Pemigatinib

A total of 800 NMuMG or SNU-1 cells; 2,000 MCF7, MDA-MB-231, KATO-III, SNU-16, or SUM52PE cells; 3,000 MFM-223 or NCI-H716 cells; or 4,000 MDA-MB-134-VI cells per well were seeded in 96-well plates using DMEM/F-12 supplemented with penicillin–streptomycin and 10% FBS for human cell lines or 3% FBS for NMuMG. After 24 h, cells were treated with FGFRi for 4 days using vehicle (DMSO), AZD4547 (AstraZeneca), or pemigatinib (HY-109099), BGJ398 (HY-13311) or debio-1347 (HY-19957, all MedChemExpress) with a range of 0.1 nM to 100 μM. Usage of AZD4547, pemigatinib, BGJ398 and debio-1347 was previously described^{63 (link)–66 (link)}. Cell viability was assayed using CellTiter-Blue Reagent (G808A, Promega) for 4 h and subsequently measuring fluorescence on the Infinite M Plex plate reader operated using the Tecan i-control software. Drug-response curves were modelled using [inhibitor] versus response with variable slope (four parameters) and least-squares regression in Prism (v.9.3.1, GraphPad Software).

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).

Pooled HLM (M0567) from male donors (20 mg mL⁻¹ in 250 mM sucrose buffer) were procured from Sigma-Aldrich (St. Louis, MO, USA) and stored at −70 °C until use. All solvents used in the study were of HPLC grade. All chemicals and reference powders were of analytical (AR) grade. Pemigatinib (99.88%) and flavopiridol (99.72%) were procured from MedChem Express Company (Princeton, NJ, USA). Acetonitrile and formic acid were purchased from Sigma-Aldrich (St. Louis, MO, USA). Water (HPLC grade) was obtained from an in-house Milli-Q plus purification equipment that was procured from Millipore (Billerica, MA, USA). Waters Acquity UPLC [serial number H10UPH model code UPH] and Acquity TQD MS [serial number QBB1203 and model code TQD] were used for chromatographic separation and mass detection of analyte peaks. The LC-MS/MS system was controlled by MassLynx 4.1 Data acquisition, processing and reporting were automatically performed using ‘QuanLynx’ included in the MassLynx Software package (Version 4.1, SCN 805). Mass tuning was assisted with IntelliStart®. Rotary pump (SV40B; Murrysville, PA, USA) was used for generating vacuum and a nitrogen generator from Peak Scientific (Renfrewshire, Scotland, UK) was used for supplying desolvation gas. Argon gas of 99.999% purity was obtained locally.