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. Author manuscript; available in PMC: 2017 Dec 13.
Published in final edited form as: Cell Rep. 2017 Dec 5;21(10):2796–2812. doi: 10.1016/j.celrep.2017.11.022

Figure 4. Melanoma cell lines with intrinsic resistance to BRAFi derive resistance by rapidly upregulating MYC following drug treatment.

Figure 4

A) The indicated cell lines were incubated with DMSO or 1 μM PLX4720 for 24 hrs, then immunoblotted for MYC. Total protein staining is shown in Figure S4A. B) As in A). Total protein staining is shown in Figure S4B. C) Quantification of immunoblots shown in A) and B) showing MYC protein levels normalized to total protein. D) JQ1 GI50 values for the indicated lines following pre-incubation with either DMSO or 1 μM PLX4720 for four days. P values indicate significance between DMSO and PLX4720 pre-treatment. E) GI50 values in A375 cells for either PLX4720 (solid line) or JQ1 (dashed line), measured at intervals during continuous culture in 1 μM PLX4720. F) MYC levels in A375 at several time points while in culture with 1 μM PLX4720. Quantification shown in Figure S4C. G) As in E), for the intrinsically resistant line, WM793. H) As in F) for WM793. Quantification shown in Figure S4D. I) Sensitization of the indicated lines to PLX4720 by expression of two shRNAs targeting MYC. Differences between shGFP and shMYC are denoted by P values. J) Signaling pathways controlling intrinsic resistance to BRAFi/MEKi in intrinsically resistant cell lines and their convergence on downstream MYC activation. As indicated, previous findings demonstrate that inhibition of the PI3K-AKT-mTOR and NOTCH1 pathways can sensitize these lines to ERK pathway inhibition. K) Sensitivity of indicated lines to the ERKi VX-11E following expression of luc or MYC and treatment DMSO or a combination of inhibitors (200 nM BEZ235 (PI3K/mTOR inhibitor) for the PI3K-AKT-mTOR pathway and shNotch1 for the Notch1 pathway for Hs 294T cells; 200 nM BEZ235, shNotch1, and 1 μM fulvestrant for Lox IMVI, A2058 and WM793 cells). Viabilities of cells treated with pathway inhibitors plus VX-11E are normalized to the viability of the same cells treated with pathway inhibitors alone to account for nonspecific toxicity. All data are means (SD) from 3 experiments. *P < 0.05; **P < 0.01; ***P < 0.005; ****P < 0.001; #, the upper bound of the assay was reached. See also Figure S4.