Effects of ibrutinib on proliferation and histamine release in canine neoplastic mast cells

Abstract The Bruton's tyrosine kinase (BTK) inhibitor ibrutinib is effective in the treatment of human chronic lymphocytic leukaemia and mantle cell lymphoma. Recent data have shown that ibrutinib also blocks IgE‐dependent activation and histamine release in human basophils (BAs) and mast cells (MCs). The aim of this study was to investigate whether BTK serves as a novel therapeutic target in canine mast cell tumours (MCTs). We evaluated the effects of ibrutinib on two canine MC lines, C2 and NI‐1 and on primary MCs obtained from canine MCTs (n = 3). Using flow cytometry, we found that ibrutinib suppresses phosphorylation of BTK and of downstream STAT5 in both MC lines. In addition, ibrutinib decreased proliferation of neoplastic MCs, with IC50 values ranging between 0.1 and 1 μM in primary MCT cells and between 1 and 3 μM in C2 and NI‐1 cells. In C2 cells, the combination “ibrutinib + midostaurin” produced synergistic growth‐inhibitory effects. At higher concentrations, ibrutinib also induced apoptosis in both MC lines. Finally, ibrutinib was found to suppress IgE‐dependent histamine release in primary MCT cells, with IC50 values ranging from 0.05 to 0.1 μM in NI‐1 cells, and from 0.05 to 1 μM in primary MCT cells. In summary, ibrutinib exerts anti‐proliferative effects in canine neoplastic MCs and counteracts IgE‐dependent histamine release in these cells. Based on our data, ibrutinib may be considered as a novel therapeutic agent for the treatment of canine MCT. The value of BTK inhibition in canine MCT patients remains to be elucidated in clinical trials.

possible or if a tumour recurs, radio-and/or chemotherapy are usually offered. 5,[10][11][12][13][14] Recently, masitinib and toceranib, two targeted drugs directed against proto-oncogene, receptor tyrosine kinase (KIT), have been approved for therapy of non-resectable or metastasized highgrade MCT. 15, 16 These drugs have been shown to be effective in several MCT patients. [15][16][17] Therefore, current efforts focus on the identification and preclinical development of novel targeted drugs that can counteract growth and activation of canine MCT cells.
In humans, the Bruton's tyrosine kinase (BTK) inhibitor ibrutinib is a promising drug that has recently received the Food and Drug Administration approval for the treatment of chronic lymphocytic leukaemia and mantle cell lymphoma. [18][19][20] In addition, BTK has been reported to be a critical signalling molecule mediating growth and survival of various tumour cells, including neuroblastoma cells and neoplastic human MCs. 21,22 Furthermore, BTK has been described to play an important role as a target in the downstream pathway of IgE receptor-cross-linked human basophils (BAs) and MCs. [23][24][25][26] More recently, studies published by our and other groups have identified ibrutinib as a potent blocker of IgE-dependent activation and histamine release in human BAs. 26,27 However, only few reports exist about the in vitro and in vivo effects of ibrutinib in dogs. Recently, it has been described that ibrutinib exerts anti-tumour effects in naturally occurring B cell non-Hodgkin-lymphoma in canine patients. 28 Based on these data, ibrutinib may be an interesting agent to test in comparative oncology contexts. We were interested to examine the effect of ibrutinib on canine neoplastic MCs. The specific aims of our study were to examine whether ibrutinib may serve as a potential new drug for treatment of canine MCT and whether ibrutinib is able to suppress histamine release in neoplastic MCs.

| Western blot analysis of BTK and STAT5 expression in MC lines
Western blot experiments were performed using C2 and NI-1 cells essentially as reported. 33 A detailed description of the technique is provided in the Method Section of the Supplement.  To evaluate potential cooperative drug effects, cells were exposed to ibrutinib, midostaurin, toceranib, masitinib (as single agent) or to drug combinations, namely "ibrutinib + midostaurin", "ibrutinib + toceranib", and "ibrutinib + masitinib," at a fixed ratio of drug concentrations. After incubation, 0.5 μCi of 3 Hthymidine was added (37 C, 16 hours). Thereafter, cells were harvested on filter membranes (Packard Bioscience, Meriden, Connecticut) in a Filtermate 196 harvester (Packard Bioscience). Filters were air-dried and the bound radioactivity was measured in a β-counter (TopCount NXT, Packard Bioscience). All experiments were performed in triplicates.

| Statistical analysis
Data were presented as mean values from at least three independent experiments with standard deviation (SD). To determine the significance in differences seen between drug-exposed and untreated cells, the Student's t test for independent samples was applied. Results were considered statistically significant when P was <0.05. Effects of drug combinations were determined using Calcusyn software (Biosoft, Ferguson, Missouri) and expressed as combination index (CI) values.
Drug effects were considered to be synergistic when the CI was <1.

| Effects of ibrutinib on pBTK and pSTAT5 expression in canine neoplastic MCs
To test the effects of ibrutinib on BTK activation and downstream STAT5 activation, we examined the phosphorylation status of BTK and STAT5 using flow cytometry in drug-exposed cells. We found that C2 cells and NI-1 cells constitutively express pBTK and pSTAT5

| Effects of ibrutinib on proliferation of canine neoplastic MCs
As determined by 3 H-thymidine uptake, ibrutinib was found to decrease proliferation of C2 cells and NI-1 cells with IC 50 values ranging between 1 and 3 μM (Figure 2A, between 0.1 and 1 μM ( Figure 2B, Table 2). Synergistic effects between ibrutinib and midostaurin on proliferation were seen in C2 cells, but not in NI-1 cells ( Figure 2C). The drug combinations "ibrutinib + masitinib" and "ibrutinib + toceranib" induced some cooperative growth-inhibitory effects in C2 and NI-1 cells, although synergistic interactions were not seen ( Figure S3).

| Effects of ibrutinib on survival of C2 cells and NI-1 cells
In a next step, we examined whether ibrutinib induces apoptosis in

| Effects of ibrutinib on IgE-dependent histamine release in NI-1 cells and primary mastocytoma cells
Ibrutinib has recently been shown to counteract IgE-dependent activation of human BAs. 26,27 To examine whether ibrutinib exerts inhibitory effects on IgE-dependent histamine release in canine neoplastic MCs, we performed experiments using NI-1 cells, known to express a

| DISCUSSION
Aggressive canine MCT is a disease with an unmet therapeutic need. [2][3][4][5] In human and canine MC neoplasms, mutated KIT can induce growth-factor independent activation and uncontrolled proliferation of neoplastic MCs. 2 BTK has also been described to be a downstream molecule in the KIT-signalling pathway, known to be critical for MC growth, proliferation and survival. 43 We were able to show that ibrutinib counteracts proliferation of C2 and NI-1 cells with IC 50  Neoplastic MCs harbouring KIT-mutations may gain resistance against TKIs; therefore, current research in canine and human MC neoplasms focuses on drug combinations to overcome this problem. 33,50 In the current study, we were interested to examine cooperative effects between ibrutinib and KIT-targeting drugs. Midostaurin is a KIT-targeting drug that is well known to suppress the growth of human KIT D816V+ MCs in vitro and in vivo. [50][51][52] We found that the drug combination "ibrutinib + midostaurin" exerts synergistic growthinhibitory effects in C2 cells (CI < 1), but not in NI-1 cells. A possible explanation for the weaker effect of the drug combination "ibrutinib + midostaurin" in NI-1 cells may be the presence of several different mutations in KIT. Notably, whereas C2 cells contain only one KIT mutation, NI-1 cells are known to display six different KIT mutations, which may result in a faster proliferation of NI-1 cells and possibly also in a more resistant phenotype. 30 We also tested the effects of other drug combinations in C2 and NI-1 cells. Toceranib and masitinib are two KIT-targeting drugs that are used to treat canine MCT patients in daily practice. We found that the drug combinations "ibrutinib + toceranib" and "ibrutinib + masitinib" exert cooperative growth-inhibitory effects in both MC lines. However, synergistic growth-inhibitory effects were not seen.
In patients suffering from MC neoplasms, clinical symptoms may also arise from effects of MC-derived mediators. 4,7 In the current study, we tested the effects of ibrutinib on IgE-dependent histamine release in NI-1 cells and primary MCT cells. C2 cells could not be employed in these experiments because they do not express a functionally active IgE receptor. 53 The IC 50 values obtained with ibrutinib in NI-1 cells as well as in primary mastocytoma cells were found to be within a "pharmacological" range (0.05-1 μM). Since concentrations required for blocking histamine release were significantly lower than for blocking proliferation and survival, ibrutinib may also have clinical implications for other IgE-dependent diseases in dogs, such as canine atopic dermatitis or food allergies. [54][55][56][57] Our findings are reinforced by previous data showing that BTK is expressed in human BAs and MCs and that BTK inhibition by ibrutinib is associated with reduced IgEdependent mediator release in these cells. [23][24][25][26][27] In summary, our data show that ibrutinib exerts anti-proliferative and survival-reducing effects in canine neoplastic MCs. Moreover, we found that BTK inhibition by ibrutinib resulted in a concentrationdependent suppression of IgE-dependent histamine release in NI-1 cells and primary MCT cells. These observations suggest that targeting BTK may represent a potential therapeutic approach in canine MCT.
The clinical impact of this concept remains to be elucidated.
of the manuscript. All authors have read and approved the final