Defining the Sensitivity Landscape of 74,389 <i>EGFR</i> Variants to Tyrosine Kinase Inhibitors

Abstract

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, ge tinib, 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, ge tinib, 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 ve 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 ve commonly used TKIs. The approach used here should be applicable to other genes and targeted drugs. Signi cance StatementMutation-directed cancer precision medicine requires pre-interpretation of variants response on targeted drugs. For Asian lung adenocarcinoma patients, epidermal growth factor receptor (EGFR) is a commonly mutated gene with many rare mutations that lack drug response interpretations. We conducted a systematic cytotoxicity screening of EGFR mutations on ve tyrosine kinase inhibitors. We found that patients with rare EGFR mutations are most likely to bene t from osimertinib therapy compared to afatinib, erlotinib, ge tinib, or icotinib therapy. This study provides the rst case of deep mutational scanning that simultaneously assayed substitution, deletion, and insertion variants. This approach is applicable for other oncogenes and targeted drugs.