The effect of Bruton’s tyrosine kinase (BTK) inhibitors on collagen-induced platelet aggregation, BTK, and tyrosine kinase expressed in hepatocellular carcinoma (TEC)

Jun Chen,1 Taisei Kinoshita,1 Tarikere Gururaja,1 Juthamas Sukbuntherng,1 Danelle James,1 Daniel Lu,1 Jennifer Whang,1 Matthias Versele,2 and Betty Y. Chang1

ABSTRACT

Objectives: Bruton’s tyrosine kinase (BTK) and tyrosine kinase expressed in hepatocellular carcinoma (TEC) are expressed by human platelets. These kinases participate in platelet activation through the collagen receptor glycoprotein VI and may perform overlapping functions. In clinical studies, BTK inhibitors (ibrutinib, acalabrutinib, tirabrutinib, zanubrutinib) have been associated with increased bleeding risk, which may result from inhibition of BTK alone or of both BTK and TEC, although the role of TEC in bleeding risk remains unclear. Methods: Here, in vitro catalytic and binding activities of ibrutinib and acalabrutinib were determined with four assay systems. Platelet aggregation assays determined inhibitor potency and its relationship to selectivity between BTK and TEC.
Results: Neither inhibitor was substantially more selective for BTK over TEC. The potencies at which BTK inhibitors suppressed platelet aggregation correlated with the potencies in ontarget BTK assays, including those in cells. At clinically-relevant plasma concentration, ibrutinib, acalabrutinib, and tirabrutinib inhibited collagen-induced platelet aggregation to a similar extent, despite differing in vitro IC50s.
Conclusions: Our results suggest BTK inhibition is the primary driver for inhibition of platelet aggregation. The subtle differences between these inhibitors suggest only randomized, double-blind, placebo-controlled clinical studies can fully address the bleeding risks of different BTK inhibitors.

Keywords: Bruton’s tyrosine kinase (BTK), BTK inhibitor, TEC, platelet, aggregation.

INTRODUCTION

Bruton’s tyrosine kinase (BTK) plays a critical role in B-cell receptor (BCR) signaling and is involved in B-cell differentiation, proliferation, adhesion, migration, and survival (1-5). In addition to its activities in B cells, BTK is expressed in platelets and mediates signaling from the cell-surface receptor Glycoprotein VI (GPVI) (6-8). Studies using BTK-deficient platelets from patients with X-linked agammaglobulinemia (XLA) and from BTK-knockout (KO) mice showed significantly diminished platelet aggregation in response to low concentrations of collagen but near-normal platelet aggregation at high collagen concentrations (7, 9). BTK has also been shown to work in concert with tyrosine kinase expressed in hepatocellular carcinoma (TEC) in platelets to mediate collagen-induced platelet aggregation via phospholipase C activation (7-10). Platelets from BTK/TEC double KO mice were completely unresponsive or severely suppressed upon stimulation with a wide range of collagen concentrations (9). These findings suggest that TEC may partially compensate for loss of BTK function at least in mice; however, the role of TEC in collageninduced platelet activation in humans has not been fully established.
Ibrutinib, a first-in-class, once-daily, inhibitor of BTK, is approved for treatment of various B-cell malignancies (11-16) as well as chronic graft-versus-host disease following failure of 1 line of systemic therapy. Ibrutinib provides sustained BTK engagement and is effective after daily dosing (17). Ibrutinib treatment has been associated with an increased risk of bleeding-related adverse events (AEs), with most of them being grade ≤2 and seldom grade ≥3 (13, 14, 18). Platelets from patients receiving ibrutinib exhibited reduced collagen or collagen-related peptide-induced aggregation (10, 18, 19), while other receptor systems, such as thrombin receptor, adrenergic receptor, and P2Y receptors, were largely unaffected (20). Because platelets play a central role in hemostasis following vascular injury (21) and maintaining vascular integrity (22, 23), bleeding-related AEs observed among patients on ibrutinib are generally believed to be associated with platelet inhibition (7, 10, 19). Indeed, ibrutinib has been shown to inhibit collagen-induced platelet aggregation in healthy donors, donors taking warfarin, and donors with severe renal dysfunction with IC50 values between 0.8 µM and 4.6 µM (manuscript in preparation). This concept is further supported by studies showing that addition of untreated platelets reverses ibrutinib-induced inhibition of platelet aggregation in vitro (10, 24) and a report that platelet transfusion successfully treated ibrutinib-related central nervous system bleeding (25).
Bleeding has also been reported for other BTK inhibitors either approved or in clinical development, including acalabrutinib/Calquence® (26), (27), tirabrutinib (ONO/GS-4059) (28), and zanubrutinib (29). Some of these BTK inhibitors have been studied in in vitro or ex vivo platelet assays and demonstrated variable potency and selectivity. It has been suggested that off-target inhibition of TEC by BTK inhibitors may be responsible for the increased incidence of bleeding seen in patients (26). In addition, past studies only evaluated the BTK inhibitors’ potency, measured by IC50 in platelet assays in vitro, and did not take the varying clinically meaningful human plasma exposures exhibited by each inhibitor into account. For instance, acalabrutinib and tirabrutinib treatments are associated with substantially higher plasma exposure than ibrutinib is in patients (26, 28). Therefore, a simple comparison of the in vitro potency defined by IC50 of BTK inhibitors could lead to inaccurate conclusions in clinical settings.
Herein, we characterized ibrutinib and several other approved or clinical-stage covalent BTK inhibitors and estimated their relative potency against platelets by analysing the relationship between in vitro potency in platelet aggregation and plasma exposure in patients. We also analysed the correlation between the cellular potency of BTK inhibitors on platelet aggregation and activites against BTK or TEC. Our results indicate that, when adjusted to account for each drug’s pharmacokinetic (PK) plasma exposure profile in humans, all tested covalent BTK inhibitors had comparable inhibitory activities in collageninduced platelet activation, which correlated well with inhibition of BTK rather than TEC.

MATERIALS AND METHODS

BTK inhibitors

All BTK inhibitors were synthesized at Pharmacyclics LLC (Sunnyvale, CA, USA) including ibrutinib, acalabrutinib, tirabrutinib, RN486, and zanubrutinib.

Biochemical kinase assays

Kinase activities for BTK and TEC were measured with the following four assays: Caliper LabChip® microfluidic mobility shift assay performed by Nanosyn, Inc. (Santa Clara, CA, USA); HotSpotSM miniaturized radioisotope filter-binding assay performed by Reaction Biology Corp. (Malvern, PA, USA); LanthaScreen™ TR-FRET assay performed by Aptuit (Verona, Italy); and KINOMEscan® active site-directed competition binding assay performed by DiscoverX (Fremont, CA, USA). LabChip®, HotSpotSM, and LanthaScreen™ TR-FRET platforms measure catalytic activity, and KINOMEscan® determines binding affinity. LabChip® is a mobility shift assay that uses fluorophore-labeled substrate and employs electrophoresis to separate the more negatively charged phosphorylated product from the unphosphorylated substrate. Phosphorylated product is quantified by measuring fluorescence intensity (30). HotSpotSM is a radioisotope filter-binding assay that uses 32P-γATP or 33P-γ-ATP. The reaction mixtures are spotted onto filter papers, which bind the radioisotope-labeled catalytic product. Unreacted phosphate is removed by washing the filter papers. LanthaScreen™ TR-FRET is based on time-resolved fluorescence resonance energy transfer (TR-FRET). This system uses a peptide substrate labelled with an acceptor fluorophore, and an antiphosphopeptide detection antibody labelled with a donor fluorophore. As a result, only phosphorylated substrates will exhibit TR-FRET (31). The KINOMEscan® screening platform uses an active site-directed competition binding assay to measure interactions between test compounds and kinases. It does not require ATP and therefore reports only the thermodynamic interaction affinities. Full-length recombinant proteins or peptides partial fragments containing kinase domains were used in the assays, and the kinase reactions were performed at each protein’s Km.

Cellular activity assays

DoHH2 B lymphoma cells (DSMZ, Braunschweig, Germany) or whole blood in sodium heparin anticoagulant was pretreated with inhibitors for 15 minutes. Goat anti-human IgD or IgM (Southern Biotech, Birmingham, AL, USA) was added at 50 µg/mL for 18 hours to stimulate B cells before staining with antibodies against CD20 (2H7) and CD69 (FN50) (BD Biosciences, San Jose, CA, USA) for 30 minutes. After red blood cell lysis and fixation of the remaining cells in paraformaldehyde (VWR, Radnor, PA, USA), the expression of CD69 on B cells was examined on a BD FACSCanto II and analysed with FlowJo software (Tree Star Inc., Ashland, OR, USA). Representative plots and gating stratagies for CD69 expression in B cells in whole blood are shown in Supplemental Fig S1. To measure cell proliferation, TMD8 cells were plated into 96-well plates and treated for 72 hr. Proliferation was assessed using the CellTiter-Glo® assay (Promega, Madison, WI, USA).

Human platelet preparation and aggregation assay with light transmission aggregometer (LTA)

Fresh venous blood samples were collected from healthy drug-free volunteers with their informed consent. Platelet-rich plasma (PRP) was isolated by centrifugation of whole blood at 150×g for 10 min. Platelet-poor plasma (PPP) was prepared by additional centrifugation after removal of PRP at 2500×g for 15 min. The platelet concentration in PRP was counted and adjusted to 3×105/µL by dilution with PPP. Collagen-induced platelet aggregation was measured using a LTA (Aggregation Profiler, Model PAP-8E, Bio/Data Corporation, Horsham, PA, USA), and recorded for 11 minutes. Samples were pretreated with compounds for 15–45 minutes before stimulation with collagen (lyophilized preparation of soluble calf skin [Collagen Type I], Bio/DATA Corporation). Since the response of platelet aggregation to collagen concentration differs among donors. we first determined the collagen concentration that stimulated half-maximal platelet aggregation response for each donor and this concentration was used for subsequent assays with inhibitors for the same donor. In general, two groups of donors were identified, and the collagen concentrations used for each group were 100 µg/mL and 75 µg/mL.

Statistical analysis

Data are presented as mean±SD for in vitro studies. CalcuSyn software (Biosoft, Great Shelford, Cambridge, United Kingdom) was used to calculate IC50 for inhibition of platelet aggregation by inhibitors. GraphPad Prism (GraphPad Software, La Jolla, CA, USA; Version 7) was used for correlation analysis using the Pearson correlation calculation and two-sided P values. IC50 values for on- and off-target cellular activity of different compounds were calculated using GraphPad Prism. Comparison of differences among multiple compounds was analysed by non-parametric one-way ANOVA, followed by a Mann-Whitney test to determine differences between compounds.

RESULTS

The activity of BTK inhibitors on BTK and TEC in biochemical and cellular assays

The potency and selectivity profiles of BTK inhibitors in in vitro assays using recombinant enzymes have been notably inconsistent in previous reports, presumably due to utilization of different assay platforms and assay conditions. We therefore examined ibrutinib and acalabrutinib for their biochemical activity and affinity against BTK and TEC as well as their relative selectivity (IC50 TEC/BTK) in three catalytic activity assay platforms, Caliper LabChip®, LanthaScreenTM TR-FRET, and HotSpotSM, and one binding assay platform, KINOMEscan®, to evaluate the variation among different assay platforms. As shown in Table 1, the activity, affinity, and selectivity of the two compounds towards TEC and BTK differed in different platforms. However, ibrutinib was consistently more potent than acalabrutinib in all three catalytic activity platforms that measured on-target BTK activity, with IC50 values ranging from 0.13 to 1.18 nM, in contrast with 2.79 to 135 nM for acalabrutinib.
Ibrutinib also demonstrated stronger binding affinity for BTK than acalabrutinib in the KINOMEscan® assay, with Kd values of 0.89 nM and 11.45 nM, respectively. The selectivity profiles of ibrutinib and acalabrutinib for BTK versus TEC varied across the four assays. In the LabChip® and KINOMEscan® assay, the difference in the TEC/BTK ratio between ibrutinib and acalabrutinib was less than two-fold, with acalabrutinib being slightly more selective for BTK, although the differences did not reach statistical significance (Table 1). In contrast, acalabrutinib was much less selective than ibrutinib in the LanthaScreenTM TRFRET and HotSpotSM assays and the differences were statistically significant.
Focusing on the LabChip® platform, we extended our profiling to include two additional BTK inhibitors, tirabrutinib and RN486, for further characterization and assay validation. RN486, a reversible BTK inhibitor known to be highly selective for BTK (32), showed the highest selectivity with over a 124-fold difference between IC50 values of BTK and TEC, validating the assay sensitivity (Table 2). As expected, ibrutinib was more potent against BTK than the other covalent BTK inhibitors and had the highest potency among all four inhibitors against TEC. However, there was no significant difference among the three covalent BTK inhibitors regarding the selectivity over TEC (Table 2). Thus, we were unable to demonstrate that acalabrutinib was significantly more selective towards BTK over TEC compared with ibrutinib using multiple assay platforms.
We next examined BTK inhibitors in cell-based assays. BCR-mediated CD69 induction in B cells is known to be BTK-dependent (32, 33), although TEC may have a limited, overlapping role (34). TMD8 cells, an ABC-type B lymphoma cell line derived from diffuse large B cell lymphoma (DLBCL), are known to be dependent on BTK activity to proliferate (35). Thus, on-target cellular activity of BTK inhibitors was assessed by measuring CD69 induction in DoHH2 cells and in human whole blood and by measuring growth inhibition of TMD8 cells. Additionally, the cellular off-target activity of BTK inhibitors was evaluated with T-cell receptor (TCR)-stimulated IL-2 secretion by Jurkat acute T leukemic cells, and with proliferation assays of Jurkat and A549 lung adenocarcinoma cells.
BTK on-target assays, with ibrutinib demonstrating the greatest potency. Of note, ibrutinib reduced IL-2 production by Jurkat T cells, while no growth inhibitory activity was seen even when increased to more than 3 M. Inhibition of IL-2 production by ibrutinib likely resulted from its known activity against ITK (Table 2), another member of the TEC kinase family expressed in T cell lineages.

Potency of BTK inhibitors on collagen-induced platelet aggregation relative to their pharmacokinetic properties

The potency of BTK inhibitors against collagen-induced platelet aggregation was examined with LTA and blood samples from healthy volunteers. The sensitivity of platelets to collagen stimulation is variable among donors, and the same concentration of collagen does not always generate the same magnitude of platelet responses across donors (Supplemental Fig S2). Therefore, we predetermined the stimulating collagen concentration that produced a half-maximal aggregation response for each donor. Under these conditions, ibrutinib demonstrated a comparable potency in platelet aggregation across donors, which also fell within ibrutinib’s clinically achievable plasma exposure range. Importantly, potency of other BTK inhibitors relative to ibrutinib also remained consistent. We thus decided to use this strategy to evaluate all BTK inhibitors in this study using ibrutinib as the control in every assay.
These observations may be extended to zanubrutinib, another covalent BTK inhibitor in late-stage clinical trials in B lymphoma. Zanubrutinib had an IC50 value of 0.44 ± 0.10 μM in our LTA assay and its Cmax level after 80 mg once-daily dosing was reportedly comparable to that of ibrutinib given at 560 mg once-daily dosing (0.37 μM) (36), again demonstrating a close relationship between Cmax and IC50 in the LTA assay. Of note, it is unlikely that the wide differences in the IC50 values of BTK inhibitors, as measured by LTA, resulted from differences in plasma protein binding (PPB) properties. For example, according to their respective drug labels (as of May 2018) ibrutinib and acalabrutinib have PPB values of 97.3% and 97.5%, respectively, despite a more than 5-fold difference in the LTA assay (27,
Platelet aggregation affected by BTK inhibitors is associated with inhibition of BTK but not
We next expanded our study to a total of 12 BTK inhibitors to further define the contribution of BTK and TEC activities to collagen-induced platelet aggregation. Tested inibitors included both covalent and reversible inhibitors, which were approved, in development by other companies, or obtained from our proprietary BTK inhibitor library. The potency of BTK inhibitors in collagen-induced platelet aggregation, BTK and TEC enzymatic activities, and the activity on whole-blood BCR-induced B-lymphocyte CD69 expression of these compounds were measured and their correlations were examined. As shown in Fig 2A and B, a clear correlation existed between potency in platelet aggregation and CD69 induction in B cells and, to a lesser extent, biochemical BTK activities. In contrast, biochemical TEC activity did not correlate with platelet aggregation at all (Fig 2C), suggesting that BTK, rather than TEC, plays a predominant role in mediating signaling to induce platelet aggregation in human samples. This analysis also revealed that the compounds’ inhibitory activity on B lymphocyte CD69 expression correlated with BTK enzymatic activity, but not with TEC enzymatic activity (Supplemental Fig S3), consistent with the report that BTK plays a predominant role in signaling pathways through BCR in B cells (32, 33).

DISCUSSION

Bleeding is one of the most common adverse events associated with BTK inhibitors including ibrutinib, acalabrutinib, and zanubrutinib, and inhibition of platelet aggregation has been suggested as a direct cause of bleeding in patients (38). The activity of BTK inhibitors on platelet activation has often been discussed as a result of BTK inhibition, based on studies of genetic BTK deficiency in humans (X-linked agammaglobulinemia) (7). There are reports that suggest TEC may also be involved in bleeding associated with BTK inhibitors, but these reports are based primarily on studies in BTK and/or TEC knock-out mice (9). In this study, we comprehensively characterized marketed (ibrutinib and acalabrutinib) and clinical-stage (zanubrutinib and tirabrutinib) covalent BTK inhibitors in various assays in vitro and in cells, including in platelets freshly isolated from healthy human donors. For in vitro assays, we examined the inhibitors’ relative potency toward on-target BTK compared with TEC using four assay platforms to draw unbiased conclusions. In cell assays, we tested BTK inhibitors in platelet aggregation assays and examined the correlation to well-defined human BCR-dependent assays in primary B cells and in the human B cell line, as well as in BTK/TEC enzymatic activities. We further evaluated their potency in platelets in association with their drug exposure in humans.
Biochemical kinase assays using recombinant proteins generate valuable information on the intrinsic potency of kinase inhibitors towards direct targets in relatively simple reconstituted systems. However, the results of these assays often differ between assay platforms. It is therefore desirable to examine inhibitors in more than one assay platform for a generalized evaluation that better reflects the activity of inhibitors. We compared the potency and selectivity in different assay platforms between ibrutinib and acalabrutinib.
Further, we compared the activities of ibrutinib, acalabrutinib, tirabrutinib, and RN486 using the LapChip platform and in several cellular assays. Among the four inhibitors, ibrutinib was the most potent in on-target BTK assays (Tables 1–3). Consistent with previous publications, acalabrutinib and tirabrutinib were significantly more selective than ibrutinib on ITK, TXK, and BMX; however, no significant selectivity advantage for BTK over TEC was identified between ibrutinib and acalabrutinib with four different biochemical assay platforms in the present study, which was contrary to a previous report by Byrd, et al. (26). Although many variables between laboratories might account for this difference, one possible explanation for the discrepancy is that the conclusion made by Byrd et al was based on activities of BTK and TEC measured with different commercial assay platforms and procedures. Specifically, the potency of ibrutinib and acalabrutinib on BTK was tested using an IMAP (Immobilized Metal Ion Affinity-based Fluorescence Polarization) assay platform and on TEC using a LanthaScreenTM platform. As demonstrated in our study, substantial variations exist between different assay platforms, and comparison of results from different assay platforms may lead to different conclusions.
All 3 covalent BTK inhibitors tested in this study were very potent in both BTK biochemical and B-cell based assays, with the IC50s in the nM range and ibrutinib being the most potent inhibitor (Tables 1–3). Although they were less potent in affecting platelet aggregation partially due to the presence of a high amount of protein in platelet-rich plasma, ibrutinib was still more active than acalabrutinib and tirabrutinib with 5.3- and 9.0-fold differences, respectively. These results might imply that ibrutinib induces a higher incidence or more severe bleeding in patients compared with the other inhibitors. However, ibrutinib has a low plasma exposure relative to acalabrutinib and tirabrutinib; for example, the Cmax of acalabrutinib after 100 mg twice-daily dosing was 1.78 M, 4.8-fold higher than ibrutinib’s 0.37 M after 560 mg once-daily dosing (Table 4), eliminating the apparent IC50-based advantage for acalabrutinib while still necessitating twice-daily dosing regimen due to a shorter half-life in the plasma (1.13 h as opposed to 4–6 h for ibrutinib) (26). In fact, the Cmax levels of these 3 covalent inhibitors were close to their respective IC50s for platelet aggregation and well within their variation range (standard derivation) (Fig 1 and Table 4), suggesting they all would have similar impacts on platelet aggregation and possibly bleeding risk. Indeed, treatment with acalabrutinib is associated with bleeding events in approximately 50% of patients with hematological malignancies (27). These observations may also apply to zanubrutinib, another covalent BTK inhibitor in late-stage clinical trials in B lymphoma, as described in the Results section. Thus, our study suggests a similar effect on platelets by the BTK covalent inhibitors, although further clinical confirmation is needed.
Using BTK, TEC, or BTK/TEC double knockout mice, Atkinson et al (9) demonstrated that TEC partially compensated for the loss of BTK in platelet aggregation when platelets were stimulated with a low concentration of collagen and completely compensated for the loss of BTK when stimulated with a high concentration of collagen. However, our study on human platelets clearly demonstrates that inhibition of BTK, not TEC, correlated with the potency of BTK inhibitors on platelet aggregation (Fig 2). Both BTK and TEC are reported to regulate PLCγ2 (7, 9, 39, 40); however, others (32, 41) found that RN486, a highly selective BTK inhibitor with little TEC activity, potently inhibited phosphorylation of PLCγ2 in human B cells and platelets after stimulation through BCR or GPVI, suggesting that TEC did not seem to play a substantial compensatory role. This result further supports that BTK is an important factor mediating signaling from collagen stimulation in human platelets.
In summary, when drug exposure in patients was taken into consideration, all tested BTK inhibitors showed comparable activities in platelet aggregation despite their varying in vitro activities. BTK, but not TEC, was likely a major target of BTK inhibitors in human platelet inhibition. Importantly, this study demonstrates that simple IC50 comparisons Zanubrutinib in biochemical and cell-based assays do not predict the impact of BTK inhibitors on platelet functions in patients, and it is important to compare the potency (IC50 in the platelet assay) of each drug with the observed clinical plasma exposure. Direct head to head comparisons in randomized, double-blind, placebo-controlled clinical studies would be necessary to address the difference between BTK inhibitors and their effects on bleeding adjusted by drug exposure.

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