Restricting Glutamine Uptake Enhances NSCLC Sensitivity To Third-generation EGFR-TKI HS10296

Background and Objectives:Secondary drug resistance is the main cause of the failure of epidermal growth factor receptor-tyrosine kinase inhibitors(EGRF-TKIs)in the targeted treatment of NSCLC.Recent studies have indicated that EGFR mutation in non-small cell lung cancer(NSCLC)cells can significantly up-regulate the expression of glutamine transporter and increase the metabolism of glutamine,while the glutamine metabolites can activate the downstream signals of EGFR,including mTOR,erk1/2,STAT3,etc.,which has become an important reason for NSCLC is insensitive to EGFR-TKIs.Therefore,we speculate that inhibiting glutamine transporter may increase the sensitivity of NSCLC to EGFR-TKIs and delay the development of drug resistance.The purpose of this study was to investigate the effect of NSCLC cells on the sensitivity of HS10296 under the limitation of glutamine uptake.Methods: 1.CCK8 and Annexin V/PI were used to detect the effects of HS10296 and/or V9302 on proliferation and apoptosis of NSCLC.2.Proteomics was used to detect the effect of HS10296 on energy metabolism-related proteins in NSCLC.3.siRNA transfection was used to observe the effect of down-regulation of SLC1A5 on cell proliferation.4.Colony formation experiment was used to detect the effect of drugs on the ability of cell cloning.5.Immunofluorescence assay and Western Blotting were used to detect the expression of apoptotic caspase 3,caspase 8,PARP and autophagy-related proteins LC3 B and P62.6.DAPI staining was used to detect the effects of drugs on the nucleus.7.Transmission electron microscopy was used to observe the changes of the submicroscopic structures of autophagosomes and nuclei.8.The level of autophagic flow was detected by tandem fluorescent protein mCherryGFP-LC3 B.9.The tumor-bearing model of nude mice was used to detect the anti-tumor effect of V9302 on HS10296 in vivo.Results: 1.HS10296 inhibited the proliferation of H1975 and A549 cells in a concentrationand time-dependent manner and induced apoptosis,among which A549 cells with wild-type EGFR were less sensitive to HS10296.2.The expression of SLC1A5 was up-regulated in NSCLC cells stimulated by HS10296 at low concentration for 18 hours.3.SLC1A5 was highly expressed in A549 cells of wild-type EGFR.4.Glutamine deletion or SLC1A5 inhibition/gene silencing significantly inhibited the proliferation of non-small cell lung cancer cells and reduced the uptake of glutamine;5.SLC1A5 inhibitor V9302 combined with HS10296 has a synergistic inhibitory effect on NSCLC proliferation.6.V9302 enhances the effect of HS10296 on NSCLC cell apoptosis.7.V9302 combined with HS10296 inhibited autophagy in NSCLC cells.8.V9302 combined with HS10296 may induce apoptosis by inhibiting autophagy.9.V9302 enhanced the antitumor effect of HS10296 in nude mice.Conclusion:Inhibition of glutamine transporter SLC1A5 can enhance the antitumor effect of HS10296 in vivo and in vitro.