Fig 1: Rapid feedback activation of FGFR causes resistance to SHP2 inhibitors.(A) Immunoblot of p-ERK in Hep3B cells treated with SHP099 at indicated concentrations (1 µM, 3 µM, 10 µM, or 30 µM) for 15 min, 30 min, 60 min, or 120 min, or 0.5 µM BGJ398 for 2 h. (B) Immunoblot of p-ERK and tubulin in Detroit 562, KYSE-520, Hep3B, and JHH-7 cells treated with either SHP394 at indicated concentrations (0.3 µM, 1 µM, or 3 µM) for 15 min or 2 h or an RTK inhibitor (1 µM erlotinib for Detroit 562 and KYSE-520; 0.1 µM FGF401 for JHH-7 and Hep3B) for 2 h. Lane numbers were added for the convenience of comparison. (C) Detroit 562 cells were treated with 1 µM SHP394 for 15 min or 2 h and Hep3B and JHH-7 cells were treated with either SHP394 at indicated concentrations (0.3 µM or 1 µM) for 15 min or 2 h or 0.5 µM BGJ398 for 2 h. RAS-GTP levels were determined by a G-LISA RAS activation assay. n. s., not significant; ***, p < 0.001; ****, p < 0.0001 (by t-test). (D) Hep3B and JHH-7 cells were treated with SHP394 at indicated concentrations (1 µM or 3 µM) or 0.1 µM FGF401 for 15 min or 2 h or 100 ng/ml FGF19 for 15 min. Pan-tyrosine phosphorylation levels of FGFR4 were determined by the Phospho-FGF Receptor 4 (panTyr) Sandwich ELISA assay. ***, p < 0.001; ****, p < 0.0001 (by t-test). (E) Hep3B and JHH-7 cells were pre-treated with 1 µM SHP394 for 2 h to induce the rebound of p-ERK levels, followed by addition of 10 µM SHP394 (also 1 µM SHP394 treatment for JHH-7), 0.1 µM FGF401 for Hep3B, or 0.5 µM BGJ398 for JHH-7 for either 15 min or 2 h. Cell lysates were collected and p-ERK1/2 levels were determined by immunoblotting. (F) Immunoblot of p-ERK and tubulin in JHH-7 and Hep3B cells treated with 0.5 µM RMC-4550 (site 1 inhibitor), site 2 inhibitor SHP244 (30 or 100 µM), or the combination of RMC-4550 and SHP244 for either 15 min or 2 h. Ratios of p-ERK/ERK were calculated as described in Materials and Methods.