The N550K/H mutations in FGFR2 confer differential resistance to PD173074, dovitinib, and ponatinib ATP-competitive inhibitors

Abstract

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.

Figure 1

Identified FGFR2 mutations confer dovitinib resistance in stable BaF3.FGFR2 cells. (A) The ribbon diagram of the A-loop phosphorylated activated WT FGFR2 kinase structure (PDB ID: 2PVF) [36] showing the locations of the drug-resistant mutations. The A-loop and kinase hinge are colored light purple and light orange, respectively. The drug-resistant mutations are rendered as ball and stick. The AMP-PCP (the ATP analog) is shown in both ball-and-stick representation and a semitransparent surface. (B) Western blot analysis of stable BaF3 cells expressing WT or mutant FGFR2 using an anti-FGFR2 antibody (BEK-C17) or anti-tubulin antibody as loading control. (C) Stable BaF3-FGFR2 cells were seeded in 96-well plates in media containing 5 µg/ml heparan sulfate and 1 nM FGF10, and dovitinib was added in concentrations ranging from 3 nM to 10 µM. The cells were incubated at 37°C for 72 hours and proliferation was measured using the ViaLight proliferation kit and the IC₅₀ was calculated using GraphPad Prism.

Figure 2

The molecular mechanisms by which mutations confer resistance to dovitinib. (A and B) Gatekeeper V565I mutation confers resistance to dovitinib through steric hindrance. Binding of dovitinib [in yellow, partial, taken from the CHK-1 and inhibitor 4-(aminoalkylamino)-3-benzimidazole-quinolinones complex structure, PDB ID: 2GDO] [37] is modeled onto the A-loop phosphorylated activated WT FGFR2 kinase structure (PDB ID: 2PVF) [36]. Hydrogen bonds between the kinase and dovitinib are colored yellow. The molecular surface of dovitinib is shown to emphasize the steric clash with the mutated I565. (C–F) Mutations at N550 and E566 confer dovitinib resistance by activating the kinase through disengagement of the molecular brake. The molecular brake is engaged at the kinase hinge region in the unphosphorylated unactivated WT FGFR2 kinase (C; PDB ID: 2PSQ) [36] and is disengaged by A-loop tyrosine phosphorylation (D; PDB ID: 2PVF) [36] or by mutations at N550 (E; PDB ID: 2PWL) [36] and E566 (F; PDB ID: 2PY3) [36]. (G) Some mutations confer dovitinib resistance by activating the kinase through strengthening the hydrophobic spine of the FGFR2 kinase. The hydrophobic spine is shown as a semitransparent surface. Residues comprising the hydrophobic spine are rendered as sticks. The drug-resistant mutations targeting residues V565, M536, M538, I548, and L618 are colored and labeled green, and the others are labeled white.

Figure 3

Dovitinib-resistant mutations increase the intrinsic kinase activity of FGFR2. (A) The substrate phosphorylation activities of WT and mutated FGFR2 kinase domain harboring the drug-resistant mutations were compared using native PAGE (panel I) coupled with time-resolved mass spectrometry (MS; panels II and III). For accuracy, only the early time point (30- and 60-second) MS data, which are in the linear phase of the kinase assay, were processed. The percentage of at least one site phosphorylation on the substrate (panel III) was estimated using the peak intensity data generated by mass spectrometry. (B) Stable BaF3.FGFR2 cells were seeded at 10,000 cells/well in 96-well plates in media containing 5 µg/ml heparan sulfate and 1 nM FGF10. The cells were incubated at 37°C for 72 hours and proliferation was measured using the ViaLight proliferation kit. The increase in proliferation compared to FGFR2 WT cells is presented. (C) Stable BaF3 cells expressing WT or mutant FGFR2 were stimulated for 7.5 minutes with media containing 5 µg/ml heparan sulfate and 16 nM FGF10. After the stimulation period, the cells were lysed in lysis buffer containing phosphatase inhibitors, and 150 µg of total protein was separated on an SDS-PAGE, transferred to a nitrocellulose membrane, and probed with an anti-pan-phospho-FGFR, anti-FGFR2 (BEK-C17), and anti-tubulin antibodies. The ratio of phospho to total FGFR2 was calculated by densitometry using Odyssey 3.0 software.

Figure 4

Dovitinib resistance mutations are similarly resistant to PD173074 but are almost all sensitive to ponatinib. (A) Stable BaF3-FGFR2 cells were seeded in 96-well plates in media containing 5 µg/ml heparan sulfate and 1 nM FGF10, and PD173074 was added in concentrations ranging from 3 nM to 10 µM. The cells were incubated at 37°C for 72 hours and proliferation was measured using the ViaLight proliferation kit and the IC₅₀ was calculated using Prism (GraphPad Software). (B) Cells were treated as in A, but 0.1 nM to 1 µM ponatinib was added to each stable cell line.

Figure 5

Change in FGFR2 phosphorylation in response to treatment with dovitinib and ponatinib. (A) Stable BaF3.FGFR2 cells were pretreated in IL-3-free BaF3 media for 90 minutes at 37°C. After the 90-minute incubation period, the media were removed and the cells were incubated for 7.5 minutes at 37°C with media containing 5 µg/ml heparan sulfate and 16 nM FGF10. Cells were lysed in lysis buffer containing phosphatase inhibitors and 150 µg of total protein was separated on an SDS-PAGE, transferred to a nitrocellulose membrane, and probed using an anti-pan-phospho-FGFR, anti-FGFR2 (BEK-C17), and anti-tubulin antibodies. (B) Densitometry analysis of the change in phosphorylation due to pretreatment with dovitinib and ponatinib. The ratio of phospho to total FGFR2 was calculated by densitometry using Odyssey 3.0 software and the concentration required to decrease the phosphorylation to 50% compared to WT.

Figure 6

The V565I gatekeeper FGFR2 kinase mutant is also refractory to inhibition by ponatinib due to steric conflicts. Ponatinib taken from the Abl-ponatinib complex structure (PDB ID: 3OY3) [38] was modeled onto the A-loop phosphorylated activated WT FGFR2 kinase structure (PDB ID: 2PVF) [36]. The molecular surface of ponatinib (in yellow) is shown to emphasize the steric clash with the mutated I565.

Figure 7

The N550K mutation confers resistance to PD173074, but not dovitinib or ponatinib, when expressed in FGFR inhibitor-sensitive JHUEM-2 cells. (A) Stably transfected JHUEM-2 cells were seeded in 96-well plates 24 hours before drug addition. Dovitinib, PD173074, and ponatinib were added in increasing concentrations from 1 nM to 10 µM. The cells were incubated at 37°C for 72 hours and proliferation was measured using the CyQUANT Cell Proliferation Assay Kit and the IC₅₀ was calculated. (B) Western blots demonstrating FGFR2 expression levels in stably transfected JHUEM-2 lines. EV, empty vector. (C) Both dovitinib and ponatinib are potent inhibitors of the WT FGFR2 kinase, whereas only ponatinib effectively inhibits the N550H mutant FGFR2. Due to the steric clash, neither dovitinib nor ponatinib is capable of inhibiting the V565I “gatekeeper” mutant. The control lane 1 shows extent of phosphorylation in the absence of inhibitors. In lanes 2 to 7, increasing concentrations of inhibitors were added into the autophosphorylation reactions to inhibit the kinase autophosphorylation. The kinase/inhibitor molar ratios of lanes 2 to 7 are 1:0.2, 1:0.5, 1:1, 1:2, 1:5, and 1:10, respectively. The control lane 0 is the kinase in the absence of ATP/MgCl2.