Fig. 3. Overcoming BRAF^V600E-mediated resistance in EGFR-mutant cells.

a Growth series of PC9 derived cell lines counted for 5 days every 24 h (see Methods). b Immunoblotting of PC9^BRAF-V600E OS 100 nM, PC9^NRAS-Q61K OS 100 nM, and PC9 (EV). Osimertinib-preselected cells were cultured for 0, 7, and 21 days without osimertinib treatment and plated 48 h before lysis. c Cell viability assay of PC9 cells expressing the annotated constructs treated for 72 h with osimertinib is shown. The relative AUC (see Methods) of BRAF^V600E OS 100 nM and NRAS^Q61K OS 100 nM after osimertinib withdrawal for >40 days are shown. d Clonogenicity assay of PC9 cells expressing the annotated constructs treated for 14 days with indicated compounds before staining. e RNA-seq based expression of E2F gene set genes (rows) in PC9 derived cell lines (columns) after 48 h treatment with indicated inhibitors. Expression was normalized as z-score per gene. f Synergy screen of osimertinib and trametinib combination treatment in PC9 derived cell lines for 72 h are displayed. g Immunoblotting of PC9 cells expressing the annotated constructs is shown. Treatment with indicated compounds 48 h before lysis. h Relative tumor volume of xenograft mice injected with PC9^BRAF-V600E OS 100 nM cells in % compared to day 0 of the treatment regimen (see Methods). Error bars indicate mean ± SD. Two-tailed paired t tests (all except (h); two-tailed Welch’s t tests with Bonferonni-correction), ***p < 0.001, **p < 0.01, *p < 0.05, ^n.s.p > 0.05. EGFR epidermal growth factor receptor, BRAF B-rapidly accelerated fibrosarcoma, NRAS neuroblastoma rat sarcoma, EV empty vector.