Growth-inhibitory effects of four tyrosine kinase inhibitors on neoplastic feline mast cells exhibiting a Kit exon 8 ITD mutation

Abstract

Systemic mastocytosis (SM) in felines is a rare neoplasm defined by increased growth and accumulation of immature mast cells (MC) in various organs including the spleen. Although in many cases splenectomy is an effective approach, relapses may occur. In these patients, treatment options are limited. Recent data suggest that various Kit tyrosine kinase inhibitors (TKI) interfere with growth of neoplastic MC in humans. In the current study, we examined the effects of four TKI, imatinib, midostaurin, nilotinib, and dasatinib, on growth of spleen-derived feline neoplastic MC in three SM patients. Expression of Kit in neoplastic MC was confirmed by flow cytometry and/or Western blotting. In all three cases, a 12-bp internal tandem duplication in exon 8, resulting in a four amino acid-insertion between residues Thr418 and His419 in Kit, was detectable. As assessed by (3)H-thymidine incorporation experiments, all four TKI were found to inhibit the growth of feline neoplastic MC in a dose-dependent manner. The growth-inhibitory TKI effects were found to be associated with morphologic signs of apoptosis in MC. In conclusion, various Kit-targeting TKI can inhibit the in vitro growth and survival of feline neoplastic MC in SM.

Fig. 1

Expression of Kit/CD117 in feline neoplastic mast cells. (A) In two patients with systemic mastocytosis (left image: feline patient #1 and right image: feline patient #2 in Table 1), primary neoplastic mast cells were stained with a PE-labeled anti-Kit antibody (grey graph) or an IgG1 isotype control antibody (open graph). Expression of Kit was analyzed by flow cytometry on a FACScan. (B) Immunocytochemical detection of Kit in neoplastic feline mast cells. A cytospin preparation of primary neoplastic mast cells prepared in feline patient #3 was stained with a polyclonal anti-Kit antibody as described in the text. As visible, neoplastic mast cells were found to stain positive for Kit.

Fig. 2

Kit mutation analysis in feline neoplastic mast cells. Genomic DNA from feline neoplastic mast cells was extracted, amplified using primers specific for a Kit exon 8 internal tandem duplication (ITD), purified, cloned into plasmids, sequenced, and analyzed by PCR (A) as well as by chromatography (B). (A) Shows ethidium bromide-stained agarose gel electrophoresis results from amplification products from the three feline mastocytoma patients examined (lanes #1, #2, and #3) as well as from a normal cat genomic DNA that served as control (lane #4). The identity of the products was all confirmed by sequencing. A chromatogram of the Kit ITD (obtained in patient #1) compared to wild-type Kit is shown in (B).

Fig. 3

TKI inhibit growth and survival of feline neoplastic mast cells. (A) Feline neoplastic mast cells obtained from patient #2 were cultured in control medium (Co) or in the presence of various concentrations of dasatinib, imatinib, midostaurin, and nilotinib (as indicated) at 37 °C for 48 h. Then, cells were examined for ³H-thymidine uptake. Results show the percentage of ³H-thymidine uptake compared to medium control (=100%) and represent the mean ± SD of triplicates. (B) TKI-induced apoptosis in feline neoplastic mast cells. Neoplastic mast cells isolated from patient #2 (upper panel) and patient #3 (lower panel) were cultured in control medium (control) or with various TKI (dasatinib, imatinib, nilotinib, and midostaurin) at 0.5 μM (black bars) or 1 μM (open bars) for 24 h (left graph) or 48 h (right graph). Then, the numbers (percentage) of apoptotic cells were assessed by light microscopy. (C) Wright–Giemsa-stained mast cells in patient #2 after incubation in control medium (left panel) or in dasatinib at 1 μM for 24 h (right panel). As visible dasatinib induced apoptosis in neoplastic feline mast cells. (D) TKI-induced dephosphorylation of Kit in feline neoplastic mast cells. Cells isolated from primary neoplastic mast cells were incubated with the tyrosine kinase inhibitors imatinib, nilotinib, midostaurin, and dasatinib (each 1 μM) at 37 °C for 4 h. Then, the expression of phosphorylated Kit (pKit) and of total Kit was analyzed by Western blotting and immunopreciptiation (IP) as described in the text.

Fig. 3

TKI inhibit growth and survival of feline neoplastic mast cells. (A) Feline neoplastic mast cells obtained from patient #2 were cultured in control medium (Co) or in the presence of various concentrations of dasatinib, imatinib, midostaurin, and nilotinib (as indicated) at 37 °C for 48 h. Then, cells were examined for ³H-thymidine uptake. Results show the percentage of ³H-thymidine uptake compared to medium control (=100%) and represent the mean ± SD of triplicates. (B) TKI-induced apoptosis in feline neoplastic mast cells. Neoplastic mast cells isolated from patient #2 (upper panel) and patient #3 (lower panel) were cultured in control medium (control) or with various TKI (dasatinib, imatinib, nilotinib, and midostaurin) at 0.5 μM (black bars) or 1 μM (open bars) for 24 h (left graph) or 48 h (right graph). Then, the numbers (percentage) of apoptotic cells were assessed by light microscopy. (C) Wright–Giemsa-stained mast cells in patient #2 after incubation in control medium (left panel) or in dasatinib at 1 μM for 24 h (right panel). As visible dasatinib induced apoptosis in neoplastic feline mast cells. (D) TKI-induced dephosphorylation of Kit in feline neoplastic mast cells. Cells isolated from primary neoplastic mast cells were incubated with the tyrosine kinase inhibitors imatinib, nilotinib, midostaurin, and dasatinib (each 1 μM) at 37 °C for 4 h. Then, the expression of phosphorylated Kit (pKit) and of total Kit was analyzed by Western blotting and immunopreciptiation (IP) as described in the text.