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1.
Nat Med ; 25(7): 1073-1081, 2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-31270502

RESUMO

Bladder cancer is lethal in its advanced, muscle-invasive phase with very limited therapeutic advances1,2. Recent molecular characterization has defined new (epi)genetic drivers and potential targets for bladder cancer3,4. The immune checkpoint inhibitors have shown remarkable efficacy but only in a limited fraction of bladder cancer patients5-8. Here, we show that high G9a (EHMT2) expression is associated with poor clinical outcome in bladder cancer and that targeting G9a/DNMT methyltransferase activity with a novel inhibitor (CM-272) induces apoptosis and immunogenic cell death. Using an immunocompetent quadruple-knockout (PtenloxP/loxP; Trp53loxP/loxP; Rb1loxP/loxP; Rbl1-/-) transgenic mouse model of aggressive metastatic, muscle-invasive bladder cancer, we demonstrate that CM-272 + cisplatin treatment results in statistically significant regression of established tumors and metastases. The antitumor effect is significantly improved when CM-272 is combined with anti-programmed cell death ligand 1, even in the absence of cisplatin. These effects are associated with an endogenous antitumor immune response and immunogenic cell death with the conversion of a cold immune tumor into a hot tumor. Finally, increased G9a expression was associated with resistance to programmed cell death protein 1 inhibition in a cohort of patients with bladder cancer. In summary, these findings support new and promising opportunities for the treatment of bladder cancer using a combination of epigenetic inhibitors and immune checkpoint blockade.


Assuntos
Histona-Lisina N-Metiltransferase/antagonistas & inibidores , Receptor de Morte Celular Programada 1/antagonistas & inibidores , Neoplasias da Bexiga Urinária/tratamento farmacológico , Animais , Linhagem Celular Tumoral , Cisplatino/uso terapêutico , Proteína Potenciadora do Homólogo 2 de Zeste/fisiologia , Feminino , Antígenos de Histocompatibilidade , Humanos , Camundongos , Neoplasias da Bexiga Urinária/imunologia , Neoplasias da Bexiga Urinária/patologia
2.
Cancer Res ; 79(3): 625-638, 2019 02 01.
Artigo em Inglês | MEDLINE | ID: mdl-30563891

RESUMO

Because of the refractory nature of mutant KRAS lung adenocarcinoma (LUAD) to current therapies, identification of new molecular targets is essential. Genes with a prognostic role in mutant KRAS LUAD have proven to be potential molecular targets for therapeutic development. Here we determine the clinical, functional, and mechanistic role of inhibitor of differentiation-1 (Id1) in mutant KRAS LUAD. Analysis of LUAD cohorts from TCGA and SPORE showed that high expression of Id1 was a marker of poor survival in patients harboring mutant, but not wild-type KRAS. Abrogation of Id1 induced G2-M arrest and apoptosis in mutant KRAS LUAD cells. In vivo, loss of Id1 strongly impaired tumor growth and maintenance as well as liver metastasis, resulting in improved survival. Mechanistically, Id1 was regulated by the KRAS oncogene through JNK, and loss of Id1 resulted in downregulation of elements of the mitotic machinery via inhibition of the transcription factor FOSL1 and of several kinases within the KRAS signaling network. Our study provides clinical, functional, and mechanistic evidence underscoring Id1 as a critical gene in mutant KRAS LUAD and warrants further studies of Id1 as a therapeutic target in patients with LUAD. SIGNIFICANCE: These findings highlight the prognostic significance of the transcriptional regulator Id1 in KRAS-mutant lung adenocarcinoma and provide mechanistic insight into how it controls tumor growth and metastasis.


Assuntos
Adenocarcinoma de Pulmão/metabolismo , Proteína 1 Inibidora de Diferenciação/metabolismo , Neoplasias Pulmonares/metabolismo , Proteínas Proto-Oncogênicas p21(ras)/genética , Adenocarcinoma de Pulmão/genética , Adenocarcinoma de Pulmão/patologia , Animais , Processos de Crescimento Celular/fisiologia , Linhagem Celular Tumoral , Feminino , Humanos , Proteína 1 Inibidora de Diferenciação/genética , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patologia , Camundongos , Mutação , Metástase Neoplásica , Proteínas Proto-Oncogênicas c-fos/genética , Proteínas Proto-Oncogênicas c-fos/metabolismo , Proteínas Proto-Oncogênicas p21(ras)/metabolismo
3.
Nat Commun ; 9(1): 5454, 2018 12 21.
Artigo em Inglês | MEDLINE | ID: mdl-30575740

RESUMO

CRISPR/Cas9 technology offers novel approaches for the development of new therapies for many unmet clinical needs, including a significant number of inherited monogenic diseases. However, in vivo correction of disease-causing genes is still inefficient, especially for those diseases without selective advantage for corrected cells. We reasoned that substrate reduction therapies (SRT) targeting non-essential enzymes could provide an attractive alternative. Here we evaluate the therapeutic efficacy of an in vivo CRISPR/Cas9-mediated SRT to treat primary hyperoxaluria type I (PH1), a rare inborn dysfunction in glyoxylate metabolism that results in excessive hepatic oxalate production causing end-stage renal disease. A single systemic administration of an AAV8-CRISPR/Cas9 vector targeting glycolate oxidase, prevents oxalate overproduction and kidney damage, with no signs of toxicity in Agxt1-/- mice. Our results reveal that CRISPR/Cas9-mediated SRT represents a promising therapeutic option for PH1 that can be potentially applied to other metabolic diseases caused by the accumulation of toxic metabolites.


Assuntos
Oxirredutases do Álcool/antagonistas & inibidores , Sistemas CRISPR-Cas , Terapia Genética/métodos , Hiperoxalúria Primária/terapia , Oxalatos/urina , Oxirredutases do Álcool/genética , Animais , Modelos Animais de Doenças , Edição de Genes , Células HEK293 , Humanos , Masculino , Camundongos , Nefrocalcinose/prevenção & controle
4.
J Med Chem ; 61(15): 6518-6545, 2018 Aug 09.
Artigo em Inglês | MEDLINE | ID: mdl-29953809

RESUMO

Using knowledge- and structure-based approaches, we designed and synthesized reversible chemical probes that simultaneously inhibit the activity of two epigenetic targets, histone 3 lysine 9 methyltransferase (G9a) and DNA methyltransferases (DNMT), at nanomolar ranges. Enzymatic competition assays confirmed our design strategy: substrate competitive inhibitors. Next, an initial exploration around our hit 11 was pursued to identify an adequate tool compound for in vivo testing. In vitro treatment of different hematological neoplasia cell lines led to the identification of molecules with clear antiproliferative efficacies (GI50 values in the nanomolar range). On the basis of epigenetic functional cellular responses (levels of lysine 9 methylation and 5-methylcytosine), an acceptable therapeutic window (around 1 log unit) and a suitable pharmacokinetic profile, 12 was selected for in vivo proof-of-concept ( Nat. Commun. 2017 , 8 , 15424 ). Herein, 12 achieved a significant in vivo efficacy: 70% overall tumor growth inhibition of a human acute myeloid leukemia (AML) xenograft in a mouse model.


Assuntos
Antineoplásicos/farmacologia , Metilases de Modificação do DNA/antagonistas & inibidores , Desenho de Drogas , Inibidores Enzimáticos/farmacologia , Histona-Lisina N-Metiltransferase/antagonistas & inibidores , Animais , Antineoplásicos/química , Antineoplásicos/metabolismo , Antineoplásicos/farmacocinética , Linhagem Celular Tumoral , Metilases de Modificação do DNA/química , Metilases de Modificação do DNA/metabolismo , Inibidores Enzimáticos/química , Inibidores Enzimáticos/metabolismo , Inibidores Enzimáticos/farmacocinética , Antígenos de Histocompatibilidade/química , Antígenos de Histocompatibilidade/metabolismo , Histona-Lisina N-Metiltransferase/química , Histona-Lisina N-Metiltransferase/metabolismo , Humanos , Camundongos , Simulação de Acoplamento Molecular , Conformação Proteica , Ensaios Antitumorais Modelo de Xenoenxerto
5.
Oncotarget ; 9(16): 12842-12852, 2018 Feb 27.
Artigo em Inglês | MEDLINE | ID: mdl-29560114

RESUMO

Long Non-Coding RNAs (lncRNAs) are functional RNAs longer than 200 nucleotides in length. Several lncRNAs are involved in cell proliferation and are deregulated in several human tumors. Few lncRNAs have been described to play a role in Acute Lymphoblastic Leukemia (ALL). In this study, we carried out a genome wide lncRNA expression profiling in ALL samples and peripheral blood samples obtained from healthy donors. We detected 43 lncRNAs that were aberrantly expressed in ALL. Interestingly, among them, linc-PINT showed a significant downregulation in T and B-ALL. Re-expression of linc-PINT in ALL cells induced inhibition of leukemic cell growth that was associated with apoptosis induction and cell cycle arrest in G2/M phase. linc-PINT induced the transcription of HMOX1 which reduced the viability of ALL cells. Intriguingly, we observed that treatment with anti-tumoral epigenetic drugs like LBH-589 (Panobinostat) and Curcumin induced the expression of linc-PINT and HMOX1 in ALL. These results indicate that the downregulation of linc-PINT plays a relevant role in the pathogenesis of ALL, and linc-PINT re-expression may be one of the mechanisms exerted by epigenetic drugs to reduce cell proliferation in ALL.

6.
PLoS One ; 12(12): e0190275, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-29281720

RESUMO

The combination of defined factors with small molecules targeting epigenetic factors is a strategy that has been shown to enhance optimal derivation of iPSCs and could be used for disease modelling, high throughput screenings and/or regenerative medicine applications. In this study, we showed that a new first-in-class reversible dual G9a/DNMT1 inhibitor compound (CM272) improves the efficiency of human cell reprogramming and iPSC generation from primary cells of healthy donors and patient samples, using both integrative and non-integrative methods. Moreover, CM272 facilitates the generation of human iPSC with only two factors allowing the removal of the most potent oncogenic factor cMYC. Furthermore, we demonstrated that mechanistically, treatment with CM272 induces heterochromatin relaxation, facilitates the engagement of OCT4 and SOX2 transcription factors to OSKM refractory binding regions that are required for iPSC establishment, and enhances mesenchymal to epithelial transition during the early phase of cell reprogramming. Thus, the use of this new G9a/DNMT reversible dual inhibitor compound may represent an interesting alternative for improving cell reprogramming and human iPSC derivation for many different applications while providing interesting insights into reprogramming mechanisms.


Assuntos
Reprogramação Celular , Genoma Humano , Histona-Lisina N-Metiltransferase/antagonistas & inibidores , Células-Tronco Pluripotentes Induzidas/citologia , Proteínas Repressoras/antagonistas & inibidores , Fatores de Transcrição/metabolismo , Células Cultivadas , Antígenos de Histocompatibilidade , Humanos , Reação em Cadeia da Polimerase em Tempo Real
7.
Nat Commun ; 8: 15424, 2017 05 26.
Artigo em Inglês | MEDLINE | ID: mdl-28548080

RESUMO

The indisputable role of epigenetics in cancer and the fact that epigenetic alterations can be reversed have favoured development of epigenetic drugs. In this study, we design and synthesize potent novel, selective and reversible chemical probes that simultaneously inhibit the G9a and DNMTs methyltransferase activity. In vitro treatment of haematological neoplasia (acute myeloid leukaemia-AML, acute lymphoblastic leukaemia-ALL and diffuse large B-cell lymphoma-DLBCL) with the lead compound CM-272, inhibits cell proliferation and promotes apoptosis, inducing interferon-stimulated genes and immunogenic cell death. CM-272 significantly prolongs survival of AML, ALL and DLBCL xenogeneic models. Our results represent the discovery of first-in-class dual inhibitors of G9a/DNMTs and establish this chemical series as a promising therapeutic tool for unmet needs in haematological tumours.


Assuntos
Antineoplásicos/farmacologia , Metilases de Modificação do DNA/antagonistas & inibidores , Desenho de Drogas , Inibidores Enzimáticos/farmacologia , Neoplasias Hematológicas/tratamento farmacológico , Histona-Lisina N-Metiltransferase/antagonistas & inibidores , Animais , Antineoplásicos/química , Antineoplásicos/uso terapêutico , Apoptose/efeitos dos fármacos , Apoptose/genética , Apoptose/imunologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Proliferação de Células/genética , Cristalografia por Raios X , Metilases de Modificação do DNA/química , Metilases de Modificação do DNA/genética , Metilases de Modificação do DNA/metabolismo , Relação Dose-Resposta a Droga , Avaliação Pré-Clínica de Medicamentos , Inibidores Enzimáticos/química , Inibidores Enzimáticos/uso terapêutico , Epigênese Genética/efeitos dos fármacos , Feminino , Neoplasias Hematológicas/genética , Neoplasias Hematológicas/imunologia , Neoplasias Hematológicas/mortalidade , Antígenos de Histocompatibilidade/química , Antígenos de Histocompatibilidade/genética , Antígenos de Histocompatibilidade/metabolismo , Histona-Lisina N-Metiltransferase/química , Histona-Lisina N-Metiltransferase/genética , Histona-Lisina N-Metiltransferase/metabolismo , Humanos , Interferons/imunologia , Interferons/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Microssomos Hepáticos , Simulação de Acoplamento Molecular , Análise de Sobrevida , Resultado do Tratamento , Ensaios Antitumorais Modelo de Xenoenxerto
8.
Nat Commun ; 7: 11889, 2016 06 14.
Artigo em Inglês | MEDLINE | ID: mdl-27297662

RESUMO

NKX2 homeobox family proteins have a role in cancer development. Here we show that NKX2-3 is overexpressed in tumour cells from a subset of patients with marginal-zone lymphomas, but not with other B-cell malignancies. While Nkx2-3-deficient mice exhibit the absence of marginal-zone B cells, transgenic mice with expression of NKX2-3 in B cells show marginal-zone expansion that leads to the development of tumours, faithfully recapitulating the principal clinical and biological features of human marginal-zone lymphomas. NKX2-3 induces B-cell receptor signalling by phosphorylating Lyn/Syk kinases, which in turn activate multiple integrins (LFA-1, VLA-4), adhesion molecules (ICAM-1, MadCAM-1) and the chemokine receptor CXCR4. These molecules enhance migration, polarization and homing of B cells to splenic and extranodal tissues, eventually driving malignant transformation through triggering NF-κB and PI3K-AKT pathways. This study implicates oncogenic NKX2-3 in lymphomagenesis, and provides a valid experimental mouse model for studying the biology and therapy of human marginal-zone B-cell lymphomas.


Assuntos
Proteínas de Homeodomínio/genética , Linfócitos/metabolismo , Linfoma de Zona Marginal Tipo Células B/genética , Receptores de Antígenos de Linfócitos B/genética , Transdução de Sinais/genética , Fatores de Transcrição/genética , Animais , Moléculas de Adesão Celular/genética , Moléculas de Adesão Celular/metabolismo , Perfilação da Expressão Gênica , Proteínas de Homeodomínio/metabolismo , Humanos , Estimativa de Kaplan-Meier , Tecido Linfoide/metabolismo , Linfoma de Zona Marginal Tipo Células B/metabolismo , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fosforilação , Receptores de Antígenos de Linfócitos B/metabolismo , Quinase Syk/genética , Quinase Syk/metabolismo , Fatores de Transcrição/metabolismo
9.
Br J Haematol ; 155(1): 73-83, 2011 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-21810092

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 Jovem
10.
PLoS One ; 6(2): e17012, 2011 Feb 28.
Artigo em Inglês | MEDLINE | ID: mdl-21386967

RESUMO

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 (n = 48). 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 (p = 0.001) and mortality (p<0.001) rate being an independent prognostic factor for disease-free survival (DFS) (p = 0.006) and overall survival (OS) (p = 0.005) 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 Jovem
11.
Cancer Sci ; 101(2): 425-32, 2010 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-19874313

RESUMO

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-5a
12.
Mol Cancer ; 8: 69, 2009 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-19723306

RESUMO

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 Reversa
13.
Cancer Res ; 69(10): 4443-53, 2009 May 15.
Artigo em Inglês | MEDLINE | ID: mdl-19435910

RESUMO

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.


Assuntos
Quinase 6 Dependente de Ciclina/genética , Epigênese Genética/genética , Inativação Gênica , MicroRNAs/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , Animais , Divisão Celular , Linhagem Celular Tumoral , Cromatina/genética , Cromatina/fisiologia , Proteínas de Ligação a DNA/deficiência , Proteínas de Ligação a DNA/genética , Modelos Animais de Doenças , Intervalo Livre de Doença , Regulação para Baixo , Feminino , Humanos , Masculino , Camundongos , Camundongos Knockout , Leucemia-Linfoma Linfoblástico de Células Precursoras/mortalidade , Leucemia-Linfoma Linfoblástico de Células Precursoras/patologia , Prognóstico , RNA Mensageiro/genética , Taxa de Sobrevida , Transcrição Genética
14.
J Clin Oncol ; 27(8): 1316-22, 2009 Mar 10.
Artigo em Inglês | MEDLINE | ID: mdl-19164206

RESUMO

PURPOSE: To identify microRNAs (miRNAs) epigenetically regulated in acute lymphoblastic leukemia (ALL). METHODS: We first examined ALL-derived cell lines for the presence of abnormal levels of two different histone modifications (trimethylation of H3 lysine 4 [K4H3me3] and dimethylation of H3 lysine 9 [K9H3me2]) in the 5'UTR regions around CpG islands of 78 miRNAs by chromatin immunoprecipitation (ChIP)-on-ChIP analysis. Methylation status (methylation-specific polymerase chain reaction [PCR]) and expression (quantitative PCR) of miRNAs showing a pattern of histone modifications linked to a closed chromatin structure were analyzed in a panel of six ALL cell lines and in 353 ALL patients. RESULTS: CpG islands around 13 miRNAs disclosed high levels of K9H3me2 and/or low levels of K4H3me3, a pattern of histone modifications underlying a closed chromatin structure associated with repressive gene expression. Complete consistency in the correlation between both histone marks, the presence of DNA methylation around these miRNAs, and their expression patterns was confirmed in the six ALL cell lines. Treatment with 5-Aza-2'-deoxycytidine upregulated the expression levels of these genes, suggesting that epigenetic mechanisms deregulate the expression of these miRNAs. A total of 65% of the ALL samples had at least one miRNA methylated (methylated group). Estimated disease-free survival (DFS) and overall survival (OS) at 14 years were 78% and 71% for nonmethylated patients and 24% and 28% for methylated patients (P = .00001 for both). Multivariate analysis demonstrated that methylation profile was an independent prognostic factor for predicting DFS (P = .0001) and OS (P = .0001). CONCLUSION: Aberrant miRNA methylation is a common phenomenon in ALL that affects the clinical outcome of these patients.


Assuntos
Epigênese Genética , MicroRNAs/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Linhagem Celular Tumoral , Ilhas de CpG , Metilação de DNA , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Análise Multivariada , Prognóstico
15.
Mol Cancer Res ; 6(12): 1830-40, 2008 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-19074828

RESUMO

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.


Assuntos
Regulação Leucêmica da Expressão Gênica/genética , Leucemia Mielogênica Crônica BCR-ABL Positiva/genética , Leucemia Mielogênica Crônica BCR-ABL Positiva/fisiopatologia , MicroRNAs/fisiologia , Fatores Estimuladores Upstream/genética , Antígenos CD34/metabolismo , Divisão Celular/fisiologia , Linhagem Celular Tumoral , Regulação para Baixo/fisiologia , Perfilação da Expressão Gênica , Genes abl/genética , Humanos , Leucemia Mielogênica Crônica BCR-ABL Positiva/patologia , MicroRNAs/química , Conformação de Ácido Nucleico , Transfecção , Regulação para Cima/fisiologia
16.
Cancer Sci ; 99(9): 1865-8, 2008 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-18549404

RESUMO

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.


Assuntos
Metilação de DNA , Cromossomo Filadélfia , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , Proteínas Wnt/genética , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Criança , Pré-Escolar , Intervalo Livre de Doença , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Leucemia-Linfoma Linfoblástico de Células Precursoras/metabolismo , Prognóstico , Fatores de Risco , Transdução de Sinais , Proteínas Wnt/metabolismo
17.
Eur J Cancer ; 43(18): 2736-46, 2007 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-18032022

RESUMO

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.


Assuntos
Biomarcadores Tumorais/genética , Metilação de DNA , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , Proteínas Proto-Oncogênicas/genética , Proteínas Wnt/genética , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Biomarcadores Tumorais/metabolismo , Linhagem Celular Tumoral , Criança , Pré-Escolar , Feminino , Humanos , Lactente , Masculino , Pessoa de Meia-Idade , Leucemia-Linfoma Linfoblástico de Células Precursoras/metabolismo , Prognóstico , Regiões Promotoras Genéticas , Proteínas Proto-Oncogênicas/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Proteínas Wnt/metabolismo , Proteína Wnt-5a
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