Nilotinib inhibits the Src-family kinase LCK and T-cell function in vitro.

Dear Editor:

In a recent issue of the Journal of Cellular and Molecular Medicine, it was reported that the tyrosine kinase inhibitor nilotinib (AMN-107, Tasigna®; Norvartis Pharmaceuticals, Basel, Switzerland), used for the treatment of chronic myeloid leukaemia, is able to inhibit the function of normal human T lymphocytes in vitro[1]. In addition, this group demonstrated nilotinib inhibits T-cell receptor (TCR) activation and the phosphorylation of signalling proteins involved in TCR activation. We have also investigated the effects of nilotinib on T cells and have expanded on the findings of Chen et al. by demonstrating that like imatinib [2], nilotinib is able to inhibit LCK, a Src-family kinase that plays a critical role in TCR activation [3].

Experiments were performed using normal human peripheral blood mononuclear cells (PBMCs) isolated by density centrifugation. Experimental use of human material was approved by the Royal Adelaide Hospital Ethics Committee and blood was collected with informed consent. 5’6 carboxyfluorescein diacetate succin-imidyl ester (CFSE) staining of human PBMCs was performed as described previously [4]. In the presence of varying concentrations of nilotinib and imatinib (Novartis Pharmaceuticals), T cells were stimulated with 10 μg/ml phytohaemagglutinin (PHA) or Concanavalin A (ConA) (Sigma, St. Louis, MO, USA) or directly using 75 ng/ml of an anti-CD3 antibody (Mabtech, Stockholm, Sweden). Following a 5-day incubation, the proliferation of T cells determined by analysing CFSE dye dilution in cells stained positive by an anti-CD3 PE antibody (BD Biosciences, San Jose, CA, USA) (Fig. 1A). T-cell data were then analysed by Modfit analysis program (Verity Software, Topsham, ME, USA) and the proliferation index determined for each sample. Graphing these values allowed IC50 values for each drug to be determined. Inhibition of LCK by nilotinib and imatinib was analysed by evaluating the effects of each drug on LCK phosphorylation of a substrate peptide. Active LCK kinase (Cell Signaling, Danvers, MA, USA) was incubated with varying concentrations of both drugs in a standard kinase reaction buffer containing a mix of unlabelled and γ32p labelled ATP and a Src-family kinase peptide substrate (Upstate Biotechnology, Lake Placid, NY, USA). The level of γ32pATP labelled substrate, and hence LCK activity, was determined by blotting reactions on p81 filter paper (Whatman, Kent, UK) and determining radioactivity of each sample using a bench top scintillation β-counter.

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Nilotinib inhibits T-cell proliferation and LCK activity. Following PHA stimulation, nilotinib was able to strongly inhibit the proliferation of T cells as determined by CFSE tracking (A). Using this CFSE data, the proliferation index at various drug concentrations was determined and used to calculate IC50 values for the inhibition of proliferation (B). Values represent the mean from five donors each analysed in different experiments and significant donor variability was seen as represented by large standard deviations. The effects of varying concentrations of nilotinib and imatinib on LCK kinase activity was determined and normalized to a percentage of maximum kinase activity when no drug was present allowing IC50 values to be determined (C). Data presented represent the mean of three independent experiments.

Nilotinib inhibited T-cell proliferation at IC50's of 2–5 μM depending on the stimulus used (Fig. 1B). The IC50 values were roughly half that we observed with imatinib. We also observed a similar effect of nilotinib on T-cell activation marker expression and cytokine production (data not shown). LCK kinase activity was inhibited by nilotinib with an IC50 of 550 nM (Fig. 1C). Imatinib inhibited LCK at a similar concentration as reported in the literature [2] with an IC50 of 1250 nM, approximately twice the IC50 of nilotinib. In contrast to our findings, another group [5] found nilotinib to inhibit LCK weakly with an IC50 of 5200 nM. While this is somewhat less potent inhibition to that we obtained, the different IC50s could be due to technical differences in the kinase assays used. Abl has also been implicated in T-cell function [6-8] and it is possible that Abl inhibition by nilotinib may cause, or add to LCK blockade for the inhibitory effect on T-cell function. Nilotinib has 20-fold increased potency against Abl compared with imatinib [9] and we found it to have twice the potency against LCK. As nilotinib inhibited T cells approximately twice as strongly as imatinib, not 20 times as strongly, our data would suggest LCK inhibition may be the main mechanism by which the drug inhibits T-cell activation. Our findings agree well with those of Chen et al.[1] and expand on their results by demonstrating nilotinib inhibits the activity of the Src-family kinase LCK, and propose inhibition of LCK as the likely mechanism by which nilotinib interrupts TCR signalling and the function of T cells.