Functional Research Of Resistance Mutations To EGFR Or MET Tyrosine Kinase Inhibitors In Lung Adenocarcinoma Patients

BackgroundThe epidermal growth factor receptor(EGFR)tyrosine kinase inhibitors(TKIs)raise the first-line therapy median progression free survival(mPFS)of lung adenocarcinoma patients with activating EGFR mutations to 14 months.However,drug resistance is inevitable,and over half of resistant patients acquired EGFR T790M mutation in exon 20.The second generation EGFR TKIs such as afatinib and dacomitinib show higher potency to EGFR activating mutations,but not the acquired T790M mutation.Then the third generation EGFR TKIs osimertinib got an mPFS of 9.9-12.3 months in treated patients with acquired T790M mutations.What a pity is that,resistance to osimertinib is also unavoidable:the mechanisms are quite complicated and still need further exploration.Mesenchymal to epithelial transition factor(MET)amplification,as an acquired resistant mechanism to EGFR TKIs,can also be an oncogene alteration in lung cancer patients.Crizotinib has been approved as an inhibitor to anaplastic lymphoma kinase(ALK)rearrangement,but it was originally developed as a kind of MET inhibitors.NCCN guideline has recommended patients with high-level MET amplification and MET exon 14 skipping mutations to take crizotinib.Except crizotinib,there are many MET TKIs under research,such as INC280,XL 184 BMS777607.However,we still known little about the resistance mechanisms to these MET TKIs.MethodsThe liquid biopsy and next generation sequencing(NGS)help us detect novel mutations in patients who resistant to EGFR TKIs or MET TKIs.Then we used the lentivirus stably infected tool cells(NIH-3T3 and Ba/F3)and subcutaneous in vivo models to further explore the function of these novel mutations and screen the possible drugs to overcome them.And we also use the computer to analyze the effects of mutations on the binding of EGFR protein and osimertinib.ResultsIn the first section,two novel tertiary EGFR mutations L792H(c.T2375A)and G796R(c.G2386C)in cis with T790M but in trans with each other respectively were identified by cfDNA NGS testing in a patient who progressed on osimertinib treatment after initial response.Mouse Ba/F3 cells and NIH-3T3 cells stably expressing L858R/T790M mutation(in cis)either with the L792H or G796R mutation were generated as the cell models to further investigate the impact of the two novel mutations on EGFR TKIs in vitro.And we found the introduction of L792H and G796R mutations into the background of EGFR L858R/T790M resulted in significant loss of cellular sensitivity to EGFR TKIs now approved and many other drugs combinations.Structural modelling showed that mutations in cis with T790M either forced the ligand(osimertinib)to rotate out(breaking binding)or pulled the hinge loop(breaking the hinge).In the second section,we explored the effect of mouse MET D1226N and Y1228H mutations to multiple MET TKIs base on the former researches of our team.We successfully constructed the subcutaneous tumors model in nude mice by inoculated mouse MET D1226N and Y1228H stablely transfected NIH-3T3 cells,and tested MET TKIs.And we found that these two novel mutations resistant to INC280 and crizotinib but sensitive to XL184.ConclusionsWe firstly identified the two novel EGFR mutations,G796R and L792H,which,when in cis with T790M,confer resistance to the third-generation EGFR TKI osimertinib both in vitro and in silico.Moreover,we also found XL 184 can overcome the MET mutations D1226N and Y1228H in vitro and in vivo,which provide effect regimen for patients with these mutations.