RESUMEN
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.
Asunto(s)
Proteínas Cromosómicas no Histona/genética , ADN-Topoisomerasas de Tipo II/genética , Leucemia Mieloide Aguda/genética , Proteínas Oncogénicas/genética , Inhibidores de Topoisomerasa/uso terapéutico , Translocación Genética/genética , Secuencia de Bases , Resistencia a Antineoplásicos , Femenino , Humanos , Leucemia Mieloide Aguda/inducido químicamente , Leucemia Mieloide Aguda/tratamiento farmacológico , Proteínas de Unión a Poli-ADP-RibosaAsunto(s)
Antineoplásicos/efectos adversos , Subunidad alfa 2 del Factor de Unión al Sitio Principal/genética , Genes abl , Leucemia Mieloide Aguda/inducido químicamente , Proteínas Proto-Oncogénicas/genética , Factores de Transcripción/genética , Adulto , Electroforesis en Gel de Agar , Femenino , Humanos , Cariotipificación , Leucemia Mieloide Aguda/genética , Proteína 1 Compañera de Translocación de RUNX1RESUMEN
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.
Asunto(s)
Subunidad alfa 2 del Factor de Unión al Sitio Principal/genética , Leucemia Mieloide Aguda/genética , Mitoxantrona/efectos adversos , Translocación Genética , Subunidad alfa 2 del Factor de Unión al Sitio Principal/metabolismo , Análisis Citogenético , Bases de Datos Genéticas , Regulación Neoplásica de la Expresión Génica , Humanos , Hibridación Fluorescente in Situ , Leucemia Mieloide Aguda/inducido químicamente , Leucemia Mieloide Aguda/diagnóstico , Masculino , Persona de Mediana Edad , Mitoxantrona/uso terapéutico , Esclerosis Múltiple/complicaciones , Esclerosis Múltiple/tratamiento farmacológico , Proteínas de Fusión Oncogénica/genética , Proteínas de Fusión Oncogénica/metabolismo , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Proteínas Represoras/genética , Proteínas Represoras/metabolismo , Análisis de Secuencia de ADN , Inhibidores de Topoisomerasa II , Proteínas Supresoras de Tumor/genética , Proteínas Supresoras de Tumor/metabolismoRESUMEN
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.
Asunto(s)
Electroforesis Capilar/métodos , Genes abl/genética , Trastornos Mieloproliferativos/diagnóstico , Trastornos Mieloproliferativos/genética , Reacción en Cadena de la Polimerasa/métodos , Análisis Mutacional de ADN , Proteínas de Fusión bcr-abl/genética , Humanos , Hibridación Fluorescente in Situ , Janus Quinasa 2/análisis , Janus Quinasa 2/genéticaRESUMEN
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.