Targeted Inhibition Of GLS1 Enhances The Antitumor Effect Of Selumetinib In KRAS-mutant Lung Adenocarcinoma And Its Molecular Imaging Evaluation

Objective:Regulated by the tumor microenvironment,the metabolic network of the tumor is reprogrammed,driven by oncogenes and tumor suppressor genes.The metabolic phenotype of tumors of different driven-genes and different tissue types is extremely heterogeneous.KRAS-driven lung adenocarcinoma(LUAD)are addicted to glutamine.Beginning with glutaminase1(GLS1)catalyzed decomposition,glutamine provides nitrogen for the synthesis of macromolecules,maintain redox homeostasis,and replenishes?-ketoglutarate(?-KG)for the tricarboxylic acid cycle.CB839,an efficient GLS1 inhibitor,has antitumor effect in several tumors.Selumetinib is an efficient and selective inhibitor targeting the MEK/ERK pathway.Due to the existence of the pathway activated by KRAS bypassing MEK,the effect of selumetinib is not ideal.To capitalize on the link between MEK signaling and glutamine metabolism in KRAS-mutant LUAD,we evaluated the baseline glutamine metabolism in KRAS-mutant LUAD,inhibited GLS1 to synergistically sensitize KRAS-mutant LUAD to selumetinib in vitro and in vivo,and assessed therapeutic response by ¹⁸F-FDG PET imaging.Furthermore,we evaluated the redox and energetic stress induced by dual inhibition of MEK and GLS1,and preliminarily explored the mechanism of synergistic sensitivity.Methods:1.Firstly,the metabolic baseline of LUAD with and without KRAS mutation were evaluated.¹⁸F-FDG and ¹⁸F-Gln PET/MR imaging were performed on the xenograft model.The effects of glutamine deprivation on the proliferation of LUAD with and without KRAS mutation were detected by CCK8 assay.The expressions of key enzymes in LUAD cells were detected by Western blot.KRAS was knockdown in A549 cell and overexpressed in H1299 cell,then the expression of GLS1 and c-Myc was analyzed by Western blot.2.KRAS-mutant cells A549,H23,H2122 and the KRAS wild-type cells H1299,H838 were treated with selumetinib,CB839,or a combination of both at escalating doses,CCK8 assay was performed to test whether CB839 would synergize with selumetinib to inhibit cell proliferation.The EDU assay was performed to test the short-term response to the combination therapy while colony formation assay was performed to test the long-term response in A549 and H1299 cells.The efficacy of combination therapy compared with monotherapy on A549 and H1299 mouse xenografts were evaluated by tumor volume curve and ¹⁸F-FDG micro-PET imaging.3.To evaluate the changes in cell metabolism,cellular lactate,ATP,glutamate production,and glucose consumption were determined.The mitochondrial membrane potential and ROS level were also evaluated using Assay Kit.To investigate the mechanism by which dual inhibition of MEK and GLS1 was more effective in A549 cells,Western blot and immunohistochemical analysis were used to evaluate the phosphorylation status of ERK,AKT,and the expression of autophagy biomarker p-ULK1,p62.Results:1.The A549 xenografts had higher glutamine uptake than H1299 xenografts.When glutamine was deprived(by culturing in medium without glutamine or treatment by CB839),cell proliferation of A549 was significantly inhibited.The expression of GLS,the key enzyme for glutamine degradation,in A549 cells was higher than that in H1299and HBE cells.After the knockdown of KRAS in A549 cell,the expression of GLS and c-Myc was significantly decreased,while that was significantly increased in H1299 cell when KRAS was overexpression.2.The combination of selumetinib and CB839significantly inhibited the proliferation of A549,H23 and H2122 cells compared with single drug treatment while it did not show added benefit when compared to selumetinib treatment alone in H1299 and H838 cells.When combined with CB839,the IC₅₀ of selumetinib was sharply decreased KRAS-mutant cells.Selumetinib and CB839 showed moderate or strong synergy in the KRAS mutant cell line,but weak synergy or only additive in the KRAS wild-type cell line.The outcomes of the EDU assay and colony formation assay were consistent with the CCK8 assay.In A549 xenografts,combination therapy induced a significantly larger reduction in tumor volume than selumetinib or CB839 alone,while in H1299 xenografts,combination therapy did not provide added benefit when compared with monotherapy.According to the ¹⁸F-FDG micro-PET imaging,100%(5 in 5)of A549 xenografts receiving combination treatment achieved PMR,the ratio of SUV_max pre-and post-therapy closely mirrored the fold change in tumor volume,at the same time the expression of GLUT1 and GLS decreased.3.In A549 cell receiving combination treatment,the mitochondrial membrane potential decreased and the ROS levels increased.Combination treatment reduced the consumption of glucose and the production of lactate,glutamate,and ATP in A549 cell.The phosphorylation of ERK was inhibited by selumetinib and that of AKT was promoted compensatorily,while the phosphorylation of ERK and AKT was significantly suppressed by dual inhibition of MEK and GLS1 in A549 cell and xenografts.At the same time,the phosphorylation of autophagy positive regulator ULK increased and the level of autophagy substrate p62 decreased following combination therapy.Conclusion:LUAD with and without KRAS mutation showed differences in baseline glutamine metabolism.KRAS mutated LUAD cells were more dependent on glutamine,and the expression of glutamine metabolizing enzyme was closely related to the level of KRAS.Targeted inhibition of GLS1 synergized with selumetinib to enhance antitumor activity in KRAS-mutant LUAD,the therapeutic response could be well identified by¹⁸F-FDG PET imaging.This combination therapy induced redox and energetic stress and suppressed the phosphorylation of AKT.These effects combined to induce autophagy,resulting in cell death.Targeting the pathways and addicted metabolism activated by the KRAS mutation,and using PET imaging to dynamically monitor therapeutic efficacy may be a feasible model for LUAD tumor management.