RESUMO
Clinical experience with tyrosine kinase inhibitors (TKIs) over the past two decades has shown that, despite the apparent therapeutic benefit, nearly 30% of patients with chronic myelogenous leukemia (CML) display primary resistance or intolerance to TKIs, and approximately 25% of those treated are forced to switch TKIs at least once during therapy due to acquired resistance. Safe and effective treatment modalities targeting leukemic clones that escape TKI therapy could hence be game changers in the professional management of these patients. Here, we aimed to investigate the efficacy of a novel therapeutic oligonucleotide of unconventional design, called ASP210, to reduce BCR-ABL1 mRNA levels in TKI-resistant CML cells, with the assumption of inducing their apoptosis. Imatinib- and dasatinib-resistant sublines of BCR-ABL1-positive MOLM-7 and CML-T1 cells were established and exposed to 0.25 and 2.5 µM ASP210 for 10 days. RT-qPCR showed a remarkable reduction of the target mRNA level by >99% after a single application. Cell viability was monitored daily by trypan blue staining. In response to the lack of driver oncoprotein BCR-ABL1, TKI-resistant CML cells underwent apoptosis regardless of the presence of the clinically relevant T315I mutation by day 5 after redosing with ASP210. The effect was selective for cancer cells, indicating a favorable safety profile for this therapeutic modality. Furthermore, the spontaneous uptake and high intracellular concentrations of ASP210 suggest its potential to be effective at relatively low doses. The present findings suggest that ASP210 is a promising therapeutic avenue for patients with CML who fail to respond to TKI therapy.NEW & NOTEWORTHY Effective treatment modalities targeting leukemic clones that escape tyrosine kinase inhibitor (TKI) therapy could be game changers in the professional management of patients displaying primary resistance, intolerance, or acquired resistance to TKIs. Although delivering authentic innovations today is more complex than ever, we developed a highly potent and safe oligonucleotide-based modality against BCR-ABL1 mRNA named ASP210 that effectively induces cell death in BCR-ABL1-positive TKI-resistant cells while sparing BCR-ABL1-negative healthy cells.
Assuntos
Apoptose , Resistencia a Medicamentos Antineoplásicos , Proteínas de Fusão bcr-abl , Mesilato de Imatinib , Leucemia Mielogênica Crônica BCR-ABL Positiva , Oligonucleotídeos , Inibidores de Proteínas Quinases , Humanos , Leucemia Mielogênica Crônica BCR-ABL Positiva/tratamento farmacológico , Leucemia Mielogênica Crônica BCR-ABL Positiva/genética , Leucemia Mielogênica Crônica BCR-ABL Positiva/patologia , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Inibidores de Proteínas Quinases/farmacologia , Proteínas de Fusão bcr-abl/genética , Proteínas de Fusão bcr-abl/antagonistas & inibidores , Proteínas de Fusão bcr-abl/metabolismo , Linhagem Celular Tumoral , Oligonucleotídeos/farmacologia , Apoptose/efeitos dos fármacos , Mesilato de Imatinib/farmacologia , Mesilato de Imatinib/uso terapêutico , Dasatinibe/farmacologia , Antineoplásicos/farmacologia , Sobrevivência Celular/efeitos dos fármacos , RNA Mensageiro/genética , RNA Mensageiro/metabolismoRESUMO
Tyrosine kinase inhibitors (TKIs) targeting BCR-ABL have dramatically improved chronic myeloid leukemia therapy. While imatinib remains to be the first line therapy, about 30% of patients develop resistance or intolerance to this drug and are recommended to switch to other TKIs. Nilotinib and dasatinib are currently implemented into the first line therapy and other inhibitors have already entered the clinical practice. This opens further questions on how to select the best TKI for each patient not only during the therapy but also at diagnosis. The individualized therapy concept requires a reliable establishment of prognosis and prediction of response to the available TKIs. We tested the ex vivo sensitivity of patient primary leukocytes to imatinib, nilotinib and dasatinib - two concentrations of each inhibitor for 48h incubation - and we evaluated the usefulness of such tests for the clinical practice. Besides reflecting the actual sensitivity to the therapy, our optimized simple tests were able to predict the outcome in 90/87% of patients, for the next 12/24months, respectively. According to these results, the presented ex vivo testing could help clinicians to select the appropriate drug for each patient at diagnosis and also at any time of the therapy.
Assuntos
Antineoplásicos/farmacologia , Dasatinibe/farmacologia , Resistencia a Medicamentos Antineoplásicos , Mesilato de Imatinib/farmacologia , Leucemia Mielogênica Crônica BCR-ABL Positiva/tratamento farmacológico , Inibidores de Proteínas Quinases/farmacologia , Pirimidinas/farmacologia , Antineoplásicos/uso terapêutico , Morte Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Dasatinibe/uso terapêutico , Humanos , Mesilato de Imatinib/uso terapêutico , Leucemia Mielogênica Crônica BCR-ABL Positiva/patologia , Leucócitos/efeitos dos fármacos , Leucócitos/patologia , Inibidores de Proteínas Quinases/uso terapêutico , Pirimidinas/uso terapêutico , Resultado do TratamentoRESUMO
Heat shock proteins (Hsp) are important for the stability and function of cell proteins and thus for cell survival under physiological as well as stress conditions. Hsps were also reported to play an important role in tumorogenesis including leukemias. In this study we followed up Hsp70 and 90 protein levels in samples from patients with chronic myeloid leukemia (CML) to evaluate these Hsps with regard to their ability to characterize the disease status and disease prognosis. We analyzed 68 samples of total leukocytes of CML patients with different response to therapy with tyrosine kinase inhibitors. The results of Western blot analyses showed that the level of Hsp70 did not change in the course of the disease and did not correlate with response to therapy. In contrast, Hsp90 levels showed good correlation with the disease state. Patients with good response to therapy (major molecular response-MMR, molecular remission-CMR) had low expression levels of Hsp90, similar to those in healthy individuals. High Hsp90 levels were found in patients with resistance to therapy (hematological relapse-HR, accelerated phase or blast crisis), and in leukemic cell line K562. The results of the study suggested that not the kinetics but the particular level of Hsp90 at any time point since therapy start is of prognostic value: Hsp90 level above 0.27 significantly predicted poor response to TKI therapy (relapse, progression) and the level below 0.085 good response (MMR, CMR). In conclusion, Hsp90 level in total leukocytes could serve as a risk factor at diagnosis as well as during therapy and might help in clinical decision making especially in cases where BCR-ABL monitoring is of low predictive value. Our data suggest that high expression of HSP90 contributes to the aggressivity of the disease and should be considered as an important target for specialized CML therapy.
Assuntos
Biomarcadores Tumorais , Proteínas de Choque Térmico HSP90/metabolismo , Leucemia Mielogênica Crônica BCR-ABL Positiva/metabolismo , Adulto , Idoso , Linhagem Celular Tumoral , Progressão da Doença , Feminino , Proteínas de Fusão bcr-abl/genética , Humanos , Células K562 , Leucemia Mielogênica Crônica BCR-ABL Positiva/tratamento farmacológico , Leucemia Mielogênica Crônica BCR-ABL Positiva/genética , Masculino , Pessoa de Meia-Idade , Prognóstico , Transcrição Gênica , Adulto JovemRESUMO
Chronic myelogenous leukemia (CML) is a myeloproliferative disease characterized by the BCR-ABL oncogene. Despite the high performance of treatment with tyrosine kinase inhibitors (TKI), about 30% of patients develop resistance to the therapy. To improve the outcomes, identification of new targets of treatment is needed. Here, we explored the Casein Kinase 2 (CK2) as a potential target for CML therapy. Previously, we detected increased phosphorylation of HSP90ß Serine 226 in patients non-responding to TKIs imatinib and dasatinib. This site is known to be phosphorylated by CK2, which was also linked to CML resistance to imatinib. In the present work, we established six novel imatinib- and dasatinib-resistant CML cell lines, all of which had increased CK2 activation. A CK2 inhibitor, CX-4945, induced cell death of CML cells in both parental and resistant cell lines. In some cases, CK2 inhibition also potentiated the effects of TKI on the cell metabolic activity. No effects of CK2 inhibition were observed in normal mononuclear blood cells from healthy donors and BCR-ABL negative HL60 cell line. Our data indicate that CK2 kinase supports CML cell viability even in cells with different mechanisms of resistance to TKI, and thus represents a potential target for treatment.
Assuntos
Caseína Quinase II , Leucemia Mielogênica Crônica BCR-ABL Positiva , Humanos , Mesilato de Imatinib/farmacologia , Dasatinibe/farmacologia , Proteínas de Fusão bcr-abl/metabolismo , Resistencia a Medicamentos Antineoplásicos , Apoptose , Inibidores de Proteínas Quinases/farmacologia , Leucemia Mielogênica Crônica BCR-ABL Positiva/tratamento farmacológico , Morte CelularRESUMO
OBJECTIVES: The objective was to make a contribution to deepening the knowledge of the etiopathogenesis of ADHD. DESIGN: In an association study design, an analysis of polymorphisms of selected genes was conducted in 119 hyperkinetic boys and a control group of boys, aged 7-13. Furthermore several psychologically determined subgroups were identified. A connection between psychological functions (endophenotypes) and genes were looked for. RESULTS: There was a statistically significant difference found in allelic and genotype frequencies of the TaqI A polymorphism of the DRD2 gene. The frequency of the allele A1 in hyperkinetic boys and the control subjects was 0.26 and 0.15, respectively (p<0.003). A statistically significant occurrence of atypical genotypes (8/10, 7/10 and 10/11) of the DAT1 gene was also found in hyperkinetic boys and a connection between the M235 polymorphism of the angiotensinogene gene and the positive family history of psychiatric illness was found in probands (p=0.031). Significant correlations between the results of some neuropsychological tests and genes for neuro-/immunomodulators (IL-6, TNF-alpha) and the gene for the brain-derived neurotrophic factor (BDNF) were found. CONCLUSION: The study showed a statistically significant prevalence of A1 allele of the DRD gene in the hyperkinetic group. We also found a significantly higher incidence of atypical DAT genotypes in the hyperkinetic group. Furthermore we found significant connections with particular gene polymorphisms which may hypothetically represent a neurodevelopmental risk factor in the etiopathogenesis of the disorder (IL-2, IL-6, TNF-alpha, BDNF). We further found a connection of the M235 polymorphism of the AGT (angiotensinogene) gene to positive family history of psychiatric illness (p=0.031). As for cognitive characteristics, we identified three subtypes with different cognitive performance profiles. This finding shows interindividual variability of cognitive style in the group of hyperkinetic boys.
Assuntos
Transtorno do Deficit de Atenção com Hiperatividade/genética , Transtorno do Deficit de Atenção com Hiperatividade/metabolismo , Adolescente , Atenção/efeitos dos fármacos , Atenção/fisiologia , Transtorno do Deficit de Atenção com Hiperatividade/epidemiologia , Biomarcadores , Estimulantes do Sistema Nervoso Central/uso terapêutico , Criança , República Tcheca/epidemiologia , Discriminação Psicológica/fisiologia , Percepção de Forma/fisiologia , Marcadores Genéticos , Genótipo , Humanos , Masculino , Metilfenidato/uso terapêutico , Testes Neuropsicológicos , Fenótipo , Polimorfismo Genético/genética , Escalas de Graduação Psiquiátrica , Desempenho Psicomotor/efeitos dos fármacos , Desempenho Psicomotor/fisiologia , Receptores de Dopamina D2/genéticaRESUMO
Rat liver mitochondria contain a negligible amount of mitochondrial uncoupling protein UCP2 as indicated by 3H-GTP binding. UCP2 recruitment in hepatocytes during infection may serve to decrease mitochondrial production of reactive oxygen species (ROS), and this, in turn, would counterbalance the increased oxidative stress. To characterize in detail UCP2 recruitment in hepatocytes, we studied rats pretreated with lipopolysaccharide (LPS) or hepatocytes isolated from them, as an in vitro model for the systemic response to bacterial infection. LPS injection resulted in 3.3- or 3-fold increase of UCP2 mRNA in rat liver and hepatocytes, respectively, as detected by real-time RT-PCR on a LightCycler. A concomitant increase in UCP2 protein content was indicated either by Western blots or was quantified by up to three-fold increase in the number of 3H-GTP binding sites in mitochondria of LPS-stimulated rats. Moreover, H2O2 production was increased by GDP only in mitochondria of LPS-stimulated rats with or without fatty acids and carboxyatractyloside. When monitored by JC1 fluorescent probe in situ mitochondria of hepatocytes from LPS-stimulated rats exhibited lower membrane potential than mitochondria of unstimulated rats. We have demonstrated that the lower membrane potential does not result from apoptosis initiation. However, due to a small extent of potential decrease upon UCP2 recruitment, justified also by theoretical calculations, we conclude that the recruited UCP2 causes only a weak uncoupling which is able to decrease mitochondrial ROS production but not produce enough heat for thermogenesis participating in a febrile response.
Assuntos
Lipopolissacarídeos/biossíntese , Proteínas de Membrana Transportadoras/metabolismo , Proteínas Mitocondriais/metabolismo , Animais , Sequência de Bases , Primers do DNA , Canais Iônicos , Mitocôndrias Hepáticas/metabolismo , Ratos , Ratos Long-Evans , Ratos Wistar , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Proteína Desacopladora 2RESUMO
Reconstitution of novel mitochondrial uncoupling proteins, human UCP2 and UCP3, expressed in yeast, was performed to characterize fatty acid (FA)-induced H+ efflux in the resulted proteoliposomes. We now demonstrate for the first time that representatives of physiologically abundant long chain FAs, saturated or unsaturated, activate H+ translocation in UCP2- and UCP3-proteoliposomes. Efficiency of lauric, palmitic or linoleic acid was roughly the same, but oleic acid induced faster H+ uniport. We have confirmed that ATP and GTP inhibit such FA-induced H+ uniport mediated by UCP2 and UCP3. Coenzyme Q10 did not further significantly activate the observed H+ efflux. In conclusion, careful instant reconstitution yields intact functional recombinant proteins, UCP2 and UCP3, the activity of which is comparable with UCP1.
Assuntos
Proteínas de Membrana Transportadoras , Mitocôndrias/metabolismo , Proteínas Mitocondriais , Proteínas/metabolismo , Proteínas de Transporte/metabolismo , Ácidos Graxos/metabolismo , Humanos , Canais Iônicos , Proteína Desacopladora 2 , Proteína Desacopladora 3 , Leveduras/metabolismoRESUMO
Chronic myeloid leukemia (CML) is caused by constituve activity of BCR-ABL tyrosine kinase. Despite of high efficiency of imatinib, selective BCR-ABL inhibitor, about 30% of patients develop resistance. Novel markers and targets for therapy are thus necessary. MicroRNAs are small intereference RNAs whose role in physiological and malignant hematopoiesis has been shown. This study is focused on miR-451 in CML. Following our observation of miR-451 downregulation in CML, we further show its relation to BCR-ABL activity. Our data together with current literature indicate a more complex relationship of miR-451 and BCR-ABL in CML.
Assuntos
Proteínas de Fusão bcr-abl/antagonistas & inibidores , Genes abl/fisiologia , Leucemia Mielogênica Crônica BCR-ABL Positiva/genética , MicroRNAs/genética , HumanosAssuntos
Leucemia Mielogênica Crônica BCR-ABL Positiva/metabolismo , Leucemia Mieloide Aguda/metabolismo , Proteínas WT1/metabolismo , Processamento Alternativo , Perfilação da Expressão Gênica , Humanos , Leucemia Mielogênica Crônica BCR-ABL Positiva/genética , Leucemia Mieloide Aguda/genética , Isoformas de Proteínas/análise , Reação em Cadeia da Polimerase em Tempo Real , Proteínas WT1/genéticaRESUMO
UCP2 (the lowest Km values: 20 and 29 microm, respectively) for omega-6 polyunsaturated FAs (PUFAs), all-cis-8,11,14-eicosatrienoic and all-cis-6,9,12-octadecatrienoic acids, which are also the most potent agonists of the nuclear PPARbeta receptor in the activation of UCP2 transcription. omega-3 PUFA, cis-5,8,11,14,17-eicosapentaenoic acid had lower affinity (Km, 50 microm), although as an omega-6 PUFA, arachidonic acid exhibited the same low affinity as lauric acid (Km, approximately 200 microm). These findings suggest a possible dual role of some PUFAs in activating both UCPn expression and uncoupling activity. UCP2 (UCP3)-dependent H+ translocation activated by all tested FAs was inhibited by purine nucleotides with apparent affinity to UCP2 (reciprocal Ki) decreasing in order: ADP > ATP approximately GTP > GDP >> AMP. Also [3H]GTP ([3H]ATP) binding to isolated Escherichia coli (Kd, approximately 5 microm) or yeast-expressed UCP2 (Kd, approximately 1.5 microm) or UCP3 exhibited high affinity, similar to UCP1. The estimated number of [3H]GTP high affinity (Kd, <0.4 microm) binding sites was (in pmol/mg of protein) 182 in lung mitochondria, 74 in kidney, 28 in skeletal muscle, and approximately 20 in liver mitochondria. We conclude that purine nucleotides must be the physiological inhibitors of UCPn-mediated uncoupling in vivo.