The Src/ABL kinase inhibitor dasatinib (BMS-354825) inhibits function of normal human T-lymphocytes in vitro
Introduction
Dasatinib (BMS-354825) (Bristol Myers Squibb) is a dual Src/ABL kinase inhibitor that has strong activity against ABL and BCR/ABL tyrosine kinases and broad activity against Src-family tyrosine kinases [1]. Dasatinib has been proposed for the treatment of malignancies arising from overactive tyrosine kinase activity such as chronic myeloid leukemia (CML), where the mutant tyrosine kinase BCR/ABL drives the cancer phenotype [2], [3]. Dasatinib has been designed to improve on the clinical results of the current BCR/ABL inhibitor imatinib mesylate (STI-571, Novartis Pharmaceuticals), as it has increased potency against BCR/ABL, and activity against most of the BCR/ABL mutants that confer imatinib resistance [1]. Phase 2 trial data has shown dasatinib to be well tolerated and effective in the management of early phase CML in patients resistant or intolerant to imatinib [4]. This has lead to approval for the use of dasatinib for the treatment of imatinib resistant CML. Dasatinib has also shown promise for the treatment of gastrointestinal stromal tumors, Philadelphia chromosome positive acute lymphoblastic leukemia and lung and prostate cancers [5], [6], [7], [8].
Whilst Src-family kinases may be involved in cancer and cancer development, they have many roles in the functioning of normal cells [9]. One such role is in the activation of normal T-lymphocytes where the Src-family kinase LCK plays a critical role in TCR signalling [10]. Several other Src-family kinases are also thought to play a role in T-cell activation including FYN and YES [11], [12]. The broad inhibitory activity of dasatinib against Src-family kinases includes activity against LCK, FYN and YES with IC50s of 1.1, 0.2 and 0.41 nM respectively [13], [14]. Potent LCK and Src-kinase inhibitors have been proposed as potential therapeutic agents for the treatment of T-cell related disorders, and dasatinib has been shown previously to affect T-cell proliferation [15]. Imatinib has been shown to inhibit LCK with an IC50 of 600–920 nM [13], [16], [17]. The activity of imatinib against LCK has been proposed as the likely mechanism behind documented in vitro effects of imatinib on proliferation, activation and cytokine production by T-cells, and effects in murine models of T-cell function [17], [18], [19], [20]. There are also a few examples of immune modulation in patients taking imatinib that could be attributed to T-cell inhibition [21], [22]. Our aim was to examine the effects of dasatinib on the function of normal T-cells by evaluating its effects on T-cell proliferation, activation and production of cytokines. By also testing imatinib and cyclosporine, we aimed to compare the effects of dasatinib on T-cells to an ABL inhibitor with greater clinical usage and a commonly used T-cell suppressive agent.
Section snippets
Isolation of normal peripheral blood mononuclear cells
Normal human PBMCs were isolated from buffy coat samples obtained from the Australian Red Cross Blood Service or from freshly drawn blood collected in Lithium Heparin tubes. Experimental use of human material was approved by the Human Ethics Committee, Royal Adelaide Hospital. PBMCs were isolated by density gradient centrifugation using Lymphoprep (Axis-Shield), and were washed twice in HBSS before use in experiments.
Isolation of T-cells
CD3+ T-cells were isolated using a T-cell Negative Isolation Kit (Dynal,
Dasatinib blocks T-cell proliferation
CFSE labeled PBMCs were stimulated non-specifically and varying concentrations of the drugs were added to culture to assess effects on proliferation while cells containing vehicle only were used as a positive control. After 5 day culture, T-cell division was determined and the proliferation index (PI) calculated for each sample. Dasatinib potently inhibited T-cell proliferation, with complete inhibition of responses to all polyclonal stimuli observed at 25–50 nM (Figs. 1B, E, F). Proliferation
Discussion
Dasatinib had a potent inhibitory effect on all the in vitro T-cell functions that were evaluated in this study. We propose that inhibition of TCR signalling and entry into cell cycle is a likely mechanism by which T-cell function and proliferation are blocked by dasatinib. This is supported by our data as dasatinib affected T-cell signalling, activation marker expression and cell cycle entry but not viability. Our study is consistent with a previously published result where dasatinib blocked
Acknowledgments
The authors acknowledge Bristol Myers Squibb for the provision of the dasatinib used in the experiments performed within this paper. They would also like to thank the Australian Red Cross Blood Service for supplying Buffy coat blood packets, and the Detmold Imaging Centre for the flow cytometer access. Duncan Hewett provided critical reading of the manuscript. Experiments were funded in part by a Cancer Council of Australia Grant to Prof. T.P. Hughes and Dr. A.B Lyons. Stephen Blake was funded
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