Abstract 6961: Characterization of EGFR C797S-mutant, osimertinib-resistant non-small cell lung cancer cells

Abstract Osimertinib is a third-generation epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor that irreversibly binds to active-site cysteine 797 (C797). Originally, it was developed to target the T790M gatekeeper mutation which arises as acquired resistance mechanism to first-generation EGFR inhibitors such as gefitinib. Due to its efficacy and selectivity, osimertinib is now used as first-line treatment for non-small cell lung cancer (NSCLC) patients harboring the activating EGFR mutation L858R or exon 19 deletion. However, a wide range of on- and off-target resistance mechanisms have been observed in clinical settings. Developing relevant models is crucial for designing effective treatment strategies to overcome osimertinib resistance. The most frequently occurring on-target resistance mechanism against osimertinib is the C797S mutation, which has urged the development of fourth-generation EGFR inhibitors. To enable the profiling of novel EGFR inhibitors and drug combinations on C797S-mutant EGFR models, the mutation was introduced in NCI-H1975 cells via gene-editing with CRISPR/Cas9. NCI-H1975 is an NSCLC cell line harboring the L858R activating mutation and T790M resistance mutation. The cell line is resistant to first-generation EGFR inhibitors such as gefitinib, but remains sensitive to osimertinib. NCI-H1975 cells were transfected with a homology-directed template containing the C797S mutation and were edited via CRISPR/Cas9. After confirmation that the mutation was introduced in a significant number of cells by DNA sequencing (mutation frequency: 28%), the cell pool was single-cell sorted followed by expansion and analysis of single cell clones. Three cell clones were characterized in detail using whole exome sequencing, quantitative analysis of EGFR surface expression using Quantibrite beads (BD Biosciences), and cell viability assays using ATP as readout in 2D culture and 3D spheroids. Whole exome sequencing confirmed the presence of the C797S mutation in the three clones. Quantification of cell surface EGFR expression levels revealed two times higher EGFR expression in the clones compared to the parental cell line. In 2D cell viability assays, the C797S-mutant clones were 316-fold less sensitive to osimertinib compared to the parental cell line. In contrast, the fourth-generation EGFR inhibitors BLU-945 and JBJ-09-063 reduced viability of the parental and C797S-mutant clones with similar potency. While potencies in the clones were similar between 2D culture and 3D spheroids, BLU-945 and JBJ-09-063 reached a higher maximum effect in 3D spheroids compared to 2D culture. The successful generation and characterization of the NCI-H1975 C797S-mutant clones provides a valuable in vitro model in which additional next-generation EGFR inhibitors and drug combinations can be profiled to identify therapeutic strategies to overcome osimertinib resistance. Citation Format: Janneke J. Melis, Kirsten J. Kevenaar, Jonne van Eck, Janneke M. van den End, Yvonne Grobben, Jeffrey J. Kooijman, Guido J. Zaman. Characterization of EGFR C797S-mutant, osimertinib-resistant non-small cell lung cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 6961.