RESUMEN
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.
Asunto(s)
Proteínas Reguladoras de la Apoptosis/genética , Linfoma de Burkitt/tratamiento farmacológico , Linfoma de Burkitt/genética , Resistencia a Antineoplásicos/efectos de los fármacos , Silenciador del Gen/efectos de los fármacos , Inhibidores de Histona Desacetilasas/uso terapéutico , Proteínas de la Membrana/genética , Proteínas Proto-Oncogénicas/genética , Animales , Antibióticos Antineoplásicos/farmacología , Proteína 11 Similar a Bcl2 , Línea Celular Tumoral , Doxorrubicina/farmacología , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Inhibidores de Histona Desacetilasas/farmacología , Humanos , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Transgénicos , Regiones Promotoras Genéticas , Proteínas Proto-Oncogénicas c-myc/genética , Células Tumorales CultivadasRESUMEN
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.
Asunto(s)
Quinasa 6 Dependiente de la Ciclina/genética , Epigénesis Genética , MicroARNs/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , Receptor Tipo 1 de Factor de Crecimiento de Fibroblastos/genética , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Antineoplásicos/farmacología , Apoptosis/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Niño , Preescolar , Aberraciones Cromosómicas , Islas de CpG/genética , Quinasa 6 Dependiente de la Ciclina/antagonistas & inhibidores , Quinasa 6 Dependiente de la Ciclina/biosíntesis , Metilación de ADN , ADN de Neoplasias/genética , Regulación hacia Abajo , Femenino , Regulación Neoplásica de la Expresión Génica , Humanos , Lactante , Masculino , MicroARNs/fisiología , Persona de Mediana Edad , Piperazinas/farmacología , Leucemia-Linfoma Linfoblástico de Células Precursoras/metabolismo , Leucemia-Linfoma Linfoblástico de Células Precursoras/patología , Pronóstico , Piridinas/farmacología , Pirimidinas/farmacología , ARN Neoplásico/genética , ARN Neoplásico/fisiología , Receptor Tipo 1 de Factor de Crecimiento de Fibroblastos/antagonistas & inhibidores , Receptor Tipo 1 de Factor de Crecimiento de Fibroblastos/biosíntesis , Transducción de Señal/efectos de los fármacos , Células Tumorales Cultivadas , Adulto JovenRESUMEN
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.
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Proteínas de Unión al ADN/genética , Regulación Leucémica de la Expresión Génica , Metaloproteínas/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras B/diagnóstico , Leucemia-Linfoma Linfoblástico de Células Precursoras B/genética , Proteínas Adaptadoras Transductoras de Señales , Adolescente , Adulto , Factores de Edad , Anciano , Anciano de 80 o más Años , Subgrupos de Linfocitos B/metabolismo , Diferenciación Celular/genética , Línea Celular Tumoral , Niño , Preescolar , Humanos , Inmunofenotipificación , Lactante , Cariotipificación , Proteínas con Dominio LIM , Persona de Mediana Edad , Leucemia-Linfoma Linfoblástico de Células Precursoras B/metabolismo , Leucemia-Linfoma Linfoblástico de Células Precursoras B/mortalidad , Leucemia-Linfoma Linfoblástico de Células T Precursoras/genética , Pronóstico , Proteínas Proto-Oncogénicas , Análisis de Supervivencia , Resultado del Tratamiento , Adulto JovenRESUMEN
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.
Asunto(s)
Antraciclinas/uso terapéutico , Antineoplásicos/uso terapéutico , Leucemia Promielocítica Aguda/tratamiento farmacológico , Mutación , Tretinoina/uso terapéutico , Tirosina Quinasa 3 Similar a fms/genética , Adolescente , Adulto , Anciano , Niño , Femenino , Humanos , Leucemia Promielocítica Aguda/genética , Leucemia Promielocítica Aguda/mortalidad , Masculino , Persona de Mediana Edad , Pronóstico , Inducción de Remisión , Análisis de Supervivencia , Resultado del Tratamiento , Adulto JovenRESUMEN
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.
Asunto(s)
Células Dendríticas/inmunología , Células Dendríticas/trasplante , Efecto Injerto vs Leucemia/inmunología , Leucemia Mieloide Aguda/terapia , Técnicas de Cultivo de Célula , Estudios de Factibilidad , Humanos , Separación Inmunomagnética , Inmunoterapia , Leucemia Mieloide Aguda/inmunología , Trasplante de Células Madre de Sangre Periférica , Factores de Tiempo , Trasplante Autólogo , Células Tumorales CultivadasRESUMEN
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.
Asunto(s)
Epigénesis Genética , Leucemia Mieloide Aguda/genética , Proteínas Proto-Oncogénicas/genética , Transducción de Señal/fisiología , Proteínas Wnt/genética , Adulto , Anciano , Ciclina D1/genética , Metilación de ADN , Femenino , Humanos , Leucemia Mieloide Aguda/metabolismo , Leucemia Mieloide Aguda/mortalidad , Masculino , Persona de Mediana Edad , Regiones Promotoras Genéticas , Proteínas Proto-Oncogénicas/fisiología , Proteínas Wnt/fisiología , Proteína Wnt-5aRESUMEN
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).
Asunto(s)
Leucemia Mieloide de Fase Crónica/tratamiento farmacológico , Piperazinas/uso terapéutico , Pirimidinas/uso terapéutico , Adolescente , Adulto , Anciano , Anemia/inducido químicamente , Antineoplásicos/efectos adversos , Antineoplásicos/uso terapéutico , Benzamidas , Esquema de Medicación , Femenino , Estudios de Seguimiento , Humanos , Mesilato de Imatinib , Leucemia Mieloide de Fase Crónica/patología , Masculino , Persona de Mediana Edad , Neutropenia/inducido químicamente , Piperazinas/efectos adversos , Pirimidinas/efectos adversos , España , Trombocitopenia/inducido químicamente , Factores de Tiempo , Resultado del Tratamiento , Adulto JovenRESUMEN
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.
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Perfilación de la Expresión Génica , Leucemia Mielógena Crónica BCR-ABL Positiva/genética , MicroARNs/genética , Piperazinas/farmacología , Pirimidinas/farmacología , Antineoplásicos/farmacología , Antineoplásicos/uso terapéutico , Benzamidas , Análisis por Conglomerados , Resistencia a Antineoplásicos/genética , Humanos , Mesilato de Imatinib , Leucemia Mielógena Crónica BCR-ABL Positiva/tratamiento farmacológico , Leucemia Mielógena Crónica BCR-ABL Positiva/patología , Piperazinas/uso terapéutico , Pirimidinas/uso terapéutico , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
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.
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Neoplasias Colorrectales/genética , Metilación de ADN , Epigénesis Genética , Regulación Neoplásica de la Expresión Génica , MicroARNs/genética , Acetilación , Antimetabolitos Antineoplásicos/farmacología , Azacitidina/farmacología , Línea Celular Tumoral , Neoplasias Colorrectales/metabolismo , Neoplasias Colorrectales/patología , Islas de CpG , Silenciador del Gen , Inhibidores de Histona Desacetilasas , Histona Desacetilasas/metabolismo , Histonas/metabolismo , Humanos , Metástasis Linfática , MicroARNs/metabolismo , Fenilbutiratos/farmacología , Regiones Promotoras Genéticas , Células Tumorales CultivadasRESUMEN
MicroRNAs (miRNA) are small noncoding, single-stranded RNAs that inhibit gene expression at a posttranscriptional level, whose abnormal expression has been described in different tumors. The aim of our study was to identify miRNAs potentially implicated in chronic myeloid leukemia (CML). We detected an abnormal miRNA expression profile in mononuclear and CD34(+) cells from patients with CML compared with healthy controls. Of 157 miRNAs tested, hsa-miR-10a, hsa-miR-150, and hsa-miR-151 were down-regulated, whereas hsa-miR-96 was up-regulated in CML cells. Down-regulation of hsa-miR-10a was not dependent on BCR-ABL1 activity and contributed to the increased cell growth of CML cells. We identified the upstream stimulatory factor 2 (USF2) as a potential target of hsa-miR-10a and showed that overexpression of USF2 also increases cell growth. The clinical relevance of these findings was shown in a group of 85 newly diagnosed patients with CML in which expression of hsa-miR-10a was down-regulated in 71% of the patients, whereas expression of USF2 was up-regulated in 60% of the CML patients, with overexpression of USF2 being significantly associated with decreased expression of hsa-miR-10a (P = 0.004). Our results indicate that down-regulation of hsa-miR-10a may increase USF2 and contribute to the increase in cell proliferation of CML implicating a miRNA in the abnormal behavior of CML.
Asunto(s)
Regulación Leucémica de la Expresión Génica/genética , Leucemia Mielógena Crónica BCR-ABL Positiva/genética , Leucemia Mielógena Crónica BCR-ABL Positiva/fisiopatología , MicroARNs/fisiología , Factores Estimuladores hacia 5'/genética , Antígenos CD34/metabolismo , División Celular/fisiología , Línea Celular Tumoral , Regulación hacia Abajo/fisiología , Perfilación de la Expresión Génica , Genes abl/genética , Humanos , Leucemia Mielógena Crónica BCR-ABL Positiva/patología , MicroARNs/química , Conformación de Ácido Nucleico , Transfección , Regulación hacia Arriba/fisiologíaRESUMEN
The clinical significance of aberrant promoter methylation of the canonical Wnt pathway antagonist genes (sFRP1, sFRP2, sFRP4, sFRP5, Wif1, Dkk3, and Hdpr1) and also putative tumor-suppressor gene Wnt5a, belonging to the non-canonical Wnt signaling pathway, was investigated in a large series of 75 patients with Philadelphia chromosome-positive acute lymphoblastic leukemia by methylation-specific polymerase chain reaction. At least one methylated gene was observed in cells from 66% (49/75) of patients (methylated group). Disease-free survival and overall survival at 9 years were 51 and 40%, respectively, for the unmethylated group and 3 and 2%, respectively, for the methylated group (both P < 0.0001). Multivariate analysis demonstrated that the Wnt methylation profile was an independent prognostic factor predicting disease-free survival (P = 0.007) and overall survival (P = 0.039). Abnormal DNA methylation of promoter-associated CpG islands in the Wnt signaling pathway is very common in Philadelphia chromosome-positive acute lymphoblastic leukemia and potentially defines subgroups with distinct clinical characteristics.
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Metilación de ADN , Cromosoma Filadelfia , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , Proteínas Wnt/genética , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Niño , Preescolar , Supervivencia sin Enfermedad , Femenino , Humanos , Masculino , Persona de Mediana Edad , Leucemia-Linfoma Linfoblástico de Células Precursoras/metabolismo , Pronóstico , Factores de Riesgo , Transducción de Señal , Proteínas Wnt/metabolismoRESUMEN
In order to determine new signal transduction pathways implicated in chronic myeloid leukaemia (CML), we performed a gene expression profile comparison between CD34+ cells from CML patients and healthy donors. Functional studies were performed using the Mo7e and Mo7e-p210 cell lines. Expression of CCND1 (Cyclin D1), as well as the chaperone HSPA8, which is important for regulation of CCND1, were significantly upregulated in CD34+ CML cells. Upregulation of HSPA8 was dependent, at least in part, on STAT5 (signal transducer and activator of transcrition 5)-dependent transcriptional activation, as demonstrated by chromatin immunoprecipitation. The presence of HSPA8 in the nuclear protein fraction as well as its binding to CCND1 suggests that it may contribute to stabilization of the CCND1/CDK4 complex, which, in turn, may participate in proliferation of CML cells. Treatment of CML cells with the specific HSPA8 inhibitor 15-deoxyspergualin induced inhibition of CML cell viability but did not induce apoptosis. In conclusion, our studies suggest that STAT5-mediated activation of HSPA8 induces nuclear translocation and activation of the CCND1/CDK4 complex leading to increased proliferation of CML cells, deciphering a new pathway implicated in CML and supporting a potential role of chaperone inhibitors in the treatment of CML.
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Proteínas del Choque Térmico HSC70/metabolismo , Leucemia Mielógena Crónica BCR-ABL Positiva/metabolismo , Antígenos CD34/metabolismo , Línea Celular Tumoral/metabolismo , Supervivencia Celular , Ciclina D , Ciclinas/metabolismo , Perfilación de la Expresión Génica , Proteínas del Choque Térmico HSC70/genética , Humanos , Leucemia Mielógena Crónica BCR-ABL Positiva/genética , Análisis por Micromatrices , Transducción de SeñalRESUMEN
Repetitive elements are heavily methylated in normal tissues, but hypomethylated in malignant tissues, driving the global genomic hypomethylation found in cancer. This hypomethylation results in chromosomal instability, a well-characterized feature of the advanced phases of chronic myeloid leukemia (CML). We investigated methylation changes of DNA repetitive elements (LINE1, Alu, Satellite-alpha and Satellite-2) during the progression of CML from chronic phase (CP) to blast crisis (BC). CP-CML samples were significantly more hypomethylated for all repetitive sequences compared with normal samples. Furthermore, a more profound level of hypomethylation was observed among BC samples compared with CP samples. Our data suggest that repetitive DNA hypomethylation are closely associated with CML progression.
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Metilación de ADN , Leucemia Mielógena Crónica BCR-ABL Positiva/genética , Secuencias Repetitivas de Ácidos Nucleicos , Elementos Alu , Crisis Blástica/genética , ADN Satélite , Humanos , Leucemia Mieloide de Fase Crónica/genéticaRESUMEN
The aim of our study was to determine the potential mechanism(s) implicated in Imatinib resistance in patients with Ph+ ALL. Resistance of Ph+ ALL cells to Imatinib-induced apoptosis was associated with lack of inhibition of Akt phosphorylation. Addition of the PI3K inhibitor LY294002 to Imatinib significantly increased apoptosis of Ph+ ALL cells. Interestingly, expression of PTEN was reduced in Ph+ ALL cells which was due to PTEN promoter hypermethylation. Treatment of Ph+ ALL cells with 5-Aza-2'-deoxycytidine was associated with an increased expression of PTEN and an increase in cell apoptosis. These results suggest that Imatinib resistance in patients with ALL may be dependent at least in part to PTEN down-regulation due to the abnormal promoter hypermethylation and support the potential role of de-methylating agents for the treatment of patients with Ph+ ALL.
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Antineoplásicos/farmacología , Apoptosis/efectos de los fármacos , Metilación de ADN , Fosfohidrolasa PTEN/genética , Piperazinas/farmacología , Leucemia-Linfoma Linfoblástico de Células Precursoras/tratamiento farmacológico , Regiones Promotoras Genéticas , Pirimidinas/farmacología , Benzamidas , Línea Celular Tumoral , Regulación hacia Abajo , Resistencia a Antineoplásicos , Proteínas de Fusión bcr-abl/fisiología , Humanos , Mesilato de Imatinib , Fosfatidilinositol 3-Quinasas/fisiología , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras/patología , Proteínas Proto-Oncogénicas c-akt/fisiologíaAsunto(s)
Proteínas de Unión al ADN/metabolismo , Regulación Leucémica de la Expresión Génica , Silenciador del Gen , Leucemia Mieloide Aguda/metabolismo , MicroARNs/metabolismo , Factores de Transcripción/metabolismo , Animales , Línea Celular Tumoral , Proteínas de Unión al ADN/genética , Humanos , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/patología , Proteína del Locus del Complejo MDS1 y EV11 , Ratones , MicroARNs/genética , Proto-Oncogenes/genética , Factores de Transcripción/genéticaRESUMEN
Wnt5a is a member of the Wnt family of proteins that signals through the non-canonical Wnt/Ca(2+) pathway to suppress cyclin D1 expression and negatively regulate B cell proliferation suggesting that it acts as an tumour suppressor for lymphoid leukemogenesis. Although canonical Wnt pathway is a 'hot spot' for methylation in acute lymphoblastic leukaemia (ALL), the role of Wnt5a abnormalities has never been evaluated in this clinical setting. The methylation status of the WNT5A promoter was analysed by methylation-specific PCR (MSP) and sequencing in six ALL-derived cell lines (TOM-1, NALM-20, MY, LOUCY, JURKAT and TANOUE) and in 307 ALL patients. WNT5A and CYCLIN D1 expressions were assessed by quantitative RT-PCR. We observed WNT5A hypermethylation in all cell lines and in cells from 43% (132/307) of ALL patients. WNT5A methylation was associated with decreased WNT5A mRNA expression (P<0.001) and this expression was restored after exposure to the demethylating agent 5-Aza-2'-deoxycytidine. Moreover, WNT5A hypermethylation correlated with upregulation of CYCLIN D1 expression (P=0.002). Disease-free survival (DFS) and overall survival (OS) at 13 and 14 years, respectively, were 59% and 53% for unmethylated patients and 28% and 31% for hypermethylated patients (P=0.0003 and P=0.003). Multivariate analysis demonstrated that WNT5A methylation was an independent prognostic factor predicting DFS (P=0.003) and OS (P=0.04). We have demonstrated that WNT5A, a putative tumour suppressor gene in ALL, 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 this group of patients.
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Biomarcadores de Tumor/genética , Metilación de ADN , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , Proteínas Proto-Oncogénicas/genética , Proteínas Wnt/genética , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Biomarcadores de Tumor/metabolismo , Línea Celular Tumoral , Niño , Preescolar , Femenino , Humanos , Lactante , Masculino , Persona de Mediana Edad , Leucemia-Linfoma Linfoblástico de Células Precursoras/metabolismo , Pronóstico , Regiones Promotoras Genéticas , Proteínas Proto-Oncogénicas/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Proteínas Wnt/metabolismo , Proteína Wnt-5aRESUMEN
Tumor associated antigens (TAA) provide attractive targets for cancer-specific immunotherapy. PRAME is a TAA gene up-regulated in advanced phases of chronic myeloid leukemia (CML). To date, molecular mechanisms for the expression of PRAME have never been studied. We found that some Ph'-positive cell lines did not express PRAME. The expression of PRAME was restored in these cell lines by treatment with 5'-aza-2'-deoxycytidine, suggesting that the expression of PRAME is mainly suppressed by hypermethylation. Bisulfite sequencing analysis of the CpG sites of the PRAME exon 2 in these cancer cell lines revealed a close relationship between the methylation status of the PRAME gene and its expression. A methylation-specific PCR analysis demonstrated that hypomethylation of PRAME was significantly more frequent in CML blast crisis (70%) than in chronic phase (36%) (P=0.01) and was correlated with high expression levels of PRAME transcripts (P<0.0001). These results suggest that hypomethylation of PRAME up-regulates its expression in CML and might play a significant role in the progression of the disease.
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Antígenos de Neoplasias/genética , Epigénesis Genética , Leucemia Mielógena Crónica BCR-ABL Positiva/genética , Secuencia de Bases , Metilación de ADN , Cartilla de ADN , Humanos , Reacción en Cadena de la Polimerasa , ARN Mensajero/genéticaRESUMEN
BACKGROUND AND OBJECTIVES: We examined common polymorphisms in the genes for glutathione S-transferase (GST), cytochrome P450 (CYP), quinone oxoreductase (NQO1), methylene tetrahydrofolate reductase (MTHFR), and thymidylate synthetase (TYMS) and the role of gender associated with the susceptibility to de novo acute leukemia (AL). DESIGN AND METHODS: We conducted a case-control study analyzing the prevalence of the polymorphisms CYP1A1*2A, CYP2E1*5B, CYP3A4*1B, del{GSTT1}, del{GSTM1}, NQO1*2, MTHFR C6777, and TYMS 2R/3R in 443 patients with AL [302 with acute myeloblastic leukemia (AML) and 141 with acute lymphoblastic leukemia (ALL)] and 454 control volunteers, using polymerase chain reaction (PCR)-based methods. RESULTS: We found a higher incidence of del{GSTT1} in patients with AML than among controls (25.6% vs. 13.7%, OR=2.2, p<0.001) and a higher incidence of NQO1*2 homozygosity (NQO1*2hom.) in males with the M3 FAB subtype than in control males (8.6% vs. 2.2%, OR=4.9, p=0.02). The del{GSTT1} and NQO1*2hom. polymorphisms increased the risk of ALL (OR=2.2 and 3.0, p=0.001 and 0.003, respectively). The higher risk conferred by NQO1*2hom. and del{GSTT1} mainly affected males (OR=6.1 and 2.4; p=0.002 and 0.005, respectively). INTERPRETATION AND CONCLUSIONS: Males harboring NQO1*2hom. and del{GSTT1} polymorphisms showed a higher risk than females of developing AL. Thus, gender might influence the risk of AL associated with these genetic polymorphisms.
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Leucemia/epidemiología , Polimorfismo Genético , Factores Sexuales , Enfermedad Aguda , Adolescente , Adulto , Factores de Edad , Anciano , Anciano de 80 o más Años , Estudios de Casos y Controles , Transformación Celular Neoplásica/genética , Niño , Preescolar , Citocromo P-450 CYP1A1/genética , Citocromo P-450 CYP2E1/genética , Citocromo P-450 CYP3A , Sistema Enzimático del Citocromo P-450/genética , Análisis Mutacional de ADN , Susceptibilidad a Enfermedades , Femenino , Frecuencia de los Genes , Predisposición Genética a la Enfermedad , Glutatión Transferasa/genética , Humanos , Lactante , Leucemia/genética , Leucemia/fisiopatología , Leucemia Mieloide/epidemiología , Leucemia Mieloide/genética , Leucemia Mieloide/fisiopatología , Masculino , Metilenotetrahidrofolato Reductasa (NADPH2)/genética , Persona de Mediana Edad , NAD(P)H Deshidrogenasa (Quinona)/genética , Reacción en Cadena de la Polimerasa/métodos , Leucemia-Linfoma Linfoblástico de Células Precursoras/epidemiología , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras/fisiopatología , Riesgo , España/epidemiología , Timidilato Sintasa/genéticaRESUMEN
BACKGROUND AND OBJECTIVES: Cancer testis antigens (CTA) provide attractive targets for cancer-specific immunotherapy. Although CTA genes are expressed in some normal tissues, such as the testis, this immunologically protected site lacks MHC I expression and as such, does not present self antigens to T cells. To date, CTA genes have been shown to be expressed in a range of solid tumors via demethylation of their promoter CpG islands, but rarely in chronic myeloid leukemia (CML) or other hematologic malignancies. DESIGN AND METHODS: In this study, the methylation status of the HAGE CTA gene promoter was analyzed by quantitative methylation-specific polymerase chain reaction (MSP) and sequencing in four Philadelphia-positive cell lines (TCC-S, K562, KU812 and KYO-1) and in CML samples taken from patients in chronic phase (CP n=215) or blast crisis (BC n=47). HAGE expression was assessed by quantitative reverse transcriptase-polymerase chain reaction. RESULTS: The TCC-S cell line showed demethylation of HAGE that was associated with over-expression of this gene. HAGE hypomethylation was significantly more frequent in BC (46%) than in CP (22%) (p=0.01) and was correlated with high expression levels of HAGE transcripts (p<0.0001). Of note, in CP-CML, extensive HAGE hypomethylation was associated with poorer prognosis in terms of cytogenetic response to interferon (p=0.01) or imatinib (p=0.01), molecular response to imatinib (p=0.003) and progression-free survival (p=0.05). INTERPRETATIONS AND CONCLUSION: The methylation status of the HAGE promoter directly correlates with its expression in both CML cell lines and patients and is associated with advanced disease and poor outcome.
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Antígenos de Neoplasias/sangre , Islas de CpG , ARN Helicasas DEAD-box/genética , ARN Helicasas DEAD-box/fisiología , Metilación de ADN , Epigénesis Genética , Regulación Neoplásica de la Expresión Génica , Leucemia Mielógena Crónica BCR-ABL Positiva/genética , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/fisiología , Regiones Promotoras Genéticas , Antineoplásicos/farmacología , Benzamidas , Línea Celular Tumoral , Humanos , Mesilato de Imatinib , Interferones/uso terapéutico , Masculino , Piperazinas/farmacología , Pronóstico , Pirimidinas/farmacología , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Testículo/metabolismoRESUMEN
The hallmark of acute lymphoblastic leukemia (ALL) is a progressive appearance of malignant cell behavior that is triggered by the evolution of altered gene function. ALL has traditionally been viewed as a genetic disease; however, epigenetic defects also play an important role. DNA promoter methylation has gained increasing recognition as an important mechanism for transcriptional silencing of tumor-suppressor genes. Hypermethylation may contribute to the pathogenesis of leukemias providing an alternative route to gene mutation. We have reported that gene methylation in ALL cells is the most important way to inactivate cancer-related genes in this disease. In fact, this epigenetic event can help to inactivate tumor-suppressive apoptotic or growth-arresting responses and has prognostic impact in B- and T-ALL. The presence in individual tumors of multiple genes simultaneously methylated is an independent factor of poor prognosis in both childhood and adult ALL in terms of disease-free survival and overall survival. Moreover, methylation status is able to redefine the prognosis of selected ALL groups with well-established prognostic features.