RESUMO
Myelodysplastic syndromes (MDS) are highly heterogeneous myeloid diseases, characterized by frequent genetic/chromosomal aberrations. Olaparib is a potent, orally bioavailable poly(ADP-ribose) polymerase 1 (PARP1) inhibitor with acceptable toxicity profile, designed as targeted therapy for DNA repair defective tumors. Here, we investigated olaparib activity in primary cultures of bone marrow mononuclear cells collected from patients with MDS (n = 28). A single treatment with olaparib induced cytotoxic effects in most samples, with median IC50 of 5.4 µM (2.0-24.8 µM), lower than plasma peak concentration reached in vivo. In addition, olaparib induced DNA damage as shown by a high proportion of γH2AX positive cells in samples with low IC50s. Olaparib preferentially killed myeloid cells causing a significant reduction of blasts and promyelocytes, paralleled by an increase in metamyelocytes and mature granulocytes while sparing lymphocytes that are not part of the MDS clone. Consistently, flow cytometry analysis revealed a decrease of CD117+/CD123+ immature progenitors (p < 0.001) and induction of CD11b+/CD16+ (p < 0.001) and CD10+/CD15+ (p < 0.01) neutrophils. Morphological and immunophenotypic changes were associated with a dose-dependent increase of PU.1 and CEBPA transcription factors, which are drivers of granulocytic and monocytic differentiation. Moreover, the combination of olaparib with decitabine resulted in augmented cytotoxic and differentiating effects. Our data suggest that olaparib may have therapeutic potential in MDS patients.
Assuntos
Proteínas Cromossômicas não Histona/genética , DNA Topoisomerases Tipo II/genética , Leucemia Mieloide Aguda/genética , Proteínas Oncogênicas/genética , Inibidores da Topoisomerase/uso terapêutico , Translocação Genética/genética , Sequência de Bases , Resistencia a Medicamentos Antineoplásicos , Feminino , Humanos , Leucemia Mieloide Aguda/induzido quimicamente , Leucemia Mieloide Aguda/tratamento farmacológico , Proteínas de Ligação a Poli-ADP-RiboseAssuntos
Antineoplásicos/efeitos adversos , Subunidade alfa 2 de Fator de Ligação ao Core/genética , Genes abl , Leucemia Mieloide Aguda/induzido quimicamente , Proteínas Proto-Oncogênicas/genética , Fatores de Transcrição/genética , Adulto , Eletroforese em Gel de Ágar , Feminino , Humanos , Cariotipagem , Leucemia Mieloide Aguda/genética , Proteína 1 Parceira de Translocação de RUNX1RESUMO
The translocation t(16;21) involving RUNX1 (AML1) and resulting in the RUNX1-CBFA2T3 fusion is a rare but recurrent abnormality mostly found in therapy-related acute myeloid leukemia (t-AML) associated with agents targeting topoisomerase II (topo II). We characterized, at the genomic level, the t(16;21) translocation in a patient who developed t-AML after treatment of multiple sclerosis with mitoxantrone (MTZ). Long template nested PCR of genomic DNA followed by direct sequencing enabled the localization of RUNX1 and CBFA2T3 (ETO2) breakpoints in introns 5 and 3, respectively. Sequencing of the cDNA with specific primers showed the presence of the expected RUNX1-CBFA2T3 fusion transcript in leukemic cells. The RUNX1 intron 5 breakpoint was located at nucleotide position 24,785. This region contained an ATGCCCCAG nucleotide sequence showing approximately 90% homology to a "hotspot" DNA region ATGCCCTAG present in intron 6 of PML previously identified in therapy-related acute promyelocytic leukemia cases arising following treatment with MTZ. This study suggests a wider distribution in the human genome, and particularly at genes involved in chromosome translocations observed in t-AML, of DNA regions (hotspot) targeted by specific topo II drugs.
Assuntos
Subunidade alfa 2 de Fator de Ligação ao Core/genética , Leucemia Mieloide Aguda/genética , Mitoxantrona/efeitos adversos , Translocação Genética , Subunidade alfa 2 de Fator de Ligação ao Core/metabolismo , Análise Citogenética , Bases de Dados Genéticas , Regulação Neoplásica da Expressão Gênica , Humanos , Hibridização in Situ Fluorescente , Leucemia Mieloide Aguda/induzido quimicamente , Leucemia Mieloide Aguda/diagnóstico , Masculino , Pessoa de Meia-Idade , Mitoxantrona/uso terapêutico , Esclerose Múltipla/complicações , Esclerose Múltipla/tratamento farmacológico , Proteínas de Fusão Oncogênica/genética , Proteínas de Fusão Oncogênica/metabolismo , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Análise de Sequência de DNA , Inibidores da Topoisomerase II , Proteínas Supressoras de Tumor/genética , Proteínas Supressoras de Tumor/metabolismoRESUMO
Detection of genetic markers improves diagnostic refinement of chronic myeloproliferative disorders (CMDs) and is helpful in discriminating reactive conditions mimicking CMDs such as reactive erythrocytosis and thrombocytosis. We set-up a multiplex real-time polymerase chain reaction assay followed by capillary electrophoresis, designed to simultaneously screen the two main genetic lesions associated with CMDs, i.e. the BCR-ABL fusion characteristic of chronic myeloid leukemia and the JAK2 V617F mutation that characterises polycythaemia vera and a proportion of cases of essential thrombocythemia and idiopathic myelofibrosis. The test was used in the diagnostic work-up of 50 patients with elevation of >or=2 myeloid cell types in their blood count at presentation and in 42 patients with isolated, non-reactive thrombocytosis. This approach refined diagnosis in 44 of 50 cases in the first series and in 22 of 42 cases with isolated thrombocytosis. We conclude that this non-isotopic and rapid assay amenable to automation may be adopted in routine genetic diagnosis of CMDs as well as for initial screening of thrombocytosis of unknown nature.
Assuntos
Eletroforese Capilar/métodos , Genes abl/genética , Transtornos Mieloproliferativos/diagnóstico , Transtornos Mieloproliferativos/genética , Reação em Cadeia da Polimerase/métodos , Análise Mutacional de DNA , Proteínas de Fusão bcr-abl/genética , Humanos , Hibridização in Situ Fluorescente , Janus Quinase 2/análise , Janus Quinase 2/genéticaRESUMO
The "golden path", produced by the Human Genome Project effort, is composed of a collection of overlapping and fully sequenced BAC/PAC clones covering almost completely the human genome. These clones can be advantageously exploited as fluorescence in situ hybridization (FISH) probes for the characterization of rearrangements frequently found in tumors. Breakpoint characterization can be further refined by generating additional smaller FISH probes through LONG-PCR amplification of specific DNA segments, 5-10 kb in size, using appropriate BAC/PAC probes as template. We report here an example of this approach that has been used to characterize a complex Ph-negative chronic myeloid leukemia (CML Ph-) case in which the BCR/ABL fusion gene was found located on chromosome 9.