Abstract
Mutations in the extracellular portion of the KIT receptor tyrosine kinase (exon 8 mutations) are strongly associated with core binding factor (CBF) - acute myeloid leukemia (AML), but the functional role of these mutations has not been elucidated. In 93% of cases, codon Asp419 is deleted and exon 8 mutations were reported to confer an impaired prognosis to patients with CBF-AML. In this study, we are the first to report pro-proliferative and antiapoptotic potential of representative KIT exon 8 mutations in a cell culture model and to show a significant difference to KIT wildtype (KIT-WT). Three representative exon 8 mutants including a single deletion of codon 419 were created by in vitro site-directed mutagenesis. The integrity of all constructs was assessed by complete nucleotide sequencing. After stable expression in IL-3 dependent Ba/F3 cells (confirmed by FACS analysis and immunoblotting), exon 8 KIT mutants were characterized by a hypersensitivity to stem cell factor (SCF) stimulation in terms of proliferation and resistance to apoptotic cell death. The differences to KIT-WT occurred in the physiological range of SCF from 1 to 10ng/ml. The proliferative response caused by stimulation with SCF was reversed in KIT-WT and exon 8 mutants in the presence of Imatinib® (Novartis) in contrast to the activation loop mutant D816V which could not be inhibited. These biological effects were confirmed by demonstrating increased phosphorylation of the KIT downstream targets mitogen-activated protein kinase (MAPK) and AKT after SCF stimulation compared to the KIT-WT receptor. Furthermore, the MEK inhibitor PD98059 and the PI3 kinase inhibitor LY294002 resulted in a dose dependent inhibition of SCF induced proliferation in exon 8 mutants. Our data show that KIT exon 8 mutations represent gain-of-function mutations by inducing receptor hypersensitivity to its ligand SCF by activation of MAPK and PI3K and might represent a new molecular target for treatment of CBF leukemias.
Author notes
Corresponding author
