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Confirmation of CDK8/19 inhibitor selectivity and activity in colorectal cancer [in vivo]

ABSTRACT: We verified the selectivity of two chemical series of inhibitors of the Mediator kinases using a combination of high-quality chemical probes and genetic knockouts. Knockout of CDK8 and/or CDK19 resulted in a range of overlapping and differential effects on gene expression or colony formation implying both shared and unique functions. Analysis of molecular responses following knockout or inhibitor treatment revealed that many of the changes measured were attributable to CDK8 inhibition. Knockout of both kinases resulted in enrichment of oncogene-associated gene expression consistent with a role gene expression associated with oncogene-dependence. Knockout of both kinases abrogated in vivo anti-tumour activity resulting from inhibitor treatment, while loss of CDK8 alone was sufficient to block inhibitor effects on colony growth in vitro. Overall, we demonstrated that our potent chemical probes are highly selective for the Mediator kinases in cancer cells and that many of the tumour-related responses to inhibitor treatment resulted from CDK8 inhibition.

ORGANISM(S): Homo sapiens

PROVIDER: GSE290001 | GEO | 2026/02/20

REPOSITORIES: GEO

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Project description:There is an unmet need for chemical tools to explore the role of the Mediator complex in human pathologies ranging from cancer to cardiovascular disease. Here we determine that CCT251545 a WNT-pathway inhibitor discovered by cell-based screening is a potent and selective chemical probe for the Mediator complex-associated protein kinases CDK8 and CDK19. CCT251545 is an ATP competitive inhibitor with >100-fold selectivity over 291 other kinases. X-ray crystallography demonstrates Type 1 binding involving insertion of the CDK8 C-terminus into the ligand binding site. In contrast to Type II inhibitors of CDK8/19 CCT251545 displays potent cell-based activity. We show that CCT251545 and close analogues not only alter WNT-pathway regulated gene expression but also other CDK8/19 targets including STAT1-regulated gene expression. Consistent with this we find that phospho-STAT1SER727 is a biomarker of CDK8 kinase activity in vitro and in vivo. Finally we demonstrate in vivo activity of CCT251545 in WNT-dependent tumours. Compound 1,2, 6 and 9 are close analogues of a 3,4,5-trisubstituted pyridine series identified from a high-throughput cell-based reporter assay of WNT signalling. They were shown to be potent and selective inhibitors of the Mediator complex-associated protein kinases CDK8 and CDK19. They are ATP competitive inhibitors with >100-fold selectivity over 291 other kinases. X-ray crystallography demonstrates Type 1 binding involving insertion of the CDK8 C-terminus into the ligand-binding site. A 3D culture model of murine intestinal-derived organoids expressing a transgenic doxycycline-inducible mutant beta-catenin expression following removal of doxycycline results in reduced WNT signalling and was compared to treatment for 24 hours with coumpound 2, 6 and 9. Compound 2 (n=3), compound 6 (n=4), compound 9 (n=3), doxycycline removal (n=4), doxycycline treatment (n=7). Samples were hybridized agianst mouse reference. Compounds 2, 6 and 9 are close analogues of a 3,4,5-trisubstituted pyridine series identified from a high-throughput cell-based reporter assay of WNT signalling. They were shown to be potent and selective inhibitors of the Mediator complex-associated protein kinases CDK8 and CDK19. They are ATP competitive inhibitors with >100-fold selectivity over 291 other kinases. X-ray crystallography demonstrates Type 1 binding involving insertion of the CDK8 C-terminus into the ligand-binding site.

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Confirmation of CDK8/19 inhibitor selectivity and activity in colorectal cancer [in vivo]

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