The Role And Mechanism Of A Novel BRD4 Degradation Agent ARV-825 On Cholangiocarcinoma

BackgroundCholangiocarcinoma(CCA)is a rare type of malignant tumor.Recently,first line treatment of unresectable CCA are combined chemotherapy of Gemcitabine and cisplatin.However,the median survival time is less than one year,so developing new targeted therapy is of great significance.BRD4 interacts with acetylated lysine in histone or non-histone proteins and participates in transcriptional activation and extension,thus becoming an ideal target for tumor diagnosis and treatment.PROTAC(proteolysis-targeting chimeric)ARV-825 recruits bromine domain and extraterminally(BET)proteins into the E3 ubiquitin ligase cereblon,causing BET protein degradation,including BRD4.At present,there is no report on the therapeutic effect and sensitivity of ARV-825 in cholangiocarcinoma.The aim of this study was to evaluate the biological function of BRD4 in cholangiocarcinoma,elucidate the therapeutic effect and molecular mechanism of ARV-825 on cholangiocarcinoma,evaluate the anti-cholangiocarcinoma effect of ARV-825,and explore the targeted therapeutic effect of BRD4.MethodsExpression of BRD4 in cholangiocarcinoma tissues and cell lines was detected by immunohistochemistry(IHC)and Western Blot(WB)assay.By the use of BRD4 degradation agent ARV-825 and traditional BRD4 inhibitors JQ1 and OTX015 on cholangiocarcinoma cells respectively,Brd U cell proliferation assay,CCK8 cell growth activity assay and plate cloning assay,the difference in expression level of BRD4 were evaluated.Caspase-Glo 3/7 cell apoptosis assay and flow cytometry assay were used to detect the effect on cell cycle and apoptosis.Effects on cell cycle and apoptosis.The anti-cancer molecular mechanism of ARV-825 was verified by WB,q-PCR and other similar experimental tests.ResultsThe expression level of BRD4 appeared to be high in cholangiocarcinoma tissues and cells on comparing with tumor adjacent tissue and normal common bile duct epithelial cells.Brd U cell proliferation assay,CCK8 cell growth activity assay and plate cloning assay showed that the proliferation ability of cholangiocarcinoma cells was significantly decreased and Caspase-Glo 3/7 apoptosis assay and flow cytometry indicated that the apoptosis of cholangiocarcinoma increased,and the cell cycle was arrested in G1 phase after inhibition of BRD4 with BRD4 degradation agents ARV-825 and BRD4 inhibitors JQ1 and OTX015.Compared with JQ1 and OTX015,treatment via ARV-825 is more effective and durable and causes BRD4 depletion to be faster.RNA-Seq,Western Blot,q-PCR and up-regulates SA-?-Gal activity confirmed that ARV-825 could inhibit the expression of downstream c-Myc,increase the level of p21,and induce cell senescence.ConclusionOn comparing BET-PROTAC ARV-825 with traditional BETi JQ1 and OTX015,it causes more significant and sustained BRD4 depletion,inhibits cell proliferation,induces apoptosis and senescence and by inhibiting the expression of c-MYC and increasing the level of p21,cell cycle arrest induced by BET-PROTAC ARV-825 may depend on the BRD4-c-Myc-p21 axis in cholangiocarcinoma cells.