Supplementary Figure S1.

1. Supplementary Figure S1. Supplementary Figure S1. Determination of the binding mode of the FGFR inhibitor CH5183284/Debio1347. Lineweaver-Burk plot of FGFR1 with different concentrations of ATP in presence of various concentrations of CH5183284/Debio 1347. Linear graphs intersected all with y-axis (i.e. Vmax unchanged) which does support an ATP-competitive mode-of-action of CH5183284/Debio 1347 for FGFR1.

2. Supplementary Figure S2. A. B. (a) (d) (b) (e) (c) Supplementary Figure S2. Effects of CH5183284/Debio 1347and cediranib on in vitro VEGF-induced tube formation (A) Inhibition of tube formation by CH5183284/Debio 1347and cediranib in a HUVEC and fibroblast co-culture system. Total vessel area was measured quantitatively for the area of capillary-like tube formation with Kurabo angiogenesis image analysis software. N=3. (B) Representative mages of tube formation of HUVEC. x4 objective. (a) DMSO treatment. (b) 0.1 µM of CH5183284/Debio 1347. (c) 1 µM of CH5183284/Debio 1347. (d) 0.01 µM of cediranib. (e) 0.1 µM of cediranib.

3. Supplementary Figure S3. Relative kinase activity of FGFR2 to mock Supplementary Figure S3. Kinase activity of FGFR2 WT, V564F, V564I, and V564L mutants. The 293 cells were transiently transfected with pCXND3 FGFR2 WT, FGFR2 V564F, FGFR2 V564I, and FGFR2 V564L. The kinases were immunoprecipitatedwith anti-FGFR2 antibody. Then, kinase activity was evaluated by examining their ability to phosphorylate substrate peptide using a time-resolved fluorescence resonance energy transfer (TR-FRET) assay. (N=2)

4. Supplementary Figure S4. CH5183284 AZD4547 CH5183284 AZD4547 0.001 0.003 0.001 0.003 0.03 0.01 0.01 0.03 0.01 0.03 0.01 0.03 (µM) 0.1 0.3 0.1 0.3 0.1 0.3 0.1 0.3 10 10 0 0 0 1 3 1 3 0 1 1 WT V564F V564I V564L pY-FGFR FGFR2 Supplementary Figure S4. Inhibition of cellular pY-FGFR2 WT, pY-FGFR2 V564F, pY-FGFR2 V564I, and pY-FGFR2 V564L. The HCT-116 cells were transiently transfected with pCXND3 FGFR2 WT, FGFR2 V564F, FGFR2 V564I, and FGFR2 V564L. A day after the transfection, the cells were treated with the indicated concentration of CH5183284/Debio 1347or AZD4547 for 2 h. Cells were extracted and analyzed by Western blotting.

5. Supplementary Figure S5. A. PD173074 cediranib % inhibition Drug conc. μM ( log10) Drug conc. μM ( log10) B. NVP-BGJ398 NVP-BGJ398 CH5183284 CH5183284 AZD4547 AZD4547 DMSO DMSO pY-FGFR TEL-FGFR2 V564F WT Supplementary Figure S5. Susceptibility of CH5183284/Debio 1347against gatekeeper mutants of FGFR2. (A) Anti-proliferative activity of PD173074 and cediranib against TEL-FGFR2 WT driven Ba/F3 cells and TEL-FGFR2 V564F-driven Ba/F3 cells. Three clones were treated with various concentrations of the indicated inhibitors for 4 days. The viable cells were counted with WST-8. (B) TEL-FGFR2 WT driven Ba/F3 and TEL-FGFR2 V564-driven Ba/F3 were treated with the indicated 1 μM of CH5183284/Debio 1347, AZD4547, and NVP-BGJ398 for 2 hours. Cells were extracted and analyzed by Western blotting.

6. Supplementary Figure S6. A. CH5183284 AZD4547 Tumor volume (mm3) Days after the inoculation Days after the inoculation CH5183284 (mg/kg) AZD4547 (mg/kg) B. Vehicle 50 100 25 50 pY-FGFR TEL-FGFR2 WT Actin Supplementary Figure S6. Antitumor activity of CH5183284/Debio 1347and AZD4547 in Ba/F3 TEL-FGFR2 WT-driven mouse model. (A) Mice bearing Ba/F3 TEL-FGFR2 WT cells were treated with CH5183284/Debio 1347or AZD4547 orally once daily for 11 days at the indicated doses. Tumor volume and body weight change for each dose group were measured. Data are shown as mean ± SD (n = 5). (B) Suppression of phospho-FGFR inhibition in xenograft tissue. Mice bearing Ba/F3 TEL-FGFR2 WT cells were treated for 11 days at 50 and 100 mg/kg of CH5183284/Debio 1347or 25 and 50 mg/kg of AZD4547, and xenograft tumors were extracted at 4 hours post-dosing and analyzed by Western blotting. (n = 3)

7. Supplementary Figure S7. CH5183284 dovitinib WT % inhibition % inhibition N549H Drug conc. μM ( log10) Drug conc. μM ( log10) AZD4547 NVP-BGJ398 PD173074 % inhibition % inhibition % inhibition Drug conc. μM ( log10) Drug conc. μM ( log10) Drug conc. μM ( log10) Supplementary Figure S7. Kinase inhibition assay of FGFR2 WT or FGFR2 N549H mutant Kinase inhibitory activity of FGFR inhibitors against FGFR2 WT or FGFR2 N549H were measured (n=3). CH5183284, dovitinib, AZD4547, NVP-BGJ398, and PD173074 showed 12, 5.2, 26, 19, 30 fold higher IC50 against FGFR2 N549H than FGFR2 WT, respectively.

8. Supplementary Table S1. Supplementary Table S1 Crystallographic data collection and refinement statistics for FGFR1 in complex with CH5183284/Debio 1347.

9. Supplementary Table S2. Supplementary Table S2 Kinase selectivity profile of CH5183284/Debio 1347. The values of % competition at 0.1 or 1 μM CH5183284/Debio 1347in DiscoveRx’sKINOMEscan for 442 kinases including mutated kinases.

10. Supplementary Table S2 (cont).

11. Supplementary Table S2 (cont).

12. Supplementary Table S2 (cont).

13. Supplementary Table S2 (cont).

14. Supplementary Table S2 (cont).

15. Supplementary Table S2 (cont).

16. Supplementary Table S2 (cont).

17. Supplementary Table S2 (cont).

18. Supplementary Table S3. Supplementary Table S3 Selective antiproliferative activity of CH5183284/Debio 1347against cancer cell lines harboring genetic alterations in FGFR

19. Supplementary Table S3 (cont).

20. Supplementary Table S3 (cont).

21. Supplementary Table S3 (cont).

22. Supplementary Table S3 (cont).

23. Supplementary Table S3 (cont).

24. Supplementary Table S3 (cont).