Recent years have shown a rapid expansion in chronic lymphocytic leukaemia (CLL) research, especially in identifying the role of antigen stimulation in the pathophysiology of the disease and the development of B cell receptor (BCR) kinase inhibitors as potential therapeutic targets. Upon stimulation through the antigen receptor, several phosphoproteins, including SYK, BTK, LYN, AKT and PI3Kδ, are recruited to the B cell signalosome. In vitro manipulation of some of these molecules can inhibit further downstream signalling and subsequent biological outputs, such as proliferation (Herman et al, 2010). Inhibitors of some of these molecules have already entered different phases of clinical trials (Byrd et al, 2013). SYK inhibitor R406, the active compound of the pro-drug R788 (fostamatinib disodium), has been shown to affect various aspects of CLL cell survival signalling and to be clinically effective in B-cell non-Hodgkin lymphoma (B-NHL), including 11 CLL/small lymphocytic leukaemia patients (Friedberg et al, 2010). However, this molecule was not further developed as a treatment for CLL for a number of reasons, mostly related to the fact that the specificity of the molecule is not confined to SYK alone (Cha et al, 2006). Subsequently, it was reported that a series of diaminopyrimidine carboxamides (DAPC) displayed substantial SYK inhibitory activity (Hisamichi et al, 2005). The aminoethylamino moiety at the 2-position of the pyrimidine ring was important for SYK inhibition, as shown by enzyme screening. If an anilino moiety was substituted at the 4th meta position the resulting compound showed high selectivity for SYK, compared to other kinases, such as ZAP70, c-Src, and PKC. Moreover, this compound has a high level of activity towards hERG together with moderate selectivity over Aurora B kinase, a kinase essential for cell proliferation (Liddle et al, 2011). Lead optimisation of the DAPC series of SYK inhibitors led to the discovery of a compound, GSK143, which is a potent and highly selective SYK inhibitor showing good efficacy in the rat Arthus model (Yamamoto et al, 1975; Liddle et al, 2011). Recently, other highly selective SYK inhibitors were also found to be effective against CLL. This includes GS-9973, which was found to be clinically effective in a phase 2 trial (Sharman et al, 2015), and P505-15, which inhibits BCR-mediated signalling and reduces CLL viability (Spurgeon et al, 2013). Therefore, GSK143 was evaluated for its impact on CLL cells in vitro. The critical role of SYK kinase in the propagation of signals from the BCR makes it an attractive target for therapies aimed at crippling BCR dependency. The development of a highly selective inhibitor of SYK therefore provided an opportunity to assay its effect on BCR-dependent CLL cells. To optimise the methods for testing the new SYK inhibitor, a PI3Kδ kinase inhibitor, GS-1101, was used. Treatment with GS-1101 showed significant reduction in the percentage viability of CLL cells and abrogated the IgM-induced survival benefit (data not shown). Using the same experimental conditions, mononuclear cells and negatively selected CLL cells were incubated with GSK143 at concentrations ranging from 10 nmol/l to 10 μmol/l. CLL cells were incubated with fludarabine in parallel as a control and viability was assessed every 24 h for 3 days. The viability and cell number with GSK143 > 100 nmol/l was decreased compared to control cells, but not as pronounced as with fludarabine (Fig 1A). A dose response curve using normalised cytotoxicity as the end point gave a 50% inhibitory concentration of 323 nmol/l [95% confidence interval (CI)- 5·74 to 18261]. This is comparable to dose- and time-dependent cytotoxicity mediated by R406 (GSK 143- mean 51·3–43·1%, R406 - 54·8–44%) and GS-1101 (Quiroga et al, 2009; Herman et al, 2010). Similar to the GS-1101 results, there was no significant difference in cytotoxicity between prognostic groups based on their cytogenetics and IGHV mutational status (Fig 1B). GSK143 also abrogated the pro-survival signal delivered by BCR stimulation given in the form of the F(ab)2 portion of anti-IgM (Fig 1C), which is comparable with the effects noticed with R406 and P505-15 (Quiroga et al, 2009; Spurgeon et al, 2013). To determine whether proximal SYK-associated signalling events were affected by GSK143, CLL cells were first evaluated for SYK phosphorylation status. SYK phosphorylation induced by anti-IgM or -IgD stimulation was partially abrogated by pre-incubating cells in GSK143. There was a difference in mean fluorescence intensity following IgD stimulation of 81·2 (95% CI 33·03–129·4, P = 0·0095) when cells were pre-incubated with GSK143 (Fig 2A). To ascertain whether the loss of SYK phosphorylation translated into a change in function, CLL cells were treated with GSK143 and assayed for calcium flux after BCR ligation. Pre-incubation with GSK143 profoundly inhibited the calcium flux induced by stimulation with either anti-IgM or anti-IgD. There was a mean decrease of 9·9% (95% CI 5·4–14·3, P = 0·0009) in the percentage of fluxed calcium cells pre-incubated with GSK143 (Fig 2B). In conclusion, these experiments demonstrated that the highly selective SYK inhibitor GSK143 severely impairs CLL viability in vitro, even in the presence of pro-survival signals. The compound is effective in reducing BCR-triggered SYK phosphorylation and subsequent signalling as evidenced by diminished calcium flux. This study provides justification for clinical use of GSK143 in CLL patients. The authors thank the patients who provided samples for this work. The authors also thank GlaxoSmithKline Pharmaceuticals for providing GSK143. A.M.V., A.R., G.M.D. and P.H. designed the experiments analysed data, wrote the article and reviewed and approved the final version; M.D. provided GSK143 and approved the final version; and D.N., R.M.T., T.M., planned and contributed to components of the experimental work reviewed and modified versions of article and approved the final version. MD is an employee/shareholder of GSK.
