Zebularine Promotes Anti-tumor Immunity Through Sensitization Of The CGAS-STING Pathway

Epigenetic modification such as DNA methylation plays pivotal roles in many cellular functions and its abnormality is closely related to tumor occurrence and development.Some chemical inhibitors targeting DNA methyltransferases（DNMTs）have been approved for treating leukemia,and tested in clinical trials for treating solid tumors.Functions of nucleotide DNMTis（DNMT inhibitors）are dependent on DCK（Deoxycytidine kinase）to inhibit DNA methyltransferase activity,which leads to DNA demethylation.However,in addition to demethylation,the other mechanisms might contribute to the anti-tumor effects of DNMTis,including changes in cell signaling pathways and cell cycle activity,anti-cell proliferation,and changes in stem cell functions,etc.Particularly,DNMTis have been shown to have an immune-regulatory role as shown by many studies,especially in the regulation of interferons.The cGAS-STING pathway is essential for pathogenic DNA-induced immune responses.Once accumulated DNA is detected inside of the cytoplasm,which may be caused by pathogen infections or cell damage,cGAS produces cyclic GMP-AMP（cGAMP）,the second messenger,to activate STING and its downstream signaling cascades,leading to the production of interferons.Thus,cGAS/STING-mediated DNA sensing pathway is one of the most important ways to regulate interferons and has become a promising therapeutic target for cancer treatments.Our prelimilary data have showed that DNMTis were capable of activating the production of interferons thus might also participate in the regulation of cGAS-STING pathway,which has not been well studied,and the effect of DNMTis on tumor immunity needs to be further explored.Therefore,my thesis studied the role of zebularine,a DNA methyltransferase inhibitor,in regulating the cGAS-STING pathway and anti-tumor immunity.We also studied the therapeutic potential of combined use of zebularine and cGAMP in treating cancers.The data are summarized as follows:1.Zebularine treatment of cancer cells led to increased basal levels of type I interferon and significantly induces the production of type I interferon and interferon stimulating genes in cells by HT-DNA or cGAMP,but not Poly（I:C）.By quantitative reverse transcription polymerase chain reaction（qRT-PCR）,Western blotting,enzyme linked immunosorbent assay（ELISA）and fluorescence activated cell sorting（FACS）,it showed that with the treatment of zebularine in various types of tumor cell lines significantly up-regulated the expression of type I interferons and interferon stimulating genes（ISGs）induced by HT-DNA and cGAMP but not Poly（I:C）.In addition,zebularine treatment alone led to increased basal levels of type I interferon.These results indicate that zebularine may play a role in sensitizing cGAS-STING pathway.2.Enhanced sensitivity of cGAS-STING pathway by zebularine is dependent on cGAS or STING gene.To study the role of cGAS-STING,we used the CRISPR-Cas9 gene editing technology for generating cGAS or STING gene knockout mice.With the treatment of zebularine,cGAS or STING deficient cells were unable to induce IFNβcompared with the wild-type AGS or B16F10 cells.These data suggest that sensitizing of cGAS-STING pathway by zebularine is dependent on the cGAS or STING gene.3.DCK-depenedent zebularine’s demethylation function upregulates STING’s expression by reducing the methylation degree of the STING promoter.Knock-out or expression silencing of DNMTs by the CRISPR-Cas9 technology and RNA interference technology showed that the deletion or silence of DNMTs significantly enhanced the expression of IFNβafter HT-DNA or cGAMP stimulation;Knockout of glycoside kinase（DCK）by the CRISPR-Cas9 technology and evaluation of DNA methylation by methylation-sensitive melt curve analysis(MS-MCA showed that zebularine’s demethylation effect depends on the presence of DCK,and zebularine can significantly enhance the expression of IFNβor STING;In addition,the methylation in STING gene promoter,as shown by bisulfite sequencing（BSP）showed that zebularine can inhibit the methylation of the STING promoter in tumor cells,thereby enhancing its expression.These results indicate that the mechanism of zebularine-mediated sensitizing of cGAS-STING pathway is likely by reducing the methylation of STING promoter and restoring the expression.4.Zebularine increases the accumulation of DNA and cGAMP in the cytoplasm of tumor cells,but not by inducing DNA damage.The existence of cytoplasmic DNA in AGS cells was detected by immunofluorescence.Zebularine can increase the accumulation of DNA and cGAMP in the cytoplasm.The cytoplasmic DNA and cGAMP isolated from the cytoplasm were able to enhance the expression of IFNβ.In addition,the expression of the DNA damage marker,γh2ax,was examined by Western blotting,and the data showed that zebularine did not induce DNA damage as the DNA damaging agent control,Ara-C,did.These data suggest that zebularine can increase the accumulation of cytoplasmic DNA and cGAMP in tumor cells.5.Zebularine or cGAMP used alone or in combination can significantly inhibit tumor growth and prolong the survival of tumor-bearing mice.The combined effect is significantly better than each one alone,and can further enhance the anti-tumor effect of PD-L1 antibody.The various types of tumors were established on C57BL/6 mice by inoculation of B16F10,MC38 and Panc02 cells,and zebularine or cGAMP alone or combination was used to treat the tumor-bearing mice.It was found that the combined use of zebularine and cGAMP worked better than either one used alone in terms of inhibiting tumor growth and prolonging the survival of tumor-bearing mice.In addition,we also studied the treatments of zebularine,cGAMP and PD-L1 antibody alone or in combination.The data showed that zeburaline can significantly improve the anti-tumor effects of PD-L1antibody,with the best effect obtained when all the three drugs were combined.These data support the important role of zebularine in anti-tumor immunity.6.Zebularine’s anti-tumor effect depends on the cGAS and STING genes in tumor cells or mice.By comparing the anti-tumor effects of zebularine or cGAMP alone or in combination in B16F10-Wt、B16F10-Cgas^-/-and B16F10-Sting^-/-cell lines,or Wt,Cgas^-/-and Sting^-/-C57BL/6 mouse models,the anti-tumor effect was abolished in either tumor models established by Cgas^-/-and Sting^-/-tumor cells,or the Cgas^-/-and Sting^-/-mouse tumor models.The data suggest that zebularine’s anti-tumor effect depends on the presence of cGAS and STING genes in vitro and in vivo.7.Zebluarine significantly increases the expression of ISGs in tumor or immune cells,as well as the number of CD8 T cells and NK cells.The expression of ISGs in tumor tissues,purified tumor cells（excluding immune cells）and extracted tumor infiltrating lymphocytes（TILs）was analyzed by qRT-PCR.It was shown that zebularine significantly increased the expression of ISGs in tumor cells or especially TILs,which was dependent on the cGAS and STING;In addition,analyses by flow cytometry found that zebluarine or cGAMP alone or combined treatment can significantly increase the populations of CD8 T cells and NK cells in TILs,which was also dependent on cGAS and STING.These data suggest that Zebularine induces the expression of ISGs,and further increases CD8 T cells and NK cells infiltration in tumors.Conclusion:In this work,we found zebularine was capable of causing various changes in tumor cells,including accumulation of DNA species in cytoplasm and sensitization of cGAS-STING pathway,which was associated with demethylation and upregulation of the STING gene.In a cGAS-STING dependent manner,zebularine exhibited therapeutic effects in multiple syngeneic tumor models.Synergizing with cGAMP and immune checkpoint blockade antibodies,zebularine facilitated tumor infiltration of CD8 T cells and NK cells.This study unveils a novel immunomodulatory mechanism of zebularine and provides the foundation for further therapeutic investigation in cancer treatments.