Abstract CDK8 has been identified as being frequently upregulated in various types of human cancer such as colorectal cancer, certain sub-types of breast cancer and acute myeloid leukemia. In different tumor cell lines RNA interference mediated knockdown of CDK8 results in significant inhibition of tumor cell growth in vitro and in vivo. CDK8, therefore, represents an interesting target for the development of novel anti-cancer drugs. Various compounds targeting CDK8/Cyclin C with high potency in low nanomolar range have been reported. These compounds differ with respect to their binding modes, but all of them show high potency in biochemical assays, inhibition of tumor cell growth, and for many in vivo activities in different tumor cell xenograft models have been demonstrated. CDK8 binds to Cyclin C resulting in the active protein kinases complex. CDK8/Cyclin C interacts with MED12 and MED13, two proteins that regulate CDK8/Cyclin C activity and its interaction with the mediator complex. Mediator consists of 26 subunits and the interaction of the CDK8/Cyclin C/MED12/Med13 complex with mediator enables CDK8/Cyclin C to regulate gene transcription via C-terminal phosphorylation of RNA-polymerase II. This mechanism mediates an indirect regulation of transcription factors such as ß-catenin. In addition of its function via phosphorylation of RNA-polymerase II, CDK8/Cyclin C can also directly regulate the activity of transcription factors by phosphorylation. Best known examples are STAT1 and STAT5 which are phosphorylated by CDK8/Cyclin C on serine 727 and Serine 726, respectively. Recent data indicates that mutations of MED12 can result in a reduced sensitivity of CDK8/Cyclin C towards certain published CDK8/Cyclin C inhibitor in a cellular context. In this study we performed real-time quantitative PCR (qPCR) analyses to characterize the effect of different type I and type II CDK8/Cyclin C inhibitors on the cellular expression of selected genes controlled by two CDK8/Cyclin C dependent pathways, namely the WNT/ß-catenin- and the STAT-pathway. Furthermore, results of qPCR analyses of tumors derived from human xenograft in vivo models treated with type I and type II CDK8/Cyclin C inhibitors will be presented. Citation Format: Laura M. Jordt, Frank Totzke, Joachim Lauterwasser, Jan E. Ehlert, Koen Hekking, Bas Aerts, Cynthia Obodozie, Holger Weber, Gerhard Müller, Michael H. Kubbutat. Real-time quantitative PCR based analysis of transcriptional effects of CDK8/Cyclin C inhibitors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2318.