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P.L.R. Nicolson et al.
ibrutinib-treated platelets was unchanged when compared to that of vehicle-treated platelets (Figure 4B).
Btk-specific concentrations of ibrutinib block GPVI mediated aggregation in patients with X-linked agammaglobulinemia
The B-cell immunodeficiency XLA is caused by muta- tions in the BTK gene. Using knowledge of patients’ muta- tions (Online Supplementary Table S2), and an antibody to the N-terminus of Btk, we selected unrelated patients lack- ing Btk protein to test for off-target effects of ibrutinib (Figure 5Ai). Strikingly, the concentration-response curve for inhibition of CRP-induced aggregation by ibrutinib was shifted to the left in the XLA patients when compared to that of the healthy donors (Figure 5Aii-iii), whereas the curve for inhibition of PLCg2 phosphorylation was unchanged (Figure 5Aiv). Since the only known difference between XLA patients and controls is the absence of Btk, this demonstrates an off-target effect of ibrutinib that was unmasked in the absence of Btk protein. This off-target action occurred over a similar concentration range to that required for inhibition of Btk. The GPCR agonists ADP and PAR1 peptide stimulated robust aggregation in XLA patients, as previously demonstrated16 (data not shown).
One possible explanation for the increased sensitivity of XLA patients to ibrutinib relative to controls is that Btk also functions as an adapter protein in the GPVI signaling pathway (as the only known difference in these two groups is Btk protein). To investigate this, we transfected Btk-deficient DT40 chicken B cells with GPVI and its sig- naling partner, FcRg, in the presence of wild-type (WT) or kinase-dead (KD) Btk. Importantly, these cells express PLCg2 but do not express other Tec family kinases.22,23 The
AB
K430E mutant of Btk has been previously reported to lack kinase activity.24 Cells lacking Btk or GPVI were unrespon- sive to collagen. Cells transfected with WT or KD Btk reconstituted NFAT signaling to a similar degree (Figure 6A,B), demonstrating that Btk also functions as an adapter protein in the GPVI signaling pathway. A low dose of ibrutinib had no effect on cells transfected with WT or KD Btk whereas a high dose of ibrutinib blocked NFAT signal- ing in both WT and KD transfected cells (Figure 6C).
Together these results demonstrate that Btk functions as an adapter protein, as well as a kinase, in XLA platelets and in transfected DT40 cells.
Acalabrutinib inhibits Btk Y223 phosphorylation and platelet aggregation, secretion and Ca2+ mobilization by glycoprotein VI
Studies were extended to a second-generation Btk inhibitor, acalabrutinib, which, like ibrutinib, irreversibly binds to Btk at C481 and is highly plasma protein-bound. Acalabrutinib has a higher selectivity over other tyrosine kinases relative to ibrutinib, including Src, Syk and Tec,25 but a 5-fold lower IC50 for Btk.17 In patients the mean peak free drug concentration of acalabrutinib is 1.3 mM.17
Acalabrutinib has a similar, dose-dependent effect on platelet aggregation to that of ibrutinib. At a concentration of 2 mM in washed platelets, acalabrutinib induced a slight delay in aggregation (Online Supplementary Figure S3A) but had no effect on the overall magnitude of response (Figure 7Aii). The difference in the dose-dependency relative to ibrutinib is consistent with the lower IC50 of acalabrutinib for Btk. Similar to ibrutinib, inhibition of platelet aggrega- tion by CRP occurs at acalabrutinib concentrations that are one order of magnitude higher than those which cause
C
Figure 6. Kinase-dead Btk is sufficient for glycoprotein VI signaling. Btk-defi- cient DT40 cells were transfected with either wild-type (WT) or kinase-dead (KD) Btk with or without GPVI/FcRg. All cells were transfected with a NFAT- luciferase reporter plasmid. Cells were stimulated with collagen (10 mg/mL) in the presence of serum. (A) Luciferase activity was measured and is shown as mean ± SEM of five identical experi- ments. (B) Representative western blot showing equal Btk expression in WT and KD-transfected cells. (C) Cells were stimulated with collagen (10 mg/mL) in the presence or absence of ibrutinib (0.5 mM – 10 mM). Serum was excluded during stimulation to avoid plasma binding of the drugs. Luciferase activity between vehicle and drug-treated sam- ples was measured and is shown as the mean ± SEM of three independent experiments. *P<0.05, **P<0.01.
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haematologica | 2018; 103(12)