We sought to identify fibroblast growth factor receptor 2 (FGFR2) kinase domain 
mutations that confer resistance to the pan-FGFR inhibitor, dovitinib, and 
explore the mechanism of action of the drug-resistant mutations. We cultured 
BaF3 cells overexpressing FGFR2 in high concentrations of dovitinib and 
identified 14 dovitinib-resistant mutations, including the N550K mutation 
observed in 25% of FGFR2(mutant) endometrial cancers (ECs). Structural and 
biochemical in vitro kinase analyses, together with BaF3 proliferation assays, 
showed that the resistance mutations elevate the intrinsic kinase activity of 
FGFR2. BaF3 lines were used to assess the ability of each mutation to confer 
cross-resistance to PD173074 and ponatinib. Unlike PD173074, ponatinib 
effectively inhibited all the dovitinib-resistant FGFR2 mutants except the V565I 
gatekeeper mutation, suggesting ponatinib but not dovitinib targets the active 
conformation of FGFR2 kinase. EC cell lines expressing wild-type FGFR2 were 
relatively resistant to all inhibitors, whereas EC cell lines expressing mutated 
FGFR2 showed differential sensitivity. Within the FGFR2(mutant) cell lines, 
three of seven showed marked resistance to PD173074 and relative resistance to 
dovitinib and ponatinib. This suggests that alternative mechanisms distinct from 
kinase domain mutations are responsible for intrinsic resistance in these three 
EC lines. Finally, overexpression of FGFR2(N550K) in JHUEM-2 cells 
(FGFR2(C383R)) conferred resistance (about five-fold) to PD173074, providing 
independent data that FGFR2(N550K) can be associated with drug resistance. 
Biochemical in vitro kinase analyses also show that ponatinib is more effective 
than dovitinib at inhibiting FGFR2(N550K). We propose that tumors harboring 
mutationally activated FGFRs should be treated with FGFR inhibitors that 
specifically bind the active kinase.