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Pluripotent stem cells (PSCs) transition between cell states in vitro, reflecting developmental changes in the early embryo. PSCs can be stabilized in the naive state by blocking extracellular differentiation stimuli, particularly FGF–MEK signalling. Here, we report that multiple features of the naive state in human and mouse PSCs can be recapitulated without affecting FGF–MEK signalling or global DNA methylation. Mechanistically, chemical inhibition of CDK8 and CDK19 (hereafter CDK8/19) kinases removes their ability to repress the Mediator complex at enhancers. CDK8/19 inhibition therefore increases Mediator-driven recruitment of RNA polymerase II (RNA Pol II) to promoters and enhancers. This efficiently stabilizes the naive transcriptional program and confers resistance to enhancer perturbation by BRD4 inhibition. Moreover, naive pluripotency during embryonic development coincides with a reduction in CDK8/19. We conclude that global hyperactivation of enhancers drives naive pluripotency, and this can be achieved in vitro by inhibiting CDK8/19 kinase activity. These principles may apply to other contexts of cellular plasticity.Lynch et al. demonstrate that inhibiting CDK8 and CDK19 kinases increases Mediator-driven recruitment of RNA Pol II to promoters and enhancers, therefore stabilizing the naive transcriptional program.

The Mediator kinase module regulates eukaryotic transcription by phosphorylating transcription-related targets and by modulating the association ofMediator and RNA polymerase II. The activity of its catalytic core, cyclin-dependent kinase 8 (CDK8), is controlled by Cyclin C and regulatory subunit MED12, with its deregulation contributing to numerous malignancies. Here, we combine in vitro biochemistry, cross-linking coupled to mass spectrometry, and in vivo studies to describe the binding location of the N-terminal segment of MED12 on the CDK8/Cyclin C complex and to gain mechanistic insights into the activation of CDK8 by MED12. Our data demonstrate that the N-terminal portion of MED12 wraps around CDK8, whereby it positions an “activation helix” close to the T-loop of CDK8 for its activation. Intriguingly, mutations in the activation helix that are frequently found in cancers do not diminish the affinity of MED12 for CDK8, yet likely alter the exact positioning of the activation helix. Furthermore, we find the transcriptome-wide gene-expression changes in human cells that result from a mutation in the MED12 activation helix to correlate with deregulated genes in breast and colon cancer. Finally, functional assays in the presence of kinase inhibitors reveal that binding of MED12 remodels the active site of CDK8 and thereby precludes the inhibition of ternary CDK8 complexes by type II kinase inhibitors. Taken together, our results not only allowus to propose a revised model of how CDK8 activity is regulated by MED12, but also offer a path forward in developing small molecules that target CDK8 in its MED12-bound form.

Salt stress has devastating effects on agriculture, yet the key regulators modulating the transcriptional dynamics of salt-responsive genes remain largely elusive in plants. Here, we discover that salt stress substantially induces the kinase activity of Mediator cyclin-dependent kinase 8 (CDK8), which is essential for its positive role in regulating salt tolerance. CDK8 is identified to phosphorylate AT-hook motif nuclear-localized protein 10 (AHL10) at serine 314, leading to its degradation under salt stress. Consistently, AHL10 is found to negatively regulate salt tolerance. Transcriptome analysis further indicates that CDK8 regulates over 20% of salt-responsive genes, half of which are co-regulated by AHL10. Moreover, AHL10 is revealed to recruit SU(VAR)3-9 homologs (SUVH2/9) to AT-rich DNA sequences in the nuclear matrix-attachment regions (MARs) of salt-responsive gene promoters, facilitating H3K9me2 deposition and repressing salt-responsive genes. Our study thereby has identified the CDK8-AHL10-SUVH2/9 module as a key molecular switch controlling transcriptional dynamics in response to salt stress. The authors demonstrate that salt stress activates CDK8, which phosphorylates AHL10 and promotes its degradation. AHL10 is involved in recruiting SUVH2/9 to repress salt-responsive genes. The CDK8-AHL10-SUVH2/9 module is critical for transcriptional dynamics in response to salt stress.

Mediator kinases CDK19 and CDK8, pleiotropic regulators of transcriptional reprogramming, are differentially regulated by androgen signaling, but both kinases are upregulated in castration-resistant prostate cancer (CRPC). Genetic or pharmacological inhibition of CDK8 and CDK19 reverses the castration-resistant phenotype and restores the sensitivity of CRPC xenografts to androgen deprivation in vivo. Prolonged CDK8/19 inhibitor treatment combined with castration not only suppressed the growth of CRPC xenografts but also induced tumor regression and cures. Transcriptomic analysis revealed that Mediator kinase inhibition amplified and modulated the effects of castration on gene expression, disrupting CRPC adaptation to androgen deprivation. Mediator kinase inactivation in tumor cells also affected stromal gene expression, indicating that Mediator kinase activity in CRPC molded the tumor microenvironment. The combination of castration and Mediator kinase inhibition downregulated the MYC pathway, and Mediator kinase inhibition suppressed a MYC-driven CRPC tumor model even without castration. CDK8/19 inhibitors showed efficacy in patient-derived xenograft models of CRPC, and a gene signature of Mediator kinase activity correlated with tumor progression and overall survival in clinical samples of metastatic CRPC. These results indicate that Mediator kinases mediated androgen-independent in vivo growth of CRPC, supporting the development of CDK8/19 inhibitors for the treatment of this presently incurable disease.

Chemoproteomic studies have revealed that a Wnt-pathway inhibitor, CCT251545, is a potent and selective small-molecule chemical probe that inhibits the Mediator complex–associated protein kinases CDK8 and CDK19 through a type 1 binding mode and modulates the growth of Wnt-dependent tumors. There is unmet need for chemical tools to explore the role of the Mediator complex in human pathologies ranging from cancer to cardiovascular disease. Here we determine that CCT251545, a small-molecule inhibitor of the WNT pathway discovered through cell-based screening, is a potent and selective chemical probe for the human Mediator complex–associated protein kinases CDK8 and CDK19 with >100-fold selectivity over 291 other kinases. X-ray crystallography demonstrates a type 1 binding mode involving insertion of the CDK8 C terminus into the ligand binding site. In contrast to type II inhibitors of CDK8 and CDK19, CCT251545 displays potent cell-based activity. We show that CCT251545 and close analogs alter WNT pathway–regulated gene expression and other on-target effects of modulating CDK8 and CDK19, including expression of genes regulated by STAT1. Consistent with this, we find that phosphorylation of STAT1 SER727 is a biomarker of CDK8 kinase activity in vitro and in vivo . Finally, we demonstrate in vivo activity of CCT251545 in WNT-dependent tumors.

Glioblastoma (GBM) is the most malignant form of glioma. Glioma stem cells (GSCs) contribute to the initiation, progression, and recurrence of GBM as a result of their self-renewal potential and tumorigenicity. Cyclin-dependent kinase 8 (CDK8) belongs to the transcription-related CDK family. Although CDK8 has been shown to be implicated in the malignancy of several types of cancer, its functional role and mechanism in gliomagenesis remain largely unknown. Here, we demonstrate how CDK8 plays an essential role in maintaining stemness and tumorigenicity in GSCs. The genetic inhibition of CDK8 by shRNA or CRISPR interference resulted in an abrogation of the self-renewal potential and tumorigenicity of patient-derived GSCs, which could be significantly rescued by the ectopic expression of c-MYC , a stem cell transcription factor. Moreover, we demonstrated that the pharmacological inhibition of CDK8 significantly attenuated the self-renewal potential and tumorigenicity of GSCs. CDK8 expression was significantly higher in human GBM tissues than in normal brain tissues, and its expression was positively correlated with stem cell markers including c-MYC and SOX2 in human GBM specimens. Additionally, CDK8 expression is associated with poor survival in GBM patients. Collectively, these findings highlight the importance of the CDK8-c-MYC axis in maintaining stemness and tumorigenicity in GSCs; these findings also identify the CDK8-c-MYC axis as a potential target for GSC-directed therapy.

CDK8 and CDK19 paralogs are regulatory kinases associated with the transcriptional Mediator complex. We have generated mice with the systemic inducible Cdk8 knockout on the background of Cdk19 constitutive knockout. Cdk8/19 double knockout (iDKO) males, but not single Cdk8 or Cdk19 KO, had an atrophic reproductive system and were infertile. The iDKO males lacked postmeiotic spermatids and spermatocytes after meiosis I pachytene. Testosterone levels were decreased whereas the amounts of the luteinizing hormone were unchanged. Single-cell RNA sequencing showed marked differences in the expression of steroidogenic genes (such as Cyp17a1, Star, and Fads ) in Leydig cells concomitant with alterations in Sertoli cells and spermatocytes, and were likely associated with an impaired synthesis of steroids. Star and Fads were also downregulated in cultured Leydig cells after iDKO. The treatment of primary Leydig cell culture with a CDK8/19 inhibitor did not induce the same changes in gene expression as iDKO, and a prolonged treatment of mice with a CDK8/19 inhibitor did not affect the size of testes. iDKO, in contrast to the single knockouts or treatment with a CDK8/19 kinase inhibitor, led to depletion of cyclin C (CCNC), the binding partner of CDK8/19 that has been implicated in CDK8/19-independent functions. This suggests that the observed phenotype was likely mediated through kinase-independent activities of CDK8/19, such as CCNC stabilization.

NPR1 (NONEXPRESSER OF PR GENES1) functions as a master regulator of the plant hormone salicylic acid (SA) signaling and plays an essential role in plant immunity. In the nucleus, NPR1 interacts with transcription factors to induce the expression of PR (PATHOGENESIS-RELATED) genes and thereby promote defense responses. However, the underlying molecular mechanism of PR gene activation is poorly understood. Furthermore, despite the importance of NPR1 in plant immunity, the regulation of NPR1 expression has not been extensively studied. Here, we show that SA promotes the interaction of NPR1 with both CDK8 (CYCLIN-DEPENDENT KINASE8) and WRKY18 (WRKY DNA-BINDING PROTEIN18) in Arabidopsis (Arabidopsis thaliana). NPR1 recruits CDK8 and WRKY18 to the NPR1 promoter, facilitating its own expression. Intriguingly, CDK8 and its associated Mediator subunits positively regulate NPR1 and PR1 expression and play a pivotal role in local and systemic immunity. Moreover, CDK8 interacts with WRKY6, WRKY18, and TGA transcription factors and brings RNA polymerase II to NPR1 and PR1 promoters and coding regions to facilitate their expression. Our studies reveal a mechanism in which NPR1 recruits CDK8, WRKY18, and TGA transcription factors along with RNA polymerase II in the presence of SA and thereby facilitates its own and target gene expression for the establishment of plant immunity.

Uterine fibroids (UFs) are the most common non-cutaneous tumors in women worldwide. UFs arise from genetic alterations in myometrial stem cells (MM SCs) that trigger their transformation into tumor-initiating cells (UF SCs). Mutations in the RNA polymerase II Mediator subunit MED12 are dominant drivers of UFs, accounting for 70% of these clinically significant lesions. Biochemically, UF driver mutations in MED12 disrupt CDK8/19 kinase activity in Mediator, but how Mediator kinase disruption triggers MM SC transformation remains unknown. Here, we show that pharmacologic inhibition of CDK8/19 in MM SCs removes a barrier to myogenic differentiation down an altered pathway characterized by molecular phenotypes characteristic of UFs, including oncogenic growth and extracellular matrix (ECM) production. These perturbations appear to be induced by transcriptomic changes, arising in part through epigenomic alteration and super-enhancer reprogramming, that broadly recapitulate those found in MED12-mutant UFs. Altogether, these findings provide new insights concerning the biological role of CDK8/19 in MM SC biology and UF formation. Key messages Mediator kinase inhibition in myometrial stem cells (MM SCs) induces spontaneous differentiation. Transcriptional changes upon Mediator kinase inhibition recapitulate those of MED12 mutant uterine fibroids (UFs). Such transcriptional changes are partially mediated by super-enhancer reprogramming. Mediator kinase functions to enforce cell states and its loss induces cellular plasticity.

Accurate genome duplication underlies genetic homeostasis. Metazoan Mdm2 binding protein (MTBP) forms a main regulatory platform for origin firing together with Treslin/TICRR and TopBP1 (Topoisomerase II binding protein 1 (TopBP1)-interacting replication stimulating protein/TopBP1-interacting checkpoint and replication regulator). We report the first comprehensive analysis of MTBP and reveal conserved and metazoa-specific MTBP functions in replication. This suggests that metazoa have evolved specific molecular mechanisms to adapt replication principles conserved with yeast to the specific requirements of the more complex metazoan cells. We uncover one such metazoa-specific process: a new replication factor, cyclin-dependent kinase 8/19-cyclinC (Cdk8/19-cyclin C), binds to a central domain of MTBP. This interaction is required for complete genome duplication in human cells. In the absence of MTBP binding to Cdk8/19-cyclin C, cells enter mitosis with incompletely duplicated chromosomes, and subsequent chromosome segregation occurs inaccurately. Using remote homology searches, we identified MTBP as the metazoan orthologue of yeast synthetic lethal with Dpb11 7 (Sld7). This homology finally demonstrates that the set of yeast core factors sufficient for replication initiation in vitro is conserved in metazoa. MTBP and Sld7 contain two homologous domains that are present in no other protein, one each in the N and C termini. In MTBP the conserved termini flank the metazoa-specific Cdk8/19-cyclin C binding region and are required for normal origin firing in human cells. The N termini of MTBP and Sld7 share an essential origin firing function, the interaction with Treslin/TICRR or its yeast orthologue Sld3, respectively. The C termini may function as homodimerisation domains. Our characterisation of broadly conserved and metazoa-specific initiation processes sets the basis for further mechanistic dissection of replication initiation in vertebrates. It is a first step in understanding the distinctions of origin firing in higher eukaryotes.