Characterizing CDK8/19 Inhibitors through a NFκB-Dependent Cell-Based Assay.

Li Li,Porter Porter,Chen Chen,Ji Ji,Broude Broude,Roninson Roninson

doi:10.3390/cells8101208

Li Li, Porter Porter + Show 4 more

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https://doi.org/10.3390/cells8101208

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Cell-based assays for CDK8/19 inhibition are not easily defined, since there are no known cellular functions unique to these kinases. To solve this problem, we generated derivatives of 293 cells with CRISPR knockout of one or both of CDK8 and CDK19. Double knockout (dKO) of CDK8 and CDK19 together (but not individually) decreased the induction of transcription by NFκB (a CDK8/19-potentiated transcription factor) and abrogated the effect of CDK8/19 inhibitors on such induction. We generated wild type (WT) and dKO cell lines expressing luciferase from an NFκB-dependent promoter. Inhibitors selective for CDK8/19 over other CDKs decreased TNFα-induced luciferase expression in WT cells by ~80% with no effect on luciferase induction in dKO cells. In contrast, non-selective CDK inhibitors flavopiridol and dinaciclib and a CDK7/12/13 inhibitor THZ1 (but not CDK4/6 inhibitor palbociclib) suppressed luciferase induction in both WT and dKO cells, indicating a distinct role for other CDKs in the NFκB pathway. We used this assay to characterize a series of thienopyridines with in vitro bone anabolic activity, one of which was identified as a selective CDK8/19 inhibitor. Thienopyridines inhibited luciferase induction in the WT but not dKO cells and their IC50 values in the WT reporter assay showed near-perfect correlation (R2 = 0.98) with their reported activities in a bone anabolic activity assay, confirming that the latter function is mediated by CDK8/19 and validating our assay as a robust and quantitative method for CDK8/19 inhibition.

Highlights

Small-molecule inhibitors of the Mediator kinase CDK8 and its paralog CDK19 are being actively developed by different groups for therapeutic applications in various cancers and other chronic diseases [1]
Suppressed luciferase induction in both WT and double knockout (dKO) cells, indicating a distinct role for other CDKs in the NFκB pathway. We used this assay to characterize a series of thienopyridines with in vitro bone anabolic activity, one of which was identified as a selective CDK8/19 inhibitor
We showed that shRNA knockdown of CDK8 and CDK19 together decreases the induction and diminishes the inhibitory effects of CDK8/19 inhibitors [15]

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Introduction

Small-molecule inhibitors of the Mediator kinase CDK8 and its paralog CDK19 are being actively developed by different groups for therapeutic applications in various cancers and other chronic diseases [1]. The first CDK8/19 inhibitor has entered clinical trials in estrogen receptor-positive breast cancer (ClinicalTrials.gov Identifier: NCT03065010) and another inhibitor in acute myeloid leukemia (ClinicalTrials.gov Identifier: NCT04021368). Evaluation and optimization of such inhibitors requires quantitative, robust and selective cell-based assays for CDK8/19 inhibition. The development of such assays is complicated, by the lack of known cellular functions that are unique to CDK8/19. There are no known protein substrates that would be phosphorylated exclusively by CDK8/19 [2]. The most widely used phosphorylation substrate to assay CDK8/19 kinase activity is a transcription factor STAT1.

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