Screening And Identification Of Osimertinib Resistance Gene In Non-small Cell Lung Cancer By CRISPR/Cas9 Library

Background: Epidermal growth factor receptor tyrosine kinase inhibitor(EGFR-TKI)provides significant antitumor activity in advanced non-small cell lung cancer patients with active EGFR mutations.The third generation of EGFR-TKI osimertinib was developed to overcome the T790 M mutation,the most common resistance mechanism of EGFR-TKI.The clinical response rate was 59%~71%.However,over time,osimertinib,as the third generation of EGFR-TKI targeting anti-cancer drug,inevitably produces drug resistance,and some mechanisms of third generation EGFR-TKI resistance have been reported in the literature.Nevertheless,the mechanism of acquired resistance to third-generation EGFR-TKI has not been fully understood so far.Therefore,efficient and rapid screening of the potential resistance genes of osimertinib will provide theoretical basis for further elucidation of the mechanism of acquired resistance to third-generation EGFR-TKI,design of new targeted drugs or clinical combined use of targeted drugs.Objective: To screen the potential resistance genes of osimertinib in non-small cell lung cancer cell line NCI-H1975,and to further study the function and mechanism of the selected genes,so as to provide a theoretical basis for further elucidating the acquired resistance mechanism of the third generation of EGFR-TKI.Methods: CRISPR/Cas9 genomic library plasmid was used to amplify and package lentivirus.H1975 cells were infected after testing the viral titer(MOI < 0.3).After screening for purinomycin resistance,NCL-H1975 cells were screened by osimertinib.Survival cells were collected,and candidate genes corresponding to sgRNA were obtained by genome sequencing and bioinformatics analysis.Candidate genes were knocked out from cells and potential drug-resistant cell lines were established.Western-blot was used to detect the expression of EGFR and apoptotic signaling pathways such as c-PARP,AKT and ERK downstream of EGFR.CCK-8 cell growth experimnt and clone formation experiment were used to verify the effect of knockout candidate gene cell lines on oxetine resistance.Results: Throughout the experiment,we successfully expanded the CRISPR/Cas 9 human genomic libraries.The A libraries coverage rate were 653 %,and the B libraries were 408 %.The MOI value of our infected NCI-H1975 cells was controlled at 0.255.We got 12 potentially resistant genes from genes sequencing analysis with screened cell after libraries' Lentivirus infection and osimertinib screening,and selected the FDX1 gene for functional identification.When the gene FDX1 is knocked out in NCI-H1975 cells,The sequencing results showed thatFDX1 gene was absent,and the protein expression level of FDX1 was significantly inhibited by Western blot.The level of c-PARP expression was significantly different in sg-AAVS1 and sgFDX1 cells(P<0.01);The inhibition of osimertinib on p-EGFR,p-AKT,p-ERK was reduced after FDX1 knocked out(P<0.01).Cloning formation and CCK8 experiments showed that osimertinib inhibition on cloning formation rate and proliferation rate activity was decreased significantly on sg-FDX1 cells compared with sg-AAVS1 cells(P<0.05).Conclusion: This study successfully established a screening platform forCRISPR/Cas9-human genomic library and screened 12 potentially resistant genes of the third-generation EGFR-TKI drug osimertinib.The functional identification of FDX1,one of the potential drug-resistant genes,screened from the 12 potentially resistant genes showed that knocking out the FDX1 gene in NCI-H 1975 cells mayberesistanttothethirdgenerationofNSCLCtargeteddrugosimertinib.