The Role And Mechanism Of 2-DG In Reversing Osimertinib-acquired Resistance Of NSCLC Cell Line

Introduction: The epidermal growth factor receptor-tyrosine kinase inhibitors(EGFR-TKIs)targeting EGFR mutations have become an important part of targeted therapy in non-small cell lung cancer(NSCLC)patients.After the first-and second-generation of EGFR-TKIs widely used in clinical,the third-generation of EGFR-TKIs named Osimertinib also became increasingly mature.As this drug can specifically inhibit the T790M(+)EGFR mutation,it was initially used for sequential treatment after the previous generation of TKIs resistance appearing.And the FLAURA trial showed that Osimertinib can significantly improve the PFS of EGFR positive-NSCLC patients compared with Gefitinib or Erlotinib in first-line treatment.Based on this the FDA and EMA have approved Osimertinib used in first-line treatment for EGFR-mutated NSCLC patients.Like all the EGFR-TKIs,acquired resistance is also a bottleneck limiting the clinical application of Osimertinib.Currently,it has been found that the resistance mechanism of Osimertinib includes EGFR dependent resistance and EGFR independent resistance.The most common resistance mechanism of the former is the C797 S mutation,while the latter mainly refers to the activation of KRAS,BRAF and other bypasses.Currently,there is no recognized and effective method for the treatment of patients with acquired resistance to Osimertinib.Exploring the universal characteristics of tumor cells such as proliferation,metabolism and apoptosis and intervening them is an important research direction to overcome drug resistance.Our team long-term commitment to explore the combination strategies overcoming acquired resistance of targeted therapy in lung cancer.Our previous studies have found that classical hypoglycemic drug metformin can significantly reverse EGFR-TKIs resistance in lung cancer cells,and interfering “autophagy”,one of the important energy sources in lung cancer cells,can also reverse the targeted drug resistance.These findings indicate that the acquired resistance of EGFR–TKIs may be related to the metabolism characteristics of lung cancer cells,and interfering the metabolic pathway may be one of the possible ways to overcome acquired resistance of EGFR–TKIs.Existing researches has demonstrated that Osimertinib can inhibit the glycolysis of sensitive lung cancer cells,and the combination of 2-deoxyglucose(2-DG)can increase the sensitivity of T790 M mutation lung cancer cells to Afatinib.However,the metabolic characteristics of lung cancer cells with acquired resistance to Osimertinib have not been studied,and whether 2-DG can still be useful in reversing acquired resistance to Osimertinib in lung cancer cells and its related mechanism still need to be clarified and verified.In this study we will discuss the problems mentioned above in an attempt to provide a new method to overcome the acquired resistance to Osimertinib.Objectives: To investigate the metabolic characteristics of lung cancer cells after acquired resistance to Osimertinib,and to clarify the role and mechanism of 2-DG in reversing Osimertinib-acquired resistance of non-small cell lung cancer cell line.Methods: The acquired resistance NSCLC cell line H1975-OR was established by induction in vitro.MTT assay,clonal formation assay,Ki67 protein fluorescence staining,and lactic acid metabolism assay were used to evaluate the resistance of cell lines to Osimertinib and the metabolic characteristics after acquired resistance.Western blot,flow apoptosis assay,lactic acid metabolism assay,and small interfering RNA transfection assay were used to detect the role and possible mechanism of 2-DG in reversing Osimertinibacquired resistance of NSCLC cell line.Results: Compared with the Osimertinib sensitive NSCLC cell line(H1975),the Osimertinib-acquired resistance NSCLC cell line(H1975-OR)had significantly increased level of glycolysis and its glycolysis inhibited by Osimertinib was significantly decreased.2-DG could significantly inhibit the glycolysis level of H1975-OR,and reverse the resistance of H1975-OR to Osimertinib.The apoptosis of H1975-OR cell line induced by Osimertinib was increased when combined with 2-DG,and in H1975-OR the expression of pro-apoptotic protein BIM was up-regulated while the expression of anti-apoptotic protein Bcl-2 was decreased.After interfering with the expression of BIM,the effect of 2-DG in reversing the resistance of H1975-OR to Osimertinib was weakened.Conclusions: 2-DG can reverse the acquired resistance of NSCLC cells to Osimertinib,and the mechanism may be related to the inhibition of cell glycolysis and the activation of BIM/BCL-2 signaling pathway which inducing apoptosis.