Clonal dynamics of BRAF-driven drug resistance in EGFR-mutant lung cancer

Abstract

Activation of MAPK signaling via BRAF mutations may limit the activity of EGFR inhibitors in EGFR-mutant lung cancer patients. However, the impact of BRAF mutations on the selection and fitness of emerging resistant clones during anti-EGFR therapy remains elusive. We tracked the evolution of subclonal mutations by whole-exome sequencing and performed clonal analyses of individual metastases during therapy. Complementary functional analyses of polyclonal EGFR-mutant cell pools showed a dose-dependent enrichment of BRAF^V600E and a loss of EGFR inhibitor susceptibility. The clones remain stable and become vulnerable to combined EGFR, RAF, and MEK inhibition. Moreover, only osimertinib/trametinib combination treatment, but not monotherapy with either of these drugs, leads to robust tumor shrinkage in EGFR-driven xenograft models harboring BRAF^V600E mutations. These data provide insights into the dynamics of clonal evolution of EGFR-mutant tumors and the therapeutic implications of BRAF co-mutations that may facilitate the development of treatment strategies to improve the prognosis of these patients.

Fig. 1. Clinicopathological characteristics for the study cohort and clonal evolution.

a Spectrum and distribution of BRAF co-mutations in patients with EGFR-mutant lung adenocarcinoma. b Kaplan–Meier curve of the time elapsed from the detection of the EGFR mutation until the detection of the acquired BRAF mutation (as events) in days. c Kaplan–Meier curve of overall survival for patients P01, P04, P12–P15 that were available for survival analysis. d Overview of the biopsies and key molecular findings by NGS for patient P04. Flow chart (top right) summarizes lines of therapy approaches overtime after the acquisition of BRAF^V600E mutation. e, f Clustering of WES-derived mutations based on their CCFs between pairs of tumor biopsies to detect clusters of shared clonal and private mutations. Candidate mutations in EGFR and BRAF are highlighted. g Subclonal composition in individual biopsies indicating two subclones (C1, C3) in the peritoneal metastasis and single clones in the liver metastases. h Clonal evolution of reconstructed cell populations presented as a phylogenetic tree. The computationally inferred most common ancestor C0 is common to all subsequent clones and highlighted mutations are present in descendent clones. (i) Visualization of evolutionary genetic distances between normal tissue and longitudinal biopsies. WES whole-exome sequencing, NGS next-generation sequencing, PD progressive disease, PR partial response, D + T dabrafenib+trametinib, O + D( + T) osimertinib+dabrafenib(+trametinib), O + CTX + B osimertinib+chemotherapy+bevacizumab, O + Tc TACE osimertinib+transarterial chemoembolization, C clone, CCF cancer cell fraction.

Fig. 2. Selection of BRAF^V600E-positive clones in EGFR-mutant cells.

a Immunoblotting of PC9 cells expressing the annotated constructs, treated with (+) or without (−) osimertinib (48 h). Hsp90 is used as a loading control. b Clonogenicity assays of PC9 derived cell lines treated with osimertinib for 7 and 14 days or DMSO control for 7 days are displayed. c Quantitative analysis of (b) normalized to PC9 (EV). d Limited dilution assay of PC9-derived cell lines treated for 21 days before analysis. e, f qRT-PCR analysis of mRNA expression in e BRAF and f NRAS in PC9 derived cell lines normalized to EV. g Immunoblotting of PC9 cells expressing the annotated constructs that were treated as in (a). h Viability curves of PC9 cells expressing the annotated constructs treated with osimertinib (72 h) are shown. The relative area under the curve (AUC) in % compared to a theoretical non-responding AUC. Error bars indicate mean ± SD. Two-tailed paired t tests, ***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.

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.