RESUMEN
In acute myeloid leukemia (AML), internal tandem duplication mutations in the FLT3 tyrosine kinase receptor (FLT3-ITD) account for up to 25% of cases and are associated with a poor outcome. In order to better target this AML subtype, a comprehensive view of how FLT3-ITD impacts AML cell biology is required. Here, we found that FLT3-ITD expression increased basal autophagy in AML cells, and that both pharmacological and genetic inhibition of the receptor reduced autophagy in primary AML samples and cell lines. Conditional expression of shRNAs against key autophagy proteins demonstrated that autophagy is required for AML cell proliferation in vitro and for leukemic cells survival in a mouse model of xenograft. Importantly, autophagy inhibition also overcame FLT3 inhibitor resistance both in vitro and in vivo. The transcription factor ATF4 was identified as an essential actor of FLT3-ITD-induced autophagy. Cellular levels of ATF4 were highly dependent on FLT3-ITD activity, and downregulation of ATF4 inhibited autophagy-dependent AML cell proliferation and improved overall mouse survival similarly to autophagy inhibition. These results suggest that targeting autophagy or ATF4 in patients expressing FLT3 mutations may represent a novel promising and innovative therapeutic strategy for AML.
Asunto(s)
Factor de Transcripción Activador 4/metabolismo , Autofagia , Biomarcadores de Tumor/metabolismo , Resistencia a Antineoplásicos , Leucemia Mieloide Aguda/patología , Tirosina Quinasa 3 Similar a fms/metabolismo , Factor de Transcripción Activador 4/genética , Animales , Biomarcadores de Tumor/genética , Proliferación Celular , Humanos , Leucemia Mieloide Aguda/tratamiento farmacológico , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/metabolismo , Ratones , Ratones Endogámicos NOD , Ratones SCID , Mutación , Inhibidores de Proteínas Quinasas/farmacología , Secuencias Repetidas en Tándem , Células Tumorales Cultivadas , Ensayos Antitumor por Modelo de Xenoinjerto , Tirosina Quinasa 3 Similar a fms/genéticaRESUMEN
Phosphorylation by Akt on Ser 280 was reported to induce cytoplasmic retention and inactivation of CHK1 with consequent genetic instability in PTEN-/- cells. In acute myeloid leukemia cells carrying the FLT3-internal tandem duplication (ITD) mutation, we observed high rates of FLT3-ITD-dependent CHK1 Ser 280 phosphorylation. Pharmacological inhibition and RNA interference identified Pim1/2, not Akt, as effectors of this phosphorylation. Pim1 catalyzed Ser 280 phosphorylation in vitro and ectopic expression of Pim1/2-induced CHK1 phosphorylation. Ser 280 phosphorylation did not modify CHK1 localization, but facilitated its cell cycle and resistance functions in leukemic cells. FLT3, PIM or CHK1 inhibitors synergized with DNA-damaging agents to induce apoptosis, allowing cells to bypass the etoposide-induced G2/M arrest. Consistently, etoposide-induced CHK1-dependent phosphorylations of CDC25C on Ser 216 and histone H3 on Thr11 were decreased upon FLT3 inhibition. Accordingly, ectopic expression of CHK1 improved the resistance of FLT3-ITD cells and maintained histone H3 phosphorylation in response to DNA damage, whereas expression of unphosphorylated Ser 280Ala mutant did not. Finally, FLT3- and Pim-dependent phosphorylation of CHK1 on Ser 280 was confirmed in primary blasts from patients. These results identify a new pathway involved in the resistance of FLT3-ITD leukemic cells to genotoxic agents, and they constitute the first report of CHK1 Ser 280 regulation in myeloid malignancies.
Asunto(s)
Leucemia Mieloide Aguda/metabolismo , Proteínas Quinasas/metabolismo , Proteínas Proto-Oncogénicas c-pim-1/metabolismo , Línea Celular Tumoral , Quinasa 1 Reguladora del Ciclo Celular (Checkpoint 1) , Duplicación de Gen , Humanos , Espacio Intracelular/metabolismo , Leucemia Mieloide Aguda/genética , Fosforilación , Transporte de Proteínas , Serina/metabolismo , Transducción de Señal , Tirosina Quinasa 3 Similar a fms/metabolismoRESUMEN
Several receptor tyrosine kinases (TKs) are involved in the pathogenesis of acute myeloid leukemia (AML). Here, we have assessed the expression of the Recepteur d'Origine Nantais (RON) in leukemic cell lines and samples from AML patients. In a series of 86 AML patients, we show that both the full length and/or the short form (sf) of RON are expressed in 51% and 43% of cases, respectively. Interestingly, sfRON is not expressed in normal CD34+ hematopoietic cells and induces part of its oncogenic signaling through interaction with the Src kinase Lyn. sfRON-mediated signaling in leukemic cells also involves mTORC1, the proapoptotic bcl2-family member, BAD, but not the phosphatidylinositol 3-kinase/Akt pathway. Furthermore, the expression of sfRON was specifically downregulated by 5-azacytidine (AZA). Conversely, AZA could induce the expression of sfRON in sfRON-negative leukemic cells suggesting that the activity of this drug in AML and myelodysplastic syndromes could involve modulation of TKs. cMET/RON inhibitors exhibited an antileukemic activity exclusively in AML samples and cell lines expressing sfRON. These results might support clinical trials evaluating cMET/RON inhibitors in AML patients expressing sfRON.
Asunto(s)
Resistencia a Antineoplásicos/efectos de los fármacos , Leucemia Mieloide Aguda/tratamiento farmacológico , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Proto-Oncogénicas c-met/antagonistas & inhibidores , Proteínas Tirosina Quinasas Receptoras/metabolismo , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Antimetabolitos Antineoplásicos/farmacología , Apoptosis/efectos de los fármacos , Azacitidina/farmacología , Western Blotting , Proliferación Celular/efectos de los fármacos , Células Cultivadas , Femenino , Citometría de Flujo , Células Madre Hematopoyéticas/efectos de los fármacos , Células Madre Hematopoyéticas/metabolismo , Humanos , Inmunoprecipitación , Indoles/farmacología , Leucemia Mieloide Aguda/metabolismo , Masculino , Diana Mecanicista del Complejo 1 de la Rapamicina , Persona de Mediana Edad , Complejos Multiproteicos/genética , Complejos Multiproteicos/metabolismo , Fosfatidilinositol 3-Quinasas/genética , Fosfatidilinositol 3-Quinasas/metabolismo , Fosforilación/efectos de los fármacos , Piperazinas/farmacología , Proteínas Proto-Oncogénicas c-akt/genética , Proteínas Proto-Oncogénicas c-akt/metabolismo , Proteínas Proto-Oncogénicas c-met/genética , Proteínas Proto-Oncogénicas c-met/metabolismo , ARN Mensajero/genética , ARN Interferente Pequeño/genética , Reacción en Cadena en Tiempo Real de la Polimerasa , Proteínas Tirosina Quinasas Receptoras/antagonistas & inhibidores , Proteínas Tirosina Quinasas Receptoras/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transducción de Señal/efectos de los fármacos , Sulfonamidas/farmacología , Serina-Treonina Quinasas TOR/genética , Serina-Treonina Quinasas TOR/metabolismo , Adulto Joven , Proteína Letal Asociada a bcl/genética , Proteína Letal Asociada a bcl/metabolismo , Familia-src Quinasas/genética , Familia-src Quinasas/metabolismoRESUMEN
Myeloproliferative neoplasms are frequently associated with aberrant constitutive tyrosine kinase (TK) activity resulting from chimaeric fusion genes or point mutations such as BCR-ABL1 or JAK2 V617F. We report here the cloning and functional characterization of two novel fusion genes BCR-RET and FGFR1OP-RET in chronic myelomonocytic leukemia (CMML) cases generated by two balanced translocations t(10;22)(q11;q11) and t(6;10)(q27;q11), respectively. The two RET fusion genes leading to the aberrant activation of RET, are able to transform hematopoietic cells and skew the hematopoietic differentiation program towards the monocytic/macrophage lineage. The RET fusion genes seem to constitutively mimic the same signaling pathway as RAS mutations frequently involved in CMML. One patient was treated with Sorafenib, a specific inhibitor of the RET TK function, and demonstrated cytological and clinical remissions.
Asunto(s)
Diferenciación Celular/efectos de los fármacos , Leucemia Mielomonocítica Crónica/patología , Monocitos/citología , Proteínas Proto-Oncogénicas c-ret/genética , Secuencia de Bases , Cartilla de ADN , Humanos , Hibridación Fluorescente in Situ , Leucemia Mielomonocítica Crónica/genética , Mutación Puntual , Reacción en Cadena de la Polimerasa/métodos , Translocación GenéticaRESUMEN
The impact of ten-eleven-translocation 2 (TET2) mutations on response to azacitidine (AZA) in MDS has not been reported. We sequenced the TET2 gene in 86 MDS and acute myeloid leukemia (AML) with 20-30% blasts treated by AZA, that is disease categories wherein this drug is approved by Food and Drug Administration (FDA). Thirteen patients (15%) carried TET2 mutations. Patients with mutated and wild-type (WT) TET2 had mostly comparable pretreatment characteristics, except for lower hemoglobin, better cytogenetic risk and longer MDS duration before AZA in TET2 mutated patients (P=0.03, P=0.047 and P=0.048, respectively). The response rate (including hematological improvement) was 82% in MUT versus 45% in WT patients (P=0.007). Mutated TET2 (P=0.04) and favorable cytogenetic risk (intermediate risk: P=0.04, poor risk: P=0.048 compared with good risk) independently predicted a higher response rate. Response duration and overall survival were, however, comparable in the MUT and WT groups. In higher risk MDS and AML with low blast count, TET2 status may be a genetic predictor of response to AZA, independently of karyotype.
Asunto(s)
Antimetabolitos Antineoplásicos/uso terapéutico , Azacitidina/uso terapéutico , Proteínas de Unión al ADN/genética , Leucemia Mieloide Aguda/genética , Mutación , Síndromes Mielodisplásicos/genética , Proteínas de Neoplasias/genética , Proteínas Proto-Oncogénicas/genética , Anciano , Anciano de 80 o más Años , Metilación de ADN/efectos de los fármacos , ADN de Neoplasias/genética , Proteínas de Unión al ADN/fisiología , Dioxigenasas , Supervivencia sin Enfermedad , Femenino , Hemoglobinas/análisis , Humanos , Estimación de Kaplan-Meier , Cariotipificación , Leucemia Mieloide Aguda/sangre , Leucemia Mieloide Aguda/tratamiento farmacológico , Leucemia Mieloide Aguda/patología , Recuento de Leucocitos , Masculino , Persona de Mediana Edad , Síndromes Mielodisplásicos/sangre , Síndromes Mielodisplásicos/tratamiento farmacológico , Síndromes Mielodisplásicos/patología , Proteínas de Neoplasias/fisiología , Proteínas Proto-Oncogénicas/fisiología , Análisis de Secuencia de ADN , Resultado del TratamientoRESUMEN
Telomerase catalytic subunit (hTERT) exerts important cellular functions including telomere homeostasis, genetic stability, cell survival and perhaps differentiation. However, the nature of external or internal signals, which regulate hTERT expression in tissues, remains poorly understood. Thus, whereas it has been described that hTERT gene is regulated along the differentiation of primitive myeloid progenitors, the effect of specific cytokines on telomerase expression in each myeloid lineage is currently unknown. Based on these considerations, we have investigated hTERT expression in erythroid cells treated with erythropoietin (EPO) and transforming growth factor beta (TGFbeta), as putative positive and negative regulators, respectively. We describe here that EPO activates hTERT gene transcription in in vitro-expanded primary erythroid precursors as well as in UT7 erythroleukemia cells. In UT7 cells, this study shows also that EPO acts through a JAK2/STAT5/c-myc axis. In contrast, TGFbeta blocks EPO signaling downstream of c-myc induction through a Smad3-dependent mechanism. Finally, hTERT appears to be efficiently regulated by EPO and TGFbeta in an opposite way in erythropoietic cells, arguing for a role of telomerase in red blood cell production.
Asunto(s)
Células Precursoras Eritroides/metabolismo , Eritropoyetina/metabolismo , Regulación Leucémica de la Expresión Génica , Telomerasa/biosíntesis , Factor de Crecimiento Transformador beta/metabolismo , Antígenos CD34/biosíntesis , Apoptosis , Línea Celular , Línea Celular Tumoral , Proliferación Celular , Supervivencia Celular , Humanos , Modelos Biológicos , Plásmidos/metabolismo , Proteínas Proto-Oncogénicas c-myc/metabolismoRESUMEN
Activation of the Wnt/beta-catenin pathway has recently been shown to be crucial to the establishment of leukemic stem cells in chronic myeloid leukemia. We sought to determine whether beta-catenin was correlated to clonogenic capacity also in the acute myeloid leukemia (AML) setting. Eighty-two patients were retrospectively evaluated for beta-catenin expression by Western blot. beta-Catenin was expressed (although at various protein levels) in 61% of patients, and was undetectable in the remaining cases. In our cohort, beta-catenin expression was correlated with the clonogenic proliferation of AML-colony forming cells (AML-CFC or CFU-L) in methylcellulose in the presence of 5637-conditioned medium, and more strikingly with self-renewing of leukemic cells, as assessed in vitro by a re-plating assay. In survival analyses, beta-catenin appeared as a new independent prognostic factor predicting poor event-free survival and shortened overall survival (both with P<0.05). Furthermore, variations in beta-catenin protein levels were dependent on post-transcriptional mechanisms involving the Wnt/beta-catenin pathway only in leukemic cells. Indeed, beta-catenin negative leukemic cells were found to increase beta-catenin in response to Wnt3a agonist in contrast to normal counterparts. Altogether, our data pave the way to the evaluation of Wnt pathway inhibition as a new rationale for eradicating the clonogenic pool of AML cells.
Asunto(s)
Leucemia Mieloide Aguda/mortalidad , Leucemia Mieloide Aguda/patología , Células Madre Neoplásicas/fisiología , beta Catenina/genética , Línea Celular Tumoral , Células Clonales , Femenino , Regulación Leucémica de la Expresión Génica , Humanos , Leucemia Monocítica Aguda/metabolismo , Leucemia Monocítica Aguda/mortalidad , Leucemia Monocítica Aguda/patología , Leucemia Mieloide Aguda/metabolismo , Leucemia Mielomonocítica Aguda/metabolismo , Leucemia Mielomonocítica Aguda/mortalidad , Leucemia Mielomonocítica Aguda/patología , Masculino , Persona de Mediana Edad , Células Madre Neoplásicas/patología , Valor Predictivo de las Pruebas , Pronóstico , Estudios Retrospectivos , Transducción de Señal , Análisis de Supervivencia , Proteínas Wnt/metabolismoRESUMEN
The mechanism by which leukemic cells interfere with normal hematopoiesis remains unclear. We show here that, whereas the leukemic KG1a cells are naturally devoid from cellular cytotoxicity, once activated by TNFalpha, they display cytolytic activity toward various cellular targets including CFU-GM. This mechanism is dependent on stimulation of the granzyme B/perforin system. In addition, KG1a cells expressed the NKG2D receptor and its signal-transducing adaptator DAP 10, which were functional as confirmed by redirected lysis experiments. Interestingly, flow cytometry analysis of 20 samples of patients with acute myeloid leukemia (AML) (FAB M0-M5) revealed the expression of NKG2D (40%) and other natural cytotoxicity receptors (40% for NKp30, 74% for NKp44, 39% for NKp46) by a pool >15% of leukemic cells. Furthermore, CD34+ hematopoietic progenitors undergoing granulomonocytic differentiation expressed NKG2D ligands. Altogether, we propose a model in which, upon stimulation by TNFalpha, leukemic cells may exert cytotoxicity against myeloid progenitors. This finding may have important clinical implications in the context of diseases characterized by TNFalpha accumulation, such as AML or myelodisplasic syndromes.
Asunto(s)
Citotoxicidad Inmunológica , Leucemia Mieloide/patología , Células Progenitoras Mieloides/citología , Receptores Inmunológicos/fisiología , Factor de Necrosis Tumoral alfa/farmacología , Enfermedad Aguda , Línea Celular Tumoral , Técnicas de Cocultivo , Granzimas , Hematopoyesis , Humanos , Leucemia Mieloide/inmunología , Glicoproteínas de Membrana/genética , Subfamilia K de Receptores Similares a Lectina de Células NK , Perforina , Proteínas Citotóxicas Formadoras de Poros , Receptores Inmunológicos/análisis , Receptores de Células Asesinas Naturales , Serina Endopeptidasas/genética , Regulación hacia ArribaAsunto(s)
Antimetabolitos Antineoplásicos/farmacología , Citarabina/farmacología , Esfingomielina Fosfodiesterasa/metabolismo , Familia-src Quinasas/metabolismo , Apoptosis , ADN/biosíntesis , ADN/efectos de los fármacos , Activación Enzimática/efectos de los fármacos , Células HL-60 , Humanos , Proteínas Quinasas JNK Activadas por Mitógenos , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Estrés Oxidativo/fisiología , Especies Reactivas de Oxígeno/metabolismo , Transducción de Señal , Células U937RESUMEN
Anthracyclines such as daunorubicin (DNR) generate radical oxygen species (ROS), which account, at least in part, for their cytotoxic effect. We observed that early ceramide generation (within 6-10 min) through neutral sphingomyelinase stimulation was inhibitable by the antioxidants N-acetylcysteine and pyrrolidine dithiocarbamate, which led to a decrease in apoptosis (>95% decrease in DNA fragmentation after 6 h). Furthermore, we observed that DNR triggers the c-Jun N-terminal kinase (JNK) and the transcription factor activated protein-1 through an antioxidant-inhibitable mechanism. Treatment of U937 cells with cell-permeant ceramides induced both an increase in ROS generation and JNK activation, and apoptosis, all of which were antioxidant-sensitive. In conclusion, DNR-triggered apoptosis implicates a ceramide-mediated, ROS-dependent JNK and activated protein-1 activation.
Asunto(s)
Antineoplásicos/farmacología , Apoptosis , Ceramidas/biosíntesis , Daunorrubicina/farmacología , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Acetilcisteína/farmacología , Antioxidantes/farmacología , Interacciones Farmacológicas , Transporte de Electrón/efectos de los fármacos , Activación Enzimática , Depuradores de Radicales Libres/antagonistas & inhibidores , Depuradores de Radicales Libres/farmacología , Humanos , Peróxido de Hidrógeno/metabolismo , Proteínas Quinasas JNK Activadas por Mitógenos , Mitocondrias/efectos de los fármacos , Mitocondrias/metabolismo , Pirrolidinas/farmacología , Esfingomielina Fosfodiesterasa/metabolismo , Tiocarbamatos/farmacología , Factor de Transcripción AP-1/metabolismo , Células U937RESUMEN
Daunorubicin induces apoptosis in myeloid leukemia cells by activation of neutral sphingomyelinase and ceramide generation occurring 4-10 min after daunorubicin addition. We show here that daunorubicin is able to increase the phosphoinositide 3-kinase activity and enhance intracellular phosphoinositide 3-kinase lipid products prior to ceramide generation. Daunorubicin activates Akt, a downstream phosphoinositide 3-kinase effector. Interestingly, the phosphoinositide 3-kinase inhibitors wortmannin and LY294002 accelerate daunorubicin-induced apoptosis in U937 cells. The phosphoinositide 3-kinase/Akt pathway has been involved in cell survival following serum deprivation, tumor necrosis factor alpha, anti-Fas and UV radiations. Our results suggest that anti-tumor agents such as daunorubicin may also activate anti-apoptotic signals that could contribute to drug resistance.
Asunto(s)
Daunorrubicina/farmacología , Fosfatidilinositol 3-Quinasas/metabolismo , Enfermedad Aguda , Androstadienos/farmacología , Apoptosis , Ceramidas/metabolismo , Cromonas/farmacología , Activación Enzimática , Inhibidores Enzimáticos/farmacología , Humanos , Cinética , Leucemia Mieloide , Morfolinas/farmacología , Fosfatos de Fosfatidilinositol/metabolismo , Células U937 , WortmaninaRESUMEN
Several studies have suggested that diacylglycerol can affect the induction of apoptosis induced by toxicants and ceramide. The present study demonstrates that clinically relevant concentrations of the chemotherapeutic drugs daunorubicin and mitoxantrone (0.2-1 microM) transiently stimulated concurrently with sphingomyelin-derived ceramide generation and diacylglycerol and phosphorylcholine production within 4 to 10 min via phospholipase C hydrolysis of phosphatidylcholine. Pretreatment of cells with the xanthogenate compound D609, a potent inhibitor of phosphatidylcholine-phospholipase C, led to significant inhibition of drug triggered diacylglycerol and phosphorylcholine production and to a sustained increase in ceramide levels for a period up to 2 h. Moreover, D609 pretreatment induced both cell death and ceramide generation at daunorubicin and mitoxantrone concentrations previously shown to be ineffective (i.e., 0.1 microM). These results underline the importance of diacylglycerol in the regulation of programmed cell death and strongly argue for a balance between apoptotic (ceramide) and survival (diacylglycerol) signal transducers.