| Background and ObjectiveLung cancer is one of the malignant tumor with the highest tumor-related mortality.Non-small cell lung cancer(NSCLC)accounts for about 80-85% of all lung cancer types.With the in-depth study of molecular diagnostic techniques,the therapeutic mode of advanced NSCLC patients with driver genes has turned to targeted therapy based on the type of driver genes.The echinoderm microtubule associated like4-anaplastic lymphoma kinase(EML4-ALK)fusion gene is a type of lung cancer driver gene,accounts for 3%-5% of NSCLC.The PROFILE 1014 study showed that the first-generation anaplastic lymphoma kinase tyrosine kinase inhibitor(ALK-TKI)Crizotinib was used to treat patients with EML4-ALK fusion gene-positive NSCLC with an objective response rate(ORR)of up to 74%.This confirmed the status of crizotinib as the first-line treatment for ALK-positive NSCLC patients.But most patients progressed after about 11 months of treatment.Secondary mutations in the ALK kinase domain are one of the major resistance mechanisms ofCrizotinib.The second and third generation ALK-TKIs can overcome most of the secondary mutations caused by Crizotinib resistance.In particular,the third generation of ALK-TKI Lorlatinib,a novel,highly selective and efficient TKI,has good antitumor activity against all Crizotinib resistant mutations including G1202 R.Previous studies have shown that although Lorlatinib was used in ALK-positive NSCLC patients who were resistant to an ALK-TKI,the ORR was 57%.However,patients also developed acquired resistance after about 14 months of treatment.There are two major clinical problems in the treatment of patients with advanced ALK-positive NSCLC.First,the exact resistance mechanisms of the third-generation ALK-TKI Lorlatinib have not yet been elucidated.And after Lorlatinib resistance,the next generation of ALK-TKI is lacking.Second,chemotherapy is still one of the main choices for ALK-positive NSCLC patients who are Lorlatinib resistance,but there are few studies on the sensitivity of Lorlatinib-resistant ALK-positive NSCLC patients to different chemotherapy drugs.Therefore,to explore the resistance mechanisms of Lorlatinib,the selection of appropriate chemotherapy drugs are important issues in clinical practice.Based on the above background,the EML4-ALK fusion gene-positive,Crizotinib-resistant NSCLC cell SNU-2535 was selected,and the Lolattinib acquired resistant cell line SNU-2535 LR was established by increasing the dose of Lorlatinib.On this basis,the resistance mechanisms of Lorlatinib,the treatment strategies to overcome Lorlatinib resistance and the sensitivity of Lorlatinib-resistant cells to different chemotherapy drugs were further explored.In order to provide theoretical guidance for the continued treatments of clinical Lorlatinib-resistant ALK-positive NSCLC patients.This study has important clinical implications.Methods1.The establishment of Lorlatinib acquired resistant cell line of NSCLC: The Lorlatinib-resistant cell SNU-3535 LR was induced by increasing dose of Lorlatinib to SNU-2535 cell.The gene mutations of SNU-2535 cell were detected by next-generation sequencing(NGS).The resistance of SNU-2535 cell to Crizotinib and the resistance of SNU-2535 LR cell to Lorlatinib were detected by CCK-8 assay.The cell growth curves were drawn by CCK-8 assay.The cell cycles distribution were detected by flow cytometry.The morphological changes were observed by inverted microscope.The expressions of epithelial-mesenchymal transition(EMT)marker proteins were detected by Western blot.2.The exploring of resistance mechanisms of SNU-2535 LR cells and the sensitivity of different chemotherapy drugs after Lorlatinib resistance: The gene mutations of SNU-2535 LR cell were detected byNGS.The sensitivity of SNU-2535 LR cell to Crizotinib and the reversal effect of APR-246 on Lorlatinib-induced resistance and the sensitivity of chemotherapy drugs in SNU-2535 LR cell were detected by CCK-8 assay.The expressions of EML4-ALK fusion gene-related proteins were detected by Western blot.3.Statistical analysis: The experimental results were drawn by Graphpad Prism 7.0.The experimental datas were statistically analyzed by SPSS 20.0.All statistics were expressed as mean±standard deviation(mean±SD).The comparation of datas between two groups was used by t-test.P < 0.05 was considered to indicate that the difference was statistically significant.Results1.The establishment of Lorlatinib acquired resistant cell line of NSCLC1)The results of NGS showed that EML4-ALK fusion gene and Crizotinib resistance mutation G1269 A were positive of SNU-2535 cells.The CCK-8 results showed that the sensitivity of SNU-2535 cells to Crizotinib was worse than NCI-H3122 cells,P < 0.05.The IC50 values were(1.211 ± 0.033)and(0.187 ± 0.007)?mol/L,respectively.The resistance index(RI)was 6.476.The above results confirmed that SNU-2535 is a Crizotinib-resistant cell.2)The acquired Lorlatinib-resistant cell line SNU-2535 LR was constructed in vitro for 7 months.Compared with SNU-2535 cells,SNU-2535 LR cells were insensitive to lorlatinib,P<0.05.The IC50 values were(46.433±9.562)and(1.362±0.324)?mol/L,respectively.The resistance index was 34.092,indicating that the successful establishment of Lorlatinib-resistant cell line.3)Compared with SNU-2535 cells,the volume of SNU-2535 LR cells were smaller,the cellular antennas were increased,and the growth was relatively scattered.4)Western blot results showed that the expression of E-cadherin protein was decreased(p <0.05),the expression of Vimentin protein was significantly decreased(p <0.01),there is no difference in the expression of N-cadhrein protein(P> 0.05).It indicated that SNU-2535 LR cells did not undergo EMT phenotypic changes.5)The cell doubling time of SNU-2535 cells is about 3.425 d,and the doubling time of SNU-2535 LR cells is about 1.963 d.Compared with SNU-2535 cells,the proportion of SNU-2535 LR cells in G1 phase decreased(P<0.05).The proportion of G2 + S phase increased(P < 0.05).It indicated that the SNU-2535 LR cells have stronger proliferation ability.2.The exploring of resistance mechanisms of SNU-2535 LR cells and the sensitivity of different chemotherapy drugs after Lorlatinib resistance:1)The results of NGS showed that compared with SNU-2535 cells,the abundance of EML4-ALK fusion gene in SNU-2535 LR cells wassignificantly decreased and the G1269 A mutation was disappeared.The most important mutation of SNU-2535 LR cells was TP53,and the abundance was close to 100%.No other gene mutations and fusion gene amplification were found in the ALK kinase domain.2)The Western blot results showed that the expressions of EML4-ALK fusion gene-related proteins decreased in SNU-2535 LR cells,p<0.05.3)The CCK-8 assay results showed that SNU-2535 LR cells were insensitive to Crizotinib compared with SNU-2535 cells,P<0.05.The IC50 values were(4.726 ± 1.056)and(1.211 ± 0.033)?mol/L,respectively.It is suggested that the sensitivity of Crizotinib was not restored after the disappearance of Crizotinib resistance mutation G1269 A in SNU-2535 LR cells.4)The CCK-8 assay results showed that the combination of P53 protein activator APR-246 and Lorlatinib could partially reverse Lorlatinib-induced resistance,and the reversal multiple was 4.768.It is suggested that the combined TP53 mutation may be one of the mechanisms of SNU-2535 LR cell resistance.5)SNU-2535 LR cells were more sensitive to gemcitabine,docetaxel and paclitaxel than cisplatin and pemetrexed(both P<0.05).Compared with SNU-2535 cells,the sensitivities of SNU-2535 LR cells to gemcitabine,docetaxel and paclitaxel were significantly increased(bothP<0.05).ConclusionSuccessfully established NSCLC Lorlatinib acquired Lorlatinib-resistant cell SNU-2535 LR for the first time based on Crizotinib-resistant.The combination of p53 protein activator APR-246 and Lorlatinib could partially reverse Lorlatinib-induced resistance to SNU-2535 LR cell and the TP53 mutation maybe its possible mechanisms.Lorlatinib-resistant cells SNU-2535 LR which we had constructed were more sensitive to gemcitabine,docetaxel and paclitaxel than cisplatin and pemetrexed. |
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