MEK1/2 mutations drive resistance to trametinib in BRAFG469V-driven DIPG cells. A, Protein structure representation of BRAF showing the mutant residue (shaded orange) for the observed G469 missense mutation observed in ICR-B169. Generated in COSMIC-3D (cancer.sanger.ac.uk/cosmic3d). B, Timeline of clinical experience for the child with BRAFG469V-mutant DIPG treated with trametinib at progression. Initial therapy with everolimus and radiotherapy is shaded in orange, later treatment with trametinib in blue. Sagittal T1-weighted postgadolinium MRI images are provided at diagnosis, the initial progression, and the later progression immediately prior to death from disease. Periods of steroid treatment are noted by purple lines. The tumor is highlighted with arrows. C, Survival curves for mice bearing ICR-B169 cell-derived orthotopic xenografts, treated with trametinib (blue), compared with vehicle-treated controls (gray). Treatment window is shaded in gray. D, Experimental design for the generation of cells resistant to trametinib by the continuous exposure model. Parental ICR-B169 cells are treated with either an exponentially increasing dose of inhibitor over time (approach 1) or a constant IC80 dose (approach 2). E, Dose–response curves for trametinib tested against ICR-B169 parental cells (gray) and resistant clones T1 (MEK2I115N, pink), T3 (MEK1I141S, purple), and T6 (MEK1K57N, red) after 7 to 9 months of exposure to inhibitor. F, Dose–response curves for trametinib tested against ICR-B169 parental cells (gray) and resistant clones (dashed lines) after 2-month withdrawal of inhibitor. Concentration of compound is plotted on a log scale (x-axis) against cell viability (y-axis). Means plus standard errors are plotted from at least n = 3 experiments. **, P < 0.001; ***, P < 0.001, AUC t test. G, Emergence of MEK1/2 mutations in clones T1 (pink), T3 (purple), and T6 (red) over time, as assessed by ddPCR. The x-axes represent passage number; left y-axes are specific mutation VAFs; right y-axes plot the concentration of trametinib that cells were exposed to (gray dashed line). H, Emergence of resistance in clones over time (T1, pink; T3, purple; T6, red), plotted as days exposed to inhibitor on the x-axis, with passage numbers labeled. The y-axis is a GI50 value for trametinib in cells harvested at the given passage. I, Pathway activation in resistant clones (T1, pink; T3, purple; T6, red) assessed by a capillary electrophoresis assay and plotted as a ratio of respective phosphorylated/total protein compared to ICR-B169 parental cells (gray).