RESUMO
Chidamide is a novel selective histone deacetylase inhibitor (HDACi) with promising activity in hematological malignancies, but its role in chronic myeloid leukemia (CML) was marginally addressed. In this study, we firstly demonstrated that chidamide alone inhibited CML cells proliferation, induced apoptosis and cell cycle arrest. Further, chidamide combined with imatinib (IM) induced synergistic lethality in CML cell line KBM5, as well as IM-resistant CML cells KBM5T315I, associated with a marked reduction of Bcr-Abl kinase activity and acetyl-histone H3 expression. The combination treatment markedly inhibited constitutive activity of ß-catenin signaling in IM-resistant cells and abolished the protective effects of mesenchymal stromal cells (MSCs) to CML cells. In addition, the co-treatment significantly reduced Bcr-Abl and ß-catenin transcript levels and induced apoptosis of primary CD34+ stem/progenitor cells derived from blast crisis (BC)-CML patients, but exhibited minimal toxicity to normal CD34+ progenitors. Collectively, our data show that combination of chidamide and imatinib synergistically targets tyrosine kinase inhibitor (TKI) -resistant BC-CML cells via inhibition of Bcr-Abl and ß-catenin signaling, suggesting that this combination has the potential for treating TKI-resistant CML and improving clinical outcomes of BC-CML patients.
Assuntos
Aminopiridinas/farmacologia , Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Benzamidas/farmacologia , Resistencia a Medicamentos Antineoplásicos , Proteínas de Fusão bcr-abl/antagonistas & inibidores , Inibidores de Histona Desacetilases/farmacologia , Mesilato de Imatinib/farmacologia , Leucemia Mielogênica Crônica BCR-ABL Positiva/tratamento farmacológico , Inibidores de Proteínas Quinases/farmacologia , Apoptose/efeitos dos fármacos , Crise Blástica/tratamento farmacológico , Crise Blástica/enzimologia , Crise Blástica/genética , Crise Blástica/patologia , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Técnicas de Cocultura , Resistencia a Medicamentos Antineoplásicos/genética , Sinergismo Farmacológico , Proteínas de Fusão bcr-abl/genética , Proteínas de Fusão bcr-abl/metabolismo , Regulação Leucêmica da Expressão Gênica , Humanos , Leucemia Mielogênica Crônica BCR-ABL Positiva/enzimologia , Leucemia Mielogênica Crônica BCR-ABL Positiva/genética , Leucemia Mielogênica Crônica BCR-ABL Positiva/patologia , Células-Tronco Neoplásicas/efeitos dos fármacos , Células-Tronco Neoplásicas/enzimologia , Células-Tronco Neoplásicas/patologia , Via de Sinalização Wnt/efeitos dos fármacos , beta Catenina/genética , beta Catenina/metabolismoRESUMO
Placental-like alkaline phosphatase (PLAP) is expressed by many tumors and can be detected in sera of patients with various cancers. Its aberrant expression has been considered to be potentially useful as tumor marker. However, the biological background of the role of this aberrant alkaline phosphatase (AP) in cancer is still unclear. The expression of various forms of AP in cells of chronic myeloid leukemia (CML) has not yet been studied. AIM: To analyze the expression patterns of various AP forms in cells originated from CML patients in blast crisis and to modify their expression by vitamin E. MATERIALS AND METHODS: RNA extracted from leukemic cells was converted to cDNA and real-time reverse transcription polymerase chain reaction was performed using SYBR Green protocol with primers to tissue non-specific alkaline phosphatase (TNAP), intestinal alkaline phosphatase and CCAAT-enhancer-binding proteins alpha (C/EBPα). To analyze the modulation of expression of APs and C/EBPα, CML cells were incubated with 100 µM vitamin E. RESULTS: We have observed the aberrant expression of mRNA intestinal alkaline phosphatase in CML cells that upon sequencing demonstrated the significant alignment with PLAP sequence while no gene homology with tissue placental alkaline phosphatase (PAP) was revealed. Vitamin E decreases mRNA PLAP expression and increases mRNA TNAP expression. Moreover, along with down-regulation of aberrant PLAP and up-regulation of TNAP, vitamin E increases C/EBPα mRNA expression. CONCLUSION: The loss of TNAP in CML may contribute to pathogenesis of this disease. PLAP may be considered as a putative target in differentiation therapies in myeloid neoplasms. Our findings suggest the potential role of vitamin E as the inducer of differentiation potential of leukemic cells in CML.
Assuntos
Fosfatase Alcalina/genética , Biomarcadores Tumorais/genética , Crise Blástica/enzimologia , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Isoenzimas/genética , Leucemia Mielogênica Crônica BCR-ABL Positiva/enzimologia , Vitamina E/farmacologia , Crise Blástica/genética , Crise Blástica/patologia , Linhagem Celular Tumoral , Regulação para Baixo , Humanos , Células K562 , Leucemia Mielogênica Crônica BCR-ABL Positiva/genética , Leucemia Mielogênica Crônica BCR-ABL Positiva/patologia , Regulação para CimaRESUMO
BACKGROUND: Philadelphia chromosome-positive (Ph+) advanced leukemias, including acute myeloid leukemia (AML) and chronic myeloid leukemia (CML) in myeloid blast phase (MBP), have poor outcomes. Venetoclax has shown synergism with BCR-ABL1 tyrosine kinase inhibitors (TKI) in preclinical studies. However, clinical activity of venetoclax and TKI-based regimens is unknown. METHODS: We conducted a retrospective study on patients with Ph+ AML (n = 7) and CML-MBP (n = 9) who received venetoclax combined with TKI-based regimens at our institution. RESULTS: Median patient age was 42 years, and the median number of prior therapy cycles was 5 (range 2-8). Nine patients received decitabine-based, and 7 received intensive chemotherapy-based regimens. Ten patients (63%) received ponatinib. The overall response rate (ORR) in 15 evaluable patients was 60% (1 complete remission [CR], 6 CR with incomplete hematologic recovery [CRi], 1 morphologic leukemia-free state, and 1 partial response). The ORR was 43% in Ph+ AML and 75% in CML-MBP. The median overall survival (OS) for all patients was 3.6 months, for AML OS was 2.0 months, and for CML-MBP OS was 10.9 months. The median relapse-free survival for AML and CML-MBP was 3.6 and 3.9 months, respectively. Compared to nonresponders, patients achieving CR/CRi had higher baseline Ph+ metaphases and BCR-ABL1 PCR. CONCLUSIONS: Combination therapy of venetoclax with TKI-based regimens shows encouraging activity in very heavily pretreated, advanced Ph+ leukemias, particularly CML-MBP.
Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/administração & dosagem , Crise Blástica , Proteínas de Fusão bcr-abl , Leucemia Mielogênica Crônica BCR-ABL Positiva , Leucemia Mieloide Aguda , Adulto , Idoso , Crise Blástica/tratamento farmacológico , Crise Blástica/enzimologia , Crise Blástica/genética , Crise Blástica/mortalidade , Compostos Bicíclicos Heterocíclicos com Pontes/administração & dosagem , Intervalo Livre de Doença , Feminino , Proteínas de Fusão bcr-abl/antagonistas & inibidores , Proteínas de Fusão bcr-abl/genética , Humanos , Imidazóis/administração & dosagem , Leucemia Mielogênica Crônica BCR-ABL Positiva/tratamento farmacológico , Leucemia Mielogênica Crônica BCR-ABL Positiva/enzimologia , Leucemia Mielogênica Crônica BCR-ABL Positiva/genética , Leucemia Mielogênica Crônica BCR-ABL Positiva/mortalidade , Leucemia Mieloide Aguda/tratamento farmacológico , Leucemia Mieloide Aguda/enzimologia , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/mortalidade , Masculino , Pessoa de Meia-Idade , Inibidores de Proteínas Quinases/administração & dosagem , Piridazinas/administração & dosagem , Estudos Retrospectivos , Sulfonamidas/administração & dosagem , Taxa de SobrevidaRESUMO
Chronic myeloid leukemia (CML) is characterized by the presence of the BCR-ABL1 fusion gene, which encodes a constitutive active tyrosine kinase considered to be the pathogenic driver capable of initiating and maintaining the disease. Despite the remarkable efficacy of tyrosine kinase inhibitors (TKIs) targeting BCR-ABL1, some patients may not respond (primary resistance) or may relapse after an initial response (secondary resistance). In a small proportion of cases, development of resistance is accompanied or shortly followed by progression from chronic to blastic phase (BP), characterized by a dismal prognosis. Evolution from CP into BP is a multifactorial and probably multistep phenomenon. Increase in BCR-ABL1 transcript levels is thought to promote the onset of secondary chromosomal or genetic defects, induce differentiation arrest, perturb RNA transcription, editing and translation that together with epigenetic and metabolic changes may ultimately lead to the expansion of highly proliferating, differentiation-arrested malignant cells. A multitude of studies over the past two decades have investigated the mechanisms underlying the closely intertwined phenomena of drug resistance and disease progression. Here, we provide an update on what is currently known on the mechanisms underlying progression and present the latest acquisitions on BCR-ABL1-independent resistance and leukemia stem cell persistence.
Assuntos
Crise Blástica , Resistencia a Medicamentos Antineoplásicos , Proteínas de Fusão bcr-abl , Leucemia Mielogênica Crônica BCR-ABL Positiva , Células-Tronco Neoplásicas , Inibidores de Proteínas Quinases/uso terapêutico , Crise Blástica/tratamento farmacológico , Crise Blástica/enzimologia , Crise Blástica/genética , Crise Blástica/patologia , Progressão da Doença , Proteínas de Fusão bcr-abl/antagonistas & inibidores , Proteínas de Fusão bcr-abl/genética , Proteínas de Fusão bcr-abl/metabolismo , Humanos , Leucemia Mielogênica Crônica BCR-ABL Positiva/tratamento farmacológico , Leucemia Mielogênica Crônica BCR-ABL Positiva/enzimologia , Leucemia Mielogênica Crônica BCR-ABL Positiva/genética , Leucemia Mielogênica Crônica BCR-ABL Positiva/patologia , Células-Tronco Neoplásicas/enzimologia , Células-Tronco Neoplásicas/patologiaRESUMO
Tyrosine kinase inhibitors (TKI) have moderately improved survival in BC, but a median survival of less than 1 year is still unsatisfactory. This article reviews the various tests required for diagnosis of BC, features at diagnosis, treatment modalities (intensive chemotherapy, TKI, allo-SCT and a selection of investigational agents), options of prevention and predictors of progression. The best prognosis is observed in patients that achieve a 2nd CP. Allo-SCT probably further improves prognosis of patients in 2nd CP. The choice of TKI should be directed by the mutation profile of the patient. BC can be prevented. A careful analysis of risk factors for progression may help. Current treatment options are combined in a concluding strategy for the management of BC.
Assuntos
Crise Blástica , Leucemia Mielogênica Crônica BCR-ABL Positiva , Mutação , Inibidores de Proteínas Quinases/uso terapêutico , Transplante de Células-Tronco , Aloenxertos , Crise Blástica/diagnóstico , Crise Blástica/enzimologia , Crise Blástica/genética , Crise Blástica/terapia , Humanos , Leucemia Mielogênica Crônica BCR-ABL Positiva/enzimologia , Leucemia Mielogênica Crônica BCR-ABL Positiva/genética , Leucemia Mielogênica Crônica BCR-ABL Positiva/terapia , PrognósticoRESUMO
INTRODUCTION: Management of chronic myeloid leukemia (CML) patients in advanced phases of disease has drastically changed since the introduction of tyrosine kinase inhibitors (TKIs) which provide tailored treatment strategies. AREAS COVERED: In this review, efficacy data of different TKIs are reported and reviewed when used as single agent or in combination for the management of blast phase (BP) CML. EXPERT OPINION: Although brilliant results were achieved, the outcome of BP patients did not change when TKIs were used as single agents. Newer strategies of association of TKI with intensified chemotherapy or new agents for different pathways are strongly needed as a bridge to possible allogeneic transplant.
Assuntos
Antineoplásicos/uso terapêutico , Crise Blástica/tratamento farmacológico , Leucemia Mielogênica Crônica BCR-ABL Positiva/tratamento farmacológico , Inibidores de Proteínas Quinases/uso terapêutico , Proteínas Tirosina Quinases/antagonistas & inibidores , Crise Blástica/diagnóstico , Crise Blástica/enzimologia , Humanos , Leucemia Mielogênica Crônica BCR-ABL Positiva/diagnóstico , Leucemia Mielogênica Crônica BCR-ABL Positiva/enzimologia , Proteínas Tirosina Quinases/metabolismo , Resultado do TratamentoRESUMO
Acute myeloid leukemia (AML) is characterized by the accumulation of malignant blasts with impaired differentiation programs caused by recurrent mutations, such as the isocitrate dehydrogenase (IDH) mutations found in 15% of AML patients. These mutations result in the production of the oncometabolite (R)-2-hydroxyglutarate (2-HG), leading to a hypermethylation phenotype that dysregulates hematopoietic differentiation. In this study, we identified mutant R132H IDH1-specific gene signatures regulated by key transcription factors, particularly CEBPα, involved in myeloid differentiation and retinoid responsiveness. We show that treatment with all-trans retinoic acid (ATRA) at clinically achievable doses markedly enhanced terminal granulocytic differentiation in AML cell lines, primary patient samples, and a xenograft mouse model carrying mutant IDH1. Moreover, treatment with a cell-permeable form of 2-HG sensitized wild-type IDH1 AML cells to ATRA-induced myeloid differentiation, whereas inhibition of 2-HG production significantly reduced ATRA effects in mutant IDH1 cells. ATRA treatment specifically decreased cell viability and induced apoptosis of mutant IDH1 blasts in vitro. ATRA also reduced tumor burden of mutant IDH1 AML cells xenografted in NOD-Scid-IL2rγ(null)mice and markedly increased overall survival, revealing a potent antileukemic effect of ATRA in the presence of IDH1 mutation. This therapeutic strategy holds promise for this AML patient subgroup in future clinical studies.
Assuntos
Crise Blástica/tratamento farmacológico , Diferenciação Celular/efeitos dos fármacos , Isocitrato Desidrogenase/metabolismo , Leucemia Mieloide Aguda/tratamento farmacológico , Mutação de Sentido Incorreto , Proteínas de Neoplasias/metabolismo , Tretinoína/farmacologia , Substituição de Aminoácidos , Animais , Crise Blástica/enzimologia , Crise Blástica/genética , Crise Blástica/patologia , Proteínas Estimuladoras de Ligação a CCAAT/genética , Proteínas Estimuladoras de Ligação a CCAAT/metabolismo , Diferenciação Celular/genética , Sobrevivência Celular , Feminino , Granulócitos/metabolismo , Granulócitos/patologia , Células HL-60 , Humanos , Isocitrato Desidrogenase/genética , Leucemia Mieloide Aguda/enzimologia , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/patologia , Masculino , Camundongos , Camundongos Nus , Proteínas de Neoplasias/genética , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Leukemias expressing constitutively activated mutants of ABL1 tyrosine kinase (BCR-ABL1, TEL-ABL1, NUP214-ABL1) usually contain at least 1 normal ABL1 allele. Because oncogenic and normal ABL1 kinases may exert opposite effects on cell behavior, we examined the role of normal ABL1 in leukemias induced by oncogenic ABL1 kinases. BCR-ABL1-Abl1(-/-) cells generated highly aggressive chronic myeloid leukemia (CML)-blast phase-like disease in mice compared with less malignant CML-chronic phase-like disease from BCR-ABL1-Abl1(+/+) cells. Additionally, loss of ABL1 stimulated proliferation and expansion of BCR-ABL1 murine leukemia stem cells, arrested myeloid differentiation, inhibited genotoxic stress-induced apoptosis, and facilitated accumulation of chromosomal aberrations. Conversely, allosteric stimulation of ABL1 kinase activity enhanced the antileukemia effect of ABL1 tyrosine kinase inhibitors (imatinib and ponatinib) in human and murine leukemias expressing BCR-ABL1, TEL-ABL1, and NUP214-ABL1. Therefore, we postulate that normal ABL1 kinase behaves like a tumor suppressor and therapeutic target in leukemias expressing oncogenic forms of the kinase.
Assuntos
Crise Blástica/genética , Genes Supressores de Tumor , Genes abl , Leucemia Experimental/genética , Leucemia Mieloide de Fase Crônica/genética , Proteínas Oncogênicas v-abl/fisiologia , Proteínas de Fusão Oncogênica/fisiologia , Proteínas Proto-Oncogênicas c-abl/fisiologia , Proteínas Supressoras de Tumor/fisiologia , Animais , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Apoptose/efeitos dos fármacos , Crise Blástica/tratamento farmacológico , Crise Blástica/enzimologia , Crise Blástica/patologia , Divisão Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Citostáticos/farmacologia , Regulação Leucêmica da Expressão Gênica/efeitos dos fármacos , Instabilidade Genômica , Humanos , Mesilato de Imatinib/farmacologia , Mesilato de Imatinib/uso terapêutico , Imidazóis/farmacologia , Imidazóis/uso terapêutico , Leucemia Experimental/tratamento farmacológico , Leucemia Experimental/enzimologia , Leucemia Experimental/patologia , Leucemia Mieloide de Fase Crônica/tratamento farmacológico , Leucemia Mieloide de Fase Crônica/enzimologia , Leucemia Mieloide de Fase Crônica/patologia , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Proteínas de Neoplasias/antagonistas & inibidores , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/fisiologia , Células-Tronco Neoplásicas/efeitos dos fármacos , Células-Tronco Neoplásicas/enzimologia , Proteínas Oncogênicas v-abl/antagonistas & inibidores , Proteínas Oncogênicas v-abl/genética , Proteínas de Fusão Oncogênica/antagonistas & inibidores , Proteínas de Fusão Oncogênica/genética , Estresse Oxidativo , Inibidores de Proteínas Quinases/farmacologia , Inibidores de Proteínas Quinases/uso terapêutico , Proteínas Proto-Oncogênicas c-abl/genética , Piridazinas/farmacologia , Piridazinas/uso terapêutico , Proteínas Supressoras de Tumor/antagonistas & inibidores , Proteínas Supressoras de Tumor/genéticaRESUMO
Childhood acute lymphoblastic leukemia (ALL) blasts are characterized by inhibited apoptosis promoting fast disease progress. It is known that in chronic lymphocytic and acute myeloid leukemias the reduced apoptosis is strongly related with the activity of calpain-calpastatin system (CCS) composed of cytoplasmic proteases--calpains--performing the modulatory proteolysis of key proteins involved in cell proliferation and apoptosis, and of their endogenous inhibitor--calpastatin. Here, the CCS protein abundance and activity was for the first time studied in childhood ALL blasts and in control bone marrow CD19+ B cells by semi-quantitative flow cytometry and western blotting of calpastatin fragments resulting from endogenous calpain activity. Significantly higher µ-calpain (CAPN1) gene transcription, protein amounts and activity (but not those of m-calpain), with calpastatin amount and transcription of its gene (CAST) greatly varying were observed in CD19(+) ALL blasts compared to control cells. Significant inverse relation between the amount/activity of calpain and spontaneous apoptosis was noted. Patients older than 10 years (considered at higher risk) displayed increased amounts and activities of blast calpain. Finally, treatment of blasts with the tripeptide calpain inhibitors II and IV significantly and in dose-dependent fashion increased the percentage of blasts entering apoptosis. Together, these findings make the CCS a potential new predictive tool and therapeutic target in childhood ALL.
Assuntos
Apoptose , Linfócitos B/enzimologia , Crise Blástica/enzimologia , Calpaína/metabolismo , Proteínas de Neoplasias/metabolismo , Leucemia-Linfoma Linfoblástico de Células Precursoras B/enzimologia , Adolescente , Fatores Etários , Linfócitos B/patologia , Crise Blástica/tratamento farmacológico , Crise Blástica/patologia , Calpaína/antagonistas & inibidores , Proliferação de Células/efeitos dos fármacos , Criança , Pré-Escolar , Feminino , Humanos , Lactente , Masculino , Proteínas de Neoplasias/antagonistas & inibidores , Oligopeptídeos/farmacologia , Leucemia-Linfoma Linfoblástico de Células Precursoras B/tratamento farmacológico , Leucemia-Linfoma Linfoblástico de Células Precursoras B/patologiaRESUMO
Unbalanced (major route) additional cytogenetic aberrations (ACA) at diagnosis of chronic myeloid leukemia (CML) indicate an increased risk of progression and shorter survival. Moreover, newly arising ACA under imatinib treatment and clonal evolution are considered features of acceleration and define failure of therapy according to the European LeukemiaNet (ELN) recommendations. On the basis of 1151 Philadelphia chromosome positive chronic phase patients of the randomized CML-study IV, we examined the incidence of newly arising ACA under imatinib treatment with regard to the p210BCR-ABL breakpoint variants b2a2 and b3a2. We found a preferential acquisition of unbalanced ACA in patients with b3a2 vs. b2a2 fusion type (ratio: 6.3 vs. 1.6, p = 0.0246) concurring with a faster progress to blast crisis for b3a2 patients (p = 0.0124). ESPL1/Separase, a cysteine endopeptidase, is a key player in chromosomal segregation during mitosis. Separase overexpression and/or hyperactivity has been reported from a wide range of cancers and cause defective mitotic spindles, chromosome missegregation and aneuploidy. We investigated the influence of p210BCR-ABL breakpoint variants and imatinib treatment on expression and proteolytic activity of Separase as measured with a specific fluorogenic assay on CML cell lines (b2a2: KCL-22, BV-173; b3a2: K562, LAMA-84). Despite a drop in Separase protein levels an up to 5.4-fold increase of Separase activity under imatinib treatment was observed exclusively in b3a2 but not in b2a2 cell lines. Mimicking the influence of imatinib on BV-173 and LAMA-84 cells by ESPL1 silencing stimulated Separase proteolytic activity in both b3a2 and b2a2 cell lines. Our data suggest the existence of a fusion type-related feedback mechanism that posttranslationally stimulates Separase proteolytic activity after therapy-induced decreases in Separase protein levels. This could render b3a2 CML cells more prone to aneuploidy and clonal evolution than b2a2 progenitors and may therefore explain the cytogenetic results of CML patients.
Assuntos
Antineoplásicos/uso terapêutico , Crise Blástica/genética , Proteínas de Fusão bcr-abl/genética , Mesilato de Imatinib/uso terapêutico , Leucemia Mielogênica Crônica BCR-ABL Positiva/tratamento farmacológico , Leucemia Mielogênica Crônica BCR-ABL Positiva/patologia , Separase/metabolismo , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Crise Blástica/enzimologia , Crise Blástica/patologia , Linhagem Celular Tumoral , Aberrações Cromossômicas , Quebra Cromossômica , Evolução Clonal , Humanos , Leucemia Mielogênica Crônica BCR-ABL Positiva/enzimologia , Leucemia Mielogênica Crônica BCR-ABL Positiva/genética , Pessoa de Meia-Idade , Proteólise , Adulto JovemRESUMO
Polo-like kinase 1 (PLK1) is an important regulator of the cell cycle and is overexpressed in various solid and hematological malignancies. Small molecule inhibitors targeting PLK1, such as BI2536 or BI6727 (Volasertib) are a promising therapeutic approach in such malignancies. Here, we show a loss of specifically localized PLK1 in AML blasts in vivo, accompanied by mitotic arrest with transition into apoptosis, in bone marrow biopsies of AML patients after treatment with BI2536. We verify these results in live cell imaging experiments with the AML cell line HL-60, and demonstrate that non-neoplastic, immortalized lymphoblastoid cells are also sensitive to PLK1 inhibition. It is demonstrated that normal granulopoietic precursors have similar PLK1 expression levels as leukemic blasts. These results are in line with the adverse effects of PLK1 inhibition and underline the great potential of PLK1 inhibitors in the treatment of AML.
Assuntos
Apoptose/efeitos dos fármacos , Medula Óssea/efeitos dos fármacos , Proteínas de Ciclo Celular/antagonistas & inibidores , Leucemia Mieloide Aguda/tratamento farmacológico , Leucemia Mieloide Aguda/patologia , Mitose/efeitos dos fármacos , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Proteínas Proto-Oncogênicas/antagonistas & inibidores , Pteridinas/farmacologia , Idoso , Idoso de 80 Anos ou mais , Antimitóticos/farmacologia , Crise Blástica/tratamento farmacológico , Crise Blástica/enzimologia , Crise Blástica/patologia , Western Blotting , Medula Óssea/enzimologia , Medula Óssea/patologia , Proteínas de Ciclo Celular/metabolismo , Proliferação de Células/efeitos dos fármacos , Feminino , Humanos , Técnicas Imunoenzimáticas , Leucemia Mieloide Aguda/enzimologia , Masculino , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , RNA Mensageiro/genética , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Células Tumorais Cultivadas , Quinase 1 Polo-LikeRESUMO
Chronic myelogenous leukemia (CML) is a stem cell disorder, and leukemia stem cells (LSCs) can contribute to the relapse of the disease. Quiescent LSCs are BCR-ABL independent and resistant to imatinib; therefore, there is an unmet need to identify new therapeutic targets in LSCs. Inhibition of the mammalian target of rapamycin (mTOR) in imatinib-resistant BCR-ABL1-positive cells was effective in vitro, but in a pilot clinical trial, only a few patients responded to the treatment. In this study, we demonstrate that mTOR activation in CML CD34(+) progenitor cells is ERK dependent in chronic phase of the disease and ERK independent in blast crisis. Rapamycin effectively inhibits mTOR in all phases of CML, but does not reduce number of LSC-enriched CD34(+) blast crisis (BC) cells, neither alone nor in combination with imatinib in CML-BC cells. These results show that potential therapeutic benefits of mTOR inhibition may be the result of effects on differentiated leukemic cells and may be potentially achieved only in the chronic phase of the disease.
Assuntos
Crise Blástica/enzimologia , Leucemia Mieloide de Fase Crônica/enzimologia , Células-Tronco Neoplásicas/enzimologia , Serina-Treonina Quinases TOR/metabolismo , Animais , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Benzamidas/farmacologia , Benzamidas/uso terapêutico , Crise Blástica/tratamento farmacológico , Crise Blástica/patologia , Western Blotting , Linhagem Celular , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Ativação Enzimática/efeitos dos fármacos , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Citometria de Fluxo , Proteínas de Fusão bcr-abl/antagonistas & inibidores , Proteínas de Fusão bcr-abl/metabolismo , Humanos , Mesilato de Imatinib , Células K562 , Leucemia Mieloide de Fase Crônica/tratamento farmacológico , Leucemia Mieloide de Fase Crônica/patologia , Células-Tronco Neoplásicas/efeitos dos fármacos , Células-Tronco Neoplásicas/patologia , Piperazinas/farmacologia , Piperazinas/uso terapêutico , Inibidores de Proteínas Quinases/farmacologia , Pirimidinas/farmacologia , Pirimidinas/uso terapêutico , Transdução de Sinais/efeitos dos fármacos , Sirolimo/farmacologia , Sirolimo/uso terapêutico , Serina-Treonina Quinases TOR/antagonistas & inibidores , Células Tumorais CultivadasRESUMO
BACKGROUND AND OBJECTIVES: Tyrosine kinase inhibitors (TKI) have improved the management of patients with chronic myeloid leukemia (CML). However, a significant proportion of patients do not achieve the optimal response or are resistant to TKI. ABL kinase domain mutations have been extensively implicated in the pathogenesis of TKI resistance. Treatment with second-generation TKI has produced high rates of hematologic and cytogenetic responses in mutated ABL patients. The aim of this study was to determine the type and frequency of ABL mutations in patients who were resistant to imatinib or had lost the response, and to analyze the effect of second-generation TKI on their outcome. PATIENTS AND METHODS: The presence of ABL mutations in 45 CML patients resistant to imatinib was evaluated by direct sequencing and was correlated with the results of the cytogenetic study (performed in 39 cases). The outcome of these patients after therapy with nilotinib or dasatinib was analyzed. RESULTS: ABL mutations were detected in 14 out of 45 resistant patients. Patients with clonal cytogenetic evolution tended to develop mutations more frequently than those without clonal evolution. Nine out of the 15 patients with ABL mutation responded to a treatment switch to nilotinib (n=4), dasatinib (n=2), interferon (n=1) or hematopoietic stem cell transplantation (n=2). CONCLUSION: The frequency of ABL mutations in CML patients resistant to imatinib is high and is more frequent among those with clonal cytogenetic evolution. The change to second-generation TKI can overcome imatinib resistance in most of the mutated patients.
Assuntos
Benzamidas/uso terapêutico , Genes abl , Leucemia Mielogênica Crônica BCR-ABL Positiva/genética , Mutação , Piperazinas/uso terapêutico , Inibidores de Proteínas Quinases/uso terapêutico , Pirimidinas/uso terapêutico , Tiazóis/uso terapêutico , Benzamidas/farmacologia , Crise Blástica/tratamento farmacológico , Crise Blástica/enzimologia , Crise Blástica/genética , Células Clonais/metabolismo , Células Clonais/patologia , DNA de Neoplasias/genética , Dasatinibe , Resistencia a Medicamentos Antineoplásicos/genética , Substituição de Medicamentos , Feminino , Proteínas de Fusão bcr-abl/antagonistas & inibidores , Proteínas de Fusão bcr-abl/genética , Humanos , Mesilato de Imatinib , Cariotipagem , Leucemia Mielogênica Crônica BCR-ABL Positiva/tratamento farmacológico , Leucemia Mielogênica Crônica BCR-ABL Positiva/enzimologia , Leucemia Mielogênica Crônica BCR-ABL Positiva/patologia , Masculino , Células-Tronco Neoplásicas/metabolismo , Células-Tronco Neoplásicas/patologia , Piperazinas/farmacologia , Inibidores de Proteínas Quinases/classificação , Inibidores de Proteínas Quinases/farmacologia , Estrutura Terciária de Proteína/genética , Pirimidinas/farmacologia , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , RNA Neoplásico/biossíntese , RNA Neoplásico/genética , Análise de Sequência de DNA , Tiazóis/farmacologiaRESUMO
Separase, an endopeptidase required for the separation of sister-chromatides in mitotic anaphase, triggers centriole disengagement during centrosome duplication. In cancer, separase is frequently overexpressed, pointing to a functional role as an aneuploidy promoter associated with centrosomal amplification and genomic instability. Recently, we have shown that centrosomal amplification and subsequent chromosomal aberrations are a hallmark of chronic myeloid leukemia (CML), increasing from chronic phase (CP) toward blast crisis (BC). Moreover, a functional linkage of p210BCR-ABL tyrosine kinase activity with centrosomal amplification and clonal evolution has been established in long-term cell culture experiments. Unexpectedly, therapeutic doses of imatinib (IM) did not counteract; instead induced similar centrosomal alterations in vitro. We investigated the influence of IM and p210BCR-ABL on Separase as a potential driver of centrosomal amplification in CML. Short-term cell cultures of p210BCR-ABL-negative (NHDF, UROtsa, HL-60, U937), positive (K562, LAMA-84) and inducible (U937p210BCR-ABL/c6 (Tet-ON)) human cell lines were treated with therapeutic doses of IM and analyzed by qRT-PCR, Western blot analysis and quantitative Separase activity assays. Decreased Separase protein levels were observed in all cells treated with IM in a dose dependent manner. Accordingly, in all p210BCR-ABL-negative cell lines, decreased proteolytic activity of Separase was found. In contrast, p210BCR-ABL-positive cells showed increased Separase proteolytic activity. This activation of Separase was consistent with changes in the expression levels of Separase regulators (Separase phosphorylation at serine residue 1126, Securin, CyclinB1 and PP2A). Our data suggest that regulation of Separase in IM-treated BCR-ABL-positive cells occurs on both the protein expression and the proteolytic activity levels. Activation of Separase proteolytic activity exclusively in p210BCR-ABL-positive cells during IM treatment may act as a driving force for centrosomal amplification, contributing to genomic instability, clonal evolution and resistance in CML.
Assuntos
Antineoplásicos/farmacologia , Crise Blástica/tratamento farmacológico , Proteínas de Fusão bcr-abl/metabolismo , Leucemia Mielogênica Crônica BCR-ABL Positiva/tratamento farmacológico , Piperazinas/farmacologia , Pirimidinas/farmacologia , Benzamidas , Crise Blástica/enzimologia , Crise Blástica/genética , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Ciclina B1/genética , Ciclina B1/metabolismo , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos/genética , Endopeptidases/genética , Endopeptidases/metabolismo , Proteínas de Fusão bcr-abl/genética , Instabilidade Genômica/efeitos dos fármacos , Instabilidade Genômica/genética , Células HL-60 , Humanos , Mesilato de Imatinib , Células K562 , Leucemia Mielogênica Crônica BCR-ABL Positiva/enzimologia , Leucemia Mielogênica Crônica BCR-ABL Positiva/genética , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Fosforilação/efeitos dos fármacos , Fosforilação/genética , Proteína Fosfatase 2/genética , Proteína Fosfatase 2/metabolismo , Proteólise , Securina , Separase , Células U937RESUMO
Rising BCR-ABL1 transcripts indicate potential loss of imatinib response in CML. We determined whether the BCR-ABL1 doubling time could distinguish nonadherence from resistance as the cause of lost response. Distinct groups were examined: (1) acquired clinical resistance because of blast crisis and/or BCR-ABL1 mutations; and (2) documented imatinib discontinuation/interruption. Short doubling times occurred with blast crisis (median, 9.0 days; range, 6.1-17.6 days; n = 12 patients), relapse after imatinib discontinuation in complete molecular response (median, 9.0 days; range, 6.9-26.5 days; n = 17), and imatinib interruption during an entire measurement interval (median, 9.4 days; range, 4.2-17.6 days; n = 12; P = .72). Whereas these doubling times were consistently short and indicated rapid leukemic expansion, fold rises were highly variable: 71-, 9.5-, and 10.5-fold, respectively. The fold rise depended on the measurement interval, whereas the doubling time was independent of the interval. Longer doubling times occurred for patients with mutations who maintained chronic phase (CP: median, 48 days; range, 17.3-143 days; n = 29; P < .0001). Predicted short and long doubling times were validated on an independent cohort monitored elsewhere. The doubling time revealed major differences in kinetics according to clinical context. Long doubling times observed with mutations in CP allow time for intervention. A short doubling time for a patient in CP should raise the suspicion of nonadherence.
Assuntos
Proteínas de Fusão bcr-abl/metabolismo , Leucemia Mielogênica Crônica BCR-ABL Positiva/patologia , Antineoplásicos/administração & dosagem , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Benzamidas , Biomarcadores Tumorais , Crise Blástica/enzimologia , Gerenciamento Clínico , Progressão da Doença , Esquema de Medicação , Monitoramento de Medicamentos , Resistencia a Medicamentos Antineoplásicos , Proteínas de Fusão bcr-abl/genética , Humanos , Mesilato de Imatinib , Leucemia Mielogênica Crônica BCR-ABL Positiva/tratamento farmacológico , Leucemia Mielogênica Crônica BCR-ABL Positiva/enzimologia , Leucemia Mieloide de Fase Crônica/tratamento farmacológico , Leucemia Mieloide de Fase Crônica/enzimologia , Leucemia Mieloide de Fase Crônica/patologia , Adesão à Medicação , Piperazinas/administração & dosagem , Piperazinas/farmacologia , Piperazinas/uso terapêutico , Inibidores de Proteínas Quinases/administração & dosagem , Inibidores de Proteínas Quinases/farmacologia , Inibidores de Proteínas Quinases/uso terapêutico , Pirimidinas/administração & dosagem , Pirimidinas/farmacologia , Pirimidinas/uso terapêutico , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , RNA Neoplásico/biossíntese , RNA Neoplásico/genética , Ensaios Clínicos Controlados Aleatórios como Assunto/estatística & dados numéricos , Recidiva , Estudos Retrospectivos , Fatores de TempoRESUMO
BACKGROUND: Inhibitors of nicotinamide phosphoribosyltransferase have recently been validated as therapeutic targets in leukemia, but the mechanism of leukemogenic transformation downstream of this enzyme is unclear. DESIGN AND METHODS: Here, we evaluated whether nicotinamide phosphoribosyltransferase's effects on aberrant proliferation and survival of myeloid leukemic cells are dependent on sirtuin and delineated the downstream signaling pathways operating during this process. RESULTS: We identified significant upregulation of sirtuin 2 and nicotinamide phosphoribosyltransferase levels in primary acute myeloid leukemia blasts compared to in hematopoietic progenitor cells from healthy individuals. Importantly, specific inhibition of nicotinamide phosphoribosyltransferase or sirtuin 2 significantly reduced proliferation and induced apoptosis in human acute myeloid leukemia cell lines and primary blasts. Intriguingly, we found that protein kinase B/AKT could be deacetylated by nicotinamide phosphoribosyltransferase and sirtuin 2. The anti-leukemic effects of the inhibition of nicotinamide phosphoribosyltransferase or sirtuin 2 were accompanied by acetylation of protein kinase B/AKT with subsequent inhibition by dephosphorylation. This leads to activation of glycogen synthase kinase-3 ß via diminished phosphorylation and, ultimately, inactivation of ß-catenin by phosphorylation. CONCLUSIONS: Our results provide strong evidence that nicotinamide phosphoribosyltransferase and sirtuin 2 participate in the aberrant proliferation and survival of leukemic cells, and suggest that the protein kinase B/AKT/ glycogen synthase kinase-3 ß/ß-catenin pathway is a target for inhibition of nicotinamide phosphoribosyltransferase or sirtuin 2 and, thereby, leukemia cell proliferation.
Assuntos
Leucemia Mieloide/enzimologia , Nicotinamida Fosforribosiltransferase/metabolismo , Sirtuína 2/metabolismo , Acrilamidas/farmacologia , Apoptose/efeitos dos fármacos , Benzotiazóis/farmacologia , Crise Blástica/enzimologia , Crise Blástica/genética , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Ativação Enzimática/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Regulação Leucêmica da Expressão Gênica , Quinase 3 da Glicogênio Sintase/metabolismo , Glicogênio Sintase Quinase 3 beta , Humanos , Leucemia Mieloide/genética , Modelos Biológicos , Nicotinamida Fosforribosiltransferase/antagonistas & inibidores , Piperidinas/farmacologia , Proteínas Proto-Oncogênicas c-akt/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-akt/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Transdução de Sinais , Sirtuína 2/antagonistas & inibidores , Sirtuína 2/genética , beta Catenina/antagonistas & inibidores , beta Catenina/metabolismoRESUMO
BACKGROUND: Activation of the phosphoinositide 3-kinase (PI3K)/Akt pathway, a pro-survival pathway, plays important roles in tumor cell growth. However, the role of Akt in the pathogenesis of pediatric B-precursor acute lymphoblastic leukemia (B-pre ALL) remains to be clarified. This study was undertaken to explore the clinical relevance and molecular mechanisms underlying the activation of Akt (i.e., phosphorylated Akt, P-Akt) in pediatric B-pre ALL. PROCEDURE: We evaluated the activation status of Akt in bone marrow samples from 21 children with newly diagnosed B-pre ALL and correlated the expression level of P-Akt with clinicopathologic and prognostic features. Additionally, we transfected the myristoylated Akt cDNA into the B-pre ALL cell line, Nalm-6, and examined the effect, in vitro, of Akt activation on the response to antitumor drugs. RESULTS: P-Akt expression in B-pre ALL blast cells at diagnosis was associated significantly with poor response to induction chemotherapy including prednisolone, dexamethasone, vincristine, and adriamycin in B-pre ALL patients. Both overall survival and relapse-free survival in patients with P-Akt expression were reduced significantly more than in patients without P-Akt expression. Activation of Akt reduced the extent of apoptosis induced by the antitumor drugs in Nalm-6 listed above. Activation of Akt did not induce expression of P-glycoprotein, a drug transporter that is capable of conferring multidrug resistance. CONCLUSION: These results support the contention that Akt activation is a mechanism of chemotherapeutic resistance in B-pre ALL and suggest that Akt can be a therapeutic target for the treatment of relapsed or refractory pediatric B-pre ALL.
Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/administração & dosagem , Crise Blástica , Resistencia a Medicamentos Antineoplásicos , Leucemia-Linfoma Linfoblástico de Células Precursoras B , Proteínas Proto-Oncogênicas c-akt/metabolismo , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/metabolismo , Adolescente , Apoptose/efeitos dos fármacos , Crise Blástica/tratamento farmacológico , Crise Blástica/enzimologia , Crise Blástica/mortalidade , Linhagem Celular Tumoral , Criança , Pré-Escolar , Dexametasona/administração & dosagem , Intervalo Livre de Doença , Doxorrubicina/administração & dosagem , Ativação Enzimática/efeitos dos fármacos , Feminino , Regulação Leucêmica da Expressão Gênica/efeitos dos fármacos , Humanos , Lactente , Masculino , Fosforilação , Leucemia-Linfoma Linfoblástico de Células Precursoras B/tratamento farmacológico , Leucemia-Linfoma Linfoblástico de Células Precursoras B/enzimologia , Leucemia-Linfoma Linfoblástico de Células Precursoras B/mortalidade , Prednisolona/administração & dosagem , Taxa de Sobrevida , Vincristina/administração & dosagemRESUMO
The cytotoxic farnesyl transferase inhibitor BMS-214662 has been shown to potently induce mitochondrial apoptosis in primitive CD34+ chronic myeloid leukaemia (CML) stem/progenitor cells. Here, to enhance the BMS-214662 apoptotic effect, we further targeted the extracellular signal-regulated kinase (ERK) pathway, downstream of BCR-ABL, by treating CD34+ CML stem/progenitor cells with a highly selective adenosine triphosphate (ATP) non-competitive MEK inhibitor, PD184352. PD184352 increased the apoptotic effect of BMS-214662 in a CML blast crisis cell line, K562, and in primary chronic phase CD34+ CML cells. Compared with BMS-214662, after combination treatment we observed inhibition of ERK phosphorylation, increased Annexin-V levels, caspase-3, -8 and -9 activation and potentiated mitochondrial damage, associated with decreased levels of anti-apoptotic BCL-2 family protein MCL-1. Inhibition of K-RAS function by a dominant-negative mutant resulted in CML cell death and this process was further enhanced by the addition of BMS-214662 and PD184352. Together, these findings suggest that the addition of a MEK inhibitor improves the ability of BMS-214662 to selectively target CML stem/progenitor cells, notoriously insensitive to tyrosine kinase inhibitor treatment and presumed to be responsible for the persistence and relapse of the disease.
Assuntos
Apoptose/efeitos dos fármacos , Benzamidas/farmacologia , Benzodiazepinas/farmacologia , Crise Blástica/patologia , Inibidores Enzimáticos/farmacologia , Farnesiltranstransferase/antagonistas & inibidores , Imidazóis/farmacologia , Leucemia Mielogênica Crônica BCR-ABL Positiva/patologia , Leucemia Mieloide de Fase Crônica/patologia , MAP Quinase Quinase Quinases/antagonistas & inibidores , Proteínas de Neoplasias/antagonistas & inibidores , Células-Tronco Neoplásicas/efeitos dos fármacos , Antígenos CD34/análise , Crise Blástica/enzimologia , Ensaios de Seleção de Medicamentos Antitumorais , Sinergismo Farmacológico , Genes Dominantes , Genes ras , Células-Tronco Hematopoéticas/efeitos dos fármacos , Células-Tronco Hematopoéticas/enzimologia , Humanos , Células K562/efeitos dos fármacos , Células K562/enzimologia , Leucemia Mielogênica Crônica BCR-ABL Positiva/enzimologia , Leucemia Mieloide de Fase Crônica/enzimologia , MAP Quinase Quinase 1/genética , Células-Tronco Neoplásicas/enzimologia , Proteína Oncogênica p21(ras)/genética , Proteínas Recombinantes de Fusão/genética , Células Tumorais Cultivadas/efeitos dos fármacos , Células Tumorais Cultivadas/enzimologiaRESUMO
Indoleamine 2,3-dioxygenase (IDO), a catabolizing enzyme of tryptophan, is a novel immunosuppressive agent blocking T-cell activation in neoplastic cells, including acute myeloid leukemia (AML) cells. IDO inhibitors as 1-methyl tryptophan (1MT) can abrogate IDO enzymatic activity and may result in an effective immune response. Mononuclear cells (MNCs) were separated from peripheral blood of 25 AML patients and 25 normal adults. IDO expression was detected by RT-PCR and its enzymatic activity by a colorimetric method. MNCs were cultured and the effects of Adriamycin, 1MT and a mixture of both on blast and lymphocyte cell counts after 24 and 72 h were detected. IDO mRNA and activity were detected in 52% of patients and absent in normal subjects. There was a significant correlation between IDO mRNA expression and its enzymatic activity in AML. IDO activity was correlated positively with patient's ages and negatively with hemoglobin levels. There was a significant inhibition of blast cells proliferation with Adriamycin and more inhibition when combined with 1MT. The inhibition was more after 72 h more than 24 h of culture. However, using 1MT alone showed no significant inhibitory effect on blast cells, with a significant increase in lymphocyte counts. Our study confirms the role of indoleamine 2,3-dioxygenase in tumor-induced immune tolerance and points to the possible benefit of 1-methyl tryptophan as immunotherapeutic enhancing the anticancer effects of traditional chemotherapeutics.