The Role And Mechanism Of Histone Demethylase KDM4C In Radiotherapy And Immunotherapy Of Lung Cancer

Objective:Immune checkpoint blockade(ICB)has revolutionized the cancer treatment.However,only 20%-40%of patients benefit from this new therapy.Radiotherapy(RT)can enhance tumor immunogenicity and improve ICB response,but the clinical efficacy of combining radiotherapy with ICB is still unsatisfactory.KDM4C(Lysine-specific demethylase 4C)is a histone demethylase that plays an important role in a variety of biological processes,including DNA damage repair and genome stability.However,its role in lung cancer radioresistance and antitumor immunity is still unknown.The purpose of this study is to clarify the role and mechanism of KDM4C in lung cancer radioresistance and immunotherapy,and to provide a theoretical basis for the clinical transformation of this new target for lung cancer therapy.Methods:1)Immunohistochemical staining of tissue microarray was carried out to detect the expression of KDM4C in lung cancer,and the Kaplan-Meier method was used to analyze the correlation between the expression of KDM4C and the clinical prognosis of patients;2)Neutral comet assay,foci formation assay,and clone formation assay was performed to detect the changes of radiosensitivity;3)BALB/c nude mice were used to verify the therapeutic potential of KDM4C in vivo;4)Tandem affinity purification and mass spectrometry was carried out to identify the potential interacting proteins of KDM4C,and co-IP(co-Immunoprecipitation,co-IP)was carried out to verify the protein interaction;5)RNA sequencing technology was used to explore the downstream targets of KDM4C;6)Ch IP(Chromatin immunoprecipitation)-PCR was performed to clarify the mechanism of KDM4C in regulating downstream targets;7)Flow cytometry(FCM)was used to screen the immune cells infiltrating the tumor;8)Construction of conditioned culture model in vitro to detect the effect of targeted KMD4C on the function of CD8~+T cells;9)RNA sequencing and Ch IP-PCR was performed to clarify the specific mechanism of KDM4C in regulating downstream molecules;10)The in vivo experiment of C57 mice confirmed the efficacy and safety of combined therapy.Results:1)Tissue microarray staining results showed that KDM4C was overexpressed in lung cancer tissues compared with paired paracancerous tissues,and was associated with poor clinical prognosis of patients(P<0.001);2)Knocking down KDM4C enhanced radiosensitivity of lung cancer cells;3)Both KDM4C knockout and SD70treatment showed significant tumor-suppressive and radiosensitizing effects in an in vivo nude mouse model of subcutaneous xenografts;4)Tandem affinity purification and mass spectrometry identified the deubiquitinase USP9X(Ubiquitin Specific Peptidase 9 X-Linked)as a candidate interacting protein of KDM4C.And co-IP experiments verified the interaction between KDM4C and USP9X;5)KDM4C induces the expression of TGF-β2 by promoting the demethylation of the repressive histone H3K9me3 in the promoter region of TGF-β2;6)FCM screening found that the infiltration of intratumoral CD8~+T cells increased after KDM4C interference(P<0.01),while other immune cells did not change significantly;7)Inhibition of KDM4C could up-regulate the expression of CD8~+T cell proliferation markers(Ki67)and cytotoxic molecules(Ki67,IFN-γ,GZMB,Perforin,and CD107a),and down-regulate the exhaustion markers(PD-1 and CD39)expression.Transwell assay and"Effect-target ratio"experiments showed that inhibition of KDM4C promoted the migration of CD8~+T cells and enhanced the killing ability of CD8~+T cells on tumor cells;8)Targeting KDM4C promoted the transcriptional activation of CXCL10 by increasing the enrichment of activating histone H3K36me3 in the CXCL10 promoter region,thereby exerting the above effects on CD8~+T cells;9)SD70 exerted antitumor immunity in vivo by inhibiting the function of KDM4C.And the combination of SD70,RT and anti-PD-L1 showed the strongest antitumor effect with good safety compared with other treatment modalities.Conclusions:Abnormal recruitment of KDM4C mediated by deubiquitinating enzyme USP9X promotes the demethylation of histone H3K9me3 in the promoter region of downstream gene TGF-β2,induces the transcriptional expression of TGF-β2,and then activates the TGF-β2/Smad/ATM signaling pathway,and ultimately promote lung cancer formation and radioresistance.In addition,targeting KDM4C promotes the transcriptional activation of CXCL10 by increasing the enrichment of histone H3K36me3 in its promoter region,which ultimately leads to increased infiltration of intratumoral CD8~+T cells,promotes the proliferation,migration and activation of CD8~+T cells,and delays their senescence.More importantly,SD70,a novel specific small molecule inhibitor of KDM4C,plays a dual coordinating role with radiotherapy and immunotherapy in vivo and in vitro.Triple therapy has the strongest anti-tumor effect and a good safety profile.