Figure 3. Differential B-RAFi/MEKi sensitivities and C-RAF dependency.

(a) Survival curves of B-RAFi acquired resistant sub-lines, with indicated mechanisms of resistance, to 72 h of B-RAFi (PLX4032) treatments, showcasing differential responses at the micro-molar drug range. Results are shown relative to DMSO-treated controls (mean ± SEM, n = 5; dashed line, 50% inhibition). (b) Survival curves of cell lines, engineered by viral transduction of M229 P to be B-RAFi resistant, to 72 h of B-RAFi (PLX4032) treatments, showcasing differential responses at the micro-molar drug range. Results are shown relative to DMSO-treated controls (mean ± SEM, n = 5). Expression of indicated viral expression constructs shown in Western blots. (c) Survival curves of B-RAFi acquired resistant sub-lines, with indicated mechanisms of resistance, to 72 h of MEKi (AZD6244) treatments, showcasing differential responses at the micro-molar drug range. Results are shown relative to DMSO-treated controls (mean ± SEM, n = 5). (d) Survival curves of cell lines (engineered by viral transduction of M229 P and M238 P to over-express ^V600EB-RAF rendering these parental cells resistant to B-RAFi) to 72 h of MEKi (AZD6244) treatments, showcasing differential responses at the micro-molar drug range. Pt55 R (double B-RAF and N-RAS mutant) is a short-term melanoma culture derived from a tumor which acquired PLX4032 (vemurafenib) resistance in a treated patient. Results are shown relative to DMSO-treated controls (mean ± SEM, n = 5). (e and f) Indicated cell lines were treated with constant ratios of PLX4032 and AZD6244 and survival measured after 72h. Relative synergies, expressed as log₁₀ of CI values, are shown. (g) M249 (R4) and M395 R were seeded at single cell density and treated with indicated concentrations of PLX4032 and/or AZD6244. Inhibitors and media were replenished every two days, colonies visualized by crystal violet staining after 8 days of drug treatments, and quantified (% growth relative to cells treated with 1 μM PLX4032; representative of 2 experiments). Photographs representative of two independent experiments. (h) Survival curves of indicated cell lines after shScrambled or shC-RAF transduction (inset) and when treated with PLX4032 for 72 h. (i) Clonogenic assays of cell lines in e with 14 days (M249 R4) or 18 days (M395 R) of PLX4032 treatment. Results are representative of 2 experiments.