# Evaluating the Effectiveness of Tyrosine Kinase Inhibitors on EGFR Mutations In Vitro

## TL;DR

This study tests how different EGFR mutations respond to tyrosine kinase inhibitors in the lab, helping to understand better treatment strategies for cancer patients.

## Contribution

The study evaluates the effectiveness of multiple generations of TKIs against various EGFR mutations in vitro, revealing differential drug responses.

## Key findings

- First-generation TKI erlotinib partially inhibits T790M-containing EGFR mutants.
- Third-generation osimertinib effectively inhibits most mutants but fails against T790M/C797S combinations.
- Fourth-generation EAI045 shows broad inhibition but partial effectiveness in quiescent cells.

## Abstract

Abnormal expressions and genetic mutations of EGFR are broadly involved in the progression of many human solid tumors, which has led to the development of small molecule inhibitors (TKIs). However, patients’ tumors usually develop resistance to targeted therapeutic TKIs after a period of treatment, mostly due to secondary mutations in EGFR. In this study, we constructed a series of EGFR mutants and assessed their responses to clinical TKIs in vitro. We found that WT and mutant EGFRs responded differently to clinical TKIs. These findings provide some insights into how patients carrying typical mutations should be correctly and efficiently treated and why patients present side effects.

Abnormal expressions and genetic mutations of EGFR are broadly involved in the progression of many human solid tumors, which has led to the development of small molecule inhibitors (TKIs). However, patients’ tumors usually develop resistance to targeted therapeutic TKIs after a period of treatment, mostly due to secondary mutations in EGFR. To date, three major and prevalent point mutations in EGFR, including L858R, T790M, and C797S, impact the use of TKIs in non-small cell lung cancer patients. Although at least four generations of TKIs have been designed and developed by targeting these mutations, how each mono, dual, or triple variant responds to clinical TKIs remains largely undeciphered. To fill this gap, we constructed a series of EGFR mutants and assessed their responses to clinical TKIs in vitro. The first-generation TKI, erlotinib, completely blocked the autophosphorylation of WT, L858R, C797S, and C797S/L858R, but only partially, if at all, in EGFR containing the T790M mutation alone or in combination. The third generation, osimertinib, completely abolished the autophosphorylation of WT, T790M, L858R, and T790M/L858R. It also significantly inhibited C797S and C790S/L858R, but had no effect on T790M/C797S or T790M/C797S/L858R. EAI045, as the fourth-generation TKI, almost completely inhibited WT and all mutants in complete growth media, but EGF-mediated phosphorylation of WT, C797S, and C797S/L858R were only partially inhibited in quiescence media, while the other mutants were fully inhibited. Furthermore, the abolishment of the enhanced tolerance to Dox in cells transiently expressing T790M/L858R and T790M/C797S/L858R by EAI045 suggests that their enhanced autophosphorylation is involved in their resistant ability. These findings provide some insights into how patients carrying typical mutations should be correctly and efficiently treated and why patients present side effects (because of non-specific inhibitory effects on cells without EGFR mutations).