Targeting DNA-PK Overcomes Acquired Resistance To Third-Generation EGFR-TKi Osimertinib In Non-Small Cell Lung Cancer

Objective: This project is disigned to explore the mechanism of targeting DNA-PK overcomes acquired resistence to Osimertinib in non-small cell lung cancer,aims at finding a solution for the challenging problem of Osimertinib-resistance.Methods: 1.We modeled the acquired resistance to Osimertinib by dose escalation,the NSCLC cell line H1975 which expressing a compound EGFR L858 R and T790 M mutation as the study model.Next-Generation Sequencing(NGS)was used to detect the genomic variation of H1975-S and H1975-OR cells,intend to identify the known resistance mechanisms and explore the targetable driver of resistance.2.On the basis of the absence of any obviously targetable driverof resistance,we sought to identify pathways revealed by drugs that synergistically inhibit growth when combined with EGFR-TKIs.The CCK8 assay and Calcusyn software were used to determine the effect of drug combination,and it was found that the DNA-PK specific inhibitor NU7441 showed synergistic interaction combined with Osimertinib.Subsequently,crystal violet staining and flow cytometry analysis of apoptosis and cell cycle were used for further study.Lastly,DNA-PK was knockdown by lentiviral infection to detect the sensitivity of H1975-OR cells to Osimertinib.3.Considering DNA-PK is one of the key kinases in DNA damage repair response(DDR),and EGFR played an important role in DDR.We next to explore whether whether the DNA damage repair is affected in osimertinib-resistant cells.The cisplatin was used to induce DNA-damage,the level of ?H2AX and DNA comet tailing were used as marker for DNA damage.Firstly,Western Blotting was used to detect the level of p DNA-PKcs(S2056)in DDR,Alkaline Comet Assay was used to detect the extent of DNA damage after cells were treated and recovered from the cisplatin.Lastly Western Blotting and Alkaline Comet Assay to further verify the effect of NU7441 and Osimertinib single or combination on DNA-PK activity and the extent of DNA damage.Results:1.After Osimertinib-acquired resistant model generated,the IC50 of H1975-S cells and H1975-OR cells were 0.17?M and 8.42?M tested by CCK8,and the drug resistance index is 49.5,indicating highly resistant to Osimertinib of H1975-OR cells.Apoptosis detection,crystal violet staining and Western Blotting further confirm that Osimertinib-acquired resistant model was successfully constructed.The EGFR C797 S mutation?L718Q mutation?L792H mutation?MET amplification?HER2 amplification and RAS mutation were not detected by next-generation sequencing(NGS),and no obviously targetable driver was found.2.Across a compound cancer-focused library provided by references,PI103 was a synergistic candidate when combined with Osimertinib in H1975-OR cells.Then it was found that the combination of DNA-PK inhibitor NU7441 with Osimertinib can synergistically suppress the proliferation of Osimertinib-resistant cells.Crystal violet staining further confirmed that DNA-PK inhibitor combined with Osimertinib significantly reduced cell viability.Subsequently,Flow cytometry showed that the combination of DNA-PK inhibitor with Osimertinib can significantly promote G1 arrest in Osi-resistant cells;Finally,the sensitivity of H1975-OR cells to Osimertinib was increased when DNA-PK was down-regulation by lentiviral infection.3.Compared with H1975-S cells,the level of p DNA-PKcs(S2056)was significantly increased and longer comet tails were showed in H1975-OR cells after cisplatin induced,indicating heavier DNA damage.After 24 hours repair,the comet tailing of H1975-S cells was significantly shortened while H1975-OR cells did not change significantly,indicating deficiency in DNA damage repair of Osi-resistant cells.Subsequently,the level of p DNA-PKcs(S2056)was significantly increased in H1975-OR cells in response to DNA damage,suggesting that H1975-OR might be highly dependent on DNA-PK activity for DNA repair.Finally,The Western Blot and Alkaline Comet Assay further confirmed that the combination of DNA-PK inhibitor with Osimertinib synergistically reduce the level of p DNA-PKcs(S2056)and prolong the DNA comet tails.Conclusion: 1.Osimertinib-acquired resistant cell line(H1975-OR)is successfully established.Previously identified mechanisms of Osimertinib-acquired resistance such as MET amplification are not found in this model.2.Targeting DNA-PK combined with Osimertinib synergistically suppress the proliferation of Osimertinib-resistant cells.Inhibition of DNA-PK expression by sh RNA lead to increased sensitivity to Osimertinib in H1975-OR.3.Compared with the parental cells,Osimertinib-resistant cell line shows deficiency in DNA damage repair.DNA-PK is hyperactivated in H1975-OR in response to DNA damage,suggesting that H1975-OR might be highly dependent on DNA-PK activity for DNA repair.Targeting DNA-PK combined with Osimertinib synergistically inhibit DNA-PK antivity,further inhibit the DNA damage repair,lead to prolonged DNA damage and pomote apoptosis.