The Anti-tumor Effect Of A Novel ALK/ROS1 Dual Target Inhibitors ZX-29 In Vitro And The Mechanism Of Drug Resistance Result From By ROS1 Mutation

Anaplastic lymphoma kinase(ALK)and c-ros oncogene 1(ROS1)are the members of the receptor tyrosine kinase family,which have been identified as oncogene drivers of non-small-cell lung cancer(NSCLC).Although crizotinib,a small molecule inhibitor for ALK,ROS1 and c-MET,has a prominent effect on ALK or ROS1 NSCLC,like other targeted drugs,resistance is inevitable after treatment about 1 year.It is necessary to develop new ALK/ROS1 inhibitors and study the mechanism of drug resistance.Through screening a series of novel ALK/ROS1 double-target inhibitors,we selected the inhibitor ZX-29 for further study.We studied the anti-tumor effect of ZX-29 on the echinoderms microtubule associated protein to raise 4-anaplastic lymphoma kinase(EML4-ALK)positive NCI-H2228 cells and SLC34A2-ROS1 positive HCC78 cells by the methods of MTT,AO/EB staining,flow cytometry and protein immunoblot technology.The results showed that ZX-29 could significantly inhibit the proliferation of NCI-H2228 and HCC78,block the cells in G0/G1 phase,and promote cell apoptosis.Besides,the anti-tumor effect of ZX-29 was better than the second generation ALK inhibitor ceritinib and the first generation ROS1 inhibitor crizotinib.The kinase region mutation of ALK or ROS1 is the most common drug resistance mechanism.Among the identified mutations,ALK G1202R mutation and ROS1 G2032R mutation are the most resistance mutation.To explore whether the new inhibitor ZX-29 can overcome the cell resistance caused by genetic mutations,we constructed EML4-ALK G1202R and CD74-ROS1 G2032R mutant recombinant plasmid by the technology of molecular cloning and fusion PCR.Then the recombinant plasmid was transfected into the ALK-and ROS1-negative A549 cell lines,building the mutant stable cell model which was selected by G418.The results of MTT and protein immunoblot showed that the mutant cell models were resistant to crizotinib,indicating the mutant resistant cells were successfully constructed.Then we tested the sensitivity of the mutant resistant cells to ZX-29,the results showed that ZX-29 still effectively inhibited the cell viability of the mutant resistant cell,and the IC50 of mutant resistant cells was similar to that of wild-type cells of ALK/ROS1,indicating that ZX-29 could overcome the resistant cells caused by genetic mutations.ALK gene rearrangement has been observed in 30-5%of NSCLC,while ROS1 gene rearrangement accounts for 0.9%of NSCLC.Because of the small proportion of ROS1 lung cancer,there is little research about the biological function or resistance mechanism of ROS1.We had successfully constructed CD74-ROS1 and CD74-ROS1 G2032R mutation cell lines,so we further studied the biological function or drug resistance mechanism of ROS1 G2032R mutation.The results showed that the expression of CD74-ROS1 or CD74-ROS1 G2032R mutation in A549 cells induced epithelial-mesenchymal transition(EMT)by increasing the expression of Twistl transcription factor,dramatically enhanced the ability of invasion and migration.Moreover,we found that inhibition of Twistl could reverse EMT induced by CD74-ROS1 G2032R mutation.Combination of Twistl siRNA and crizotinib significantly reduced cell vitality,inhibited cell invasion and migration,and promoted apoptosis in A549-CD74-ROS1 G2032R mutation cells.In conclusion,ZX-29 is a novel ALK/ROS1 dual target inhibitors,and able to overcome the drug reistance resulted from ALK/ROS1 gene mutations.Our study will provide a theoretical basis for ZX-29 used as a promising anti-tumor drug.Besides,it provides a new research direction and strategy for overcoming the drug resistance.