Markers and mechanisms of resistance to toceranib phosphate (PalladiaRTM) in canine cutaneous mast cell tumor

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). Toceranib (TOC) phosphate (PalladiaRTM) is one such RTK inhibitor of KIT that has biological activity against canine MCTs. The overarching hypothesis of this dissertation is that understanding the mechanisms of TOC-resistance in canine MCT will allow us to develop rational second line and combination therapies that will overcome or prevent drug resistance.;In order to begin to study the mechanisms that confer resistant to TOC in canine MCT, TOC-resistant MCT sublines were generated from the c-kit-mutant canine C2 mastocytoma cell line. By chronically exposing C2 cells to TOC, three TOC-resistant (TR) sublines were established over a period of seven months and designated TR1, TR2, and TR3. While TOC inhibited KIT phosphorylation and cell proliferation in a dose-dependent manner in the treatment-naive, parental C2 line (IC50 1,000 nM) and phosphorylation of the KIT receptor was less inhibited compared to the TOC-sensitive C2 cells. Additionally, sensitivity to three structurally distinct KIT RTK inhibitors (imatinib, masitinib, and LY2457546) was variable among the sublines. All 3 sublines retained sensitivity to the cytotoxic agents vinblastine and lomustine. Through sequencing efforts of canine ¬c-kit, secondary point mutations in the juxtamembrane and tyrosine kinase domains of the resistant sublines were identified.;To explore the impact of these mutations on the TOC-resistant phenotype, we constructed four in silico homology models of the cytoplasmic region of TOC-sensitive and -resistant canine KIT to predict the consequent structures of the drug binding site. Utilizing computational-based small molecule docking techniques, we calculated the predicted binding energies and orientations of TOC and the three other KIT inhibitors within the KIT mutant homology models to determine the structural basis of TOC resistance in vitro in the context of canine MCT. Each of the three TOC-resistant mutants was predicted to induce a conformational change in the region of the binding site to a greater or lesser degree. The TR1 mutation, however, was predicted to have only minor effects on the binding of masitinib and imatinib while both TR2 and TR3 mutations induced a substantial decrease in predicted binding affinity. To evaluate the utility of the in silico homology model and small molecule docking methodologies in predicting response to novel KIT inhibitors, we docked ponatinib into the intracellular domain of the TOC-sensitive and each of the three TOC-resistant KIT mutant protein structures, followed by binding energy calculations. Ponatinib was predicted to bind favorably to TOC-sensitive KIT, but exhibited a substantial decrease in the favorability of the predicted binding to each of the three TOC-resistant mutants. In concordance with the predicted binding energies, ponatinib inhibited the growth of the TOC-sensitive C2 cells in a dose-dependent manner and failed to inhibit growth of the TOC-resistant cells.;Lastly, we developed an immunohistochemical-based assay to directly measure activated (phosphorylated) KIT (pKIT) in canine MCT. This assay was used to investigate whether pKIT provides a pharmacodynamic marker for monitoring response to TOC in canine MCTs in order to potentially identify patients that respond to TOC and those that are refractory and therefore might benefit from an alternative treatment. MCTs from 4/7 (57.1%) patients demonstrated a partial response to TOC therapy, 2/7 (28.6%) patients showed stable disease, and one patient demonstrated progressive disease. Of the four patients that had a partial response, 3/4 (75%) demonstrated a reduction in pKIT 6 hours after the first dose of TOC. The utility of measuring pKIT in MCT as a predictor of biological aggressiveness was determined retrospectively in a set of MCTs in order to investigate its association with two commonly used prognostic grading systems as well as other established prognostic markers (KIT localization, Ki67 expression, mitotic index, and c-kit mutation status) for MCT. Expression of phosphorylated KIT was significantly (p<0.05) correlated with mitotic index, Ki67, c-kit mutation status, and grade by the 2-tier grading system. (Abstract shortened by UMI.).