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Background. Cyclin-dependent kinase 8 (CDK8) is a multifaceted regulator and represents a catalytic component of the transcriptional Mediator complex. CDK8 activity, on the one hand, increases transcriptional elongation by the recruitment of Mediator/super elongation complexes, but, on the other hand, negatively regulates CDK7-controlled transcriptional initiation through inactivating cyclin H phosphorylation. Recently, these combined properties of CDK8 have also suggested its rate-limiting importance for herpesviral replication. Objectives. In this paper, we focused on human cytomegalovirus (HCMV) and addressed the question of whether the pharmacological inhibition or knock-down of CDK8 may affect viral replication efficiency in cell culture models. Methods. A number of human and animal herpesviruses, as well as non-herpesviruses, were used to analyze the importance of CDK8 for viral replication in cell culture models, and to assess the antiviral efficacy of CDK8 inhibitors. Results. Using clinically relevant CDK8 inhibitors (CCT-251921, MSC-2530818, and BI-1347), HCMV replication was found strongly reduced even at nanomolar drug concentrations. The EC50 values were consistent for three different HCMV strains (i.e., AD169, TB40, and Merlin) analyzed in two human cell types (i.e., primary fibroblasts and astrocytoma cells), and the drugs comprised a low level of cytotoxicity. The findings highlighted the following: (i) the pronounced in vitro SI values of anti-HCMV activity obtained with CDK8 inhibitors; (ii) a confirmation of the anti-HCMV efficacy by CDK8–siRNA knock-down; (iii) a CDK8-dependent reduction in viral immediate early, early, and late protein levels; (iv) a main importance of CDK8 for viral late-stage replication; (v) several mechanistic aspects, which point to a strong impact on viral progeny production and release, but a lack of CDK8 relevance for viral entry or nuclear egress; (vi) a significant anti-HCMV drug synergy for combinations of inhibitors against host CDK8 and the viral kinase vCDK/pUL97 (maribavir); (vii) finally, a broad-spectrum antiviral activity, as seen for the comparison of selected α-, β-, γ-, and non-herpesviruses. Conclusions. In summary, these novel data provide evidence for the importance of CDK8 as a positive regulator of herpesviral replication efficiency, and moreover, suggest its exploitability as an antiviral target for novel strategies of host-directed drug development.

Background. To date, a number of human pathogenic viruses are still unaddressed by the current repertoire of approved antiviral drugs. In order to widen this spectrum of preventive measures against virus infections, we have focused on additional host targets that exert interesting virus-supportive functions. Inhibitors of cyclin-dependent kinase 8 (CDK8) have been found to exhibit highly pronounced and relatively broad antiviral activity. Objectives. The current research question concerning the potential for broad-spectrum antiviral drug activity should be addressed in detail to understand the mechanistic basis of the antiviral target function of CDK8. Materials and Methods. We established and specifically customized six assay systems, three of these newly developed for the present study, to corroborate the range of CDK8 inhibitors’ antiviral activity against four α-, β-, and γ-herpesviruses as well as two non-herpesviruses. Results. Similar to our earlier analysis of CDK7 and CDK9 inhibitors, the clinically relevant CDK8 inhibitors currently in use demonstrated antiherpesviral activity in cell-culture-based infection models. Interestingly, the antiviral efficacy against various human and animal cytomegaloviruses was particularly strong at nanomolar concentrations, whereas other herpesviruses or non-herpesviruses showed an intermediate or low sensitivity to CDK8 inhibitors. Thus, this approach provided novel insights into the inhibitory potential of the CDK8 inhibitors, such as CCT-251921, MSC-2530818, and BI-1347, when analyzed against equine herpesvirus 1 (EHV-1, α-herpesvirus), human herpesvirus 6A (HHV-6A, β), Epstein–Barr virus (EBV, γ), murine herpesvirus 68 (MHV-68, γ), vaccinia virus (VV, non-herpes DNA virus), and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2, non-herpes RNA virus). Conclusions. Our results confirm that drug sensitivity to CDK8 inhibitors, on the one hand, is very strong for certain viruses and, on the other hand, varies widely within the spectrum of viruses and host cell types analyzed. This suggests that CDK8 may play several different roles in viral replication. The option of a refined CDK8-specific antiviral drug targeting is discussed.

The nuclear factor-κB (NFκB) family of transcription factors has been implicated in inflammatory disorders, viral infections, and cancer. Most of the drugs that inhibit NFκB show significant side effects, possibly due to sustained NFκB suppression. Drugs affecting induced, but not basal, NFκB activity may have the potential to provide therapeutic benefit without associated toxicity. NFκB activation by stress-inducible cell cycle inhibitor p21 was shown to bemediated by a p21-stimulated transcription-regulating kinase CDK8. CDK8 and its paralog CDK19, associated with the transcriptional Mediator complex, act as coregulators of several transcription factors implicated in cancer; CDK8/19 inhibitors are entering clinical development. Here we show that CDK8/19 inhibition by different small-molecule kinase inhibitors or shRNAs suppresses the elongation of NFκB-induced transcription when such transcription is activated by p21-independent canonical inducers, such as TNFα. On NFκB activation, CDK8/19 are corecruited with NFκB to the promoters of the responsive genes. Inhibition of CDK8/19 kinase activity suppresses the RNA polymerase II C-terminal domain phosphorylation required for transcriptional elongation, in a gene-specific manner. Genes coregulated by CDK8/19 and NFκB include IL8, CXCL1, and CXCL2, which encode tumor-promoting proinflammatory cytokines. Although it suppressed newly induced NFκB-driven transcription, CDK8/19 inhibition in most cases had no effect on the basal expression of NFκB-regulated genes or promoters; the same selective regulation of newly induced transcription was observed with other transcription signals potentiated by CDK8/19. This selective role of CDK8/19 identifies these kinases as mediators of transcriptional reprogramming, a key aspect of development and differentiation as well as pathological processes.

In recent years, long noncoding RNAs (lncRNAs) have received increasing attention and have been reported to be associated with various genetic abnormalities. However, the functions of many lncRNAs, including those of long exonic noncoding RNAs (lencRNAs), have not yet been elucidated. Here, we used a novel tethering luciferase assay to analyze the transcriptional regulatory functions of five lencRNAs that are upregulated in cancer. We found that the lencRNA PRKDC-210 interacts with MED12, a component of the CDK8 complex, to regulate the transcription of several genes. The transcriptional activation ability of PRKDC-210 was abolished in siRNA-treated CDK8-depleted cells. We also confirmed the enrichment of PRKDC-210 on RNA polymerase II. RNA-seq analysis of cells in which PRKDC-210 or PRKDC mRNA was knocked down using antisense oligonucleotides revealed that PRKDC-210 can affect the expression levels of genes related to fatty acid metabolism. Finally, we used a ChIRP assay to examine PRKDC-210-enriched sites in the genome. Overall, our findings demonstrate that the lencRNA PRKDC-210 promotes transcription through the CDK8 complex pathway at the transcription initiation site. We propose that PRKDC-210 can affect the transcription of adjacent genes after its transcription and splicing.

CDK8 is a key subunit of Mediator complex, a large multiprotein complex that is a fundamental part of the conserved eukaryotic transcriptional machinery. However, the biological functions of CDK8 in plant abiotic stress responses remain largely unexplored. Here, we demonstrated CDK8 as a critical regulator in the abscisic acid (ABA) signaling and drought response pathways in Arabidopsis. Compared to wild-type, cdk8 mutants showed reduced sensitivity to ABA, impaired stomatal apertures and hypersensitivity to drought stress. Transcriptomic and chromatin immunoprecipitation analysis revealed that CDK8 positively regulates the transcription of several ABA-responsive genes, probably through promoting the recruitment of RNA polymerase II to their promoters. We discovered that both CDK8 and SnRK2.6 interact physically with an ERF/AP2 transcription factor RAP2.6, which can directly bind to the promoters of RD29A and COLD-REGULATED 15A (COR15A) with GCC or DRE elements, thereby promoting their expression. Importantly, we also showed that CDK8 is essential for the ABA-induced expression of RAP2.6 and RAP2.6-mediated upregulation of ABA-responsive genes, indicating that CDK8 could link the SnRK2.6-mediated ABA signaling to RNA polymerase II to promote immediate transcriptional response to ABA and drought signals. Overall, our data provide new insights into the roles of CDK8 in modulating ABA signaling and drought responses.

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.

Mediator is a multiprotein complex that integrates the signals from transcription factors binding to the promoter and transmits them to achieve gene transcription. The subunits of Mediator complex reside in four modules: the head, middle, tail, and dissociable CDK8 kinase module (CKM). The head, middle, and tail modules form the core Mediator complex, and the association of CKM can modify the function of Mediator in transcription. Here, we show genetic and biochemical evidence that CKM-associated Mediator transmits auxin-dependent transcriptional repression in lateral root (LR) formation. The AUXIN/INDOLE 3-ACETIC ACID 14 (Aux/IAA14) transcriptional repressor inhibits the transcriptional activity of its binding partners AUXIN RESPONSE FACTOR 7 (ARF7) and ARF19 by making a complex with the CKM-associated Mediator. In addition, TOPLESS (TPL), a transcriptional corepressor, forms a bridge between IAA14 and the CKM component MED13 through the physical interaction. ChIP assays show that auxin induces the dissociation of MED13 but not the tail module component MED25 from the ARF7 binding region upstream of its target gene. These findings indicate that auxin-induced degradation of IAA14 changes the module composition of Mediator interacting with ARF7 and ARF19 in the upstream region of their target genes involved in LR formation. We suggest that this regulation leads to a quick switch of signal transmission from ARFs to target gene expression in response to auxin.

Breast cancers (BrCas) that overexpress oncogenic tyrosine kinase receptor HER2 are treated with HER2-targeting antibodies (such as trastuzumab) or small-molecule kinase inhibitors (such as lapatinib). However, most patients with metastatic HER2⁺ BrCa have intrinsic resistance and nearly all eventually become resistant to HER2-targeting therapy. Resistance to HER2-targeting drugs frequently involves transcriptional reprogramming associated with constitutive activation of different signaling pathways. We have investigated the role of CDK8/19 Mediator kinase, a regulator of transcriptional reprogramming, in the response of HER2⁺ BrCa to HER2-targeting drugs. CDK8 was in the top 1% of all genes ranked by correlation with shorter relapse-free survival among treated HER2⁺ BrCa patients. Selective CDK8/19 inhibitors (senexin B and SNX631) showed synergistic interactions with lapatinib and trastuzumab in a panel of HER2⁺ BrCa cell lines, overcoming and preventing resistance to HER2-targeting drugs. The synergistic effects were mediated in part through the PI3K/AKT/mTOR pathway and reduced by PI3K inhibition. Combination of HER2- and CDK8/19-targeting agents inhibited STAT1 and STAT3 phosphorylation at S727 and upregulated tumor suppressor BTG2. The growth of xenograft tumors formed by lapatinib-sensitive or -resistant HER2⁺ breast cancer cells was partially inhibited by SNX631 alone and strongly suppressed by the combination of SNX631 and lapatinib, overcoming lapatinib resistance. These effects were associated with decreased tumor cell proliferation and altered recruitment of stromal components to the xenograft tumors. These results suggest potential clinical benefit of combining HER2- and CDK8/19-targeting drugs in the treatment of metastatic HER2⁺ BrCa.

Salicylic acid (SA) is a plant defense signal that mediates local and systemic immune responses against pathogen invasion. However, the underlying mechanism of SA-mediated defense is very complex due to the involvement of various positive and negative regulators to fine-tune its signaling in diverse pathosystems. Upon pathogen infections, elevated level of SA promotes massive transcriptional reprogramming in which Non-expresser of PR genes 1 (NPR1) acts as a central hub and transcriptional coactivator in defense responses. Recent findings show that Enhanced Disease Susceptibility 1 (EDS1) also functions as a transcriptional coactivator and stimulates the expression of PR1 in the presence of NPR1 and SA. Furthermore, EDS1 stabilizes NPR1 protein level, while NPR1 sustains EDS1 expression during pathogenic infection. The interaction of NPR1 and EDS1 coactivators initiates transcriptional reprogramming by recruiting cyclin-dependent kinase 8 in the Mediator complex to control immune responses. In this review, we highlight the recent breakthroughs that considerably advance our understanding on how transcriptional coactivators interact with their functional partners to trigger distinct pathways to facilitate immune responses, and how SA accumulation induces dynamic changes in NPR1 structure for transcriptional reprogramming. In addition, the functions of different Mediator subunits in SA-mediated plant immunity are also discussed in light of recent discoveries. Taken together, the available evidence suggests that transcriptional coactivators are essential and potent regulators of plant defense pathways and play crucial roles in coordinating plant immune responses during plant–pathogen interactions.

Zyxin is a member of the focal adhesion complex and plays a critical role in actin filament polymerization and cell motility. Several recent studies showed that Zyxin is a positive regulator of Yki/YAP (Yes-associated protein) signaling. However, little is known about the mechanisms by which Zyxin itself is regulated and how Zyxin affects Hippo–YAP activity. We first showed that Zyxin is phosphorylated by CDK1 during mitosis. Depletion of Zyxin resulted in significantly impaired colon cancer cell proliferation, migration, anchorage-independent growth, and tumor formation in xenograft animal models. Mitotic phosphorylation is required for Zyxin activity in promoting growth. Zyxin regulates YAP activity through the colon cancer oncogene CDK8. CDK8 knockout phenocopied Zyxin knockdown in colon cancer cells, while ectopic expression of CDK8 substantially restored the tumorigenic defects of Zyxin-depletion cells. Mechanistically, we showed that CDK8 directly phosphorylated YAP and promoted its activation. Fully activated YAP is required to support the growth in CDK8-knockout colon cancer cells in vitro and in vivo. Together, these observations suggest that Zyxin promotes colon cancer tumorigenesis in a mitotic-phosphorylation-dependent manner and through CDK8-mediated YAP activation.