Overcoming Resistance To AC0010,a Third Generation Of EGFR Inhibitor By Targeting C-MET And BCL-2

Background and Objective:AC0010 is a pyrrolopyrimidine-based irreversible inhibitor of epidermal growth factor receptor(EGFR),structurally distinct from previously reported pyrimidine based irreversible EGFR inhibitors such as Osimertinib and Rociletinib.AC0010 selectively inhibits EGFR T790 M mutation in both preclinical and clinical studies.However,AC0010 treatment eventually triggers drug resistance with unknown mechanism.In this study,we used different in vitro approaches to establish AC0010 resistant cell lines and then used the RNAseq and Western Blot analyses to elucidate resistant mechanism.We found that c-MET and BCL2 are overexpressed in resitant lines.We then used both small molecular inhibitors(pharmacological)and si RNA knockdown(genetic)approaches to target c-MET and BCL2,leading to overcoming AC0010 resistance.Our study provides a sound strategy for mechanism-based drug combinations in clinical application for the treatment of lung cancer patients,resistant to AC0010.Methods:1.We used parental NCI-H1975(harboring L858R/T790 M mutations)cell line to develop resistant cell lines after a series of drug exposure and selection in either nudemice xenograft tumor(H1975-P)or cell culture(H1975-AVR)settings.2.To elucidate underlying mechanism,we performed unbiased RNAseq-based profiling analysis and RT-PCR to detect the alterations in gene expression.3.We used si RNA and Western Blot analyses to confirm altered gene expression(e.g.overexpressed c-MET and BCL-2)during the development of AC0010 resistance.4.To overcome AC0010 resistance,we used small molecular inhibitors and si RNA knockdown approaches to target altered c-MET or BCL-2.Results:1.We established two H1975 NSCLC-derived lines resistant to AC0010 after a series of drug exposure and selection in either nude-mice xenograft tumor(H1975-P)or cell culture(H1975-AVR)settings.Both lines obtained 100-fold resistance to AC0010,as compared to the parental lines.Both cell lines are also resistant to other different generations of EGFR TKIs.Six monoclonal resistant cell lines were cultured from H1975-P1 and H1975-AVR1.All monoclonal resistant cell lines have 124-433 folds resistance to AC0010 compared with parental H1975.2.To elucidate underlying mechanism,we sequenced the exon 18-21 of EGFR and confirmed L858R/T790 M mutations without detecting any new mutations in resistant lines.We further performed unbiased RNAseq-based profiling analysis,and found that H1975-P1 line has 1807 up-regulated genes and 1224 downregulated genes compared with parental cell line.By analyzing cancer related gene with highly altered expression,we found that H1975-P1 line has c-MET overexpression with 15-fold higher than patental cell,which could be the key factor to drive AC0010 resistance.3.To study H1975-AVR1's resistant mechanism,we used RNAseq-based profiling analysis and found that H1975-AVR1 line has 1700 up-regulated genes and 1228 downregulated genes compared with parental cell line.By analyzing cancer related gene with highly altered expression,we found that H1975-AVR1 cells had AKT3 and BCL-2 overexpression with over 10-fold higher than patental cells,other genes like BCL2L11,KRAS,IGF1 R have 2-5 folds higher expression.4.Weconfirmed c-MET overexpression by Western blot,and its causal relationship for H1975-P1 resistance to AC0010 via si RNA knockdown and small molecule inhibitor.Indeed,c MET overexpression is the major mechanism of H1975-P1 resistnace to AC0010.The combination of Crizotinib with AC0010 significantly sensitized H1975-P1 cells to AC0010 in both cell based growth assay and xenograft model.5.We confirmed BCL-2 overexpression in H1975-AVR1 cell line by RT-PCR and Western blot.To overcome H9175-AVR1 resistance to AC0010,we used si RNA knockdown and small molecular BCL-2 inhitibor ABT-263,and found that combination of ABT263 and AC0010 can partially overcome H1975-AVR1 resistance to AC0010 in cell based growth assay.Conclusion:Our study shows that drug resistance to AC0010 can be developed via the different mechanisms in a cell context dependent manner,and provides the proof-of-concept evidence for rational drug combinations to overcome resistance for maximal therapeutic efficacy.