Susceptibility Analysis Of Tyrosine Kinase Inhibitors To EGFR^S768I And EGFR^S768i+L858R Mutations In Non-small Cell Lung Cancer

Lung cancer is the malignant tumor with the highest mortality rate and the second-highest incidence rate worldwide.Lung cancer is divided into small-cell lung cancer（SCLC）and non-small cell lung cancer（NSCLC）.Current domestic and international research data shows that a significant proportion of epidermal growth factor receptor（EGFR）mutations occur in patients with non-small cell lung cancer,with EGFR mutations mainly concentrated in exons 18-21,the tyrosine kinase functional region.Exon 21 L858R point mutation and exon 19 deletions are classical EGFR mutations,while exon 20 S768I point mutation is a rare EGFR mutation.The previous clinical treatment plans for lung cancer patients included surgical treatment,chemotherapy,and radiation therapy.Chemotherapy and radiation therapy can improve the patient’s survival to a certain extent when the patient’s condition is intolerable to surgical treatment or when postoperative pathology suggests cancer metastasis.With the further development of clinical research,targeted therapy has become more mature.Targeted therapy targeting mutation points that undergo cancer transformation has more precise treatment characteristics and is the choice of most lung cancer patients.Tyrosine kinase inhibitors（TKIs）are considered to be targeted therapeutics for non-small cell lung cancer with EGFR mutations.There are limited data on the sensitivity of EGFR tyrosine kinase inhibitors（EGFR-TKIs）to the rare EGFR^S768I mutation,and it is more challenging to investigate the sensitivity of EGFR-TKIs to the compound mutation EGFR^S768I+L858R.Therefore,this study selected six representative tyrosine kinase inhibitors currently listed in China:Afatinib,Dacomitinib,Osimertinib,Erlotinib,Gefitinib,and Icotinib,and combined with clinical pharmacokinetics studies,selected stable blood concentration for sensitivity analysis on rare non-small cell lung cancer with EGFR^S768I and EGFR^S768I+L858R mutations.PurposeThe aim of this study was to analyze the sensitivity of six tyrosine kinase inhibitors to rare EGFR^S768I and EGFR^S768I+L858R mutations,and to provide a preliminary analysis of their mechanism of action for the refinement of clinical non-small cell lung cancer treatment.Methods（1）The human lung cancer cell line H3255(with EGFR^L858R mutation in the background)was used as the basis for the construction of stable transgenic cell lines H3255^S768I(EGFR^S768I)and H3255^S768I+L858R(EGFR^S768I+L858R)using CRISPR/Cas9 gene editing,lentiviral packaging,and transfection,after screening with insecticide and puromycin.（2）Using MTT,plate cloning,and Ed U cell proliferation experiments,the effects of six tyrosine kinase inhibitors on the viability and proliferation of H3255(EGFR^L858R),H3255^S768I(EGFR^S768I),and H3255^S768I+L858R(EGFR^S768I+L858R)cell lines were studied;The apoptosis effects of six tyrosine kinase inhibitors on H3255,H3255^S768I and H3255^S768I+L858R cell lines were further studied by flow cytometry,mitochondrial membrane potential and apoptosis experiments,and Caspase-3 activity detection.（3）Western blot was used to detect the regulation of Bad,Bcl-XL,HO-1 apoptotic genes and changes in downstream signaling pathways such as p-EGFR,EGFR,p-AKT,AKT,p-ERK,and ERK in three cell lines after treatment with six tyrosine kinase inhibitors.Results（1）The transfection rates were 88.7%for H3255^S768I and 89.4%for H3255^S768I+L858R as verified by flow cytometry,and 90.8%for H3255^S768I and 92.3%for H3255^S768I+L858R as verified by fluorescence microscopy photography.（2）The MTT results showed that six tyrosine kinase inhibitors inhibited the cell viability of H3255,H3255^S768I,and H3255^S768I+L858R cell lines,and the inhibitory effect became more significant with the prolongation of drug action time（P<0.0001）;The results of the plate clone formation experiment showed that after treatment with tyrosine kinase inhibitors,the number of cell colonies significantly decreased（P<0.0001）;The Ed U cell proliferation experiment results showed that in the three cell lines,compared with the control group,the proportion of cell proliferation in the tyrosine kinase inhibitor group was significantly reduced（P<0.0001）;The results of flow cytometry,mitochondrial membrane potential and apoptosis detection showed that six tyrosine kinase inhibitors promoted cell apoptosis in three cell lines（P<0.0001）;The caspase-3 activity detection results showed that in the three cell lines,compared with the control group,the tyrosine kinase inhibitor group activated caspase-3 activity,further promoting cell apoptosis（P<0.0001）.（3）Western blot further confirmed that six tyrosine kinase inhibitors can promote apoptosis in all three cell lines in the apoptosis related protein detection experiment;In the downstream related signal molecule protein detection results,six tyrosine kinase inhibitors exhibit different signaling pathways for the same mutation,and their regulation of downstream signals also varies among different mutations.SummarySix tyrosine kinase inhibitors are sensitive to rare EGFR^S768I and EGFR^S768I+L858Rmutations.Six tyrosine kinase inhibitors promote cell apoptosis by inhibiting cell viability,cell proliferation and cloning ability,but exert their anti-tumor effect by regulating different downstream signal pathways.For patients with EGFR^S768I mutations and compound mutations EGFR^S768I+L858R,Six first-line TKIs are reasonable treatment options.