Effect of Tyrosine Kinase Inhibitor INNO-406 on Human Mast Cells Bearing Mutated c-KIT Tyrosine Kinase.

Systemic mastocytosis (SM) is characterized by a clonal accumulation of mast cells (MC) in bone marrow and other tissues. Malignant MC have abnormal spindle shape, express aberrant surface markers, and carry, in most cases, a mutation involving codon 816 of the c-KIT gene (D816V). This mutation results in a constitutively activated c-kit receptor associated tyrosine kinase and is responsible for disease progression. Agents that affect mutated c-kit may have clinical benefit in SM, but there is none clinically available. INNO-406 is a novel multi-targeted tyrosine kinase inhibitor, previously shown to inhibit the growth of cells expressing c-kit by inhibiting its phosphorylation (Kimura, Blood, 106:3948–54, 2005). We investigated the effects of INNO-406 on mast cells with mutated c-KIT, and compared it to that of imatinib mesylate, tyrosine kinase inhibitor effective against selected c-KIT mutants. HMC-1⁵⁶⁰ cells carrying juxtamembrane domain c-KIT mutation in codon 560, and HMC-1^{560, 816} cells carrying both codon 560 mutation and tyrosine kinase domain c-KIT mutation in codon 816, were exposed to increasing concentrations (0.02 to 5 μM) of INNO-406 or imatinib in 72-hour cell proliferation assay. Both INNO-406 and imatinib effectively inhibited the growth of HMC-1⁵⁶⁰ cells, with IC⁵⁰ of 45 and 75nM, respectively. Neither drug was effective against HMC-1^{560, 816} cells at the doses tested. INNO-406 effectively induced apoptosis in HMC-1⁵⁶⁰, but not in HMC-1^{560, 816} cells, as judged by the Annexin V flow cytometry test and by PARP specific cleavage seen on western blotting. In addition, INNO-406 was effective in inhibiting phosphorylation of c-kit in HMC-1⁵⁶⁰ cells in nM range. Our results suggest similar potency of INNO-406 and imatinib in mast cells with mutated codon 560 (found in gastrointestinal stromal tumor), but no activity against cells with mutated codon 816 c-KIT (found in SM).