Resistance Mechanisms Of EGFR Small-molecule Inhibitors And Discovery Of PD-L1 Small-molecule Inhibitors

Lung cancer is the leading cause of cancer death worldwide,and non-small cell lung carcinoma(NSCLC)is the most common malignant tumor in lung cancer,accounting for 80-85% of lung cancer patients.At present,only a small number of patients with NSCLC can be diagnosed in the early stage(stage I or II)and can be treated by surgical resection.More than 60% of patients with lung cancer are locally advanced or metastatic(stage III or IV)at the time of diagnosis,which can not be completely resected.For patients who can not be completely resected,they can only be treated by chemotherapy or radiotherapy.In recent years,with the development of tyrosine kinase inhibitor(TKIs)and immunotherapy for lung cancer,great progress has been made in the treatment of lung cancer.However,tumor drug resistance is inevitable in both molecular targeted therapy and immunotherapy for lung cancer.therefore,it is of great clinical significance to study the mechanism of drug resistance in lung cancer,find overcoming strategies,or develop new immunotherapy methods.This doctoral thesis will focus on the resistance mechanism and overcoming strategies of small molecule inhibitors of epidermal growth factor receptor(EGFR),as well as the discovery of small molecule PD-L1 inhibitors.The first part is studying the mechanism of drug resistance of EGFR small molecular inhibitors and to find new strategies to overcome drug resistance.EGFR promotes tumorigenesis and development,and is an important target for the treatment of NSCLC.Tyrosine kinase inhibitors targeting EGFR(EGFR-TKIs)play an important role in the clinical treatment of NSCLC.AZD9291(osimertinib)is the first thirdgeneration EGFR-TKI,approved by FDA in the United States to effectively treat patients with T790 M drug resistance mutations in EGFR,but long-term use of AZD9291 will also produce drug resistance.therefore,it is of great clinical significance to study the mechanism of AZD9291 drug resistance and its overcoming strategies.We successfully established PC-9/gefitinib,PC-9/afatinib,HCC827/gefitinib,HCC827/afatinib and H1975/AZD9291 drug-resistant cell lines by continuously increasing the concentration of the first,second and third generation EGFR-TKIs,and selected several monoclonal drug-resistant cell lines by limited dilution method.These monoclonal drug-resistant cell lines which are resistant to gefitinib and afatinib have cross-resistance to AZD9291,western blot experiments show that there is no loss of EGFR in the drug-resistant cell lines,which is suitable to be used as a model cell line to study the mechanism of drug resistance.The downstream signal of drug resistant cells is independent of EGFR with the high expression of c-Met and P-c-Met,suggesting that the mechanism of drug resistance may be related to the activation of cMet.However,although crizotinib,an inhibitor of c-Met,cooperates with AZD9291 to inhibit the phosphorylation of ERK1/2 and AKT mediated by c-Met,it can only partially restore the sensitivity of resistant cells with overexpressing P-c-Met to AZD9291,and anti-c-Met monoclonal antibodies can not reverse the resistance of drugresistant cells to AZD9291,suggesting that it is necessary to find new strategies to overcome the resistance in drug-resistant cells with high expression of c-Met.Further studies showed that SHR-A1403,a novel antibody coupling drug targeting c-Met(antibody-drug conjugate,ADC),could effectively inhibit the proliferation of drugresistant cells overexpressing c-Met,and the proliferation inhibitory activity was not related to the level of c-Met phosphorylation.The results suggest that overcoming drug resistance with ADC targeting c-Met is a new therapeutic strategy for c-Met-mediated drug resistance to AZD9291.In addition,we found that the expression level of c-Met is a biomarker to predict the efficacy of c-Met ADC(such as SHR-A1403).The drug resistance model of EGFR L858R/T790 M was established by using AZD9291 mutant cells.It was found that YES and c-src,members of src family kinase(Src family kinases,SFKs)were highly expressed in drug-resistant cell lines.SFKs inhibitor dasatinib could effectively reverse the drug resistance of drug-resistant cells with high expression of SFKs.The results suggest that the inhibition of both SFKs and EGFR pathways may be an effective strategy to overcome drug resistance in the third generation of EGFR inhibitors such as AZD9291 resistant patients.Finally,we constructed a variety of cell models of drug resistance to AZD9291,including EGFR cis/trans-C797 S and EGFR exon 20 insertion mutation models,which are of great value for studying the resistance mechanism of EGFR small molecule inhibitors and finding new therapeutic strategies.The second part is the screening of PD-L1 small molecule inhibitors.Anti-PD-L1 monoclonal antibody is one of the most important drugs in immunotherapy of lung cancer,but it has some disadvantages,such as complex production process,high production cost,immunogenicity,relatively poor stability,inconvenient administration and so on.Therefore,it is very meaningful to find small molecule inhibitors of PD-L1 with simple production,high efficiency,low cost and good stability.In this paper,we constructed a high-throughput PD-L1 small molecule inhibitor screening model,effectively screened PD-L1 small molecule inhibitors at protein,cell and in vivo animal levels,and found a number of active PD-L1 small molecule inhibitors,which are expected to be developed into new anti-tumor drugs with independent intellectual property rights.