The transcription factor STAT5 has been implicated in the survival of human neoplastic mast cells (MC). with autonomous growth of MC.10C13 Standard treatment in MCT is surgery with wide excision margins for resectable tumors, chemo- or radiotherapy for non-resectable cases or a combined treatment for residual or locally recurrent MCT.6,14 Recently, 2 tyrosine kinase inhibitors (TKI) directed against KIT, namely masitinib and toceranib, have been approved for the treatment of mutations in canine patients suffering from PV.25 We have recently described that activated STAT5 is constitutively expressed in human MC-Val-Cit-PAB-Auristatin E neoplastic MC and triggers the proliferation and survival of these cells.26 Together, JAK2 and STAT5 are considered to be crucial mediators of growth and survival Argireline Acetate of neoplastic cells and therefore potential therapeutic targets in myeloid neoplasms.27,28 However, JAK2 and STAT5 have not been investigated in the context of canine MC neoplasms so far. The aims of this study were to examine the expression and activation of JAK2 and STAT5 in canine MCT and to explore the anti-neoplastic effects of established inhibitors of the JAK2/STAT5 pathway in these cells. For this purpose, 2 established canine MC lines, C2 and NI-1 were used both of which carry several mutations in was .05. Drug combination effects on apoptosis were evaluated by CompuSyn and considered to be synergistic when the combination index (CI) was 1, additive when CI = 1 and antagonistic when CI 1. 3.?Results 3.1. Canine neoplastic MC exhibit activated JAK2, STAT5 and KIT As determined by immunocytochemistry, C2 cells and NI-1 cells were found to express JAK2, pJAK2, pSTAT5, KIT and pKIT (Figure 1A). The presence of intracellular pSTAT5 and STAT5 MC-Val-Cit-PAB-Auristatin E as well as surface KIT in C2 and NI-1 cells was also demonstrable using flow cytometry (Figure 1B). In these experiments, higher levels of pSTAT5 were detected in C2 cells compared with NI-1 cells whereas STAT5- and KIT levels were comparable in MC-Val-Cit-PAB-Auristatin E the 2 2 cell lines. Furthermore, we were able to demonstrate the expression of pSTAT5 in primary MCT by IHC (Table 3, Figure 1C). In particular, pSTAT5 was detected in neoplastic MC in 9 of 9 canine patients examined. Open in a separate window Figure 1 Expression of JAK2, STAT5 and KIT in canine neoplastic mast cells (MC). A, C2 cells (left panel) and NI-1 cells (right panel) were stained with antibodies for JAK2, pJAK2, pSTAT5, KIT or pKIT using indirect immunocytochemistry as described in the text. B, Levels of pSTAT5, STAT5 and KIT in C2 and NI-1 cells determined using flow cytometry. Cells were incubated with an Alexa Fluor 647-conjugated anti-pSTAT5 antibody (gray histograms, upper panel), a phycoerythrin (PE)-conjugated anti-STAT5 antibody (gray histograms, middle panel) or a PE-conjugated anti-KIT antibody (gray histograms, lower panel). The isotype-matched control antibodies are also shown (open histograms). MFI, mean fluorescence intensity. C, Immunohistochemical detection of pSTAT5 in neoplastic mast cells of tumor sections obtained from canine mastocytoma patients using the monoclonal anti-pSTAT5 antibody C115C. The staining technique is described in the text. Representative examples from 3 patients are provided (grades 1, 2 and 3 according to Patnaik5, as indicated). The antibody omission control is also shown (upper left panel). 3.2. JAK2-, STAT5- and KIT-targeting drugs counteract STAT5 activation in C2 and NI-1 cells To evaluate the functional role of JAK2 and STAT5, we treated C2 and NI-1 cells with various targeted drugs. As shown in Figure 2A,B, the JAK2-targeting drugs R763, TG101348, AZD1480 and ruxolitinib (0.05-5 (M) as well as the KIT inhibitors imatinib, masitinib, midostaurin and nilotinib (0.05-5 M) were able to decrease the levels of pSTAT5 in C2 and NI-1 cells in a dose-dependent manner after 4 hours of treatment. In these experiments, C2 cells were more sensitive compared with NI-1 cells. The STAT5 blockers pimozide and piceatannol (5-50 M) showed only little effects on pSTAT5 levels in both cell lines. Using Western blot, we found that most of the JAK2-targeting drugs decrease expression of pSTAT5 whereas the STAT5 blockers only showed weak effects in C2 and NI-1 cells (Figure 2C). Open in a separate window Figure 2 Effects of targeted drugs on pSTAT5 expression in C2 and NI-1 cells. C2 (A) and NI-1 cells (B) were incubated in control medium.
The transcription factor STAT5 has been implicated in the survival of human neoplastic mast cells (MC)