In this study, we have identified a group of 19 miRNAs that may predict clinical resistance to IM in patients with newly diagnosed CML.
Assuntos
Perfilação da Expressão Gênica , Leucemia Mielogênica Crônica BCR-ABL Positiva/genética , MicroRNAs/genética , Piperazinas/farmacologia , Pirimidinas/farmacologia , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Benzamidas , Análise por Conglomerados , Resistencia a Medicamentos Antineoplásicos/genética , Humanos , Mesilato de Imatinib , Leucemia Mielogênica Crônica BCR-ABL Positiva/tratamento farmacológico , Leucemia Mielogênica Crônica BCR-ABL Positiva/patologia , Piperazinas/uso terapêutico , Pirimidinas/uso terapêutico , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
In the last years, microRNAs (miRNA) have emerged as new molecular players involved in carcinogenesis. Deregulation of miRNAs expression has been shown in different human cancer but the molecular mechanism underlying the alteration of miRNA expression is unknown. To identify tumor-supressor miRNAs silenced through aberrant epigenetic events in colorectal cancer (CRC), we used a sequential approach. We first identified 5 miRNAs down-regulated in patient with colorectal cancer samples and located around/on a CpG island. Treatment with a DNA methyltransferase inhibitor and a HDAC inhibitor restored expression of 3 of the 5 microRNAs (hsa-miR-9, hsa-miR-129 and hsa-miR-137) in 3 CRC cell lines. Expression of hsa-miR-9 was inversely correlated with methylation of their promoter regions as measure by MSP and bisulphate sequencing. Further, methylation of the hsa-miR-9-1, hsa-miR-129-2 and hsa-miR-137 CpG islands were frequently observed in CRC cell lines and in primary CRC tumors, but not in normal colonic mucosa. Finally, methylation of hsa-miR-9-1 was associated with the presence of lymph node metastasis. In summary, our results aid in the understanding of miRNA gene regulation showing that aberrant DNA methylation and histone modifications work together to induce silencing of miRNAs in CRC.
Assuntos
Neoplasias Colorretais/genética , Metilação de DNA , Epigênese Genética , Regulação Neoplásica da Expressão Gênica , MicroRNAs/genética , Acetilação , Antimetabólitos Antineoplásicos/farmacologia , Azacitidina/farmacologia , Linhagem Celular Tumoral , Neoplasias Colorretais/metabolismo , Neoplasias Colorretais/patologia , Ilhas de CpG , Inativação Gênica , Inibidores de Histona Desacetilases , Histona Desacetilases/metabolismo , Histonas/metabolismo , Humanos , Metástase Linfática , MicroRNAs/metabolismo , Fenilbutiratos/farmacologia , Regiões Promotoras Genéticas , Células Tumorais CultivadasRESUMO
BACKGROUND: Expression of the pro-apoptotic BCL-2-interacting mediator (BIM) has recently been implicated in imatinib-induced apoptosis of BCR-ABL1(+) cells. However, the mechanisms involved in the regulation of BIM in CML and its role in the clinical setting have not been established. DESIGN AND METHODS: We analysed the mRNA expression of BIM in 100 newly diagnosed patients with CML in chronic phase by Q-RT-PCR and the protein levels by Western blot analysis. Methylation status was analysed by bisulphite genomic sequencing and MSP. CML cell lines were treated with imatinib and 5-aza-2'-deoxycytidine, and were transfected with two different siRNAs against BIM and cell proliferation and apoptosis were analysed. RESULTS: We demonstrated that down-regulation of BIM expression was present in 36% of the patients and was significantly associated with a lack of optimal response to imatinib as indicated by the decrease in cytogenetic and molecular responses at 6, 12 and 18 months in comparison with patients with normal BIM expression (p<0.05). Expression of BIM was mediated by promoter hypermethylation as demonstrated by restoration of BIM expression after treatment of CML cells with 5-aza-2'-deoxycytidine. Using CML cell lines with low and normal expression of BIM we further demonstrated that the expression of BIM is required for imatinib-induced CML apoptosis. CONCLUSION: Our data indicate that down-regulation of BIM is epigenetically controlled by methylation in a percentage of CML patients and has an unfavourable prognostic impact, and that the combination of imatinib with a de-methylating agent may result in improved responses in patients with decreased expression of BIM.
Assuntos
Antineoplásicos/farmacologia , Proteínas Reguladoras de Apoptose/biossíntese , Regulação para Baixo/genética , Leucemia Mielogênica Crônica BCR-ABL Positiva/metabolismo , Proteínas de Membrana/biossíntese , Piperazinas/farmacologia , Proteínas Proto-Oncogênicas/biossíntese , Pirimidinas/farmacologia , Adulto , Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Apoptose/efeitos dos fármacos , Proteínas Reguladoras de Apoptose/genética , Azacitidina/análogos & derivados , Azacitidina/farmacologia , Proteína 11 Semelhante a Bcl-2 , Benzamidas , Proliferação de Células/efeitos dos fármacos , Metilação de DNA , Metilases de Modificação do DNA/antagonistas & inibidores , Decitabina , Relação Dose-Resposta a Droga , Regulação para Baixo/efeitos dos fármacos , Avaliação Pré-Clínica de Medicamentos/métodos , Resistencia a Medicamentos Antineoplásicos/genética , Epigênese Genética , Feminino , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/genética , Humanos , Mesilato de Imatinib , Leucemia Mielogênica Crônica BCR-ABL Positiva/patologia , Masculino , Proteínas de Membrana/genética , Pessoa de Meia-Idade , Regiões Promotoras Genéticas/genética , Proteínas Proto-Oncogênicas/genética , RNA Mensageiro/genética , RNA Neoplásico/genética , Células Tumorais CultivadasRESUMO
Whereas transcriptional silencing of genes due to epigenetic mechanisms is one of the most important alterations in acute lymphoblastic leukemia (ALL), some recent studies indicate that DNA methylation contributes to down-regulation of miRNAs during tumorigenesis. To explore the epigenetic alterations of miRNAs in ALL, we analyzed the methylation and chromatin status of the miR-124a loci in ALL. Expression of miR-124a was down-regulated in ALL by hypermethylation of the promoter and histone modifications including decreased levels of 3mk4H3 and AcH3 and increased levels of 2mK9H3, 3mK9H3, and 3mK27H3. Epigenetic down-regulation of miR-124a induced an up-regulation of its target, CDK6, and phosphorylation of retinoblastoma (Rb) and contributed to the abnormal proliferation of ALL cells both in vitro and in vivo. Cyclin-dependent kinase 6 (CDK6) inhibition by sodium butyrate or PD-0332991 decreased ALL cell growth in vitro, whereas overexpression of pre-miR124a led to decreased tumorigenicity in a xenogeneic in vivo Rag2(-/-)gammac(-/-) mouse model. The clinical implications of these findings were analyzed in a group of 353 patients diagnosed with ALL. Methylation of hsa-miR-124a was observed in 59% of the patients, which correlated with down-regulation of miR-124a (P < 0.001). Furthermore, hypermethylation of hsa-miR-124a was associated with higher relapse rate (P = 0.001) and mortality rate (P < 0.001), being an independent prognostic factor for disease-free survival (P < 0.001) and overall survival (P = 0.005) in the multivariate analysis. These results provide the grounds for new therapeutic strategies in ALL either targeting the epigenetic regulation of microRNAs and/or directly targeting the CDK6-Rb pathway.