BTK inhibition is a potent approach to block IgE‐mediated histamine release in human basophils

Abstract Background Recent data suggest that Bruton's tyrosine kinase (BTK) is an emerging therapeutic target in IgE receptor (IgER)‐cross‐linked basophils. Methods We examined the effects of four BTK inhibitors (ibrutinib, dasatinib, AVL‐292, and CNX‐774) on IgE‐dependent activation and histamine release in blood basophils obtained from allergic patients (n=11) and nonallergic donors (n=5). In addition, we examined the effects of these drugs on the growth of the human basophil cell line KU812 and the human mast cell line HMC‐1. Results All four BTK blockers were found to inhibit anti‐IgE‐induced histamine release from basophils in nonallergic subjects and allergen‐induced histamine liberation from basophils in allergic donors. Drug effects on allergen‐induced histamine release were dose dependent, with IC 50 values ranging between 0.001 and 0.5 μmol/L, and the following rank order of potency: ibrutinib>AVL‐292>dasatinib>CNX‐774. The basophil‐targeting effect of ibrutinib was confirmed by demonstrating that IgE‐dependent histamine release in ex vivo blood basophils is largely suppressed in a leukemia patient treated with ibrutinib. Dasatinib and ibrutinib were also found to counteract anti‐IgE‐induced and allergen‐induced upregulation of CD13, CD63, CD164, and CD203c on basophils, whereas AVL‐292 and CNX‐774 showed no significant effects. Whereas dasatinib and CNX‐774 were found to inhibit the growth of HMC‐1 cells and KU812 cells, no substantial effects were seen with ibrutinib or AVL‐292. Conclusions BTK‐targeting drugs are potent inhibitors of IgE‐dependent histamine release in human basophils. The clinical value of BTK inhibition in the context of allergic diseases remains to be determined.


| INTRODUCTION
Basophils (BA) and mast cells (MC) are major effector cells of anaphylactic reactions in patients suffering from IgE-dependent allergies. [1][2][3] Both cell types produce a number of biologically active mediators, including histamine, lipid mediators, and cytokines, and both cell types express high-affinity receptors for immunoglobulin E (IgE). [1][2][3][4][5][6] Once activated by IgE receptor (IgER) cross-linking or other stimuli, BA and MC liberate their proinflammatory substances, which leads to allergic inflammation and the clinical symptoms of anaphylaxis. [4][5][6][7][8] The ability of BA and MC to respond to IgE-dependent stimuli (allergens) is dependent on genetic background factors, various signal transduction molecules, and the presence of activating cytokines. [7][8][9] The type and severity of an IgE-dependent (anaphylactic) reaction depends on additional variables, including the source and configuration of the allergen, organ-specific factors, and the numbers of BA and MC involved in the reaction. [10][11][12][13][14] BA and MC may increase in number in various immunologic diseases, in certain types of (chronic) infectious diseases, and in distinct hematologic neoplasms. Likewise, in patients with systemic mastocytosis (SM), the numbers of MC increase substantially in various organs. 3,12,13 When these patients are suffering from a concomitant IgE-dependent allergy, anaphylactic reactions may be severe or even life-threatening. 12,13 IgER-dependent activation of BA and MC is accompanied by an increase in certain cell surface antigens, including CD63 and CD203c, and by activation of numerous downstream signaling pathways and molecules, including LYN, SYK, RAS, MAP kinases, PI3 kinase (PI3K), mTOR, and protein kinase C. [4][5][6][15][16][17][18][19][20][21][22][23][24][25][26][27] The Bruton's tyrosine kinase (BTK) has been identified as another important downstream target in IgER-cross-linked BA. [27][28][29][30][31] In particular, it has been described that IgER cross-linking in BA is followed by phosphorylation of SYK and that SYK, once activated, is capable of phosphorylating BTK. [27][28][29][30][31] In addition, it has been described that BTK inhibition is associated with reduced mediator release in human BA. 32 The aims of this study were to explore whether BTK can serve as a therapeutic target in BA and MC and whether the BTK blockers currently used in clinical trials are able to suppress allergen-induced (IgERdependent) activation and histamine release. In addition, we examined the effects of these drugs on growth of BA and MC. The results of our study show that BTK inhibition by ibrutinib is a potent approach to suppress allergen-induced histamine release and activation in human BA.

| Isolation of blood BA
Peripheral blood was obtained from 5 healthy individuals and 11 patients allergic to Der p 2 and/or Phl p 5. Patients were diagnosed according to standard diagnostic procedures and their molecular IgE reactivity profiles were determined by ISAC (immuno-solid-phase allergen chip) technology. 33 The patients' characteristics are shown in Table S3. Informed consent was obtained in each case. The study was approved by the ethics committee of the Medical University of Vienna (EK1641/2014) and conducted in accordance with the Declaration of Helsinki. Peripheral blood was collected in heparin-containing tubes. BA were enriched by dextran sedimentation (histamine release experiments) or were recovered together with mononuclear cells (MNC) after centrifugation over Ficoll (immunostaining experiments) as described. 34 The percentage of BA ranged from 0.1% to 1.5% in dextran preparations, and from 0.3% to 2% in MNC. Cell viability was >90% as assessed by trypan blue exclusion test.

| 1667
After incubation, cells were centrifuged at 4°C, and the cell-free supernatants and total suspensions recovered and analyzed for histamine content by RIA. Histamine release was calculated and expressed as percentage of total histamine. All experiments were performed in triplicates. In a separate set of experiments, anti-IgEinduced histamine release from BA was examined in a patient with chronic lymphocytic leukemia (CLL) treated with ibrutinib (280 mg/ day per os). In this experiment, BA were obtained before treatment with ibrutinib and 14 days after the start of therapy. ex vivo obtained BA were incubated in HRB in the absence or presence of anti-IgE antibody E-124.2.8 (0.001-10 lg/mL) at 37°C for 30 minutes. Then, histamine release was measured as described above. CD203c or a PE-labeled mAb against CD203c for 15 minutes, washed, and then permeabilized with methanol. 40 Thereafter, cells were stained with an Alexa Fluor647-conjugated antibody against pBTK or a PElabeled mAb against pSYK (30 minutes). Expression of intracellular targets in CD203c + BA was quantified by multicolor flow cytometry on a FACSCalibur as reported. 40 Apoptosis was measured in drug-exposed cells by combined AnnexinV/propidium iodide (PI) staining following a published protocol. 36,40 For cell cycle studies, drug-exposed cells were resuspended in 500 lL permeabilization buffer. Then, 40 lL PI was added and cell cycle distribution was analyzed on a FACSCalibur as described previously. 41

| Statistical analysis
To determine the level of significance in drug incubation experiments, histamine release experiments and surface staining experiments in BA and human cell lines, the paired Student's t test was applied. In case of multiple comparisons, the Bonferroni correction was performed. A P value of <0.05 was considered to indicate statistical significance.

| Effects of targeted drugs on IgER downstream signaling molecules
To study drug effects on BTK activation and to explore the specificity of these effects, we examined the phosphorylation status of various IgER downstream signaling molecules in drug-exposed and IgER-cross-linked BA as well as in untreated or drug-exposed cell lines (HMC-1.1, HMC-1.2, KU812). We found that dasatinib, ibrutinib, AVL-292, and CNX-774 counteract anti-IgE-induced expression of pBTK in BA ( Figure 1A). In addition, the SYK inhibitor P505-15 was found to block expression of pBTK in BA ( Figure 1A). These drugs were also found to block pBTK expression in unstimulated HMC-1.1, HMC-1.2, and KU812 cells ( Figure 1B). In control experiments, P505-15 also decreased expression of pSYK in IgER-crosslinked BA (not shown). We also found that ibrutinib as well as the other BTK blockers applied suppress expression of pSYK, pAKT, pS6, and pSTAT5 in HMC-1 and KU812 cells (Fig. S1). Of all drugs applied, dasatinib was found to exert most potent effects on expression of pBTK and BTK downstream kinase targets, thereby confirming the broad target interaction profile of this drug. 38

| Ibrutinib inhibits IgE-dependent histamine release in BA
The BTK blocker ibrutinib is already used in clinical practice and exhibits a favorable toxicity profile. In this study, ibrutinib was found to inhibit IgE-dependent histamine release in BA obtained from healthy  (Figure 2A, Table S4) as well as allergen-induced histamine secretion in BA of allergic patients ( Figure 2B, Table S3). The effects of ibrutinib were dose dependent, with IC 50 values ranging between 0.003 and 0.03 lmol/L (0.01AE0.01 lmol/L) in healthy donors (Table S4) and between 0.003 and 0.023 lmol/L (0.01AE0.01 lmol/L) in allergic patients (Table S3). The inhibitory effects of ibrutinib (1 lmol/L) were seen at all allergen concentrations applied (Figure 2C). We also examined ex vivo BA obtained from a patient before and during treatment with ibrutinib, and found that ibrutinib therapy is followed by downregulation of IgE-dependent histamine release in BA ( Figure 2D). These data show that ibrutinib is a potent inhibitor of IgE-dependent secretion of histamine in BA.

IgE-dependent histamine release
In a next step, we confirmed the value of BTK as a potential drug target by applying other BTK blockers. In these experiments, F I G U R E 2 Effects of ibrutinib on IgE-mediated histamine release in human basophils. Basophils (BA) obtained from three nonallergic donors (A) or patients allergic to Der p 2 (n=3) or Phl p 5 (n=3) (B) were preincubated in control medium (Co) or various concentrations of ibrutinib (0.001-1 lmol/L) at 37°C for 30 minutes. Then, cells were exposed to anti-IgE (1 lg/mL; nonallergic donors) or recombinant allergens (1 lg/mL of rDer p 2 or rPhl p 5; allergic patients) at 37°C for 30 minutes. After centrifugation, histamine concentrations were determined in supernatants and cell lysates. Histamine release is expressed as percentage of total histamine. Results show the percentage of control and represent meanAESD of three independent experiments. Asterisk (*): P<0.05 by Student's t test. (C) BA from two patients allergic to Der p 2 (left and right panel) were incubated in control medium (Co) or 1 lmol/L ibrutinib at 37°C for 30 minutes. Then, cells were incubated in histamine release buffer (HRB) in the absence or presence of rDer p 2 (0.001-10 lg/mL) at 37°C for 30 minutes. After incubation, cells were centrifuged at 4°C and cell-free supernatants and cell suspensions analyzed for histamine content. Histamine release is expressed as percentage of total histamine. Results represent the meanAESD of triplicates. (D) Left panel: BA from a patient with chronic lymphocytic leukemia (CLL) were incubated in control medium (Co) or 1 lmol/L ibrutinib at 37°C for 30 minutes. Thereafter, cells were incubated in HRB in the absence or presence of anti-IgE (0.001-10 lg/mL) at 37°C for 30 minutes. After incubation, cells were centrifuged at 4°C and cell-free supernatants and cell suspensions analyzed for histamine content. Histamine release is expressed as percentage of total histamine. Results represent the meanAESD of triplicates. Right panel: In the same patient with CLL, BA were obtained before therapy with ibrutinib (pretreatment; ■-■) and 14 days after treatment with 280 mg/day ibrutinib (post-treatment; •-•). Cells were incubated in HRB in the absence or presence of anti-IgE antibody E-124.2.8 (0.001-10 lg/mL) for 30 minutes. Then, histamine release was measured as described above. Histamine release is expressed as percentage of total histamine. Results represent the meanAESD of triplicates.
AVL-292 and CNX-774 were found to counteract IgE-mediated and allergen-induced histamine release in IgER-cross-linked BA in a dosedependent manner ( Figure 3A,B). A summary of IC 50 values obtained with BTK inhibitors is shown in Table S4 (nonallergic individuals) and   Table S5 (Der p 2-and Phl p 5-allergic donors). Dasatinib was also applied as positive control and found to block allergen-induced histamine release at higher concentrations ( Figure 3C), confirming our previous results. 38 However, at lower concentrations, dasatinib even promotes histamine release. 38 Of all drugs tested, ibrutinib was found to be the most potent inhibitor of allergen-induced histamine release in human BA (Table S5).  Table 1). However, unexpectedly, CNX-774 was found to counteract proliferation of HMC-1.1 and HMC-1.2 cells at relatively low concentrations (IC 50 in HMC-1.2: 0.1-0.5 lmol/L) ( Figure 5C and Table 1). Dasatinib, a TKI known to block wild-type KIT and KIT D816V, was found to inhibit growth of HMC-1 and KU812 cells in a dose-dependent manner. IC 50 values obtained with KU812 (0.0007 lmol/L) and HMC-1.1 cells were lower compared to that obtained with HMC-1.2 cells (1.45 lmol/L) ( Figure 5D and Table 1).

| Effects of CNX-774 on cell cycle distribution and apoptosis in human BA and MC lines
We also examined CNX-774-exposed HMC-1 and KU812 cells for alterations in cell cycle distribution and signs of apoptosis. In these experiments, CNX-774 was found to exhibit no effect on cell cycle progression in the three cell lines tested (Fig. S2A) Recent data suggest that ibrutinib blocks IgE-dependent activation and histamine release in normal blood BA. 32 In the present study, we were able to confirm this effect of ibrutinib. In addition, our data show that ibrutinib is also a potent inhibitor of allergeninduced activation and histamine release in BA obtained from allergic donors. The IC 50 values in normal BA (nonallergic donors) and allergen-exposed BA obtained from patients allergic to Der p 2 and Phl p 5 were comparable and were found to be within a pharmacologically meaningful range, suggesting that the drug may indeed be able to block histamine secretion in patients with IgE-dependent allergies. This hypothesis was confirmed by studies on ex vivo BA obtained from a CLL patient receiving ibrutinib. In this experiment, IgE-dependent histamine release in ex vivo obtained BA was almost completely suppressed during therapy compared to pretreatment results.
A number of previous and more recent data suggest that BTK is an essential IgER downstream regulator of activation and mediator secretion in BA and MC. 32 It has also been described that BTK is downstream of SYK and upstream of several other major kinase targets relevant to IgE-dependent activation of BA and MC. 43 However, the exact routes in the signaling cascades downstream of BTK in BA and MC are currently unknown. In the present study, we confirmed that ibrutinib downregulates phosphorylation and thus activation of BTK in IgER-cross-linked BA. However, we also found that ibrutinib blocks the activity (phosphorylation) of several other downstream kinases, including AKT, S6, and STAT5. As ibrutinib is known to recognize also several other kinase targets, such as LYN, FYN, SRC, and ITK, it may well be that some of the inhibitory effects of ibrutinib were not exerted through BTK but via suppression of other target kinases. 43 An alternative explanation would be that some of these targets are activated by BTK in BA and that the ibrutinibinduced effects on these additional targets were (in part) mediated through BTK inhibition. It is generally appreciated that ibrutinib is not recognizing SYK. Correspondingly, ibrutinib did not block phosphorylation of SYK in BA in our study. By contrast, the SYK inhibitor P505-15 was found to block phosphorylation of SYK in BA and to suppress histamine release, confirming our previous results. 44 As BTK is downstream of SYK, we also examined the effects of P505-15 on BTK activation. Indeed, as expected, P505-15 was found to suppress BTK activation in BA. Whether P505-15 blocks histamine secretion through BTK disruption or also in a BTK-independent manner remains unknown. We also applied other BTK inhibitors in order to confirm the role of BTK in IgE-dependent activation and histamine secretion. All BTK blockers examined, including dasatinib, were found to suppress IgE-dependent histamine secretion in BA.
With regard to dasatinib, these results confirm our previous data. 38,42 However, of all drugs tested, the most potent blocker of allergen-induced histamine secretion in BA appears to be ibrutinib.
Recent data suggest that ibrutinib inhibits IgE-dependent upregulation of CD63 and CD203c on normal BA. 32 In the current study, we confirmed this drug effect. In addition, we were able to show that ibrutinib blocks allergen-induced upregulation of CD63 and CD203c in BA obtained from patients allergic to Der p 2 and/or Phl p5, with comparable IC 50 values. In addition, we found that ibrutinib inhibits IgE-dependent upregulation of CD13 and CD164 on BA.
Unexpectedly, however, the other BTK inhibitors tested did not counteract upregulation of CD13, CD63, CD164, or CD203c on BA.
From these data, one could speculate that the effects of ibrutinib on IgE-dependent upregulation of these activation antigens were mediated by other (additional) drug targets in BA. Alternatively, the inhibitory effects of the other drugs on BTK activation were too weak to result in downregulation of these CD molecules.
The severity of an allergic reaction may not only depend on the type of allergen, IgE levels, cytokine exposure, and the microenvironment but also on the total number of effector cells involved in the reaction. [12][13][14] Conditions with high numbers of BA and MC include chronic inflammation, certain neoplastic states, and specific neoplasms, including basophilic leukemias and mastocytosis. In these patients, BA and MC progenitors have a significant proliferative potential. 3,12,13 We were therefore interested to learn whether ibrutinib and/or the other drugs tested would exert effects on growth of human BA and MC. To address this point, human BA and MC lines were employed. We found that dasatinib and CNX-774 inhibit growth of HMC-1.1 cells and KU812 cells at relatively low drug concentrations. In HMC-1.2 cells, both drugs were also effective, but the concentrations required to block cell growth were rather high.
This phenomenon may be explained by the fact that HMC-1. In summary, our data show that ibrutinib is a most potent inhibitor of allergen-induced (IgE-dependent) activation and histamine release in human BA and MC. The effects of the drug were seen in normal BA as well as in allergen-exposed BA obtained from patients allergic to Der p 2 or Phl p 5, with meaningful IC 50 values. Moreover, we found that other BTK blockers also counteract mediator contributed patient material and parts of the study design; and P.V. contributed the study conception and design, patient material, and wrote parts of the manuscript.

CONFLI CT OF INTEREST
The authors declare that they have no conflict of interest in the current study. Conflict of interest unrelated to the present study: U.J.  HMC-1 cells and KU812 cells were incubated in increasing concentrations of drugs (range for tested compounds: 0.001-10 lmol/L; range for dasatinib on KU812 cells: 0.001-10 nmol/L) at 37°C for 48 hours. Proliferation was measured by analyzing 3 H-thymidine uptake. Results are expressed as inhibitory concentration producing 50% inhibition (IC 50 ).