The Mechanism Study Of DNA Damage Response In EGFR-TKI-acquired Drug-resistant NSCLC

Objective: EGFR Tyrosine kinase inhibitor inhibitors represented by gefitinib and Osimertinib have brought innovative changes in the treatment of lung cancer many years after being approved of listing.However,the acquired resistance of targeted drugs has also become an inevitable problem.In addition to continuously developing next-generation new drugs based on drug resistance mechanisms,circumventing drug resistance by regulating the bypass signal transduction mechanism has become a new treatment method.The genome is often stimulated by internal and external environments to cause DNA damage and cause genome instability,cells will also relatively activate a series of damage response mechanisms including monitoring of damage,DNA damage repair,cell cycle regulation,and chromatin structure changes to combat DNA damage.The relationship between DNA damage response and EGFR-TKI on the growth and resistance of non-small cell lung cancer has not yet been elucidated.The objective of this study includs:1.Explore the abnormal changes in response to DNA damage in non-small cell lung cancer cells that have acquired resistance to Osimertinib.2.Explore the changes of L3MBTL1 in the acquired resistance of Osimertinib and its relationship with cell growth and resistance.3.Explore the relationship between L3MBTL1 and DNA damage response and explore the mechanism of reversing EGFR resistance.Methods: First of all,Western Blot,qPCR-PCR,Comet assay,NHEJ/HR assay and Immunofluorescence experiment were used to observe the two pairs of cell lines which sensitized and acquired resistance of Osimertinib in the laboratory to observe the difference of DNA damage and repair pathway.The difference in the level of oxygen free radicals were observed by the oxygen free radical detection assay.Chromatin structure differences were detected by Micrococcal Nuclease assay.Secondly,qPCR-PCR and Western Blot were used to detect the expression difference of L3MBTL1 in Osimertinib-sensitive cells H1975 and acquired drug-resistant cells H1975/AR cells.The immunoprecipitation was used to detect the differences in the ubiquitination level of L3MBTL1 between two cell lines.The difference of RNF8 expression was observed by Western Blot.Through,the relationship between L3MBTL1 and the abnormal changes of cell chromatin structure in drug-resistant cell lines,and its relationship with histone modification binding were determined by Micrococcal nuclease assay and Immunofluorescence.Chromatin immunoprecipitation high-throughput sequencing(CHIP-seq)was to explored the distribution and enrichment pathways of L3MBTL1 in the genome.In vitro,the changes in the proliferation,invasion,apoptosis and cell viability of L3MBTL1 knockdown were detected by Colony formation assay,Transwell assay,Cell apoptosis assay and CCK-8 assay.In vivo,the changes of L3MBTL1 on tumor growth rate,weight and volume were detected by tumor xenografts,and the biomarker of proliferation were detected by immunohistochemistry.Finally,the transcriptome sequencing technology(RNA-seq)was used to examine cells which included control group and L3MBTL1 knockdown group,pathway analysis was used to explore the correlation between L3MBTL1 and the DNA damage response mechanism;RNA-seq differential genes combined with Peaks-related genes in the L3MBTL1 promoter region of CHIP-seq were used to explore and verify the key mechanisms of L3MBTL1 affecting cell growth and drug resistance.Results: This study found that compared with sensitive cell lines,DNA damage was reduced after drug-resistant cell lines were treated with Osimertinib for 24 hours.After studying the mechanism that causes DNA damage,it is found that compared with sensitive cells,the level of oxygen free radicals is lower in drug-resistant cells,which makes them more resistant to oxidative stress.Detection of DNA damage repair pathways revealed that the non-homologous end joining(NHEJ)pathway was abnormally activated in drug-resistant cells.Compared with sensitive cells,drug-resistant cells have a denser chromatin structure after being treated with Osimertinib for 24 hours,which makes genomic DNA less sensitive to damage and faster DNA damage repair.In Osimertinib-sensitive cells H1975 and acquired drug-resistant cells H1975/AR cells,the expression of L3MBTL1 was abnormally increased,and the expression was stable after 24 hours of Osimertinib treatment.The immunoprecipitation found that the ubiquitination level of L3MBTL1 in drug-resistant cells decreased through the proteasome pathway after Osimertinib treatment.It was also observed that the expression level of RNF8,which mediates the ubiquitination of L3MBTL1,decreased after treatment in drug-resistant cells,thereby reducing the ubiquitination of L3MBTL1 by Western Blot.After overexpression and knockdown of L3MBTL1,Micrococcal nuclease assay was used to clarify that L3MBTL1 is involved in the abnormal compaction of chromatin in EGFR-TKI acquired resistance,it also was proved that L3MBTL1 binding H4K20Me2 to participates in chromatin compaction in drug-resistant cells by competing with 53BP1.The Kyoto Encyclopedia of Genes and Genomics(KEGG)analysis of 4782 genes related to Peaks in the L3MBTL1 promoter region using CHIP-seq suggests that Peaks-related genes in the L3MBTL1 promoter region are rich in EGFR-TKI resistance pathway.In the phenotypic experiment,cell proliferation and invasion decreased and apoptosis increased after knocking down L3MBTL1,and knocking down L3MBTL1 combined with Osimertinib played a synergistic effect.Si RNA,sh RNA and MBT domain inhibitor UNC669 were used to knock down L3MBTL1 combined with Osimertinib produced a combined inhibition of H1975/AR cell viability,and overexpression of L3MBTL1 enhanced the resistance of sensitive cells H1975 to Osimertinib.Tumor xenografts assay in nude mice proved that after knocking down L3MBTL1 combined with Osimertinib therapy,compared with knockdown L3MBTL1 alone and Osimertinib alone,the rate of tumor shrinkage,weight reduction and volume percentage were more obvious.Transcriptome sequencing technology(RNA-seq)was performed on the L3MBTL1 knockdown cell line,and the Gene Set Enrichment Analysis(GSEA)of the changed genes showed that the enrichment was in DNA damage response related pathways included homologous recombination,nucleotide excision repair,mismatch repair and cell apoptosis.L3MBTL1 knockdown combined with Osimertinib treatment increased DNA damage than L3MBTL1 knockdown alone and Osimertinib treatment alone were detected by Comet assay,Immunofluorescence,and Western Blot.Combining differential genes with reduced expression in RNA-seq with Peaks-related genes in the L3MBTL1 promoter region of CHIP-seq,and at the same time combining EGFR-TKI resistance pathway Peaks-related genes,PI3 K regulatory subunit PIK3R3 was screened as the target gene of L3MBTL1.The phosphorylation of PI3 K p55γ is inhibited after L3MBTL1 knockdown,and the combination of osimertinib has a synergistic effect detected by Western Blot.It is proved that L3MBTL1 inhibiting tumor growth and drug resistance by targeting PIK3R3.Conclusion: 1.Non-small cell lung cancer EGFR-TKI acquired resistance is associated with abnormal DNA damage response.2.L3MBTL1 plays a key role in the growth and resistance of non-small cell lung cancer by regulating chromatin structure and targeting EGFR-TKI resistance pathway genes.3.L3MBTL1 knockdown promotes DNA damage to increase cell apoptosis,and at the same time acts on the PI3 K regulatory subunit PIK3R3,thereby affecting the growth and resistance of EGFR-TKI to non-small cell lung cancer.