Identifying CDK8/19 as a Novel Regulator of EWS-FLI1 Function in Ewing Sarcoma

Ewing sarcoma, a pediatric bone cancer, is characterized by the translocation of the Low Complexity (LC) domain from an RNA binding protein (EWSR1, FUS, or TAF15) with the DNA binding domain of an ETS transcription factor. The translocation EWS-FLI1 accounts for 90% of Ewing sarcoma tumors and a large body of evidence suggests that EWS-FLI1, acting as an oncogenic transcription factor, is the main driver of proliferation in Ewing sarcoma. Targeted therapies for Ewing sarcoma that act through disruption of EWS-FLI1 function remain undiscovered. Using an engineered degron system I can deplete endogenous EWS-FLI1 and have identified transcripts that are activated or repressed by EWS-FLI1. From the list of EWS-FLI1 regulated transcripts I have generated a series of endogenous reporter alleles to monitor transcript levels in response to small molecules treatment. This system has been utilized to screen 380,000 compounds from the UTSW chemical library in high throughput. I have identified several independent scaffolds capable of modulating several EWS-FLI1 response genes in multiple Ewing sarcoma cell lines. Unexpectedly, our screen enriched for small molecules that resemble previously described CDK8/19 inhibitors. A role for CDK8/19 in EWS-FLI1 transcriptional activity has not been reported. To determine if the remaining small molecules capable of modulating EWS-FLI1 transcriptional activity were acting through CDK8/19 inhibition I utilized a previously described IFN-STAT1 phosphorylation assay used to test CDK8/19 activity. I identified a novel, potent small molecule capable of interrupting CDK8/19 activity. Through biochemical reconstitution of the full CDK8 module, I determined our novel CDK8/19 inhibitor is an ATP competitive active site inhibitor. Inhibition of CDK8/19 resulted in activation of genes that EWS-FLI1 represses, suggesting a role for CDK8/19 in EWS-FLI1 transcriptional repression. Chromatin profiling of CDK8 and EWS-FLI1 shows colocalization across the chromatin. In addition, treatment of Ewing sarcoma cells with either known CDK8/19 inhibitors or our novel CDK8/19 inhibitor causes growth inhibition.

Author dc:creator

• McGinnis, James Henry, III

Contributors dc:contributor

• Liszczak, Glen
• McFadden, David G.
• Rosenbaum, Daniel M.
• Tagliabracci, Vincent S.

• Cyclin-Dependent Kinases
• Oncogene Proteins, Fusion
• Proto-Oncogene Protein c-fli-1
• RNA-Binding Protein EWS
• Sarcoma, Ewing

Language dc:language

en

Identifier

1522122366

OAI identifier oai:identifier

oai:utswmed-ir.tdl.org:2152.5/10596