Adaptive Resistance to Dual BRAF/MEK Inhibition in BRAF-Driven Tumors through Autocrine FGFR Pathway Activation

FGFR inhibition synergizes with dual BRAF and MEK inhibition to enhance tumor killing through suppression of pERK. A, Top, viability of A375 cells with various FGFR inhibitors in the presence of BRAF and MEK inhibitors, as quantitated by CellTiter-Glo after 72 hours of drug treatment. Bottom, viability of Mel888 parental and dual resistant cells to BRAF and MEK inhibitors as well as to ponatinib. Viability at zero concentration for a given drug is normalized to be one-hundred percent. Mean and SEM are shown for three replicates. B, Colony formation assay with various FGFR inhibitors. BLU-554 is a FGFR4-selective inhibitor. Vemurafenib (Vem, 1.5 μmol/L) and cobimetinib (Cobi, 0.5 μmol/L) treated A375 VCR cells served as baseline control. Two different concentrations of each inhibitor [ponatinib (Pona), NVP-BGJ398, and BLU-554], 0.1 and 1 μmol/L, were tested in conjunction with vemurafenib and cobimetinib. A375 VCR cells were grown for 21 days and then stained with crystal violet. Mel888 DR cells were treated with dabrafenib (0.5 μmol/L) and trametinib (0.02 μmol/L), plus either ponatinib or NVP-BGJ398 at 0.1 and 1 μmol/L for 9 days. WM9 DR were treated with dabrafenib (1.0 μmol/L) and trametinib (0.01 μmol/L), plus either ponatinib or NVP-BGJ398 at 0.1 and 1 μmol/L for 10 days. Media were changed with fresh drug weekly. C, Western blots of A375 parental and reversibly resistant A375-VCR cells treated with ponatinib in combination with dual BRAF/MEK inhibition. Cells were grown off of drug for 24 hours, then with drug added for 4 hours, and lysed. GAPDH served as control.

Conditioned media from drug treated cells confers drug resistance to BRAF and MEK inhibitors. A, Conditioned media was collected from A375 parental cells (initial plating 1 × 10⁶million cells) treated with vehicle or vemurafenib (0.1 μmol/L) and cobimetinib (0.01 μmol/L) combination for 72 hours respectively. Fresh drug-free media was exchanged and the supernatant collected at 48 hours and 7 days later. Each conditioned media was mixed with fresh media in 2:1 ratio and used to culture A375 cells naïve to the BRAF and MEK combination. The cells were treated for 72 hours with titration of the drug combination. Cell viability was quantitated by CellTiter-Glo after 72 hours of drug treatment. Mean and SEM are shown for three replicates. B, Transcriptional profiling of FGF pathway using TaqMan real-time PCR assay. Top, A375 parental cells were treated with vemurafenib (0.1 μmol/L) and cobimetinib (0.01 μmol/L) for 72 hours. A375 VCR dual resistant cells were treated with vemurafenib (1.5 μmol/L) and cobimetinib (0.5 μmol/L) for 72 hours and compared to the parental line without drug treatment. Mean and SEM are shown for three replicates. Bottom, Mel888 parental and dual resistant cells were both treated with dabrafenib (0.5 μmol/L) and trametinib (0.02 μmol/L) for 72 hours prior to RNA extraction. In all cases, gene expression is normalized to that of the parental cells without drug treatment. C, Cellular survival of A375 parental cells treated with exogenous hFGFs and combined BRAF/MEK inhibitors for 72 hours was quantitated by CellTiter-Glo. Mean and SEM are shown for three replicates. Western blot analysis of pFGFR1 with GAPDH as control. D, ELISA was used to detect hFGF1 from supernatants of vehicle versus drug-treated A375 parental cells. E, Cellular survival of A375 parental cells treated either with a rabbit polyclonal FGF1-specific antibody (pink) or isotype control (black) and BRAF/MEK combination for 72 hours was quantitated by CellTiter-Glo. Mean and SEM are shown for three replicates.

BRAF^V600E-driven tumor cells transcriptionally upregulate FGF1 in response to dual BRAF and MEK inhibition. A, Results of a customized TaqMan human FGF pathway array (Thermo Fisher Scientific). Differential expression change comparing cells treated with vemurafenib and cobimetinib for 72 hours versus the same cell line treated with vehicle calculated using the ΔΔC_t method. Data represented average of three TaqMan experiments and the log₂ fold change was plotted. B, Cellular survival of various BRAF^V600E-driven tumor cell lines treated with exogenous hFGF1 and BRAF/MEK inhibitor combination for 72 hours was quantitated by CellTiter-Glo. Mean and SEM are shown for three replicates. C, ELISA to assay for hFGF1 in supernatant collected from several BRAF^V600E-driven tumor cell lines treated with either vehicle or BRAF/MEK combination for 72 hours. D, Phospho-RTK arrays from A375 parental, VemR, CobiR, and VCR cells.

FGFR inhibition in combination of dual BRAF and MEK inhibition induces tumor regression in xenograft models. A, Mice bearing the dual resistant A375 VCR cells were treated via oral gavage with the indicated drugs for 28 days (vemurafenib at 25 mg/kg, cobimetinib at 4 mg/kg, and ponatinib at 20 mg/kg). ***, comparing the double versus the triple combination. *, comparing either vehicle or ponatinib alone versus the triple combination. Error bars represent mean and SEM. P value was calculated using the unpaired t test. B, Mice treated with the dual vemurafenib and cobimetinib combination from A were rerandomized either to continue receiving the combination at the same dose or incorporating ponatinib at 20 mg/kg for an additional 11 days. Error bars represent mean and SEM. P value calculated using the unpaired t test. C, Cell viability and colony formation assay using the triple combination of BRAF, MEK, and FGFR inhibition on the A375 cells. Vemurafenib concentration used was 0.1 μmol/L, cobimetinib 0.01 μmol/L, and ponatinib and NVP-BGJ398 0.1 μmol/L and 0.5 μmol/L, respectively. A total of 5 × 10⁵cells were seeded initially. Media were changed with fresh drug every 3–4 days and total cells remaining were counted at each time point using the Countess II automated cell counter with Trypan blue exclusion. Mean and SEM are shown for three replicates. D, Mice bearing tumors from the A375 parental cells were grouped and treated with vehicle, vemurafenib (25 mg/kg) and cobimetinib (4 mg/kg), or the triple combination (addition of ponatinib at 20 mg/kg) for 35 days. Error bars represent mean and SEM. P value was calculated using the unpaired t test. E, IHC cross section of dual resistant A375 xenograft tumors stained with hematoxylin and eosin, pERK, and Ki-67. The mice were treated with vehicle, ponatinib, vemurafenib and cobimetinib, or the triple combination as indicated in C 4 hours prior to tumor harvesting. F, Serum was collected from A375 parental cells tumor bearing mice at baseline and after treatment with vemurafenib and cobimetinib daily for 96 hours. Serum was diluted 1:2 before running ELISA to quantitate the level of hFGF1. G, Mice bearing tumors from the Mel888 DR cells were treated with either vemurafenib (25 mg/kg) and cobimetinib (4 mg/kg), or the triple combination (addition of ponatinib at 20 mg/kg) for 32 days. Error bars represent mean and SEM. P value was calculated using the unpaired t test and adjusted for multiple comparisons.