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Leucemia Mieloide Aguda , Proteína Tumoral p73 , Humanos , Leucemia Mieloide Aguda/metabolismo , Leucemia Mieloide Aguda/genética , Pronóstico , Proteína Tumoral p73/genética , Proteína Tumoral p73/metabolismo , Femenino , Masculino , Persona de Mediana Edad , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Proteínas Supresoras de Tumor/genética , Proteínas Supresoras de Tumor/metabolismo , Anciano , Regulación Leucémica de la Expresión Génica , AdultoAsunto(s)
Calreticulina , Proteínas de Fusión bcr-abl , Leucemia Mielógena Crónica BCR-ABL Positiva , Mutación , Trombocitemia Esencial , Calreticulina/genética , Calreticulina/metabolismo , Proteínas de Fusión bcr-abl/genética , Proteínas de Fusión bcr-abl/metabolismo , Humanos , Leucemia Mielógena Crónica BCR-ABL Positiva/genética , Leucemia Mielógena Crónica BCR-ABL Positiva/metabolismo , Leucemia Mielógena Crónica BCR-ABL Positiva/patología , Masculino , Persona de Mediana Edad , Trombocitemia Esencial/genética , Trombocitemia Esencial/metabolismo , Trombocitemia Esencial/patologíaRESUMEN
Chronic myeloid leukemia (CML) is a myeloproliferative neoplasm characterized by the presence of the Philadelphia chromosome, which generates the oncogene BCR-ABL1. Protease-activated receptor 1 (PAR1) is involved in tumor progression and angiogenesis. We have previously reported that PAR1 expression is elevated in human leukemias that display a more aggressive clinical behavior, including the blast crisis of CML. In this study, we analyzed the crosstalk between the oncoprotein BCR-ABL and PAR1 in CML. Leukemic cell lines transfected with the BCR-ABL1 oncogene showed significantly higher expression levels of PAR1 compared with that of wild-type counterparts. This phenomenon was reversed by treatment with tyrosine kinase inhibitors (TKIs). Conversely, treatment with the PAR1 antagonist SCH79797 inhibited BCR-ABL expression. The PAR1 antagonist induced apoptosis in a dose- and time-dependent manner. Higher vascular endothelial growth factor (VEGF) levels were observed in cells transfected with BCR-ABL1 than in their wild-type counterparts. VEGF expression was strongly inhibited after treatment with either TKIs or the PAR1 antagonist. Finally, we evaluated PAR1 expression in CML patients who were either in the blast or chronic phases and had either received TKI treatment or no treatment. A significant decrease in PAR1 expression was observed in treatment-responsive patients, as opposed to a significant increase in PAR1 expression levels in treatment-resistant patients. Patients classified as high risk according to the Sokal index showed higher PAR1 expression levels. Our results demonstrate the crosstalk between BCR-ABL and PAR1. These data may offer important insight into the development of new therapeutic strategies for CML.
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Proteínas de Fusión bcr-abl/genética , Regulación Leucémica de la Expresión Génica , Leucemia Mielógena Crónica BCR-ABL Positiva/tratamiento farmacológico , Leucemia Mielógena Crónica BCR-ABL Positiva/genética , Inhibidores de Proteínas Quinasas/farmacología , Receptor PAR-1/genética , Adulto , Anciano , Antineoplásicos/farmacología , Apoptosis/efectos de los fármacos , Estudios de Casos y Controles , Línea Celular Tumoral , Cromonas/farmacología , Progresión de la Enfermedad , Relación Dosis-Respuesta a Droga , Femenino , Flavonoides/farmacología , Proteínas de Fusión bcr-abl/metabolismo , Humanos , Leucemia Mielógena Crónica BCR-ABL Positiva/metabolismo , Leucemia Mielógena Crónica BCR-ABL Positiva/patología , Masculino , Persona de Mediana Edad , Morfolinas/farmacología , Células Mieloides/efectos de los fármacos , Células Mieloides/metabolismo , Células Mieloides/patología , Cromosoma Filadelfia , Pirimidinas/farmacología , Pirroles/farmacología , Quinazolinas/farmacología , Receptor PAR-1/metabolismo , Transducción de Señal , Resultado del Tratamiento , Factor A de Crecimiento Endotelial Vascular/genética , Factor A de Crecimiento Endotelial Vascular/metabolismoRESUMEN
BACKGROUND: Halofuginone (HF) is a low-molecular-weight alkaloid that has been demonstrated to interfere with Metalloproteinase-2 (MMP-2) and Tumor Growth Factor-ß (TGF-ß) function and, to present antiangiogenic, antiproliferative and proapoptotic properties in several solid tumor models. Based on the fact that high levels of Vascular Endothelial Growth Factor (VEGF) and increased angiogenesis have been described in acute myeloid leukemia and associated with disease progression, we studied the in vivo effects of HF using an Acute Promyelocytic Leukemia (APL) mouse model. METHODS: NOD/SCID mice were transplanted with leukemic cells from hCG-PML/RARA transgenic mice (TM) and treated with HF 150 µg/kg/day for 21 days. The leukemic infiltration and the percentage of VEGF+ cells were evaluated by morphology and flow cytometry. The effect of HF on the gene expression of several pro- and antiangiogenic factors, phosphorylation of SMAD2 and VEGF secretion was assessed in vitro using NB4 and HUVEC cells. RESULTS: HF treatment resulted in hematological remission with decreased accumulation of immature cell and lower amounts of VEGF in BM of leukemic mice. In vitro, HF modulated gene expression of several pro- and antiangiogenic factors, reduced VEGF secretion and phosphorylation of SMAD2, blocking TGF-ß-signaling. CONCLUSION: Taken together, our results demonstrate that HF inhibits SMAD2 signaling and reduces leukemia growth and angiogenesis.
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Leucemia Promielocítica Aguda/metabolismo , Piperidinas/metabolismo , Quinazolinonas/metabolismo , Proteína Smad2/genética , Animales , Modelos Animales de Enfermedad , Humanos , Inmunofenotipificación , Ratones , Ratones Endogámicos NOD , Ratones SCID , Ratones Transgénicos , Neovascularización Patológica , Fosforilación , Proteína Smad2/metabolismo , Células Tumorales CultivadasRESUMEN
INTRODUCTION: The multitargeted tyrosine kinase inhibitor (TKI) crizotinib is active against ALK translocated non-small-cell lung cancer (NSCLC); however acquired resistance invariably develops over time. ALK mutations have previously been implicated in only a third of resistant tumors. We sought to evaluate alternative mechanisms of resistance and preclinical strategies to overcome these in a cell line driven by EML4-ALK. METHODS: We selected the NSCLC cell line NCI-H3122 (H3122: EML4-ALK E13;A20) and derived resistant variants that were able to grow in the presence of 1 µM crizotinib. These were analyzed for ALK mutations, sensitivity to crizotinib in combination with other TKIs, and for activation of alternative tyrosine kinases. RESULTS: All H3122 crizotinib resistant (CR) clones lacked amplification or mutations in the kinase domain of ALK. To evaluate if possible alternative kinases functioned as "bypass" tracks for downstream signaling activation in these resistance cells, we performed of phosho-receptor tyrosine kinase array that demonstrated that CR clones had higher phospho-EGFR signals than H3122 cells before and after exposure to crizotinib. A functional approach of dual ALK TKI (with crizotinib) with combinatory TKI inhibition was used as a secondary screen for possible targets. Crizotinib+erlotinib (reversible EGFR TKI) and crizotinib+afatinib (irreversible EGFR/ERBB2 TKI) were able to inhibit the growth of H3122 CR clones, confirming EGFR activation as a mechanism of resistance. The removal of crizotinib from the culture media re-sensitized CR cells to crizotinib. CONCLUSIONS: We identified activation of EGFR as a mechanism of resistance to crizotinib in preclinical models of ALK translocated NSCLC. If EGFR activation is confirmed as a predominant mechanism of ALK TKI-induced resistance in patient-derived tumors, the use of ALK plus EGFR TKIs could be explored for this important cohort of NSCLCs.
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Carcinoma de Pulmón de Células no Pequeñas/tratamiento farmacológico , Receptores ErbB/metabolismo , Neoplasias Pulmonares/tratamiento farmacológico , Inhibidores de Proteínas Quinasas/uso terapéutico , Pirazoles/uso terapéutico , Piridinas/uso terapéutico , Afatinib , Quinasa de Linfoma Anaplásico , Carcinoma de Pulmón de Células no Pequeñas/genética , Procesos de Crecimiento Celular/efectos de los fármacos , Línea Celular Tumoral , Crizotinib , Resistencia a Antineoplásicos/genética , Receptores ErbB/genética , Amplificación de Genes , Reordenamiento Génico , Humanos , Neoplasias Pulmonares/genética , Análisis por Micromatrices , Mutación/genética , Proteínas de Fusión Oncogénica/genética , Fosforilación , Quinazolinas/uso terapéutico , Proteínas Tirosina Quinasas Receptoras/genética , Proteínas Tirosina Quinasas Receptoras/metabolismoRESUMEN
INTRODUCTION: Most clinically available small-molecule kinase inhibitors are multi-targeted and can inhibit multiple kinases. Our driving hypothesis was that one of these multi-targeted tyrosine kinase inhibitors (TKIs) would have antiproliferative activity against ROS1 translocated non-small-cell lung cancer (NSCLC). METHODS: We selected NSCLC cell lines--A549 (KRAS G12S), NCI-H3255 (EGFR L858R), NCI-H3122 (EML4-ALK E13;A20), and HCC78 (SLC34A2-ROS1)-to evaluate the antiproliferative effects of submicromolar concentrations of the multitargeted TKIs imatinib, sorafenib, erlotinib, and crizotinib. RESULTS: Imatinib and sorafenib were unable to significantly inhibit proliferation of the aforementioned cell lines. Erlotinib only inhibited EGFR mutated NCI-H3255, as expected. Crizotinib displayed dose-dependent inhibition of anaplastic lymphoma kinase translocated NCI-H3122 and also ROS1--translocated HCC78. The SLC34A2-ROS1 translocated HCC78 cell line had phosphorylated levels of ROS1, AKT, and ERK inhibited by submicromolar doses of crizotinib, and subsequently underwent apoptosis. CONCLUSIONS: The ROS1-translocated HCC78 cell line was sensitive to inhibition by the multitargeted ALK/MET/RON/ROS1 inhibitor crizotinib. Preclinical data supports the clinical development of crizotinib for ROS1-translocated NSCLC.
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Evaluación Preclínica de Medicamentos , Neoplasias Pulmonares/tratamiento farmacológico , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Tirosina Quinasas/genética , Proteínas Tirosina Quinasas/metabolismo , Proteínas Proto-Oncogénicas/genética , Proteínas Proto-Oncogénicas/metabolismo , Pirazoles/farmacología , Piridinas/farmacología , Translocación Genética , Receptores de Activinas Tipo II/antagonistas & inhibidores , Receptores de Activinas Tipo II/metabolismo , Apoptosis/efectos de los fármacos , Western Blotting , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Crizotinib , Humanos , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patología , Fosforilación/efectos de los fármacos , Proteínas Tirosina Quinasas/antagonistas & inhibidores , Proteínas Proto-Oncogénicas/antagonistas & inhibidores , Proteínas Proto-Oncogénicas c-met/antagonistas & inhibidores , Proteínas Proto-Oncogénicas c-met/metabolismo , Proteínas Tirosina Quinasas Receptoras/antagonistas & inhibidores , Proteínas Tirosina Quinasas Receptoras/metabolismoRESUMEN
Increased fibrinolysis is an important component of acute promyelocytic leukemia (APL) bleeding diathesis. APL blasts overexpress annexin II (ANXII), a receptor for tissue plasminogen activator (tPA), and plasminogen, thereby increasing plasmin generation. Previous studies suggested that ANXII plays a pivotal role in APL coagulopathy. ANXII binding to tPA can be inhibited by homocysteine and hyperhomocysteinemia can be induced by L-methionine supplementation. In the present study, we used an APL mouse model to study ANXII function and the effects of hyperhomocysteinemia in vivo. Leukemic cells expressed higher ANXII and tPA plasma levels (11.95 ng/mL in leukemic vs 10.74 ng/mL in wild-type; P = .004). In leukemic mice, administration of L-methionine significantly increased homocysteine levels (49.0 µmol/mL and < 6.0 µmol/mL in the treated and nontreated groups, respectively) and reduced tPA levels to baseline concentrations. The latter were also decreased after infusion of the LCKLSL peptide, a competitor for the ANXII tPA-binding site (11.07 ng/mL; P = .001). We also expressed and purified the p36 component of ANXII in Pichia methanolica. The infusion of p36 in wild-type mice increased tPA and thrombin-antithrombin levels, and the latter was reversed by L-methionine administration. The results of the present study demonstrate the relevance of ANXII in vivo and suggest that methionine-induced hyperhomocysteinemia may reverse hyperfibrinolysis in APL.
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Anexina A2/metabolismo , Fibrinólisis/fisiología , Hiperhomocisteinemia/inducido químicamente , Leucemia Promielocítica Aguda , Metionina/farmacología , Animales , Anexina A2/farmacología , Coagulación Sanguínea/fisiología , Trasplante de Médula Ósea , Modelos Animales de Enfermedad , Fibrinolisina/metabolismo , Homocisteína/sangre , Leucemia Promielocítica Aguda/complicaciones , Leucemia Promielocítica Aguda/metabolismo , Leucemia Promielocítica Aguda/patología , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/farmacología , Activador de Tejido Plasminógeno/sangreRESUMEN
Promyelocytic leukemia-retinoic acid receptor alpha (PML-RARα) expression in acute promyelocytic leukemia (APL) impairs transforming growth factor beta (TGFß) signaling, leading to cell growth advantage. Halofuginone (HF), a low-molecular-weight alkaloid that modulates TGFß signaling, was used to treat APL cell lines and non-obese diabetic/severe combined immunodeficiency (NOD/SCID) mice subjected to transplantation with leukemic cells from human chorionic gonadotrophin-PML-RARα transgenic mice (TG). Cell cycle analysis using incorporated bromodeoxyuridine and 7-amino-actinomycin D showed that, in NB4 and NB4-R2 APL cell lines, HF inhibited cellular proliferation (P<0.001) and induced apoptosis (Pâ=â0.002) after a 24-hour incubation. Addition of TGFß revealed that NB4 cells were resistant to its growth-suppressive effects and that HF induced these effects in the presence or absence of the cytokine. Cell growth inhibition was associated with up-regulation of TGFß target genes involved in cell cycle regulation (TGFB, TGFBRI, SMAD3, p15, and p21) and down-regulation of MYC. Additionally, TGFß protein levels were decreased in leukemic TG animals and HF in vivo could restore TGFß values to normal. To test the in vivo anti-leukemic activity of HF, we transplanted NOD/SCID mice with TG leukemic cells and treated them with HF for 21 days. HF induced partial hematological remission in the peripheral blood, bone marrow, and spleen. Together, these results suggest that HF has anti-proliferative and anti-leukemic effects by reversing the TGFß blockade in APL. Since loss of the TGFß response in leukemic cells may be an important second oncogenic hit, modulation of TGFß signaling may be of therapeutic interest.
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Leucemia Promielocítica Aguda/metabolismo , Leucemia Promielocítica Aguda/patología , Piperidinas/farmacología , Quinazolinonas/farmacología , Transducción de Señal/efectos de los fármacos , Factor de Crecimiento Transformador beta/metabolismo , Animales , Recuento de Células Sanguíneas , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Modelos Animales de Enfermedad , Relación Dosis-Respuesta a Droga , Regulación Leucémica de la Expresión Génica/efectos de los fármacos , Humanos , Leucemia Promielocítica Aguda/sangre , Leucemia Promielocítica Aguda/genética , Ratones , Ratones SCID , Proteínas de Fusión Oncogénica/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Receptor Tipo II de Factor de Crecimiento Transformador beta , Receptores de Factores de Crecimiento Transformadores beta/metabolismo , Proteína smad3/metabolismo , Factor de Crecimiento Transformador beta/antagonistas & inhibidores , Factor de Crecimiento Transformador beta/genética , Factor de Crecimiento Transformador beta/farmacología , Regulación hacia Arriba/efectos de los fármacosAsunto(s)
Anticuerpos Monoclonales/administración & dosificación , Pérdida de Sangre Quirúrgica/prevención & control , Retinopatía Diabética/complicaciones , Desprendimiento de Retina/cirugía , Vitrectomía/métodos , Anticuerpos Monoclonales Humanizados , Retinopatía Diabética/diagnóstico , Estudios de Seguimiento , Humanos , Periodo Intraoperatorio , Inyecciones Intravítreas , Ranibizumab , Desprendimiento de Retina/diagnóstico , Desprendimiento de Retina/etiología , Resultado del TratamientoAsunto(s)
Leucemia Mieloide Aguda/genética , Tirosina Quinasa 3 Similar a fms/genética , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Brasil , Análisis Mutacional de ADN , Femenino , Pruebas Genéticas , Humanos , Inmunofenotipificación , Masculino , Persona de Mediana Edad , Mutación/genética , Adulto JovenRESUMEN
Some cases of T-cell acute lymphoblastic leukaemia (ALL) express markers found in natural-killer (NK) cells, such as CD56 and CD16. Out of 84 T-cell ALL cases diagnosed at our Institution, CD56 and/or CD16 was detected in 24 (28.5%), which we designated T/NK-ALL group. Clinical features, laboratory characteristics, survival and expression of cytotoxic molecules were compared in T/NK-ALL and T-ALL patients. Significant differences were observed regarding age (24.9 vs. 16.4 years in T/NK-ALL and T-ALL, respectively, P = 0.006) and platelet counts (177 x 10(9)/l vs. 75 x 10(9)/l in T/NK-ALL and T-ALL, respectively, P = 0.03). Immunophenotypic analysis demonstrated that CD34, CD45RA and CD33 were more expressed in T/NK-ALL patients, whereas CD8 and terminal deoxynucleotidyl transferase were more expressed in T-ALL patients (P < 0.05). The mean overall survival (863 vs. 1869 d, P = 0.02) and disease-free survival (855 vs. 2095 d, P = 0.002) were shorter in patients expressing CD56/CD16. However, multivariate analysis identified CD56/CD16 as an independent prognostic factor only for DFS. Cytotoxic molecules were highly expressed in T/NK-ALL compared to T-ALL. Perforin, granzyme B and TIA-1 were detected in 12/17, 4/17 and 7/24 T/NK-ALL patients and in 1/20, 0/20 and 1/20 T-ALL respectively (P < 0.001, P = 0.036 and P = 0.054). Therefore, the presence of CD56/CD16 was associated with distinct clinical features and expression of cytotoxic molecules in the blasts.
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Antígeno CD56/análisis , Células Asesinas Naturales/inmunología , Leucemia-Linfoma Linfoblástico de Células T Precursoras/inmunología , Receptores de IgG/análisis , Adolescente , Adulto , Factores de Edad , Antígenos CD/análisis , Antígenos CD34/análisis , Antígenos de Diferenciación Mielomonocítica/análisis , Biomarcadores/análisis , Complejo CD3/análisis , Supervivencia sin Enfermedad , Femenino , Citometría de Flujo , Granzimas/análisis , Humanos , Inmunofenotipificación , Estimación de Kaplan-Meier , Antígenos Comunes de Leucocito/análisis , Masculino , Perforina/análisis , Recuento de Plaquetas , Proteínas de Unión a Poli(A)/análisis , Leucemia-Linfoma Linfoblástico de Células T Precursoras/tratamiento farmacológico , Leucemia-Linfoma Linfoblástico de Células T Precursoras/mortalidad , Lectina 3 Similar a Ig de Unión al Ácido Siálico , Tasa de Supervivencia , Antígeno Intracelular 1 de las Células T , Resultado del Tratamiento , Adulto JovenRESUMEN
BACKGROUND: The most primitive leukemic precursor in acute myeloid leukemia (AML) is thought to be the leukemic stem cell (LSC), which retains the properties of self-renewal and high proliferative capacity and quiescence of the hematopoietic stem cell. LSC seems to be immunophenotypically distinct and more resistant to chemotherapy than the more committed blasts. Considering that the multidrug resistance (MDR) constitutive expression may be a barrier to therapy in AML, we have investigated whether various MDR transporters were differentially expressed at the protein level by different leukemic subsets. METHODS: The relative expression of the drug-efflux pumps P-gp, MRP, LRP, and BCRP was evaluated by mean fluorescence index (MFI) and the Kolmogorov-Smirnov analysis (D values) in five leukemic subpopulations: CD34+CD38-CD123+ (LSCs), CD34+CD38+CD123-, CD34+CD38+CD123+, CD34+CD38+CD123-, and CD34- mature cells in 26 bone marrow samples of CD34+ AML cases. RESULTS: : The comparison between the two more immature subsets (LSC versus CD34+CD38-CD123- cells) revealed a higher P-gp, MRP, and LRP expression in LSCs. The comparative analysis between LSCs and subsets of intermediate maturation (CD34+CD38+) demonstrated the higher BCRP expression in the LSCs. In addition, P-gp expression was also significantly higher in the LSC compared to CD34+CD38+CD123- subpopulation. Finally, the comparative analysis between LSC and the most mature subset (CD34-) revealed higher MRP and LRP and lower P-gp expression in the LSCs. CONCLUSIONS: Considering the cellular heterogeneity of AML, the higher MDR transporters expression at the most immature, self-renewable, and quiescent LSC population reinforces that MDR is one of the mechanisms responsible for treatment failure.
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Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/metabolismo , Transportadoras de Casetes de Unión a ATP/metabolismo , Leucemia Mieloide Aguda/metabolismo , Proteínas Asociadas a Resistencia a Múltiples Medicamentos/metabolismo , Proteínas de Neoplasias/metabolismo , Células Madre Neoplásicas/metabolismo , Partículas Ribonucleoproteicas en Bóveda/metabolismo , Transportador de Casetes de Unión a ATP, Subfamilia G, Miembro 2 , Biomarcadores de Tumor/metabolismo , Estudios de Casos y Controles , Diferenciación Celular , Resistencia a Antineoplásicos , Citometría de Flujo/métodos , Humanos , Leucemia Mieloide Aguda/tratamiento farmacológico , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/patología , Células Madre Neoplásicas/clasificación , Células Madre Neoplásicas/patologíaRESUMEN
The preferentially expressed antigen in melanoma (PRAME) gene is aberrantly expressed in chronic lymphoproliferative disorders (CLD). We produced and characterized an anti-PRAME monoclonal antibody (MoAb), which was then applied in a quantitative flow cytometric (QFC) method to evaluate PRAME expression in leukemic cells from the peripheral blood (PB) of 47 patients with chronic lymphocytic leukemia and seven with mantle cell lymphoma as well as in the PB mononuclear cells (PBMCs) and B lymphocytes from 15 healthy subjects. Approximately 90% of CLD, but none of the normal samples, presented more than 20% of PRAME+ lymphocytes. Moreover, the intensity of PRAME expression was significantly higher in CLD cells compared to normal B lymphocytes and PBMCs. By immunofluorescence microscopy and by permeabilized flow cytometry we demonstrated that PRAME is a membrane antigen and a cytoplasmic protein aberrantly expressed in malignant CLD. Our results suggest that the analysis of PRAME protein may contribute for the distinction between normal and leukemic cells in CLD, and that PRAME may be a potential target for therapy.