The Effect And Mechanism Of Metformin On Overcoming Immune Resistance Of STK11 Mutant Lung Cancer

Background and objectives:Lung cancer is a malignant tumor with the highest morbidity and mortality in China,Non-small cell lung cancer（NSCLC）accounts for 85%of lung cancer,most patients are diagnosed at an advanced stage,losing an opportunity for radical surgery.Immune checkpoint inhibitors（ICIs）have produced durable benefits for advanced NSCLC patients,opening a new era of cancer therapy.However,serine/threonine kinase 11（STK11）mutant NSCLC was primarily resistant to ICIs.Mechanistically,STK11 mutation leads to LKB1（Liver Kinase B1）loss,which then results in the suppression of stimulator of interferon genes（STING）,whose activation is critical for anti-cancer immune response.At present,STING agonists have been investigated for cancer immunotherapy preclinically and clinically,cyclic dinucleotides（CDNs）,or CDN derivatives are a representative class of STING agonists.However,there are challenges for its clinical practice due to low bioavailability in target tissues.Mounting evidence have found that the glucose-lowering drug metformin exerted anti-tumor effect,which was predominantly through activation of AMP-activated protein kinase（AMPK）pathway,and it was reported that axis inhibition protein 1（AXIN-1）was a crucial upstream molecule for metformin to activate AMPK.Previous studies have found that metformin is beneficial for STK11 mutant lung cancer and it can enhance the efficacy of chemotherapy in this kind of cancer.Furthermore,metformin can potentiate anti-tumor immunity by inducing the reduction of tumor hypoxia,yet it’s unclear whether metformin in combination with ICI may overcome the immune resistance of STK11 mutant NSCLC.Therefore,this paper aims to study whether metformin can overcome the immune resistance of STK11 mutant lung cancer and the underlying mechanism.Methods:Using H460 cells,A549 cells and AXIN-1 knock-out cells(AXIN-1^-/-cells),colony formation assay,cell viability assay and Ki67 staining were performed to explore the anti-tumor effect of metformin in combination with T-cells or/and pembrolizumab.We established peripheral blood mononuclear cells（PBMCs）-CDX mouse model for in vivo experiments.Western blot,Immunofluorescence staining and other experiments were conducted to explore the impact of metformin on STING expression and its downstream pathway.CRISPR/Cas9 gene editing technology was utilized to generate AXIN-1^-/-cell lines.Homology modeling of AXIN-1 and STING was constructed through I-TASSER server and docking study was performed using Hex 8.0.0 software.Immunoprecipitation（IP）-mediated endogenous ubiquitination assay was performed to detect STING ubiquitination.Untargeted metabolomics analysis was conducted through liquid chromatography（LC）-mass spectrometry（MS）/mass spectrometry（MS）system.Results:1、Metformin can overcome immune resistance of STK11 mutant NSCLC In Vitro and In Vivo.（1）We first conducted T-cell mediated killing of cancer cells experiment and demonstrated that metformin enhanced activated T-cells to kill STK11 mutant lung cancer cells through colony formation assay,Ki67 incorporation assay and cell counting Kit-8（CCK-8）assay.（2）Through Ki67 staining and In Vivo experiment which was based on a PBMCs-CDX mouse model,we found that metformin combining programmed cell death protein 1（PD-1）inhibitor can significantly decrease the proliferation of STK11 mutant lung cancer cells In Vitro,and result a significant reduction of tumor volume In Vivo.These results demonstrated that metformin can overcome immune resistance of STK11 mutant NSCLC In Vitro and In Vivo.2、Metformin relies on the scaffold protein AXIN-1 to stabilize STING and further enhance T-cell-Mediated killing of STK11 mutant lung cancer cells.（1）We performed western blot and immunofluorescence staining of tumor tissues from PBMCs-CDX mouse model,and found that metformin increases the expression levels of STING significantly.Through cycloheximide（CHX）experiment and western blot analysis,we found that metformin slowed down the degradation of STING,maintained the stabilization of STING,and activated its downstream signal.Metformin cannot maintain the stabilization of STING and enhance activated T-cells to kill STK11 mutant lung cancer cells after small interfering RNA-mediated knockdown of STING.（2）Using CRISPR/Cas9 gene editing technology to knockout AXIN-1,we found that metformin cannot enhance activated T-cells to kill STK11 mutant lung cancer cells via colony formation assay,Ki67 staining and CCK-8 assay.Simultaneously,through CHX experiment,we found that the impact of metformin on stabilization of sting relies on AXIN-1.After AXIN-1 was constructed by transfection of AXIN-1^-/-cells with lentivirus,we demonstrated that metformin stabilizes STING and enhances T-cell-Mediated killing of STK11 mutant lung cancer cells was relying on the scaffold protein AXIN-1.3、Metformin enhances the binding of AXIN-1 and STING,and competitively inhibit the binding of ring finger protein 5（RNF5）and STING mediated ubiquitination degradation of STING,thus maintaining the stability of STING.（1）Through immunofluorescence staining and laser confocal analysis,we found that AXIN-1 and STING were obviously co-located,and that can be enhanced by metformin.IP experiment verified that metformin can enhance the binding of AXIN-1 and STING.Protein-protein docking prediction showed that AXIN-1 binds with STING at K150 site.（2）We first confirmed that metformin weakened the degradation of STING ubiquitination through IP-mediated endogenous ubiquitination assay,then identified E3 ubiquitin ligase RNF5 by western blot and IP analysis,and confirmed that metformin maintained the stability of STING via AXIN-1-dependent inhibition of RNF5-mediated K48-linked ubiquitination of STING.4、Metformin treatment up-regulated multiple nucleotides in an AXIN-1-dependent manner,these nucleotide compounds can enhance the killing effect of activated T cells on STK11 mutant lung cancer cells,and may be related to the process of metformin overcoming immune resistance of STK11 mutant lung cancer.（1）We further explored the metabolic mechanism of metformin on overcoming the immune resistance of STK11 mutant lung cancer from the perspective of metabolism,through untargeted metabolomics analysis.The results showed that metformin up-regulated multiple nucleotides in an AXIN-1-dependent manner.（2）Cell experiments verified that these nucleotide compounds promoted the killing of activated T cells on STK11 mutant lung cancer cells.These results suggest that these nucleotides which were up-regulated by metformin in an AXIN-1-dependent manner,may be related to the process of metformin on overcoming immune resistance of STK11 mutant lung cancer.Conclusions:Metformin can overcome immune resistance of STK11 mutant lung cancer,and the mechanism was through inhibition of RNF5-mediated K48-linked ubiquitination of STING in an AXIN-1-dependent manner,thus maintaining the stability of STING and activating its downstream signal pathway.Moreover,through untargeted metabolomics analysis,we found that metformin up-regulated multiple nucleotides relying on AXIN-1,which may be related to the process of metformin overcoming immune resistance of STK11 mutant lung cancer.