Fig. 1. MEK inhibitors potently and selectively decrease cell viability in FN-RMS.

(A) Expression of BRAF V600E—but not the empty vector control (pBABE), Myr-AKT, or RALA Q75L—inhibits differentiation of C2C12 myoblasts serum-starved for 5 days as determined by immunofluorescence for myosin heavy chain (MHC). Scale bars, 200 μm. Quantification of differentiation index, fusion index, and myocyte width is shown at right. Data are means ± SD for 3 representative fields (indices) or 10 representative myocytes (myocyte width). *P < 0.05. (B) A bubble plot comparing the potency of the classes of compounds found in the MIPE-v4 screen in FN-RMS cell lines with potency in normal cell lines. Each bubble represents a class of drugs; the size of the bubble is proportional to the number of drugs in that class; and the color of the bubble corresponds to the potency of that class. Potency is represented as %AUC. HDAC, histone deacetylase; PLK1, Polo-like kinase; KSP, kinesin-like spindle protein. (C) A wind-rose plot shows that MEK inhibitors are potent and selective for FN-RMS, as compared to fusion-positive RMS (FP-RMS) and normal cell lines. Each cell line investigated corresponds to a spoke of the plot. The size of the wedge along each cell line spoke is proportional to the number of drugs in the class displaying potency of 80% AUC or less. The wedges are colored on the basis of the %AUC of the drugs from red (30%) to blue (80%). FGFR4, fibroblast growth factor receptor 4; HEK293T, human embryonic kidney–293T cells. (D) Quantification (left) and representative images (right) of 14-day clonogenic assays for RD, SMS-CTR, and RH30 in the presence of trametinib. Data are means ± SD for three replicates. (E) Six hours of trametinib treatment decreases ERK phosphorylation in RD (top) and SMS-CTR (bottom) as determined by immunoblot. pERK, phosphorylated ERK; tERK, total ERK.