AC220, a Potent Second Generation Class I/III Tyrosine Kinase Inhibitor, Displays a Distinct Inhibition Profile on Mutant-FLT3 as Well as -KIT Isoforms

Abstract 291

Activating mutations of the class III receptor tyrosine kinases FLT3 and KIT are associated with certain human neoplasms including hematologic malignancies, i.e. the majority of patients with systemic mast cell disorders (KIT) and subsets of acute myelogenous leukemia (AML) patients (FLT3 and KIT). Tyrosine kinase inhibitors (TKI) are urging into the clinics but the broad spectrum of FLT3 or KIT mutations, bioavailability issues of some inhibitors and the upcoming problem of primary or secondary TKI-therapy resistance lead to the search for novel second generation inhibitors to improve TKI potency and to overcome therapy resistance. AC220 was recently demonstrated to be a potent selective FLT3 WT and mutant-FLT3 ITD inhibitor with promising in vivo activity in a phase I study for AML patients with FLT3 ITD mutations. We now show, that AC220 also targets mutant-KIT and -FLT3 (other than ITDs) isoforms. Interestingly, depending on the mutation's location and kind of aminoacid substitution, the inhibitory activity is very distinct. Various FLT3 or KIT leukemia cell models were used to establish the antiproliferative and proapoptotic efficacy of AC220. To better compare differences inbetween the mutant isoforms, an isogenic BaF3 cell line model, transfected with different FLT3, KIT or BCR/ABL isoforms, was established. Dephosphorylation of mutant KIT or FLT3 isoforms after AC220 treatment was assessed by western immunoblotting. As expected, we confirmed the potent antiproliferative and proapoptotic effect of AC220 in cell models harboring ITD in the juxtamembrane domain of FLT3. We also show that AC220 effectively inhibits the V560G mutation in the juxtamembrane region of KIT in the low nanomolar range. No efficacy (up to 10 microM) was seen in BCR/ABL models. Of interest, mutations in the kinase domain of both FLT3 and KIT displayed very differing sensitivity towards AC220 in an isogenic BaF3 cell line model depending on the location and the kind of aminoacid substitution: While strong efficacy of AC220 (in the nanomolar range) was seen for tyrosine substitutions at codon 835 (FLT3) or the homologous codon 816 in KIT, only moderate or no proapoptotic efficacy was seen for other substituents such as valine or phenylalanine and a nearby located mutation at codon N822 up to a concentration of 10 microM. A potent antiproliferative effect though (with IC50 doses >1 microM) was still noted in these cells. Analysis of the phosphoproteome revealed KIT or FLT3 dephosphorylation in line with the cellular assays, arguing for an effect communicated via KIT or FLT3. Studies of the sterically/structurally related inhibitor sunitinib targeting the autoinhibited conformation of KIT recently revealed a sterical clash for D816V/H mutants providing a molecular explanation of sunitinib-resistance towards KIT D816V/H mutations. Similar sterical interactions influencing/preventing the binding of AC220 to KIT or FLT3 due to its structure are likely and need to be adressed in molecular-based studies. In conclusion, AC220 is a potent inhibitor of clinically relevant FLT3 and KIT isoforms – but, like sorafinib and sunitinib, AC220 shows a distinct resistance pattern towards some kinase domain 2 mutations. Mutation screening before start of treatment may be considered.