Figure 3: Differential B-RAFi/MEKi sensitivities and C-RAF dependency. | Nature Communications

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

From: Melanoma whole-exome sequencing identifies V600EB-RAF amplification-mediated acquired B-RAF inhibitor resistance

Figure 3

(a) Survival curves of B-RAFi-acquired resistant sublines, with indicated mechanisms of resistance, to 72 h of B-RAFi (PLX4032) treatments, showcasing differential responses at the micromolar drug range. Results are shown relative to DMSO-treated controls (mean±s.e.m., 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 micromolar drug range. Results are shown relative to DMSO-treated controls (mean±s.e.m., n=5). Expression of indicated viral expression constructs shown in western blots. (c) Survival curves of B-RAFi-acquired resistant sublines, with indicated mechanisms of resistance, to 72 h of MEKi (AZD6244) treatments, showcasing differential responses at the micromolar drug range. Results are shown relative to DMSO-treated controls (mean±s.e.m., n=5). (d) Survival curves of cell lines (engineered by viral transduction of M229 P and M238 P to overexpress V600EB-RAF rendering these parental cells resistant to B-RAFi) to 72 h of MEKi (AZD6244) treatments, showcasing differential responses at the micromolar drug range. Pt55 R (double B-RAF and N-RAS mutant) is a short-term melanoma culture derived from a tumour, which acquired PLX4032 (vemurafenib) resistance in a treated patient. Results are shown relative to DMSO-treated controls (mean±s.e.m., n=5). (e,f) Indicated cell lines were treated with constant ratios of PLX4032 and AZD6244 and survival measured after 72 h. 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 2 days, colonies visualized by crystal violet staining after 8 days of drug treatments, and quantified (% growth relative to cells treated with 1 μM PLX4032). 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 two experiments.

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