Efficacy of Combined Use of Everolimus and Second-Generation Pan-EGRF Inhibitors in KRAS Mutant Non-Small Cell Lung Cancer Cell Lines.

Abstract

Background: EGFR mutations are present in approximately 15–50% of non-small cell lung cancer (NSCLC), which are predictive of anti-EGFR therapies. At variance, NSCLC patients harboring KRAS mutations are resistant to those anti-EGFR approaches. Afatinib and allitinib are second-generation pan-EGFR drugs, yet no predictive biomarkers are known in the NSCLC context. In the present study, we evaluated the efficacy of pan-EGFR inhibitors in a panel of 15 lung cancer cell lines associated with the KRAS mutations phenotype. Methods: KRAS wild-type sensitive NCI-H292 cell line was further transfected with KRAS mutations (p.G12D and p.G12S). The pan-EGFR inhibitors’ activity and biologic effect of KRAS mutations were evaluated by cytotoxicity, MAPK phospho-protein array, colony formation, migration, invasion, and adhesion. In addition, in vivo chicken chorioallantoic membrane assay was performed in KRAS mutant cell lines. The gene expression profile was evaluated by NanoString. Lastly, everolimus and pan-EGFR combinations were performed to determine the combination index. Results: The GI50 score classified two cell lines treated with afatinib and seven treated with allitinib as high-sensitive phenotypes. All KRAS mutant cell lines demonstrated a resistant profile for both therapies (GI50 < 30%). The protein array of KRAS edited cells indicated a significant increase in AKT, CREB, HSP27, JNK, and, importantly, mTOR protein levels compared with KRAS wild-type cells. The colony formation, migration, invasion, adhesion, tumor perimeter, and mesenchymal phenotype were increased in the H292 KRAS mutated cells. Gene expression analysis showed 18 dysregulated genes associated with the focal adhesion-PI3K-Akt-mTOR-signaling correlated in KRAS mutant cell lines. Moreover, mTOR overexpression in KRAS mutant H292 cells was inhibited after everolimus exposure, and sensitivity to afatinib and allitinib was restored. Conclusions: Our results indicate that allitinib was more effective than afatinib in NSCLC cell lines. KRAS mutations increased aggressive behavior through upregulation of the focal adhesion-PI3K-Akt-mTOR-signaling in NSCLC cells. Significantly, everolimus restored sensibility and improved cytotoxicity of EGFR inhibitors in the KRAS mutant NSCLC cell lines.