Journal: Clinical Cancer Research

Article Title: PI3Kα Inhibitor and Degrader Inavolisib Can Co-opt FGFR2 to Enhance Responses in Patients with PIK3CA -Mutated Solid Tumors and in Preclinical Models

doi: 10.1158/1078-0432.CCR-25-1459

Figure Lengend Snippet: Inavolisib sensitivity depends on high FGFR2 expression. A, Legend related to panels in B–I . B, Cell lines were treated with 0.03 μmol/L of the FGFR2i or 2-μmol/L lapatinib for 1 hour followed by immunoblotting with the antibodies indicated at left. Representative results from experiments ( n = 2). C, Cell lines were treated with inavolisib or the FGFR2i at various concentrations for 1 hour. Cell lysates were immunoprecipitated with an antibody against p85β, followed by immunoblotting with the antibodies indicated at left. Representative results from experiments ( n = 2). IB, immunoblotting. D, Following RAS-GTP pulldown, cell lines were treated with the FGFR2i or lapatinib for different durations and immunoblotted with the antibodies indicated at left. Representative results from experiments ( n = 2). E, Cell lysates from cells treated with inavolisib alone or in combination with the FGFR2i or lapatinib for 4 hours were immunoprecipitated with RAS antibody and blotted with p110α antibody. F, Mechanistic model of the effects of FGFR2 and HER2 inhibition on HER3 and RAS activity. FGFR2-high–expressing cell lines induced PI3K signaling through both HER3 and WT RAS activity (top) compared with HER2-induced PI3K signaling through HER3 but not RAS activity (bottom). G, FGFR2-high–expressing cell lines, MFM223 and SUM52PE, were treated with inavolisib, FGFR2i, or lapatinib for 1 hour. Membrane fractions were analyzed by reciprocal co-IP with one another using HER3 or FGFR2 antibody and Western blotting with FGFR2, HER3, RAS, and p85β antibody. Representative results from experiments ( n = 2). H, SUM52PE, MFM223, and MFE280 cells were treated with inavolisib single-agent or in combination with the FGFR2i or lapatinib for 6 hours. Ubiquitinated proteins were pulled down from the membrane fraction with TUBE1 reagent and blotted with p110α antibody. Representative results from experiments ( n = 2). I, Western blots of the inhibitor response in PI3K signaling (pHER3 and pAKT) in PIK3CA mutant MFM223 and PIK3CA WT SUM52PE; cell lines were treated with 0.5-μmol/L inavolisib or 1-μmol/L alpelisib for different durations. Representative results from experiments ( n = 2). J, Ratio of inavolisib and alpelisib GR 50 values in FGFR2-high ( n = 12) vs. FGFR2-low ( n = 9) expressing cell lines harboring PIK3CA mutations, as assessed in a 5-day viability assay. Data are represented as median (center line) ± IQR (25th to 75th percentile, box) and ± full range (minimum to maximum, whiskers). P value was calculated using Wilcoxon rank-sum test. Representative results from experiments ( n = 2). WB, Western blotting.

Article Snippet: Antibodies to p110α (cat. No. 4249, RRID: AB_2165248), pAKT Ser473 (cat. No. 4060, RRID: AB_2315049), pS6 S235/236 (cat. No. 2211, RRID: AB_331679), HER3 (cat. No. 12708, RRID: AB_2721919), HER2 (cat. No. 2242, RRID: AB_331015), pHER2 Y1221/Y1222 (cat. No. 2243, RRID: AB_490899), pHER3 Y128 (cat. No. 4791, RRID: AB_2099709), pHER3 Y1328 (cat. No. 14525, RRID: AB_2798501), pPLCγ Y783 (cat. No. 14008, RRID: AB_2728690), pERK T202/T204 (cat. No. 9101, RRID: AB_331646), p4EBP T37/46 (cat. No. 9459, RRID: AB_330985), FGFR1 (cat. No. 9740, RRID: AB_11178519), FGFR2 (cat. No. 11835, RRID: AB_2797742), FGFR3 (cat. No. 4574, RRID: AB_2246903), FGFR4 (cat. No. 8562, RRID: AB_10891199), pFGFR Y653/654 (cat. No. 3476, RRID: AB_331369), and pFRS2A Y196 (cat. No. 3864, RRID: AB_2106222) were obtained from Cell Signaling Technology.

Techniques: Expressing, Western Blot, Immunoprecipitation, Inhibition, Activity Assay, Membrane, Co-Immunoprecipitation Assay, Mutagenesis, Viability Assay