ABSTRACT
BACKGROUND: Aurora kinases play an essential role in the orchestration of chromosome separation and cytokinesis during mitosis. Small-molecule inhibition of the aurora kinases has been shown to result in inhibition of cell division, phosphorylation of histone H3 and the induction of apoptosis in a number of cell systems. These characteristics have led aurora kinase inhibitors to be considered as potential therapeutic agents. DESIGN AND METHODS: Aurora kinase gene expression profiles were assessed in 101 samples from patients with acute myeloid leukemia. Subsequently, aurora kinase inhibitors were investigated for their in vitro effects on cell viability, histone H3 phosphorylation, cell cycle and morphology in acute myeloid leukemia cell lines and primary acute myeloid leukemia samples. RESULTS: The aurora kinase inhibitors AZD1152-HQPA and ZM447439 induced growth arrest and the accumulation of hyperploid cells in acute myeloid leukemia cell lines and primary acute myeloid leukemia cultures. Furthermore, both agents inhibited histone H3 phosphorylation and this preceded perturbations in cell cycle and the induction of apoptosis. Single cell cloning assays were performed on diploid and polyploid cells to investigate their colony-forming capacities. Although the polyploid cells showed a reduced capacity for colony formation when compared with their diploid counterparts, they were consistently able to form colonies. CONCLUSIONS: AZD1152-HQPA- and ZM447439 are effective apoptosis-inducing agents in acute myeloid leukemia cell lines and primary acute myeloid leukemia cultures. However, their propensity to induce polyploidy does not inevitably result in apoptosis.
Subject(s)
Antineoplastic Agents/pharmacology , Benzamides/pharmacology , Enzyme Inhibitors/pharmacology , Leukemia, Myeloid, Acute/drug therapy , Leukemia, Myeloid, Acute/genetics , Organophosphates/pharmacology , Protein Serine-Threonine Kinases/antagonists & inhibitors , Quinazolines/pharmacology , Apoptosis , Aurora Kinases , Blast Crisis , Cell Proliferation/drug effects , Histones/metabolism , Humans , Phosphorylation , Ploidies , Polyploidy , Tumor Cells, CulturedABSTRACT
The receptor tyrosine kinase FLT3 is a promising molecular therapeutic target in acute myeloid leukemia (AML). Activating mutations of FLT3 are present in approximately one-third of patients, while many nonmutants show evidence of FLT3 activation, which appears to play a significant role in leukemogenesis. We studied the effects of lestaurtinib (CEP701) and PKC412, 2 small molecule inhibitors of FLT3, on 65 diagnostic AML blast samples. Both agents induced concentration-dependent cytotoxicity in most cases, although responses to PKC412 required higher drug concentrations. Cytotoxic responses were highly heterogeneous and were only weakly associated with FLT3 mutation status and FLT3 expression. Importantly, lestaurtinib induced cytotoxicity in a synergistic fashion with cytarabine, particularly in FLT3 mutant samples. Both lestaurtinib and PKC412 caused inhibition of FLT3 phosphorylation in all samples. Translation of FLT3 inhibition into cytotoxicity was influenced by the degree of residual FLT3 phosphorylation remaining and correlated with deactivation of STAT5 and MAP kinase. FLT3 mutant and wild-type cases both varied considerably in their dependence on FLT3 signaling for survival. These findings support the continued clinical assessment of FLT3 inhibitors in combination with cytotoxic chemotherapy: Entry to future clinical trials should include FLT3 wild-type patients and should remain unrestricted by FLT3 expression level.
Subject(s)
Carbazoles/pharmacology , Indoles/pharmacology , Leukemia, Myeloid/drug therapy , Leukemia, Myeloid/pathology , Staurosporine/analogs & derivatives , Acute Disease , Adolescent , Adult , Aged , Apoptosis/drug effects , Blast Crisis/pathology , Drug Screening Assays, Antitumor , Female , Furans , Humans , Male , Middle Aged , Mutation , Protein Kinase C/antagonists & inhibitors , Protein-Tyrosine Kinases/antagonists & inhibitors , Signal Transduction , Staurosporine/pharmacology , Tumor Cells, Cultured , fms-Like Tyrosine Kinase 3/genetics , fms-Like Tyrosine Kinase 3/physiologyABSTRACT
Mutations of the FLT3 gene, a receptor tyrosine kinase, are the most frequent genetic alteration reported in acute myeloid leukaemia, with internal tandem duplications (ITD) or mutations within the activating loop (AL) reported at a frequency of around 24% and 6%, respectively. ITD mutations have associated with a poor prognosis. In this study we have used polymerase chain reaction (PCR), combined with restriction enzyme digestion for the detection of AL mutations, with the DNA products separated on the Agilent 2100 Bioanalyser using a DNA-500 kit. This analysis enabled the rapid identification of mutations in FLT3, approximate sizing of the ITD, an estimate of the proportion of mutant RNA and in some cases, specific heteroduplex patterns associated with triplet deletions. Our data shows that approximately 16% of the patients examined had an ITD mutation and over 13% had a mutation in the AL including triplet deletions involving codons 835/836 and point mutations in codon D839. Based on the sensitivity and speed of the bioanalyser, we suggest that this method is invaluable and provides an improvement to the current use of agarose gels for the analysis of FLT3 PCR products.
