The third-generation of epidermal growth factor receptor(EGFR)tyrosine kinase inhibitors(TKIs),represented by osimertinib,has achieved remarkable clinical outcomes in the treatment of non-small-cell lung cancer(NSCLC)with EGFR mutation.However,resistance eventually emerges in most patients and the underlying molecular mechanisms remain to be fully understood.In this study,we generated an osimertinib-acquired resistant lung cancer model from a NSCLC cell line H1975 harboring EGFR L858R and T790M mutations.We found that the capacity of DNA damage repair was compromised in the osimertinib resistant cells,evidenced by increased levels of yH2AX and higher intensity of the comet tail after withdrawal from cisplatin.Pharmacological inhibiting the activity or genetic knockdown the expression of DNA-PK,a key kinase in DNA damage response(DDR),sensitized the resistant cells to osimertinib.Combination of osimertinib with the DNA-PK inhibitor,PI-103,or NU7441,synergistically suppressed the proliferation of the resistant cells.Mechanistically,we revealed that DNA-PK inhibitor in combination with osimertinib resulted in prolonged DNA damage and cell cycle arrest.These findings shed new light on the mechanisms of osimertinib resistance in the aspect of DNA repair,and provide a rationale for targeting DNA-PK as a therapeutic strategy to overcome osimertinib-acquired resistance in NSCLC.