CDK8 inhibition induces Mediator trapping and impairment of the EWSR1::FLI1 transcriptional program in Ewing sarcoma.

Ewing sarcoma is characterized by a chromosomal translocation resulting in the fusion protein EWSR1::FLI1. We utilize endogenous EWSR1::FLI1 target gene reporters in patient-derived cell lines to perform a high-throughput phenotypic screen to identify small molecules that impair the EWSR1::FLI1 transcriptional program. We discovered that inhibitors of cyclin-dependent kinase 8 (CDK8), including a novel pyridyl imidazole, altered transcription of EWSR1::FLI1 target genes and CDK8 co-localized with EWSR1::FLI1 preferentially on single GGAA DNA binding motifs. Using pooled CRISPR screening, biochemical studies, and chromatin profiling, we discovered that CDK8 inhibitors suppressed proliferation of Ewing sarcoma cells through a gain-of-function mechanism by inducing molecular trapping of the CDK8 kinase module and Mediator complex on chromatin. This mechanism has implications for the development of small molecules to target transcription and suggests that impairment of transcriptional regulatory complex dynamics might serve as a vulnerability in cancers in which the dominant oncogenes act as dosage-sensitive transcription factors.