RESUMEN
PURPOSE: Epigenetic silencing via aberrant promoter DNA hypermethylation of normal genes has been described as a leukemogenic mechanism in myelodysplastic syndromes (MDS) and acute myeloid leukemias (AML). We hypothesized that MG98, an oligonucleotide antisense to DNA methyltransferase 1 (DNMT1), could reverse malignant phenotypes by down-regulating DNMT1 and inducing reexpression of hypermethylated genes. This phase I study was conducted to determine a biologically effective dose and describe the safety of MG98 in MDS/AML. EXPERIMENTAL DESIGN: Twenty-three patients with MDS (n = 11) and AML (n = 12) were enrolled. Biologically effective dose was defined as the dose at which > or =50% of patients experienced >50% reduction in DNMT1 expression with acceptable toxicity. Escalating doses of MG98 were administered according to two schedules (2-hour i.v. bolus followed by 5-day continuous i.v. infusion every 14 days, or 14-day continuous i.v. infusion every 21 days). RESULTS: DNMT1 down-regulation was observed in 8 patients. However, biologically effective dose was not reached. Reexpression of target genes (P15, WIT1, and ER) was observed in 12 patients but did not correlate with DNMT1 down-regulation. Escalation was stopped due to dose-limiting toxicities (bone pain, nausea, and fever). No objective clinical response was observed. Disease stabilization occurred in 6 (26%) patients. CONCLUSIONS: No pharmacodynamic or clinical activity was observed at MG98 doses and schedules administered. Despite this, pursuing DNMT1 down-regulation remains a sound approach for targeting aberrant epigenetics in AML/MDS. Future studies with different formulation and/or doses and schedules will be required to ensure efficient MG98 intracellular uptake and fully evaluate its therapeutic potential.
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ADN (Citosina-5-)-Metiltransferasas/antagonistas & inhibidores , Leucemia Mieloide Aguda/tratamiento farmacológico , Síndromes Mielodisplásicos/tratamiento farmacológico , Oligodesoxirribonucleótidos/uso terapéutico , Tionucleótidos/uso terapéutico , Anciano , Anciano de 80 o más Años , ADN (Citosina-5-)-Metiltransferasa 1 , ADN (Citosina-5-)-Metiltransferasas/genética , Femenino , Humanos , Masculino , Persona de Mediana Edad , Oligodesoxirribonucleótidos/efectos adversos , Oligodesoxirribonucleótidos/farmacocinética , ARN Mensajero/análisis , Tionucleótidos/efectos adversos , Tionucleótidos/farmacocinéticaRESUMEN
The translocation t(8;21)(q22;q22) in acute myeloid leukemia (AML) results in the expression of the fusion protein RUNX1/MTG8, which in turn recruits histone deacetylases (HDAC) to silence RUNX1 target genes [e.g., interleukin-3 (IL-3)]. We previously reported that expression of the RUNX1/MTG8 target gene IL-3 is synergistically restored by the combination of inhibitors of HDACs (i.e., depsipeptide) and DNA methyltransferases (DNMT; i.e., decitabine) in RUNX1/MTG8-positive Kasumi-1 cells. Thus, we hypothesized that DNMT1 is also part of the transcriptional repressor complex recruited by RUNX1/MTG8. By a chromatin immunoprecipitation assay, we identified a RUNX1/MTG8-DNMT1 complex on the IL-3 promoter in Kasumi-1 cells and in primary RUNX1/MTG8-positive AML blasts. The physical association of RUNX1/MTG8 with DNMT1 was shown by coimmunoprecipitation experiments. Furthermore, RUNX1/MTG8 and DNMT1 were concurrently released from the IL-3 promoter by exposure to depsipeptide or stabilized on the promoter by decitabine treatment. Finally, we proved that RUNX1/MTG8 and DNMT1 were functionally interrelated by showing an enhanced repression of IL-3 after coexpression in 293T cells. These results suggest a novel mechanism for gene silencing mediated by RUNX1/MTG8 and support the combination of HDAC and DNMT inhibitors as a novel therapeutic approach for t(8;21) AML.
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ADN (Citosina-5-)-Metiltransferasas/metabolismo , Proteínas de Unión al ADN/fisiología , Leucemia Mieloide/metabolismo , Proteínas Proto-Oncogénicas/fisiología , Factores de Transcripción/fisiología , Enfermedad Aguda , Línea Celular , Línea Celular Tumoral , Subunidad alfa 2 del Factor de Unión al Sitio Principal , ADN (Citosina-5-)-Metiltransferasa 1 , Metilación de ADN , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Regulación Neoplásica de la Expresión Génica/fisiología , Silenciador del Gen , Humanos , Interleucina-3/genética , Leucemia Mieloide/enzimología , Leucemia Mieloide/genética , Regiones Promotoras Genéticas , Proteínas Proto-Oncogénicas/genética , Proteínas Proto-Oncogénicas/metabolismo , Proteína 1 Compañera de Translocación de RUNX1 , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Transcripción Genética/fisiología , TransfecciónRESUMEN
PURPOSES: Pharmacologic downregulation of Bcl-2, an antiapoptotic protein overexpressed in cancer, might increase chemosensitivity in acute myeloid leukemia (AML). Herein, we investigated the feasibility of this approach in untreated elderly AML patients by administering oblimersen sodium (G3139), an 18-mer phosphorothioate antisense to Bcl-2, during induction and consolidation treatments. PATIENTS AND METHODS: Untreated patients with primary or secondary AML (stratified to cohort 1 or 2, respectively) who were > or = 60 years received induction with G3139, cytarabine, and daunorubicin at one of two different dose levels (45 and 60 mg/m2) and, on achievement of complete remission (CR), consolidation with G3139 and high-dose cytarabine. An enzyme-linked immunosorbent assay (ELISA)-based assay was used to measure plasma and intracellular concentrations (IC) of G3139. Bcl-2 mRNA and protein levels were quantified by real-time reverse transcriptase polymerase chain reaction and ELISA, respectively, in bone marrow samples collected before induction treatment and after 72 hours of G3139 infusion, prior to initiation of chemotherapy. RESULTS: Of the 29 treated patients, 14 achieved CR. With a median follow-up of 12.6 months, seven patients had relapsed. Side effects of this combination were similar to those expected with chemotherapy alone and were not dose limiting at both dose levels. After 72-hour G3139 infusion, Bcl-2/ABL mRNA copies were decreased compared with baseline (P = .03) in CR patients and increased in nonresponders (NRs; P = .05). Changes in Bcl-2 protein showed a similar trend. Although plasma pharmacokinetics did not correlate with disease response, the median IC of the antisense was higher in the CR patients compared with NRs (17.0 v 4.4 pmol/mg protein, respectively; P = .05). CONCLUSION: G3139 can be administered safely in combination with intensive chemotherapy, and the degree of Bcl-2 downmodulation may correlate with response to therapy.
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Daunorrubicina/administración & dosificación , Leucemia Mieloide Aguda/tratamiento farmacológico , Proteínas Proto-Oncogénicas c-bcl-2/efectos de los fármacos , Inducción de Remisión , Tionucleótidos/administración & dosificación , Tionucleótidos/farmacocinética , Anciano , Anciano de 80 o más Años , Protocolos de Quimioterapia Combinada Antineoplásica/administración & dosificación , Protocolos de Quimioterapia Combinada Antineoplásica/efectos adversos , Disponibilidad Biológica , Daunorrubicina/efectos adversos , Relación Dosis-Respuesta a Droga , Esquema de Medicación , Femenino , Humanos , Leucemia Mieloide Aguda/mortalidad , Leucemia Mieloide Aguda/patología , Masculino , Dosis Máxima Tolerada , Persona de Mediana Edad , Estadificación de Neoplasias , Oligonucleótidos Antisentido/administración & dosificación , Oligonucleótidos Antisentido/farmacocinética , Probabilidad , Pronóstico , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Medición de Riesgo , Estadísticas no Paramétricas , Análisis de Supervivencia , Resultado del TratamientoRESUMEN
PURPOSE: Down-regulation of Bcl-2 by the antisense G3139, currently under clinical evaluations, could restore chemosensitivity in otherwise resistant malignant cells. To date, the mechanism of intracellular accumulation of G3139 following in vivo administration remains to be elucidated. This study aimed to assess whether detectable intracellular concentrations of G3139 are achievable in vivo and how these relate to Bcl-2 down-regulation. EXPERIMENTAL DESIGN: Cellular uptake of G3139 was studied in leukemia myeloid cell lines and blasts collected from treated patients using a newly developed, novel, and highly sensitive ELISA-based assay. Real-time reverse transcription-PCR was used to quantify Bcl-2 mRNA changes in treated cells. RESULTS: The assay was fully validated and showed a limit of quantification of 50 pmol/L. When exposed to 0.33 to 10 mumol/L G3139, K562 cells exhibited intracellular concentrations in the range of 2.1 to 11.4 pmol/mg protein. When G3139 was delivered with cationic lipids, a 10- to 25-fold increase of the intracellular concentrations was observed. There was an accumulation of G3139 in the nuclei, and the ratio of nucleus to cytoplasm was increased 7-fold by cationic lipids. Intracellular concentrations of G3139 were correlated with Bcl-2 mRNA down-regulation. Robust intracellular concentrations of G3139 were achieved in vivo in bone marrow (range, 3.4-40.6 pmol/mg protein) and peripheral blood mononuclear cells (range, 0.47-19.4 pmol/mg protein) from acute myeloid leukemia patients treated with G3139. CONCLUSIONS: This is the first evidence that measurable intracellular levels of G3139 are achievable in vivo in acute myeloid leukemia patients and that Bcl-2 down-regulation is likely to depend on the achievable intracellular concentrations rather than on plasma concentrations.
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Leucemia Mieloide/metabolismo , Oligonucleótidos Antisentido/farmacocinética , Proteínas Proto-Oncogénicas c-bcl-2/genética , Tionucleótidos/farmacocinética , Enfermedad Aguda , Secuencia de Bases , Línea Celular Tumoral , Cromatografía Líquida de Alta Presión/métodos , Ensayo de Inmunoadsorción Enzimática/métodos , Humanos , Células K562 , Leucemia Mieloide/genética , Leucemia Mieloide/patología , Oligonucleótidos Antisentido/sangre , Oligonucleótidos Antisentido/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Reproducibilidad de los Resultados , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Tionucleótidos/sangre , Tionucleótidos/metabolismo , Factores de TiempoRESUMEN
Uveal melanoma (UM) is a highly malignant primary intraocular tumour in adults that has a high mortality rate due to haematogenous dissemination. The migration of UM cells through the basement membrane requires the presence of proteolytic enzymes, such as matrix metalloproteinases (MMPs). The expression of MMP-2, MMP-9 and membrane type-1/MMP (MT-1/MMP) in UM cells is a known risk factor for metastatic disease. We tested the effect of depsipeptide (DP) on UM cell migration and the level and activity of MMP-2, MMP-9, MT-1/MMP and tissue inhibitors of matrix metalloproteinases 1 and 2 (TIMP-1 and TIMP-2). Three primary and two metastatic (liver metastasis) UM cell lines were treated with DP (0, 1, 5 and 10 nmol/l) for 24 h. Migration of UM cells was studied in modified Boyden migration chambers for 24 h and only viable cells on both sides of the membrane were counted. Enzyme-linked immunosorbent assays (ELISAs) were used to quantify the level of MMP-2, MMP-9, MT-1/MMP, TIMP-1 and TIMP-2 after the cells had been exposed to DP (0, 1, 5 and 10 nmol/l) for 24 h. In addition, the activities of MMP-2, MMP-9 and MT-1/MMP were determined after DP treatment. A dose-dependent decrease in the migration of viable UM cells was observed for primary and metastatic cell lines (30-50% inhibition). We detected a dose-dependent: (1) decrease in the protein level of MMP-2, MMP-9 and MT-1/MMP; (2) decrease in the activity of MMP-2, MMP-9 and MT-1/MMP; and (3) increase in the protein level of TIMP-1 and TIMP-2. It can be concluded that DP is a potent inhibitor of primary and metastatic UM cell migration in vitro. Our data suggest that this inhibition is mediated by the downregulation of MMPs and the upregulation of TIMPs. DP may be a valuable adjunctive treatment modality for primary and metastatic UM in humans.
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Antineoplásicos/farmacología , Movimiento Celular/efectos de los fármacos , Depsipéptidos/farmacología , Melanoma/secundario , Neoplasias de la Úvea/patología , Animales , Línea Celular Tumoral/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Inhibidores de Histona Desacetilasas , Humanos , Neoplasias Hepáticas/secundario , Metaloproteinasa 2 de la Matriz/análisis , Metaloproteinasa 2 de la Matriz/metabolismo , Metaloproteinasa 9 de la Matriz/análisis , Metaloproteinasa 9 de la Matriz/metabolismo , Metaloproteinasas de la Matriz Asociadas a la Membrana , Melanoma/metabolismo , Metaloendopeptidasas/análisis , Metaloendopeptidasas/metabolismo , Inhibidor Tisular de Metaloproteinasa-1/análisis , Inhibidor Tisular de Metaloproteinasa-1/metabolismo , Inhibidor Tisular de Metaloproteinasa-2/análisis , Inhibidor Tisular de Metaloproteinasa-2/metabolismo , Neoplasias de la Úvea/metabolismoRESUMEN
PURPOSE: Uveal melanoma (UM) is the most common primary malignant ocular tumor in adults. No effective chemotherapy regimens are available for either intraocular or metastatic uveal melanoma. Therefore, the ability of the histone deacetylase inhibitors (HDACIs), depsipeptide, sodium butyrate (NaB) and trichostatin A (TSA), to induce apoptosis and inhibit cell growth of UM cell lines in vitro was examined. METHODS: Three primary and two metastatic UM cell lines were treated in vitro with different concentrations of histone deacetylase inhibitors (HDACIs). Cell proliferation was studied in 24-well plates. Induction of apoptosis was studied by flow cytometry. Changes in gene expression of Fas/FasL, p21(Waf/Cip1), and p27(Kip1) were studied by RT-PCR. Western blot analysis was used to study histone acetylation, Fas/FasL, p21(Waf/Cip1), p27(Kip1) and caspase-3 protein levels. Real-time PCR was used to study changes in bcl-2/bax gene expression. RESULTS: A dose-dependent increase in histone acetylation was observed in all cell lines. This corresponded to significant inhibition of cell growth and induction of apoptosis in all melanoma cell lines in a concentration-dependent manner. Western blot analysis revealed dose-dependent increases in the amount of caspase-3, Fas/FasL, p21(Waf/Cip1), and p27(Kip1) proteins. However, no changes in bcl-2/bax gene expression were detected by real-time PCR. CONCLUSIONS: HDACIs are potent inhibitors of primary and metastatic UM cell growth in vitro. The apoptosis is probably mediated through the Fas/FasL signaling pathway, whereas bcl-2 appears not to be involved. These data support further clinical evaluation of depsipeptide and other HDACIs in patients with primary and metastatic UM.
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Antibióticos Antineoplásicos/farmacología , Apoptosis/efectos de los fármacos , Depsipéptidos , Melanoma/patología , Péptidos Cíclicos/farmacología , Neoplasias de la Úvea/patología , Western Blotting , Butiratos/farmacología , Caspasa 3 , Caspasas/metabolismo , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , División Celular/efectos de los fármacos , Inhibidor p21 de las Quinasas Dependientes de la Ciclina , Inhibidor p27 de las Quinasas Dependientes de la Ciclina , Ciclinas/genética , Ciclinas/metabolismo , Relación Dosis-Respuesta a Droga , Inhibidores Enzimáticos/farmacología , Proteína Ligando Fas , Citometría de Flujo , Inhibidores de Histona Desacetilasas , Humanos , Ácidos Hidroxámicos/farmacología , Melanoma/genética , Melanoma/metabolismo , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/metabolismo , Proteínas Proto-Oncogénicas/genética , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Proto-Oncogénicas c-bcl-2/genética , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , ARN Mensajero/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Células Tumorales Cultivadas , Proteínas Supresoras de Tumor/genética , Proteínas Supresoras de Tumor/metabolismo , Neoplasias de la Úvea/genética , Neoplasias de la Úvea/metabolismo , Proteína X Asociada a bcl-2 , Receptor fas/genética , Receptor fas/metabolismoRESUMEN
The post-translational modification of the core histones is critical to the regulation of chromatin structure. Traditional methods for the determination of histone modification utilize immunoassay techniques to determine the extent and site of post-translational modification. These methods, though sensitive, require site-specific antibodies. This manuscript describes the application of reverse-phase high-pressure liquid chromatography and mass spectrometry (LC-MS) to analyze global modification levels of core histones. The method is fast, sensitive, and easily automated. Furthermore, the technique gives the global patterns of modification for all four core histones in a single experiment. The LC-MS method was optimized using histones extracted from bovine thymus. These methods were then applied to the characterization of changes in histone modification in acute myeloid leukemia (AML) cell lines treated with histone deacetylase (HDAC) inhibitors. Dose-dependent changes in the distribution of modified core histones were observed. These results were validated in primary leukemia cells from patients with refractory or relapsed AML or chronic lymphocytic leukemia (CLL) treated on a Phase I clinical trial of the HDAC inhibitor depsipeptide. An increase in the relative abundance of specific acetylated forms of histone H4 was readily observable in these patients at intervals of 4 and 24 h after treatment.
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Histonas/metabolismo , Leucemia Linfocítica Crónica de Células B/metabolismo , Leucemia Mieloide Aguda/metabolismo , Procesamiento Proteico-Postraduccional , Espectrometría de Masa por Ionización de Electrospray/métodos , Animales , Bovinos , Línea Celular Tumoral , Cromatografía Líquida de Alta Presión , Humanos , Leucemia Linfocítica Crónica de Células B/genética , Leucemia Mieloide Aguda/genéticaRESUMEN
MLL (ALL-1) chimeric fusions and MLL partial tandem duplications (PTD) may have mechanistically distinct contributions to leukemogenesis. Acute myeloid leukemia (AML) blasts with the t(9;11)(p22; q23) express MLL-AF9 and MLL wild-type (WT) transcripts, while normal karyotype AML blasts with the MLL(PTD/WT) genotype express MLL PTD but not the MLL WT. Silencing of MLL WT in MLL(PTD/WT) blasts was reversed by DNA methyltransferase (DNMT) and histone deacetylase (HDAC) inhibitors, and MLL WT induction was associated with selective sensitivity to cell death. Reduction of MLL PTD expression induced MLL WT and reduced blast colony-forming units, supporting opposing functions for MLL PTD and MLL WT whereby the MLL PTD contributes to the leukemic phenotype via a recessive gain-of-function. The coincident suppression of the MLL WT allele with the expression of the MLL PTD allele, along with the functional data presented here, supports the hypothesis that loss of WT MLL function via monoallelic repression contributes to the leukemic phenotype by the remaining mutant allele. These data from primary AML and the pharmacologic reversal of MLL WT silencing associated with a favorable alteration in the threshold for apoptosis suggest that these patients with poor prognosis may benefit from demethylating or histone deacetylase inhibitor therapy, or both.
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Metilasas de Modificación del ADN/antagonistas & inhibidores , Proteínas de Unión al ADN/genética , Duplicación de Gen , Inhibidores de Histona Desacetilasas , Leucemia Mieloide/genética , Proto-Oncogenes/genética , Factores de Transcripción/genética , Enfermedad Aguda , Muerte Celular , Islas de CpG/genética , Regulación hacia Abajo , Inhibidores Enzimáticos/farmacología , Regulación Leucémica de la Expresión Génica , Genotipo , N-Metiltransferasa de Histona-Lisina , Humanos , Leucemia Mieloide/tratamiento farmacológico , Leucemia Mieloide/patología , Proteína de la Leucemia Mieloide-Linfoide , Oligodesoxirribonucleótidos , Fenotipo , Secuencias Repetidas en Tándem , Células Tumorales CultivadasRESUMEN
Overexpression of Bcl-2 is a potential mechanism for chemoresistance in acute leukemia and has been associated with unfavorable clinical outcome. We hypothesized that down-regulation of Bcl-2 would restore chemosensitivity in leukemic cells. To test this hypothesis, we performed a phase 1 study of G3139 (Genasense, Genta, Berkeley Heights, NJ), an 18-mer phosphorothioate Bcl-2 antisense, with fludarabine (FL), cytarabine (ARA-C), and granulocyte colony-stimulating factor (G-CSF) (FLAG) salvage chemotherapy in patients with refractory or relapsed acute leukemia. Twenty patients with refractory or relapsed acute myeloid leukemia (AML) or acute lymphoblastic leukemia (ALL) were enrolled. G3139 was delivered by continuous infusion on days 1 to 10. FLAG chemotherapy was administered on days 5 to 10. Common side effects of this combination included fever, nausea, emesis, electrolyte imbalance, and fluid retention that were not dose limiting. Plasma pharmacokinetics of G3139 demonstrated steady-state concentration (Css) within 24 hours. Of the 20 patients, 9 (45%) had disease response, 6 (5 AML, 1 ALL) with complete remission (CR) and 3 (2 AML and 1 ALL) with no evidence of disease but failure to recover normal neutrophil and/or platelet counts or to remain in remission for at least 30 days (incomplete remission). Bcl-2 mRNA levels were down-regulated in 9 of the 12 (75%) evaluable patients. This study demonstrates that G3139 can be administered safely with FLAG chemotherapy and down-regulate its target, Bcl-2. The encouraging clinical and laboratory results justify the current plans for a phase 3 study in previously untreated high-risk AML (ie, age at least 60 years).