The BRD4-IRF1 Axis Regulates Chemoradiotherapy-induced PD-L1 Expression And Immune Evasion In Non-small Cell Lung Cancer

Objective: Non-small cell lung cancer(NSCLC)is the main subtype of primary lung cancer,which seriously threatens human life and health.In the management of NSCLC,radiotherapy and cisplatin-based chemotherapy have been extensively used when surgical excision is not an option.Radiotherapy and chemotherapy can not only kill tumors directly but also boost anti-tumor immune responses by increasing tumor immunogenicity.However,chemoradiotherapy-induced PD-L1 upregulation promotes immune escape and ultimately leads to therapeutic resistance and treatment failure.Here,we aimed to identify candidate regimens that might decrease treatment-induced PD-L1 expression to augment the antitumor immunity of chemoradiotherapy in NSCLC.Methods: The changes of PD-L1 in NSCLC tumor cells were detected by flow cytometry,Western Blot,RT-PCR,immunofluorescence staining,immunohistochemistry staining,etc.A drug array with more than 200 agents was used to recognize compounds that can suppress the cisplatin-induced and radiation-induced PD-L1 expression in NSCLC via the flow cytometry-based assay.The si RNA silencing and BRD4 inhibitors(JQ1 and ARV771)were used to explore the role of BRD4 protein in chemoradiation-induced tumor PD-L1 expression.The RNA-seq was used to detect the effect of JQ1 on gene transcription level in NSCLC cells.The co-immunoprecipitation experiments were performed to detect the interaction of BRD4 and IRF1 in NSCLC treated with/without radiotherapy,cisplatin,and JQ1.IRF1 knockdown was used to study its role in chemoradiationinduced tumor PD-L1 upregulation in NSCLC.The CUT&Tag analysis was conducted to detect the enrichment of BRD4/IRF1 in the PD-L1 promoter region in NSCLC treated with/without radiotherapy,cisplatin,and JQ1.In vitro co-culture assay was conducted to investigate whether the JQ1-mediated reversal of the chemoradiation-induced tumor cell surface PD-L1 upregulation in NSCLC would alleviate T cell apoptosis in co-culture systems.The anti-tumor effect of JQ1 combined with radiotherapy and cisplatin was verified in the Lewis xenograft tumor model of C57BL/6 mice.The flow cytometry phenotyping of transplanted tumor tissues and detection of IFN-? and TNF-? in tumor tissues by ELISA were aimed to find the mechanisms of enhanced anti-tumor immunity of chemoradiotherapy and JQ1.The CD8? depletion antibody was used to show whether the anti-tumor effect of JQ1 combined with chemoradiotherapy depends on CD8+ T cells.Bodyweight measurements,HE staining of the main organs,and detection of cytokines in blood from mice in animal experiments were analyzed to observe whether the JQ1-based combinatory treatments increased the toxicity.The transcriptome data analysis and tissue microarray detection in NSCLC patients were used to explore the relationship between the activation of the BRD4/IRF1/PD-L1 pathway,T cell function,and patient's prognosis.Results: First,both radiotherapy and cisplatin caused the PD-L1 upregulation in NSCLC tumor cells,and concurrent chemoradiotherapy further increased tumor PD-L1 expression.This is the first time to identify that BRD4 inhibitors reversed both the radiotherapy-induced and cisplatin-stimulated tumor PD-L1 upregulation through drug screening experiments in two NSCLC cells.Meanwhile,JQ1 blocked the IFN-?-stimulated PD-L1 expression in NSCLC.Secondly,JQ1 combined with radiotherapy and cisplatin enhanced tumor immunogenicity by facilitating upregulation of MHC-I m RNA level.Radiotherapy and cisplatin treatment promoted BRD4 to recruit IRF1 to the PD-L1 promoter and activated transcription of PD-L1.While JQ1 reversed the binding of BRD4/IRF1 to the PD-L1 promoter induced by chemoradiotherapy to hinder treatment-induced PD-L1 expression.The BRD4-targeted therapy activated tumor-infiltrating CD8+ T cells to improve the efficacy of chemoradiation and anti-PD-1 antibody without increasing treatment-related toxicity or inducing cytokine release syndrome.Moreover,CD8+ T-cell depletion assay showed that BRD4 inhibition synergized with chemoradiotherapy to show a robust antitumor immunity dependent on CD8+ T cells in vivo.Data mining in NSCLC patients of TCGA showed that BRD4 m RNA level was positively correlated with PD-L1 m RNA level,but negatively correlated with MHC-I m RNA level.In addition,the activation of the BRD4/IRF1/PD-L1 pathway is positively correlated with the level of exhausted T cells.Results of tissue microarray staining in NSCLC patients showed that BRD4 protein level was positively correlated with the proportion of PD-L1 positive cells,and the overall survival time of NSCLC patients with high expression of BRD4 and PD-L1 was shorter.Conclusion: The results indicated that chemoradiotherapy activated the BRD4/IRF1 signal to facilitate PD-L1 expression in NSCLC.It is the first drug screen project to discover and clarify that BRD4 inhibitors can reverse the chemoradiation-induced PD-L1 upregulation in NSCLC,and activate the anti-tumor immune response of CD8+ T cells to further improve the efficacy of concurrent chemoradiotherapy and the anti-PD-1 therapy.Targeting BRD4 is a safe and effective new strategy to increase the sensitivity of NSCLC to chemoradiotherapy and anti-PD-1 antibody.