Abstract A56: Canine Mast Cell Tumor and Toceranib Phosphate (Palladia™): Development of a Spontaneous Canine Model of Kinase Inhibitor Resistance

Mast cell tumors (MCTs) are one of the most common skin tumors in dogs, accounting for up to 21% of all canine cutaneous tumors, and exhibit extremely variable biologic behavior. Mutations in the juxtamembrane, kinase and ligand binding domains of the c-kit proto-oncogene have been associated with the tumorigenesis of canine MCTs, resulting in growth factor-independent and constitutive phosphorylation of the Kit receptor tyrosine kinase (RTK). Approximately one-third of canine MCTs carry a c-kit mutation. As such, small molecule inhibitors of Kit are an attractive therapeutic strategy for MCTs in dogs. Toceranib (TOC) phosphate (Palladia) is one such RTK inhibitor of Kit that has biological activity against canine MCTs. Approximately 40% of dogs experience an objective response to TOC while the remaining 60% have intrinsic resistance. Two-thirds of the responders are positive for an activating mutation in c-kit . The time to tumor progression in all responders is 18 weeks; therefore, essentially all dogs with MCTs either have intrinsic TOC resistance or eventually develop resistance. Therefore, there is a need to identify distinctive clinical and molecular features of resistance in this population. The canine C2 mastocytoma cell line, established from a spontaneously occurring MCT, contains the previously described c-kit internal tandem duplication in the juxtamembrane domain involving exon 11. TOC-resistant C2 cells were selected by growing cells in concentrations of TOC ranging from 0.02 uM to 0.3 uM and increasing in 0.025–0.05 uM increments. Three resistant sublines were established over a period of 7 months. All three resistant sublines as well as the original naive C2 cells were grown in the presence of increasing concentrations of TOC, 3 other KIT kinase inhibitors, or the cytotoxic agents vinblastine (VBL) or lomustine (CCNU) for 72 hours, followed by determination of relative viable cell number using a fluorometric bioreductive assay (Alamar Blue). To evaluate drug effects on Kit autophosphorylation, parental C2 cells and the resistant sublines were incubated for 24 hours with increasing concentrations of TOC (0 nM–100 nM) and phosphorylated and total Kit were analyzed by western blot. Western analysis of the C2 line demonstrated constitutive tyrosine phosphorylation of Kit in the absence of exogenous ligand. TOC inhibited Kit phosphorylation in a dose-dependent manner. Proliferation of the naive C2 cells was inhibited by TOC in a dose-dependent manner with an IC50 of 1,000 nM). Interestingly, sensitivity to the other 3 Kit RTK inhibitors was variable between the sublines, and all 3 sublines retained sensitivity to the cytotoxic agents VBL and CCNU. To determine whether this lack of growth inhibition was due to a lack of inhibition of autophosphorylation by TOC, the resistant sublines were incubated with increasing concentrations of TOC for 24 hours and western analysis for phophorylated and total Kit was performed. Phosphorylation of the Kit receptor was less inhibited in all three resistant sublines compared to the TOC-sensitive C2 cells, suggesting that acquisition of secondary mutations in the c-kit gene is likely responsible for the observed TOC resistance. Canine MCTs and their intrinsic or acquired resistance to the TKI TOC (Palladia) demonstrate an excellent in vivo model of a spontaneous, Kit driven neoplastic disease. This study demonstrates the development of a canine model to study drug-resistant tumor clones with a c-kit activating mutation. This model may be better utilized to study the molecular basis of and strategies to overcome drug resistance.