CDK8 inhibition sequesters Med1 at EWS-FLI enhancers [CUT&RUN]

Ewing sarcoma is characterized by a chromosomal translocation resulting in the fusion protein EWSR1-FLI1. To identify small molecules that selectively impair the oncogenic transcriptional program enacted by EWS-FLI1 we employed a novel phenotypic screen. Our screening hits were enriched for inhibitors of cyclin dependent kinase 8/19. Inhibition of CDK8/19 resulted in a proliferation defect in Ewing sarcoma cells and a reduction in growth of Ewing sarcoma xenografts. We performed a genome wide pooled CRISPR screen that suggested that small molecule inhibition of CDK8/19 acted in a gain-of-function manner that did not replicate the effects of CDK8/19 genetic knockout. Through biochemical studies and chromatin profiling, we discovered that CDK8/19 inhibitors act through a novel trapping mechanism in which the inhibitor prevents dissociation of the CDK8 kinase from core Mediator. This mechanism has broad implications for the development of small molecules to target transcriptional regulation.