{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,9,28]],"date-time":"2026-09-28T21:37:05Z","timestamp":1790631425646,"version":"4.1.0"},"reference-count":50,"publisher":"MDPI AG","issue":"13","license":[{"start":{"date-parts":[[2025,6,26]],"date-time":"2025-06-26T00:00:00Z","timestamp":1750896000000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"name":"Merit Award from the Veterans Administration","award":["BX0003368"],"award-info":[{"award-number":["BX0003368"]}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["IJMS"],"abstract":"<jats:p>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\u2019 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).<\/jats:p>","DOI":"10.3390\/ijms26136157","type":"journal-article","created":{"date-parts":[[2025,6,27]],"date-time":"2025-06-27T03:33:25Z","timestamp":1750995205000},"page":"6157","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":2,"title":["Evaluating the Effectiveness of Tyrosine Kinase Inhibitors on EGFR Mutations In Vitro"],"prefix":"10.3390","volume":"26","author":[{"given":"Hanshuang","family":"Shao","sequence":"first","affiliation":[{"name":"Department of Pathology, University of Pittsburgh, Pittsburgh, PA 15213, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-1637-8150","authenticated-orcid":false,"given":"Alan","family":"Wells","sequence":"additional","affiliation":[{"name":"Department of Pathology, University of Pittsburgh, Pittsburgh, PA 15213, USA"},{"name":"Pittsburgh VA Health System, Pittsburgh, PA 15213, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2025,6,26]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"S21","DOI":"10.1016\/j.ijrobp.2003.11.041","article-title":"Review of epidermal growth factor receptor biology","volume":"59","author":"Herbst","year":"2004","journal-title":"Int. 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