Mediator Kinase Inhibitor Selectivity and Activity in Colorectal Cancer

The Mediator complex is a regulator of gene expression,
influencing
chromatin structure and RNA polymerase II-mediated transcription.
Its activity is controlled by a protein kinase module, which includes
cyclin-dependent kinases 8 and 19, that phosphorylates RNA polymerase
II and transcription factors to regulate gene expression. Using orthogonal
approaches combining chemical and genetic tools, we demonstrated the
selectivity of our small-molecule inhibitors derived from 3,4,5-trisubstituted
pyridine and 3-methyl-1H-pyrazolo[3,4-b]pyridine chemical series in human colorectal cell culture and tumor
xenograft models. The lack of activity of our inhibitors in CDK8/19
double knockout models, with respect to molecular, proliferative,
and antitumor end points, revealed their specificity and dependence
on these kinases. Using our chemical probes and knockout models, we
explored Mediator kinase function in human colorectal cancer cells.
Phospho-proteome profiling revealed substrates enriched with transcription
and chromatin regulators, while promoter reporter experiments identified
transcription factor binding sites, including TCF/LEF and AP1, regulated
by Mediator kinases. Additionally, altered phosphorylation of several
Mediator subunits suggests a mechanism for the rapid regulation of
the Mediator complex. Overall, our results demonstrate that CDK8 and
CDK19 play pivotal roles in regulating gene expression associated
with oncogene activation and signaling pathways. Further studies are
warranted to elucidate their broader cellular roles and regulatory
mechanisms. The selective inhibitors validated in this study will
provide valuable tools for such mechanistic investigations into Mediator
kinase functions and their potential therapeutic exploitation.