Futibatinib

3T3 cells expressing each FGFR variant were cultured in DMEM-F12 medium with 1.5% FBS for 2 weeks. The remaining 3T3 cells were mixed and treated with the indicated concentrations (0.1 nM–10 µM) of inhibitors for 5 days. The inhibitors were one multikinase FGFR inhibitor (dovitinib), five FGFR1/2/3 inhibitors (AZD4547, infigratinib, E7090, futibatinib, and pemigatinib), one pan-FGFR inhibitor (erdafitinib), and one FGFR4 inhibitor (H3B-6527). The experiment was conducted in triplicate. We calculated the number of each bar code using the MANO method. Considering the different doubling times of the transduced cells, dimethyl sulfoxide (DMSO)-treated cell mixtures were used as the reference control for scaling the bar code count of each clone. The relative growth inhibition of each cell clone was calculated as the ratio of the average read number across triplicates to that of the DMSO control. All inhibitors used in the assay, except E7090 (provided from Eisai Co., Ltd., Tokyo, Japan), were commercially purchased: dovitinib, AZD4547, infigratinib, pemigatinib (all from MedChem Express, Monmouth Junction, NJ, USA), futibatinib (Cayman Chemical, Ann Arbor, MI, USA), and erdafitinib and H3B-6527 (both from Selleckchem, Houston, TX, USA).

The NIH3T3 cell line was a kind gift by Wolfgang Neubert (Max Planck Institute for Biochemistry, Martinsried, Germany) and authenticated by ATCC using Short Tandem Repeats (STR). Cells were grown in a humidified atmosphere at 37 °C in 5% CO₂ in Dulbecco’s Modified Eagle’s Medium (DMEM)—high glucose (Sigma-Aldrich, Taufkirchen, Germany) complemented with 10% FBS and 1% penicillin-streptomycin (Thermo Fisher Scientific, Schwerte, Germany) and routinely tested for mycoplasma with a DAPI test. Futibatinib was purchased from Cayman Chemicals (Ann Arbor, MI, USA); all other TKIs and gemcitabine were purchased from Selleckchem (Houston, TX, USA).