Structural and Functional Characterization of Human Transcription-regulating Kinases

Abstract

Accurate regulation of RNA polymerase II by transcriptional kinases is essential for normal development and dysregulation can promote cancer onset and progression. Small molecules targeting transcriptional kinases, such as CDKs and DYRKs, hold promise for novel treatment options in oncology and beyond. <br /> HIPKs are regulators of various cellular signalling pathways and belong to the DYRK kinase family. They are involved in the pathology of cancer, chronic fibrosis, diabetes, and multiple neurodegenerative diseases. In this study, characteristic structural features of the HIP kinase family were identified based on the HIPK3 crystal structure in the apo form at 2.5 Å resolution. Recombinant HIPKs and its close relative DYRK1A phosphorylate core components of the transcription machinery, such as the negative elongation factor SPT5, the transcription factor c-Myc, as well as pol II CTD, suggesting a direct role in transcriptional regulation. Abemaciclib, a Cdk4/Cdk6 inhibitor, which was approved by the FDA for the treatment of metastatic breast cancer, was identified in a database screen as a potent inhibitor of HIPK2, HIPK3, and DYRK1A. The crystal structures of HIPK3 and DYRK1A bound to abemaciclib were determined and demonstrated a binding mode to the kinase hinge region similar to Cdk6. Remarkably, abemaciclib inhibits DYRK1A to the same extent as Cdk4/Cdk6 in vitro, raising the question of whether transcriptional inhibition through DYRK1A contributes to the therapeutic activity of this breast cancer drug and whether abemaciclib may be a suitable treatment option for Alzheimer´s disease. <br /> CDKs are dependent on a Cyclin subunit for activation and are central regulators of cell cycle and transcription. In contrast to cell cycle CDKs that can be activated promiscuously by multiple different Cyclins, transcriptional CDKs are thought to be activated by only one particular Cyclin partner. Although this long-established dogma exists, an unbiased approach to systematically test the formation of transcriptional CDK/Cyclin complexes revealed that both Cyclins, CycK and CycT1, are able to activate Cdk9, Cdk12 and Cdk13 in vitro. Cdk9/CycK has been a little appreciated phenomenon ever since the discovery of Cdk9 and we recently discovered a Cdk13/CycT1 complex mediating RNA surveillance in melanoma (Insco et al., Science, 2023). The crystal structure of the novel Cdk12/CycT1 complex at 2.0 Å resolution revealed a highly identical CDK/Cyclin interface compared to cognate Cdk12/CycK. Cdk13/CycT1 co-exists with Cdk13/CycK in HEK293T and A375 cells and ChIP-seq analysis confirmed co-localization of Cdk13 and CycT1 at transcription start sites of similar genes. Furthermore, Hex1 and Brd4 regulate Cdk9/CycT1, but no other transcriptional CDKs. Finally, small-molecular compounds inhibit CDKs independent of the bound Cyclin and Targeted protein degraders are highly specific for their targeted Cdk or Cyclin. The discovery of these novel CDK/Cyclin pairs challenges a long-standing dogma of CDK biology and may facilitate advances in the development of therapeutics targeting transcriptional CDKs.