Establishment of a Novel Canine Mastocytoma Cell Line, NI-1: a Model for Studying Resistance Against KIT Tyrosine Kinase Inhibitors In Canine Neoplastic Mast Cells

Abstract 4936

Advanced mast cell (MC) disorders are characterized by uncontrolled growth of neoplastic MC in various organ systems, resistance to conventional cytoreductive drugs, and a poor prognosis. In most patients, transforming mutations in the KIT proto-oncogene are detectable and are considered to contribute to resistance. MC lines are an important model for analyzing drug resistance in neoplastic MC. We have established a novel canine mastocytoma cell line, NI-1 from a canine patient suffering from mast cell leukemia. NI-1 cells were found to harbour several homozygous KIT mutations including two single nucleotide mutations, one at nucleotide 107 (C to T, leading to a missense mutation, P36L) and one at nucleotide 1187 (A to G, leading to a missense mutation, Q396R), a 12 bp duplication at nucleotide 1263, and a 12 bp deletion at nucleotide 1550, the latter reflecting a transcriptional variant. NI-1 cells contained histamine and formed tumors in NOD-SCID IL-2Rgamma^null (NSG) mice. As assessed by ³H-thymidine uptake, a number of targeted drugs were found to inhibit the proliferation of NI-1 cells at pharmacologically relevant concentrations. Among these drugs were the KIT kinase blockers midostaurin (IC₅₀ <0.1 μM), dasatinib (IC₅₀ <0.1 μM), sunitinib (IC₅₀ <0.1 μM), tozasertib (0.1-0.5 μM), imatinib (IC₅₀ 0.25–0.37 μM), sorafenib (IC₅₀ 0.25–0.5 μM), masatinib (0.1-1.0 μM) as well as drugs targeting important KIT-downstream signaling molecules (PI3 kinase, mTOR), i.e. RAD001 (IC₅₀ <0.1 μM) and NVP-BEZ235 (IC₅₀ 0.01–0.05 μM). In most instances, drug-induced growth inhibition was found to be accompanied by apoptosis. No clear effects were seen with drugs that are not capable of blocking KIT or KIT-downstream kinases in MC, i.e. bosutinib, erlotinib, gefitinib, and lapatinib (IC₅₀ >2 μM). However, the HDAC inhibitor vorinostat was found to block growth of NI-1 cells (0.1-0.5 μM). Drug response profiling also revealed that NI-1 cells differ markedly from canine C2 mastocytoma cells harbouring an exon 11 KIT mutation, and the human mast cell lines HMC-1.1 (exon 11 mutation in KIT) and HMC-1.2 (exon 11 and exon 17 KIT mutations). In contrast to C2 cells, NI-1 cells were found to be largely resistant against the growth-inhibitory effects of masatinib, sorafenib, and sunitinib. By contrast, NI-1 cells were found to be even more sensitive against the growth-inhibitory effects of the mTOR blocker RAD001 and the PI3-kinase/mTOR inhibitor NVP-BEZ235. Together, our data suggest that certain KIT mutations may be associated with relative resistance of neoplastic MC against KIT-targeting drugs, a phenomenon that has also been described for human MC and the KIT mutant D816V that mediates resistance against masatinib and imatinib. The novel MC line NI-1 may serve as a novel tool to investigate the mechanisms of resistance against TKI in neoplastic MC.