Influence Of ACK1 Inhibitor On Drug Sensitivity To Osimertinib In EGFR Mutant Non-small Cell Lung Cancer

Objective: The emergence of acquired resistance to the third generation epidermal growth factor receptor(EGFR)inhibitor,osimertinib(AZD9291 or TAGRISSO),is an unavoidable huge clinical challenge.The involvement of activated Cdc42-associated kinase 1(ACK1),a non-receptor tyrosine kinase with an oncogenic function,in regulating cell response to osimertinib has not been investigated.In this project,we focused on studying the effect of ACK1 inhibitor on the anti-tumor activity of osimertinib in EGFR mutant NSCLC cells,and explored its mechanism to delay and even overcome the acquired resistance of osimertinib.Methods: Different drug effects on the growth inhibition of EGFR mutant NSCLC cells were evaluated by SRB and colony formation experiments.Intracellular ACK1 protein and m RNA alterations were detected with western blot(WB)and real time quantitative PCR(RT-q PCR),respectively.Using si RNA and sh RNA interference technology,knocked down the expression level of ACK1 in lung cancer cells for subsequent colony formation experiments to explore the effect of changes in ACK1 expression level on the anti-tumor activity of osimertinib.Apoptosis of lung cancer cells treated with different drugs was monitored with flow cytometry for annexin V-positive cells.WB experiments were used to detect the expression levels of apoptosis proteins such as PARP,Caspase-3,Caspase-8 and related pathway proteins such as EGFR,p-EGFR,ERK,and p-ERK.ACK1 inhibitor effects on delaying osimertinib acquired resistance in vitro were determined using colony formation.Xenografts in nude mice was established by the osimertinib-sensitive lung cell line PC-9 to explore the in vivo mechanism of ACK1 inhibitor effects on delaying osimertinib acquired resistance.The effect of ACK1 inhibitor on cell senescence was assayed by β-galactosidase staining.Results: Inhibition of ACK1 with the novel ACK1 inhibitor,(R)-9b synergized with osimertinib in inhibiting the growth of EGFR mutant NSCLC cell lines.Similar results were also generated with ACK1 gene knockdown.The combination of(R)-9b and osimertinib enhanced induction of apoptosis.In both colony formation in vitro and nude mice xenograft tumor models in vivo long-term resistance delay assays,the combination of(R)-9b and osimertinib clearly delayed the emergence of osimertinib-resistance.Further,the(R)-9b and osimertinib combination was also effective in inhibiting the growth of EGFR mutant NSCLC cell lines with acquired resistance to osimertinib,which possess elevated levels of ACK1,and the growth of osimertinib-resistant tumors in vivo.In some resistant cell lines,the combinations induced senescence in addition to induction of apoptosis.Conclusion: ACK1 inhibitor(R)-9b enhances the growth inhibitory effects of osimertinib on EGFR mutant NSCLC cells.Osimertinib up-regulates the expression levels of ACK1 m RNA and protein in EGFR mutant NSCLC cells.Using ACK1 si RNA or ACK1 sh RNA to knock down the expression level of ACK1 enhances the growth inhibitory effect of osimertinib on EGFR mutant NSCLC cells.ACK1inhibitor(R)-9b combined with osimertinib promotes apoptosis and inhibit the EGFR/ERK signaling pathway in EGFR mutant NSCLC cells.In addition,the ACK1inhibitor(R)-9b delays and overcomes the acquired resistance of EGFR-mutant NSCLC cells to osimertinib in vitro and in vivo.The expression levels of ACK1 are significantly increased in osimertinib-resistant NSCLC cell lines.ACK1 inhibitor(R)-9b combined with osimertinib enhances the growth inhibitory effect on osimertinib-resistant EGFR-mutant NSCLC cells and tumors,and promotes cell senescence.Therefore,these novel findings suggest that ACK1 inhibition might be a potential and innovative strategy for delaying and overcoming osimertinb acquired resistance.The availability of the ACK1 small molecule inhibitor,(R)-9b,warrants future testing of our findings in the clinic.