RESUMEN
Cytogenetic lesions often alter kinase signaling in acute myeloid leukemia (AML) and the addition of kinase inhibitors to the treatment arsenal is of interest. We have screened a kinase inhibitor library and performed combination testing to find promising drug-combinations for synergistic killing of AML cells. Cytotoxicity of 160 compounds in the library InhibitorSelect™ 384-Well Protein Kinase Inhibitor I was measured using the fluorometric microculture cytotoxicity assay (FMCA) in three AML cell lines. The 15 most potent substances were evaluated for dose-response. The 6 most cytotoxic compounds underwent combination synergy analysis based on the FMCA readouts after either simultaneous or sequential drug addition in AML cell lines. The 4 combinations showing the highest level of synergy were evaluated in 5 primary AML samples. Synergistic calculations were performed using the combination interaction analysis package COMBIA, written in R, using the Bliss independence model. Based on obtained results, an iterative combination search was performed using the therapeutic algorithmic combinatorial screen (TACS) algorithm. Of 160 substances, cell survival was ⩽50% at <0.5µM for Cdk/Crk inhibitor, KP372-1, synthetic fascaplysin, herbimycin A, PDGF receptor tyrosine kinase inhibitor IV and reference-drug cytarabine. KP372-1, synthetic fascaplysin or herbimycin A obtained synergy when combined with cytarabine in AML cell lines MV4-11 and HL-60. KP372-1 added 24h before cytarabine gave similar results in patient cells. The iterative search gave further improved synergy between cytarabine and KP372-1. In conclusion, our in vitro studies suggest that combining KP372-1 and cytarabine is a potent and synergistic drug combination in AML.
Asunto(s)
Protocolos de Quimioterapia Combinada Antineoplásica/farmacología , Citarabina/agonistas , Compuestos Heterocíclicos de 4 o más Anillos/farmacología , Leucemia Mieloide Aguda/tratamiento farmacológico , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Proto-Oncogénicas c-akt/antagonistas & inhibidores , Tetrazoles/farmacología , Adulto , Antimetabolitos Antineoplásicos/química , Antimetabolitos Antineoplásicos/farmacología , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Citarabina/farmacología , Ensayos de Selección de Medicamentos Antitumorales , Sinergismo Farmacológico , Femenino , Fluoresceína/metabolismo , Colorantes Fluorescentes/metabolismo , Ensayos Analíticos de Alto Rendimiento , Humanos , Concentración 50 Inhibidora , Leucemia Mieloide Aguda/metabolismo , Leucemia Mieloide Aguda/patología , Masculino , Proteínas Proto-Oncogénicas c-akt/metabolismo , Bibliotecas de Moléculas Pequeñas , Espectrometría de Fluorescencia , Células Tumorales CultivadasRESUMEN
To find drugs suitable for repositioning for use against leukemia, samples from patients with chronic lymphocytic, acute myeloid and lymphocytic leukemias as well as peripheral blood mononuclear cells (PBMC) were tested in response to 1266 compounds from the LOPAC(1280) library (Sigma). Twenty-five compounds were defined as hits with activity in all leukemia subgroups (<50% cell survival compared with control) at 10 µM drug concentration. Only one of these compounds, quinacrine, showed low activity in normal PBMCs and was therefore selected for further preclinical evaluation. Mining the NCI-60 and the NextBio databases demonstrated leukemia sensitivity and the ability of quinacrine to reverse myeloid leukemia gene expression. Mechanistic exploration was performed using the NextBio bioinformatic software using gene expression analysis of drug exposed acute myeloid leukemia cultures (HL-60) in the database. Analysis of gene enrichment and drug correlations revealed strong connections to ribosomal biogenesis nucleoli and translation initiation. The highest drug-drug correlation was to ellipticine, a known RNA polymerase I inhibitor. These results were validated by additional gene expression analysis performed in-house. Quinacrine induced early inhibition of protein synthesis supporting these predictions. The results suggest that quinacrine have repositioning potential for treatment of acute myeloid leukemia by targeting of ribosomal biogenesis.