Defining the Sensitivity Landscape of 74,389 EGFR Variants to Tyrosine Kinase Inhibitors

Background Tyrosine kinase inhibitors (TKIs) therapy is a standard treatment for patients with advanced non-small-cell lung carcinoma (NSCLC) when activating epidermal growth factor receptor ( EGFR ) mutations are detected. However, except for the well-studied EGFR mutations, most EGFR mutations lack treatment regimens. Methods We constructed two EGFR variant libraries containing substitutions, deletions, or insertions using the saturation mutagenesis method. All the variants were located in the EGFR mutation hotspot (exons 18–21). The sensitivity of these variants to afatinib, erlotinib, gefitinib, icotinib, and osimertinib was systematically studied by determining their enrichment in massively parallel cytotoxicity assays using an endogenous EGFR-depleted cell line, PC9. Results A total of 3,914 and 70,475 variants were detected in the constructed EGFR Substitution-Deletion (Sub-Del) and exon 20 Insertion (Ins) libraries, accounting for 99.3% and 55.8% of the designed variants, respectively. Of the 3,914 Sub-Del variants, 813 were highly enriched in the reversible TKI (erlotinib, gefitinib, icotinib) cytotoxicity assays and 51 were enriched in the irreversible TKI (afatinib, osimertinib) cytotoxicity assays. For the 70,475 Ins variants, insertions at amino acid positions 770–774 were highly enriched in all the five TKI cytotoxicity assays. Moreover, the top 5% of the enriched insertion variants included a glycine or serine insertion at high frequency. Conclusions We present a comprehensive reference for the sensitivity of EGFR variants to five commonly used TKIs. The approach used here should be applicable to other genes and targeted drugs. ### Competing Interest Statement The authors have declared no competing interest. * AA : Amino acid Afa : Afatinib ATCC : American Type Culture Collection CIViC : Clinical Interpretation of Variants in Cancer COSMIC : Catalogue of Somatic Mutations in Cancer DMS : Deep mutational scanning DMSO : Dimethyl sulfoxide EGFR : Epidermal growth factor receptor EGFR-TKI : EGFR-tyrosine kinase inhibitor Erl : Erlotinib FACS : Fluorescent-activated cells sorting FDR : False discovery rate gDNA : Genomic DNA Gef : Gefitinib Ico : Icotinib Ins : Insertion MANO : Mixed-all-nominated-mutants-in-one MITE : Mutagenesis by Integrated TilEs NEB : New England Biolabs NSCLC : Non-small-cell lung carcinoma Osi : Osimertinib sgRNA : Single guide RNA STR : Short tandem repeat Sub-Del : Substitution-Deletion TKI : Tyrosine kinase inhibitor VUS : Variants of unknown significance