title: Development of an in vitro model of acquired resistance to toceranib phosphate (Palladia®) in canine mast cell tumor.
abstract: BACKGROUND
  Mast cell tumors (MCTs) are the most common skin tumors in dogs and exhibit variable biologic behavior. Mutations in the c-kit proto-oncogene are associated with the tumorigenesis of MCTs, resulting in growth factor-independent and constitutive phosphorylation of the KIT receptor tyrosine kinase (RTK). Toceranib (TOC) phosphate (Palladia®) is a KIT RTK inhibitor that has biological activity against MCTs. Despite these benefits, patients ultimately develop resistance to TOC. Therefore, there is a need to identify distinguishing clinical and molecular features of resistance in this population.
  
  
  RESULTS
  The canine C2 mastocytoma cell line contains an activating mutation in c-kit. Three TOC-resistant C2 sublines (TR1, TR2, TR3) were established over seven months by growing cells in increasing concentrations of TOC. TOC inhibited KIT phosphorylation and cell proliferation in a dose-dependent manner in the treatment-naïve, parental C2 line (IC50 < 10 nM). In contrast, the three sublines were resistant to growth inhibition by TOC (IC50 > 1,000 nM) and phosphorylation of the KIT receptor was less inhibited compared to the TOC-sensitive C2 cells. Interestingly, sensitivity to three structurally distinct KIT RTK inhibitors was variable among the sublines, and all 3 sublines retained sensitivity to the cytotoxic agents vinblastine and lomustine. Sequencing of c-kit revealed secondary mutations in the juxtamembrane and tyrosine kinase domains of the resistant sublines. These included point mutations in TR1 (Q574R, M835T), TR2 (K724R), and TR3 (K580R, R584G, A620S). Additionally, chronic TOC exposure resulted in c-kit mRNA and KIT protein overexpression in the TOC-resistant sublines compared to the parental line. C2, TR1, TR2, and TR3 cells demonstrated minimal P-glycoprotein (P-gp) activity and no functional P-gp.
  
  
  CONCLUSIONS
  This study demonstrates the development of an in vitro model of acquired resistance to targeted therapy in canine MCTs harboring a c-kit-activating mutation. This model may be used to investigate the molecular basis of and strategies to overcome TOC resistance.
authors.affiliation: Program in Cell and Molecular Biology, Colorado State University, Fort Collins, CO, USA. charles.halsey@nih.gov.
authors.forename: Charles H C
authors.initials: CH
authors.lastname: Halsey
authors.forename: Daniel L
authors.initials: DL
authors.lastname: Gustafson
authors.forename: Barbara J
authors.initials: BJ
authors.lastname: Rose
authors.forename: Amber
authors.initials: A
authors.lastname: Wolf-Ringwall
authors.forename: Robert C
authors.initials: RC
authors.lastname: Burnett
authors.forename: Dawn L
authors.initials: DL
authors.lastname: Duval
authors.forename: Anne C
authors.initials: AC
authors.lastname: Avery
authors.forename: Douglas H
authors.initials: DH
authors.lastname: Thamm
mesh_terms: D018435:ATP Binding Cassette Transporter, Subfamily B / Q000378:metabolism; D000818:Animals; D000970:Antineoplastic Agents / Q000494:pharmacology*; D017209:Apoptosis / Q000187:drug effects; D045744:Cell Line, Tumor; D004285:Dogs; D007211:Indoles / Q000494:pharmacology*; D034801:Mastocytoma / Q000188:drug therapy*; D017354:Point Mutation; D019009:Proto-Oncogene Proteins c-kit / Q000235:genetics / Q000378:metabolism; D011758:Pyrroles / Q000494:pharmacology*; D012333:RNA, Messenger / Q000378:metabolism; D014747:Vinblastine / Q000494:pharmacology
chemical_list: D018435:ATP Binding Cassette Transporter, Subfamily B; D000970:Antineoplastic Agents; D007211:Indoles; D011758:Pyrroles; D012333:RNA, Messenger; C464577:toceranib phosphate; D014747:Vinblastine; D019009:Proto-Oncogene Proteins c-kit