Abstract
CDK9 is the kinase subunit of positive transcription elongation factor b (P-TEFb) that enables RNA polymerase (Pol) II's transition from promoter-proximal pausing to productive elongation. Although considerable interest exists in CDK9 as a therapeutic target, little progress has been made due to lack of highly selective inhibitors. Here, we describe the development of i-CDK9 as such an inhibitor that potently suppresses CDK9 phosphorylation of substrates and causes genome-wide Pol II pausing. While most genes experience reduced expression, MYC and other primary response genes increase expression upon sustained i-CDK9 treatment. Essential for this increase, the bromodomain protein BRD4 captures P-TEFb from 7SK snRNP to deliver to target genes and also enhances CDK9's activity and resistance to inhibition. Because the i-CDK9-induced MYC expression and binding to P-TEFb compensate for P-TEFb's loss of activity, only simultaneously inhibiting CDK9 and MYC/BRD4 can efficiently induce growth arrest and apoptosis of cancer cells, suggesting the potential of a combinatorial treatment strategy.
Highlights
The proper control of eukaryotic gene expression is fundamental for normal development and cellular response to environmental challenges
The C-terminal domain (CTD) Serine-2 phosphorylation, which is a hallmark of productive elongation and RNA processing, is catalyzed by the positive transcription elongation factor b (P-TEFb), which is composed of CDK9 and its cyclin partner T1 (CycT1) or the minor forms T2a and T2b
High throughput screening combined with crystal structure-enabled lead compound optimization has led to the identification of a novel and potent CDK9-selective inhibitor called i-CDK9. i-CDK9 has a N2′-(trans-4-aminocyclohexyl)-5′-chloro-N6-(3-fluorobenzyl)-2,4′-bipyridine-2′,6-diamine scaffold that is structurally distinct from flavopiridol and all the other known non-selective cyclin-dependent kinases (CDKs) inhibitors (Figure 1A)
Summary
The proper control of eukaryotic gene expression is fundamental for normal development and cellular response to environmental challenges. The control frequently occurs at the level of transcription, where RNA polymerase (Pol) II is employed to execute a series of interconnected stages that collectively constitute the transcription cycle. The early stages of this cycle involving the recruitment of Pol II to gene promoters and assembly of active pre-initiation complexes were considered the primary step where transcription is controlled (Kuras and Struhl, 1999; Ptashne, 2005). The extensive and dynamic modifications of the Pol II C-terminal domain (CTD) have been linked to specific stages of the transcription cycle and mRNA processing. The CTD Serine-2 phosphorylation, which is a hallmark of productive elongation and RNA processing, is catalyzed by the positive transcription elongation factor b (P-TEFb), which is composed of CDK9 and its cyclin partner T1 (CycT1) or the minor forms T2a and T2b. P-TEFb phosphorylates the SPT5 subunit of DSIF and the NelfE subunit of NELF, which antagonizes the
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