NAD~+ Metabolism Maintains Inducible PD-L1 Expression To Drive Tumor Immune Evasion

BACKGROUNDS&AIMSNAD~+,a key mediator of intracellular energy metabolism and signal transduction,is involved in a wide range of biological processes,such as DNA repair,inflammation,and protein acetylation.NAD~+levels decrease with age,so taking NAD~+supplements can help fight aging.Higher NAD~+/NADH and NADP~+/NADPH ratios are found in cancer cells compared to noncancerous cells,suggesting the important role of NAD~+in this metabolic conversion.NAMPT and NAD~+biogenesis is frequently upregulated in several human malignancies and plays a critical role in tumor initiation,progression,and relapse。However,the role of NAD~+metabolism mediated by NAMPT in the regulation of tumor immune escape is poorly understood.Tumor escape refers to the phenomenon that tumor cells survive and proliferate in vivo by escaping from the recognition and attack of the body’s immune system through various mechanisms.The immune system takes charge of immune monitoring.When the malignant cells appear in the body,the immune system can recognize and eliminate these"non-self"cells specifically through the immune mechanism to resist the occurrence and development of tumors.However,malignant cells can evade the immune surveillance of the body through a variety of mechanisms in some cases,and proliferate rapidly in the body to form tumors.On the one hand,the body can resist the occurrence of tumor through natural and acquired immunity;On the other hand,tumor cells can evade the recognition and attack of the body’s immune system through a variety of mechanisms.Tumorigenesis and prognosis depend on the overall effect of these two aspects.Therefore,the further study on the mechanism of immune escape of tumor provides new insights for tumor immunotherapy.PD-L1 and PD-1 play vital roles in tumor immune evasion,but only a fraction of cancer patients responds to immune checkpoint blockade.The rationale for the anti-PD-1/anti-PD-L1 treatment is that IFNγsecreted by infiltrating T cells will finally result in tumor immune escape by upregulating negative immune checkpoints like PD-L1.However,PD-1/PD-L1 immune checkpoint blockade fails to generate sustained benefits in most patients.It is urgent to explore the regulation mechanism of PD-L1 expression in response to the IFNγsecreted by activated T cells and discover additional theranostic markers for PD-1/PD-L1-based therapies.Therefore,depending on the immune checkpoint PD-L1 we drill down to the influence and mechanism of NAD~+metabolism on tumor evasion so as to provide a new treatment strategy for tumors that are resistant to immunotherapy.METHODS1.To interrogate specific functions of NAMPT,we disrupted NAMPT in wild-type murine liver cancer cells(Hepa1-6)by short hairpin-mediated RNA interference(sh Nampt)and CRISPRCas9-targeted mutation(sg Nampt),with nontargeting constructs as controls(sh Ctrl and sg Ctrl).2.To study the relationship between NAD~+metabolism and tumor immune evasion,Nampt-deficient or control cells were inoculated subcutaneously into the flanks of immunodeficient mice and immunocompetent mice.3.In vivo and in vitro,NAMPT inhibitor FK866 was treated or NAMPT metabolite NMN was supplemented to detect the changes in NAD~+metabolism,gene expression and protein levels(PD-L1,STAT1,IRF1,TET1,etc.)of cells in vitro.Meanwhile,tumor growth in vivo and changes in immune cell composition in tumor microenvironment of the xenograft were monitored.4.To further confirm T cell populations mediating this potent rejection,CD8~+T cells,CD4~+T cells,or both subsets in mice inoculated subcutaneously with sh Nampt or sh Ctrl cells were depleted via their related neutralizing antibodies.The depletion of CD4~+T cells,CD8~+T cells,or both in tumors and spleens of mice was confirmed by flow cytometry.5.Correlation between NAMPT and T cell-related immune checkpoint molecule expression were analyzed in human cancer datasets and HCC clinical samples,in order to further investigate the mechanism of T cell-dependent tumor rejection mediated by NAD~+metabolism.6.The expression of PD-L1(sh PD-L1)was interfered in HCC cell lines,and the cells were implanted into C57BL/6 mice to observe the size of tumors formed in the groups of control and sh PD-L1.NMN was supplemented to see if it could reverse tumor load and alter the infiltration of immune cells.7.Gene enrichment analysis and Reactome pathway analysis were used to explore whether there is a great positive correlation between NAMPT and IFNγ(JAK/STAT signaling pathway)in liver cancer in TCGA database.8.The expression of STAT1 was inhibited by STAT1 inhibitor or stable interference of STAT1 in hepatocellular carcinoma cell lines,and then NAD~+precursor was supplemented to observe the changes of IFNγsignaling pathway.9.Hydroxymethylated DNA immunoprecipitation sequencing(h Me DIP-seq)and pyrosequencing were used to explore whether the genome methylation levels have impact on IFNγsignaling pathway,and to interrogate the mechanism between them.10.The protein expression levels of NAMPT-TET1-p STAT1-IRF1-PD-L1 axis were investigated in tissues from cancer patients via IHC staining and survival analysis of tissue array.11.Mice were engrafted with engineered tumor cells to investigate whether the combination of NAD~+supplements with PD-(L)1 antibody can reduce immunosuppressive drug resistance and further enhance anti-tumor effects.RESULTS1.NAD~+metabolism controls CD8~+T cell-dependent tumor rejection.2.NAD~+metabolism drives inducible PD-L1 expression in tumor cells.3.NAD~+metabolism regulates PD-L1 induction through Stat1-dependent IFNγsignaling pathway.4.Hydroxymethylated DNA immunoprecipitation sequencing(h Me DIP-seq)showed that the global genomic 5hm C levels were reduced after Nampt deficiency in the presence of IFNγand NAD~+metabolism may mediate PD-L1 expression via regulating the demethylation of Irf1 in response to IFNγstimulation.5.NAD~+metabolism maintains expression and activity of methylcytosine dioxygenase Tet1 viaα-KG.6.IFNγ-activated Stat1 recruits Tet1 binding to Irf1 to regulate Irf1 demethylation,leading to PD-L1 Expression.7.NAMPT-TET1-p-STAT1-IRF1-PD-L1 axis is activated in human cancer and predicts poor prognosis.8.NAMPT predicts the efficacy of PD-(L)1 checkpoint blockade and NAD~+replenishment sensitizes anti-PD-(L)1 therapy-resistant tumors to immunotherapy.CONCLUSIONSHere,we find nicotinamide phosphoribosyltransferase(NAMPT),the rate-limiting enzyme of the NAD~+biogenesis,drives interferonγ(IFNγ)-induced PD-L1 expression in multiple types of tumors and governs tumor immune evasion in a CD8~+T cell-dependent manner.Mechanistically,NAD~+metabolism maintains activity and expression of methylcytosine dioxygenase Tet1 viaα-ketoglutarate(α-KG).IFNγ-activated Stat1facilitates Tet1 binding to Irf1 to regulate Irf1 demethylation,leading to downstream PD-L1 expression on tumors.Importantly,high NAMPT-expressing tumors are more sensitive to anti-PD-L1 treatment and NAD~+augmentation enhances the efficacy of anti-PD-L1 antibody in immunotherapy-resistant tumors.Collectively,these data delineate an NAD~+metabolism-dependent epigenetic mechanism contributing to tumor immune evasion,and NAD~+replenishment combined with PD-(L)1 antibody provides a promising therapeutic strategy for immunotherapy-resistant tumors.