Differential sensitivity of RT, EST, and differently substituted codon 816 mutant KIT to KIT inhibitors.

Antiphosphotyrosine blots of immunoprecipitated KIT expressed in COS cells show a low level of spontaneous phosphorylation of wild-type (WT) KIT (lane 1), which increases in response to SCF stimulation (lane 2). Both inhibitors at 0.1 to 1 μM prevent this ligand-induced phosphorylation (lanes 3-4). RT mutant KIT with Val560Gly substitution shows a high level of SCF-independent phosphorylation (lane 5), and the phosphorylation is inhibited by both inhibitors at physiologically achievable (0.1-1 μM) concentrations (lanes 6-7). The EST Asp816Val mutation commonly found in human mastocytosis also shows high spontaneous phosphorylation (lane 8) but is resistant to inhibition by either KIT inhibitor at 1 to 10 μM (lanes 9-10). Substitution of phenylalanine or tyrosine for aspartate 816, rarely found in human mastocytosis, also results in high spontaneous phosphorylation (lanes 11, 14), but these 2 mutant variants respond to the inhibitors at 1 to 10 μM (lanes 12-13, 15-16) unlike Asp816Val KIT. However, they are still an order of magnitude less sensitive than are RT mutant or wild-type KIT and are not valid therapeutic targets with currently available drugs.