The Mechanism And Co-targeting Strategies Of Cyclin Dependent Kinase 7 Inhibitors In Cholangiocarcinoma

Background and Object Cholangiocarcinoma(CCA)is a fatal disease with poor prognosis and still lack of effective treatment strategies.Most patients are in the progressive stage when being definitely diagnosed and miss the chance of receiving radical resection operation or liver transplatation.Also,CCA is insensitive to chemotherapy and as a result,the median survival time of these patients is still less than 48 months.So,it is emergency for researchers to discover potent anti-tumor drugs and develop promising therapeutic strategies for CCA treatment.In 2016,a research team reported that the expression of 48 transcription factors in CCA were different from that in normal tissues,by integrated analyzing the gene expression data sets of CCA in the Gene Expression Omnibus database.They also found that cell cycle was the most significant enriched pathway in CCA.So,with the aim of finding novel targets in CCA,we screened a small-molecular library of inhibitors targeting different targets of the cell cycle in three CCA cell lines.The result of the screening demonstrated that all the three CCA cell lines were sensivity to Cyclin Dependent Kinase 7(CDK7)inhibitors.CDK7,which participates in the regulation of cell cycle progression and regulates transcriptional initiation and elongation,is a member of CDK family.Besides,several researches reported that CDK7 was involved in mediating transcriptional addiction to a vital cluster of genes which was often related to super-enhancers.Also,CDK7 is usually overexpressed in tumor issues.So,CDK7 can be an important potential target in cancer treatment.In this study,we aimed to evaluate the efficacy of CDK7 in CCA preclinical models,elucidate the mechanism and develop possible combination approaches to provide promising therapeutic strategies for clinic.Methods Data from TCGA and GEO was used to analyze the m RNA expression of CDK7 in CCA.Immunohistochemistry was conducted in tissue array to validate the protein expression of CDK7 and the correlation between CDK7 and MCL1 in CCA tissue.After treating human CCA cell lines with drugs or si RNAs,CCK-8 was used to test cell viability.The results of CCK-8 were used to calculate Combination Index(CI)by Compu Syn software.Flow cytometry was used to detect cell apoptosis and Caspase3/7 activity kit and Brd U ELISA kit were used.RNA sequencing(RNA-Seq)was conducted to find downstream targets.Real-time q PCR and Western blot were conducted to measure the expression of m RNA and protein of targeted genes.By establishing Xenograft tumor model in nude mice,we measure the influence of THZ1 in vivo.Results Through analysis of public CCA datasets and immunohistochemistry of a CCA tissue array,we demonstrated that CDK7 was overexpressed in CCA tissues compared to normal bile duct or surrounding liver tissues.Inhibiting CDK7 by small molecular inhibitors or si RNA in CCA cell lines could impair cell viability,reduce cell proliferation and induce apoptosis.Druging nude mice with THZ1 delayed cell growth without unbearable toxicity.THZ1 treatment down regulated CDK7-mediated phosphorylation of RNP2,indicative of transcription inhibition.The results of RNA sequencing showed a striking impact on DNA templated transcriptional programs.THZ1 treatment down regulated a number of oncogenic transcription factors and survival proteins like FOSL1,RUNX1 and MCL1.Combining CDK7 inhibitors or si RNA with ABT-263 synergized in impairing cell viability and inducing cell apoptosis in CCA cells through inhibiting MCL1 expression.Conclusions THZ1,as a CDK7 inhibitor,impaired CCA cell viability and induced cell apoptosis.THZ1 treatment inhibited transcription prominently.Through inhibiting MCL1 expression,THZ1 synergized with ABT263.Our results demonstrated CDK7 could be a potential target in treating CCA.Combinations of CDK7 inhibition and BCL2/BCL-XL inhibition may offer a novel therapeutic strategy for CCA.