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Introduction: CDK8 and its paralogue CDK19 are highly similar cyclin-dependent kinases that function as components of the kinase module of the Mediator complex. Despite their structural similarity, emerging data suggest that these proteins may have distinct functions in the regulation of transcription. Recently, we identified two chemical series of CDK8/19 ligands (1, 2); however, as these ligands have similar binding affinities for CDK8 and CDK19 it has not been possible, until now, to determine if the biologic effects observed were driven by inhibition of one or both kinases. Here, we describe a functional genomics approach that utilizes these ligands, and others described in the literature, to identify substrates of CDK8/19 and isoform-dependent functions of these kinases. Materials and Methods: Experiments were conducted on COLO205 and SW620 human colorectal cancer cell lines and SW620 cells harbouring CRISPR knockouts of CDK8 and/or CDK19. Transcription factor reporter assays were carried out to define signalling pathways and biologic processes modulated by CDK8/19 ligands. Cells were reverse-transfected with both an inducible transcription-factor-responsive construct encoding firefly luciferase under the control of a basal promoter element, and a construct constitutively expressing the Renilla luciferase. At 42 h post-transfection, cells were treated with one of our CDK8/19 ligands, CCT251545 or MSC2530818, or the CDK8/19 inhibitors Senexin B (3) or Selvita 120-34 (4), at a 10 x EC 50 concentration. Reporter activity was quantified following 6 h continuous exposure. In addition, gene set enrichment analysis (GSEA) was employed to examine changes in gene expression resulting from ligand treatment or CDK8/19 knockout, including changes in the expression of those genes regulated by transcription factors identified in the pathway reporter assay. Results: The pathway reporter assay identified transcription factor activity that was modulated by compound treatment and/or CDK8/19 knockout. Enriched transcription factors included key players in Notch signalling, Wnt signalling, the interferon gamma response, the cellular stress response, and MAPK/Jnk signalling, all of which are known to be modulated directly or indirectly by CDK8. Some of the transcription factors were differentially regulated by the test compounds, as well as by CDK8, CDK19 and CDK8/CDK19 knockout. Data from the CRISPR-mediated CDK8/19 knockout line demonstrated that the majority of altered gene expression, and corresponding decreased phosphorylation of STAT1 SER727 , was driven by loss of CDK8 with some limited compensatory activity from CDK19. These observations suggest that there may be some functional differences between the two kinases and that the main biologic effects of ligand treatment result from CDK8 inhibition. Conclusions: These experiments have identified potential substrates of CDK8/19 and determined that the majority of the effects on gene expression or biomarker phosphorylation observed in colorectal cancer cell lines after treatment with CDK8/19 ligands are driven by the inhibition of CDK8. References: 1. Mallinger A. et al. J Med Chem 2015;58:1717-35; 2. Czodrowski P, Mallinger A. et al. J Med Chem 2016;59:9337-49; 3. Roninson IB. Porter DC.; Wentland MP WO2013116786 A1, 201; 4. Rzymski T, Mikula M, et al. Oncotarget 2017;8:20:33779-95. Citation Format: Olajumoke O. Popoola, Maria J. Ortiz-Ruiz, Konstantinos Mitsopoulos, Aurelie Mallinger, Robert Te-Poele, Kai Schiemann, Paul Workman, Dirk Wienke, Julian Blagg, Suzanne A. Eccles, Paul A. Clarke. Exploring isoform-dependent functions of the mediator kinases in colorectal cancer [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2017 Oct 26-30; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2018;17(1 Suppl):Abstract nr A187.