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

; Norvartis Pharmaceuticals, Basel,Switzerland), used for the treatment of chronic myeloidleukaemia, is able to inhibit the function of normal human T lym-phocytes in vitro[1]. In addition, this group demonstrated nilo-tinib inhibits T-cell receptor (TCR) activation and the phosphory-lation of signalling proteins involved in TCR activation. We havealso investigated the effects of nilotinib on T cells and haveexpanded on the findings of Chen et al.by demonstrating that likeimatinib [2], nilotinib is able to inhibit LCK, a Src-family kinasethat plays a critical role in TCR activation [3]. Experiments were performed using normal human peripheralblood mononuclear cells (PBMCs) isolated by density centrifuga-tion. Experimental use of human material was approved by theRoyal Adelaide Hospital Ethics Committee and blood was collectedwith informed consent. 5 6 carboxyfluorescein diacetate succin-imidyl ester (CFSE) staining of human PBMCs was performed asdescribed previously [4]. In the presence of varying concentra-tions of nilotinib and imatinib (Novartis Pharmaceuticals), T cellswere stimulated with 10 g/ml phytohaemagglutinin (PHA) orConcanavalin A (ConA) (Sigma, St. Louis, MO, USA) or directlyusing 75 ng/ml of an anti-CD3 antibody (Mabtech, Stockholm,Sweden). Following a 5-day incubation, the proliferation of T cellsdetermined by analysing CFSE dye dilution in cells stained positiveby an anti-CD3 PE antibody (BD Biosciences, San Jose, CA, USA)(Fig. 1A). T-cell data were then analysed by Modfit analysis pro-gram (Verity Software, Topsham, ME, USA) and the proliferationindex determined for each sample. Graphing these values allowedIC50 values for each drug to be determined. Inhibition of LCK bynilotinib and imatinib was analysed by evaluating the effects ofeach drug on LCK phosphorylation of a substrate peptide. ActiveLCK kinase (Cell Signaling, Danvers, MA, USA) was incubated withvarying concentrations of both drugs in a standard kinase reactionbuffer containing a mix of unlabelled and -32p labelled ATP and a Src-family kinase peptide substrate (UpstateBiotechnology, Lake Placid, NY, USA). The level of -32pATPlabelled substrate, and hence LCK activity, was determined byblotting reactions on p81 filter paper (Whatman, Kent, UK) anddetermining radioactivity of each sample using a bench top scin-tillation -counter.Nilotinib inhibited T-cell proliferation at IC50’s of 2–5 Mdepending on the stimulus used (Fig. 1B). The IC50 values wereroughly half that we observed with imatinib. We also observed asimilar effect of nilotinib on T-cell activation marker expressionand cytokine production (data not shown). LCK kinase activity wasinhibited by nilotinib with an IC50 of 550 nM (Fig. 1C). Imatinibinhibited LCK at a similar concentration as reported in the litera-ture [2] with an IC50 of 1250 nM, approximately twice the IC50 ofnilotinib. In contrast to our findings, another group [5] found nilo-tinib to inhibit LCK weakly with an IC50 of 5200 nM. While this issomewhat less potent inhibition to that we obtained, the differentIC50s could be due to technical differences in the kinase assaysused. Abl has also been implicated in T-cell function [6–8] and itis possible that Abl inhibition by nilotinib may cause, or add to LCKblockade for the inhibitory effect on T-cell function. Nilotinib has20-fold increased potency against Abl compared with imatinib [9]and we found it to have twice the potency against LCK. As nilotinibinhibited T cells approximately twice as strongly as imatinib, not20 times as strongly, our data would suggest LCK inhibition maybe the main mechanism by which the drug inhibits T-cell activa-tion. Our findings agree well with those of Chen et al.[1] andexpand on their results by demonstrating nilotinib inhibits theactivity of the Src-family kinase LCK, and propose inhibition ofLCK as the likely mechanism by which nilotinib interrupts TCR sig-nalling and the function of T cells.