Biochimica et Biophysica Acta 1854 (2015) 1617–1629

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Review

CDK8 kinase—An emerging target in targeted cancer therapy☆ Tomasz Rzymski a,1, Michał Mikula b, Katarzyna Wiklik a, Krzysztof Brzózka a,⁎ a b

Selvita SA, Kraków, Poland Maria Sklodowska-Curie Memorial Cancer Center and Institute of Oncology, Warsaw, Poland

a r t i c l e

i n f o

Article history: Received 18 March 2015 Received in revised form 15 May 2015 Accepted 16 May 2015 Available online 22 May 2015 Keywords: CDK8 kinase Cancer Novel therapies Kinase inhibitors Transcription regulation

a b s t r a c t Cyclin-dependent kinase (CDK) inhibitors have been developed as potential anticancer therapeutics and several nonselective compounds are currently in advanced clinical trials. This review is focused on the key biological roles of CDK8 kinase, which provide a proof-of-principle for continued efforts toward effective cancer treatment, targeting activity of this CDK family member. Among currently identified kinase inhibitors, several displayed significant selectivity for CDK8 and notably the effectiveness in targeting cancer specific gene expression programs. Structural features of CDK8 and available ligands were discussed from a perspective of the rational drug design process. Current state of the art confirms that further development of CDK8 inhibitors will translate into targeted therapies in oncology. This article is part of a Special Issue entitled:Inhibitors of Protein Kinases. © 2015 Elsevier B.V. All rights reserved.

1. Cyclin-dependent kinases are deregulated in cancer Uncontrolled cell divisions are one of the hallmarks of cancer and various strategies for inhibition of cell cycle have proven to be successful in oncology. Pioneering work of Lee Hartwell, Tim Hunt and Paul Nurse discovered master regulators of the cell cycle machinery, protein cyclins and their regulatory partners cyclin-dependent kinases (CDKs). CDKs are serine/threonine (Ser/Thr) kinases, which become active only in association with their regulatory partners (i.e., cyclins or other proteins) [1]. It is well established that cancer-related genetic aberrations occur frequently in cyclins, CDKs, CDK inhibitors, as well as their substrates like retinoblastoma protein (Rb). These events were postulated to override cell cycle restriction points and as a consequence could induce uncontrolled proliferation of tumor cells. Indeed, various alterations in the CDK-related cell control cascade have been observed with high frequency in cancer [2,3]. CDK4 and CDK6 proteins could be overexpressed and mutations in CDKs resulting in the loss of binding to inhibitory proteins have also been identified with low frequency in cancer. Deregulated expression is more evident for partnering cyclins, where aberrant cyclin D1 expression occurs to high extent in many cancers [4]. Well documented is the association with B-cell malignancies where translocation that juxtaposes the cyclin D1 gene to the immunoglobulin heavy chain gene, leads to cyclin D1 overexpression in mantle cell lymphoma. Several other cyclins particularly cyclin A, D2, D3 and E have also been found to be elevated in cancer [5]. ☆ This article is part of a Special Issue entitled: Inhibitors of Protein Kinases. ⁎ Corresponding author. E-mail addresses: [email protected] (T. Rzymski), [email protected] (K. Brzózka). 1 Tel.: +48 784024024.

http://dx.doi.org/10.1016/j.bbapap.2015.05.011 1570-9639/© 2015 Elsevier B.V. All rights reserved.

Notably, the most evident in neoplasm are alterations of regulatory CDK/cyclin proteins, including members of Cdc25 phosphatase family and cyclin kinase inhibitors (CKI) [6]. Cdc25 are potent oncogenes and control entry and progression through various phases of the cell cycle by unscheduled dephosphorylation of CDKs. The inhibitory activity of CKI is perturbed in a high percentage of cancers as a result of deletion, point mutation and epigenetic changes. Finally, Rb, a tumor suppressor protein and the most important CDK substrate is truncated or deleted in several major cancers [7]. Overall, alterations of at least one of CDK regulators and effectors are found in virtually all types of cancer.

2. Inhibition of cyclin-dependent kinases modulates transcription Consequently, CDKs appeared to be an attractive and, as later proven, druggable protein targets for cancer therapy. CDK kinase inhibitors have emerged as a novel class of targeted small molecule agents with great therapeutic potential in multiple indications, confirmed by a recent approval of palbociclib, a selective CDK4/6 inhibitor for a combination treatment of ER positive, Her negative breast cancer. Currently, numerous pharmacological CDK inhibitors have been described, and some of these, have shown signs of efficacy toward various types of cancer in vitro and in vivo [2]. Several compounds advanced further into clinical tests and have also displayed preliminary evidence of antitumor activity. The antitumor activity of CDK inhibitors has been primarily attributed to their capacity to induce cell cycle block. Notably, recent evidences seem to indicate that transcriptional modulation by these agents may be at least equally important [8]. Among twenty CDKs characterized in

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human genome, several members, particularly CDK7 to CDK13, have been described as primarily or solely involved in transcriptional control [9] (Table 1). Recently, high interest has focused on these CDKs and several lines of evidences support their anti-cancer potential. It has been noted that pan-CDK inhibitors have the potential to both induce cell cycle block, as well as to trigger apoptosis. Analysis of the possible mechanism underlying apoptosis seems to indicate, that impeded mRNA synthesis significantly contributes to the effectiveness of multitargeted CDK inhibitors [20,21]. Several CDK inhibitors block CDK9 activity to much higher extent than other members of the family. This activity has been attributed to the induction of apoptosis by suppressing transcription of short-lived antiapoptotic proteins, such as Mcl-1 in chronic lymphocytic leukemia cells [22,8]. Still, it is unclear to what extent CDK9 is required for transcription of all RNA polymerase (RNAP) II-dependent genes. Results from genetic silencing and pharmacological inhibitors are not consistent, indicating variability related to the discrete changes in kinetics and range of CDK9 depletion. From the perspective of development of novel therapies, there are two problems with current CDK inhibitors. The first is narrow therapeutic window by their indiscriminate toxicity to proliferating normal cells and tissues. The second is limited efficacy of selective agents targeting cell cycle regulating CDKs and incomplete understanding of the mechanism of action for pan-CDK inhibitors, which are otherwise efficacious in pre-clinical experiments. Currently, the most promising class of cell cycle targeting agents are compounds with a dual CDK4/CDK6 activity where the proper patient stratification criteria involves ER+/HER2 − breast cancer patients with overexpression of cyclin D and inactivating

Table 1 List of cyclin-dependent kinases involved in transcriptional control together with their respective transcription associated substrates. Kinase/and primary cyclin complex CDK7/cyclin H/MAT1

Function

Dual role in cell cycle control by phosphorylation of cell cycle regulating CDKs within the activation segment (T-loop), and in transcription as a component of the general transcription factor TFIIH, which phosphorylates the C-terminal domain (CTD) of the RNAP II CDK8/cyclin Forms a kinase submodule of C mediator complex, involved in both transcriptional activation and repression by phosphorylation of CTD of the RNAP II and transcriptional factors. Gene-specific rather than global modulator of transcription. CDK9/cyclin Component of the multiprotein T complex P-TEFb which controls the elongation phase of transcription by RNAP II by hyperphosphorylation of the CTD CDK12/cyclin Dual role as a transcription K elongation CTD kinase and splicing, possibly by phosphorylating SRSF1/SF2 CDK13/cyclin Dual role as a transcription K elongation CTD kinase and splicing, possibly by phosphorylating SRSF1/SF2 CDK19/cyclin High degree of structural and C functional homology to CDK8

Substrates

Ser5 and Ser7 of CTD [10], CDK9 [10]

Ser2 and Ser5 of CTD [11,12], STAT1 [13], H3Ser10 [11], E2F1 [14,15], SMAD1 and 3 [16], Notch ICN1 [17,18],

Ser2 of CTD [19], DSIF and NELF [19]

Ser2 of CTD

Ser2 of CTD

Ser5 of CTD Notch ICN1 [18]

mutations in p16 [23]. Further work is also needed for identification of appropriate patient selection criteria for other classes of CDK-targeting compounds. Notably, functional redundancy between components of the core cell cycle machinery could be limited when combined with transcriptional CDKs [24]. Efforts aimed at the identification of CDK-subtype selective compound with a standalone efficacy have led to the development of selective CDK7 and CDK9 inhibitors [25–28]. Transcriptional repression by these agents results in rapid depletion of MCL1 and the induction of apoptosis in primary chronic lymphocytic leukemia (CLL) cells, however toxic effects of global transcriptional manipulation in non-tumor cells seem to limit their therapeutic application as single agents.

3. Mechanism of RNAP II-dependent transcriptional control by CDKs In eukaryotes the process of genomic DNA sequence transcription into RNA molecule is catalyzed by the three RNAPs, namely I, II and III. Of them, RNAP II transcribes all protein-coding, and some classes of non-coding (nc) RNAs including miRNA. Transcription occurs in the context of chromatin where genomic DNA is organized into an array of nucleosomes composed of 8 histone proteins (2 each of H2A, H2B, H3, and H4) that can be covalently modified. These nucleosomes and their higher order arrangement present a barrier to the steps of transcription; thus chromatin provides a step in regulating access to the genome for the transcription machinery [29]. During gene-specific transcription RNAP II is recruited to gene promoters. Its binding is in part controlled by sequencespecific, DNA-binding transcription factors (TFs). RNAP II contains highly unstructured c-terminal domain (CTD) consisting of multiple repeats of a heptapeptide, with the number of repeats ranging from 26 repeats in yeast to 52 in mammals. The CTD is the target of posttranslational modifications, including phosphorylation, and serves as a scaffold to dock transcription-associated proteins. CTD phosphorylation sites and level changes dynamically during transcription generating a complex regulatory code referred as the “CTD code” [30]. Three CDKs, CDK7, CDK8 and CDK9 are involved in phosphorylating CTD and control of RNAP II processing through initiation, elongation, and termination stages of transcription. Although such a division is descriptive, certain CDKs driven CTD modifications orchestrate the recruitment and interaction of factors along a transcribed gene what allows establish frames for the subsequent steps of transcription. TFs bind directly to DNA and control transcription by modulating initiation step that includes the recruitment of RNAP II with unphosphorylated CTD together with several general transcription factors (GTFs) and mediator complex to form the pre-initiation complex (PIC). After PIC assembly CDK7 subunit of GTF transcription factor II (TFIIH) phosphorylates Ser5 and Ser7 residues of the CTD [10]. The elongation step involves the initial abortive formation of a nascent transcript, an early paused state, and a transition to productive elongation where the CDK9 subunit of the positive transcription elongation factor b (P-TEFb) phosphorylates Ser2 of CTD and RNAP II associated negative elongation factor (NELF) and DRB-sensitivity inducing factor (DSIF) to release the transcriptional complex to a productive elongation stage with the simultaneous processing of the transcript [19]. Phosphorylation levels of Ser5 are abundant at the promoter and decrease toward the 3′ end while Ser2 levels are low at the transcription start site and increase toward the 3′ end of genes. Several lines of evidence support the notion on CDKs crosstalk during transcription process. For example, it was shown that Ser5 priming of CTD by CDK7 is prerequisite for P-TEFb recruitment. Furthermore CDK7 activity is required for activation of CDK9 on chromatin in vivo and CDK7 impairment leads to reduced phosphorylation of chromatin-bound RNAP II on CTD Ser2 [10].

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4. CDK8 emerged as an oncogene involved in modulation of transcription Much later it has been discovered that CDK8, another member of the transcriptional subtype of the family, shows remarkable cancer tissue specific expression profile and rather more selective contribution to the regulation of gene expression levels123132 (Fig. 1). CDK8 is a part of mediator complex that functions as a bridge between basal transcriptional machinery and gene-specific transcriptional factors. CDK8 submodule consisting of 4 proteins CDK8, MED12, MED13 and CCNC can reversibly associate with the mediator complex. The uncertainty on CDK8 role in the initiation step of transcription stems from the observations suggesting mutually exclusive mediator's CDK8 module presence and RNAP II occupancy at certain genes [33,34]. Biochemical experiments from Taatjes laboratory showed that when bound to mediator, the CDK8 module blocked mediator-RNAP II binding. Importantly, the CDK8 kinase activity was not required to trigger repression and the presence of either MED12 or MED13 was indispensable for transcriptional RNAP II repression by CDK8 module [31]. On the other hand, the evidence exists on mediator role in recruitment of RNAP II elongation factors to genes. For example, mediator can recruit, a pTEFb containing, complex called Super Elongation Complex (SEC), in the mechanisms of mediator's subunit MED26 direct interaction [35] and through SEC's recruitment to CDK8 module [36]. For the latter instance, knockdown of CDK8 in HCT116 cells lowered RNAP II CTD Ser5 and Ser2 phosphorylation, impaired transcription elongation and

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the recruitment of SEC factors selectively to immediate early response (IER) genes activated during serum response [36]. Similarly, RNAP II elongation and the recruitment of SEC at hypoxia-inducible genes depend on CDK8 where a hypoxia-inducible factor I (HIF1)A transactivation domain binds CDK8 module that bridges SEC to RNAP II and helps to stimulate RNAP II pause-release to elongation [37]. Apart from the direct involvement into transcription process the CDK8 was shown to aid this process on chromatin level through nucleosomes phosphorylation at histone H3S10 [11], a mark associated with transcriptional activation of IER genes [38]. Additional work revealed an interaction of CDK8 with acetyltransferase 2A (also known as GCN5L) where both proteins as a complex cooperatively phospho-acetylated histone H3 to generate the dual H3S10p/K14Ac mark [39]. Interestingly, the kinase activity of CDK8 module toward H3S10 phosphorylation has been shown to be modulated by a class of long ncRNA termed ncRNAactivated (ncRNA-a) transcribed from enhancers. Knocking down a given ncRNA-a resulted in a specific decrease in H3S10 phosporylation at target genes [40]. Same study implicated Med12 subunit, a part of CDK8 module, as an ncRNA-a binding factor that allows DNA looping between the ncRNA-a enhancers and their gene targets. This observation indicates the importance of CDK8 module in regulating distant enhancer–promoter interactions what may provide means for specific genes expression. However, the most prominent and extent knowledge on chromatin-centered CDK8 activity is linked with both repression and activation of TFs coupled to specific cellular pathways. The evidence has accumulated underlying CDK8 module roles in already mentioned

Fig. 1. Different signaling pathways employ CDK8 as an effector acting in a context-dependent manner to modulate gene expression levels. Moving clockwise; (1) CDK8 phosphorylates and blocks cyclin-H, a regulatory component of a GTF TFIIH, what represses RNAP II transcription initiation, on the other hand, CDK8 is able to phosphorylate RNAP II CTD at Ser2 and Ser5 at regulated genes. (2) CDK8 also acts on nucleosome level by the mechanism involving phosphorylation of Ser10 of histone H3, a permissive histone mark associated with gene expression activation. (3) CDK8 directly phosphorylates E2F1 protein what leads to expression of b-catenin dependent genes. (4) CDK8 also phosphorylates chromatin bound STAT1 at Ser 727 and activates INFg-dependent gene expression. (5) CDK8 phosphorylates NOTCH1 (ICN1) intracellular domain what leads to ICN1 ubiquitination (ub) and degradation, and ultimately inhibits NOTCH1-dependent gene expression.

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serum [36] and hypoxia response network [37], and other pathways including the p53 network [41], the Wnt–β-catenin pathway [14,15], the Notch1 [17] and STAT1 signaling [13], and pathways regulated by SMAD TFs [16]. 5. CDK8 is differentially expressed in cancer CDK8 was found to be ubiquitously expressed during early embryonic development. Although CDK8 has been found to be dispensable for viability of immortalized human embryonic kidney cells 293FT, homozygous CDK8 −/− knockout resulted in an early embryonic lethality at the stage of 8-cells before compaction [42]. This is an intriguing observation in the context of reported role of CDK8 and many subunits of the mediator complex in maintenance of embryonic and cancer stem cells pluripotency [43,44]. In adult, differentiated tissues expression of CDK8 is rather low, whereas elevated expression could be observed in colorectal cancer (CRC) across various datasets. In the Cancer Genome Atlas (TCGA) 222 colorectal samples with available microarray data, high expression could be observed in 23% cases (Z-score threshold N2), whereas in 365 samples analyzed with the next generation RNA sequencing elevated expression could be observed in 42% CRC cases. CDK8 protein levels were also found to be significantly elevated in adenocarcinoma when compared with adjacent normal tissue, although no correlation was found either with grade or stage of examined tumors [45]. CDK8 gene resides at a region 13q12.13–13q12.2. This region is often amplified in CRC cell lines and primary tumors, with a frequency second to the most amplified MYC bearing amplification fragment 8q24. Functional studies on cancer cell lines indicated that CDK8 could a major oncogenic driver for this amplification [15]. Although CDK8 copy number gain is rather common, it was confirmed to be a result of polysomy rather than focal amplification. GIST 2.0 analysis of TCGA indicates that 58% and 6% out of 615 CRC samples carry mono and biallelic, amplification of CDK8 locus, respectively. (TCGA Research Network: http://cancergenome.nih.gov/). Interestingly, in few CRC cases, CDK8 is excluded from 13q12.13-2 amplification what raised important question whether CDK8 is indeed a primary target of this aberration [46]. 6. Role of CDK8 in regulation of β-catenin activity The role of CDK8 in cancer is documented by far the best in regulation of β-catenin-dependent expression 4715. Hyperactivation of this pathway occurs in almost all CRC as a result of inactivating mutations in adenomatous polyposis coli (APC), which is a negative regulator of β-catenin, and mutations leading to increased stability of β-catenin. Upregulation of β-catenin can contribute to tumorigenesis in many experimental cancer models and its high nuclear levels in CRC could predict bad prognosis, providing a rationale for targeting this pathway in oncology [48–51]. Even though a few approved drugs were found to non-selectively modulate β-catenin-pathway such as non-steroidal anti-inflammatory drugs (NSAID), there has been also a growing interest in the development of more selective strategies aiming at β-cateninpathway inhibitors as an effective anticancer therapy. Clearly this concept proved more challenging than initially anticipated, primarily due to the functional redundancy, low drugability and often context dependent roles of proteins in the pathway. Cells engineered to express β-catenin driven reporters showed to be valuable tools for identification of novel small molecule inhibitors [49,52]. Similar strategy was applied in the loss of function screening for novel protein modulators β-catenindependent expression. Notably, selective CDK8 knockdown was found to be sufficient for significant repression of β-catenin activity in CRC cell lines with both APC deletion and stabilizing mutation in β-catenin [15]. Kinase activity of CDK8 turned out to be essential for β-catenin-dependent transformation and sensitivity to CDK8 knockdown could be largely determined based on the high CDK8 level and copy number gain. Association between CDK8 and β-catenin pathway was also

observed in clinical CRC specimens, where nuclear β-catenin levels, used as a hallmark of the activated pathway, significantly correlated with the high CDK8 expression. Several questions still remain to be answered. From a pharmacological intervention point of view it would be essential to show whether inhibition of CDK8 kinase activity is sufficient for effective repression of β-catenin pathway? It has been convincingly demonstrated for cells with disturbed mediator complex by silencing of CDK8, CCNC and MED12, whereas preliminary studies using selective CDK8 inhibitor Senexin A resulted only in partially suppressed β-catenin activity [53]. Further studies are also required to clarify which CDK8-mediated phosphorylation events promote transcription. Direct phosphorylation of β-catenin could not be excluded and ChIP confirmed CDK8 occupancy on MYC promoter containing β-catenin/TCF elements, however further detailed mechanistic studies are required [15]. Alternative mechanism involving CDK8-dependent regulation of Rb-associated transcription factor 1 (E2F1) has been proposed [47]. E2F1 is situated downstream of various signaling cascades, where it regulates genes required for cell-cycle progression by acting either as a transcriptional activator or as a repressor. Active E2F1 was found to repress the activity of β-catenin transcription and promote cell apoptosis and this activity was found to be antagonized by a direct CDK8 phosphorylation at Ser375 [14]. Interesting, so far several microarray transcriptional profiling experiments failed to identify broad deregulation of β-catenin signaling in cells with disturbed CDK8 [36,37,43,54]. If obvious technical limitations of such studies could be disregarded, these results indicate that CDK8 controls transcription also outside the β-catenin signaling pathway. This notion is also supported by the observation that CDK8induced oncogenic transformation of fibroblast cells was only partially suppressed by inhibition of β-catenin signaling [15]. 7. CDK8 immediate early response in cancer CDK8 knockdown studies on CRC cells indicated significant disturbances in RNAP II dependent transcriptional machinery, particularly at IER genes. These genes are classified into this family based on the rapid, however transient upregulation following an external stimulus such as growth factors, hormones or stress [55]. In cancer many pathways that regulate IER genes are constitutively active such as MAPK and ERK1/2 signaling pathways, therefore IER expression is typically higher during cancer progression and often correlates with poor prognosis and resistance to standard therapies. Many IER genes are potential targets for inhibition in oncology, however due to the redundancy of upstream regulatory elements, many strategies proved to be either ineffective or are prone to development of drug resistance. CDK8 was found to act at the level of transcriptional elongation which involves recruitment of both CDK7 and P-TEFb complex [36]. In contrast to similar experiments conducted for CDK9 knockdown cells, changes in phosphorylation of RNAP II CTD in CDK8 deficient cells were restricted only to IER genes, without affecting global CTD phosphorylation marks. Thus, in this context inhibition of CDK8 could be explored as a specific strategy for repression of mitogenic signals in cancer. 8. CDK8 regulates DNA-damage response and paracrine activities in cancer Assembly of the CDK8-mediator complex has been also shown for p53 target genes including a damage-inducible cell-cycle inhibitor p21 (CDKN1A) after stimulation with DNA-damaging agents such as radiation therapy, doxycycline and fluorouracil in cancer cells [41]. Early studies supported the view that p21 acts as a tumor suppressor promoting cell cycle arrest in response to various stimuli, however recent evidences indicate that depending on the context p21 can well strongly promote cancer progression [56]. Genotoxic response is not only restricted to cancer cells. Stromal cells respond to chemotherapy by increased paracrine activity, creating a microenvironment providing

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a chemo-resistant reservoir that may subsequently fuel tumor relapse [57]. This tumor promoting phenotype was found to be mediated at least in part by p21 [53,58]. In contrast to cell cycle-control members of the CDK family, p21 stimulated CDK8 activity. Specific CDK8 inhibitor Senexin A blocked p21 downstream induction of NF-κB pathway and counteracted doxorubicin inducted paracrine activity [53]. Consequently, this inhibitor increased efficacy of chemotherapy against various cancer cells, particularly when grown in the presence of normal cells. A number of additional CDK8-dependent paracrine mediators have been identified that might account for the survival of cancer cells in tumor microenvironment. STAT proteins upon phosphorylation mediate paracrine signals into changes in gene expression. These phosphorylation events most often are catalyzed by the family of receptor-associated Janus kinases (JAKs) which converts them from a latent cytoplasmic form into nuclear active dimers [59]. Additional phosphorylation events occur at C-terminal transactivation domains (TAD) of STATs and result in increased transcriptional activity. JAK inhibitors are potent suppressors of tyrosine as well as followed TAD serine phosphorylation events in the canonical signaling, however in noncanonical TAD serine phosphorylation of STATs occurs without concomitant tyrosine phosphorylation. CDK8 was shown to specifically activate STAT1 at the TAD while recruited to gene promoters in response to stimulation with interferon γ (INFg) [13]. Interestingly, attenuation of STAT1s Serine 727 site resulted in impaired RNAP II elongation and lowered gene expression of INFg dependent genes. This work implicated CDK8 as a controller of STATs and selective regulator of INFg target-gene expression. The aforementioned pathway is typically associated with antiviral defense and tumor-suppressive functions, however emerging results indicate that STAT1 pathway may well confer cellular resistance to DNA-damaging agents and mediate aggressive tumor growth [60–66]. CDK8-inducted phosphorylation of STAT1 at serine 727 was also shown to be suppressive for the natural Killers (NK) cells activity [67]. STAT1-S727A-mutated NK cells displayed increased release of cytotoxic proteins such as granzyme B and perforin and in general higher cytotoxicity toward cancer cells. This work provides a rationale for further studies with CDK8 inhibitors also as cancer immunotherapy. Various ligands for transforming growth factor-β (TGFβ) receptors govern the development and tissue homeostasis and also modulate tumor microenvironment. Activation of the TGFβ receptors and bone morphogenetic protein (BMP) result in the formation of different SMAD TF complexes to trigger downstream gene expression [68]. CDK8 was shown to phosphorylate an interdomain linker region in SMADs transcriptional activator proteins what resulted in their transcriptional activation and also primed them for ubiquitin mediated degradation [16]. This result highlights the importance of CDK8 in SMADs activation and turnover to ensure precise temporal control of TGF β- and BMP-responsive genes. 9. CDK8 regulates NOTCH signaling The Notch1 signaling is important for cell–cell communication, that triggers gene expression program that control cell differentiation including T cell lineage commitment from common lymphoid precursor [69]. The Notch1 protein is a transmembrane receptor that upon activation undergoes cleavage and release of the cytoplasmic ICN1 part, which then translocates to the nucleus activating transcription of specific genes. CDK8 phosphorylates ICN1 what results in increased ubiquitination and proteasome-driven degradation [17]. Recently, the role of this dependency was underlined in hematopoietic malignancies, specifically the T-cell acute lymphoblastic leukemia (T-ALL), where cyclin C was shown to function as tumor suppressor controlling ICN1 levels and hyperactivation of oncogenic Notch1 pathway through its kinase partners CDK8, CDK19 and CDK3 [18]. Collectively, the presented evidence supports the idea that during transcription the CDK8 functions in context-specific matter; its

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biological role may differ among cell types or in response to distinct stimuli. This context could be therefore exploited to specifically target CDK8-dependent transcription process. Contrary to the two other transcription-associated CDKs, CDK7 and CDK9, with important role in global RNAP II transcription initiation and elongation, where targeting their activity could alter expression of thousands of genes, CDK8 presents potentially attractive target for small molecule intervention to precisely act on context-related gene expression. 10. The structure of CDK8/cyclin C Rational design of CDK inhibitors led to development of many potent and selective molecules, what would be otherwise impossible without detailed structural data. Crystal structure of CDK8 was determined for the first time in 2011 [70]. Up to date, 10 structures of CDK8 complexed with cyclin C (CDK8/CycC) [70,97] were deposited in the Protein Data Bank, PDB [71] and provide valuable information regarding peculiarities of CDK8 structure, differences between an active and inactive conformation of CDK8, structural changes upon ligand binding of type I and type II inhibitors, interactions between CDK8 and cyclin C and mechanism of activation (Table 2, Fig. 2). Furthermore, experimentally determined structure of a target is a standard for structure-based drug design (SBDD) [72]. Therefore such wealth of structural data is invaluable for drug design targeting CDK8. CDK8 shows typical for a protein kinase, bilobal architecture comprising the N-lobe (residues 1–96) and the C-lobe (residues 97–353) with the catalytic cleft between the two lobes [73]. In respect of secondary structure, CDK8 shows high similarity to regions well conserved in structures of the CDK family members [70]. However, there can be found some peculiarities of CDK8 [70] including (i) the DMG motif followed by Phe instead of the DFG followed by Leu found in other CDKs, (ii) an extended C-terminal domain which is suspected to play a role in selective ligand binding [74], (iii) helices αGH1 to αGH3, cluster close by, (iv) insertion consisting of nine residues (240EDIKTSNPY248) in front of the αG helix, and (v) an additional N terminal helix αB preceding the recognized αC helix (Fig. 3, left panel). The latter plays a pivotal role in cyclin C recognition and attributes to an exceptionally high affinity between CDK8 and cyclin C [70]. This is due to the fact, that other members of the CDK family such as CDK9 or CDK2 are lacking αB helix and therefore surface contact area between CDK and cyclin is reduced (Fig. 3). Generally, however, the structure of CDK8/CycC complex resembles CDK9/CycT in that the two subunits interact mainly through N-terminal domains [70] (Fig. 3, left and middle panels). The activation mechanism of CDK8 became more tangible in the light of the recently solved an apo structure (4G6L) [85], however few questions await answers. The apo structure of CDK8/CycC complex in spite of the lack of the phosphorylated residue within the activation segment (T-loop) has characteristics of an active “αC helix pushed-in” conformation which enables formation of a conserved salt bridge between Glu66CDK8 (αC helix) and Lys52CDK8. However, the visible fragment of the T-loop (missing 10 residues, 186–195) displays an unusual conformation as for an open conformation and seems to obstruct binding of a potential substrate (partially open). Moreover CDK8 contains three arginines Arg65CDK8, Arg150CDK8 and Arg178CDK8, well conserved in the CDK family [70] which in the CDK2 structure are stabilized by phosphothreonine (pThr160CDK2) and serine residue of CDK8 (Ser182CDK8) that, intriguingly, is overlapping with pThr160CDK2 [75] (Fig. 4, left and right panels). The aforementioned Ser182CDK8 however, was not identified as a phosphorylation site in CDK8 [76]. Instead, it was speculated that glutamate residue in cyclin C (Glu99Cyc) mimics phosphoresidue and interacts with arginines [70]. Indeed, the interaction of Glu99CycC with Arg150CDK8 and/or Arg65CDK8 (CDK8/CycC complexes displaying DMG-in conformation, CDK8/CycC complexed with compound SKR-2) can be found in CDK8/CycC complexes (Fig. 4, right panel). Interestingly, CDK2/CycA was also found to be able to

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Table 2 List of CDK8 structures available in PDB. SRT—short residence time (b1.4 min), DRT—detectable residence time (between SRT and LRT), LRT—long residence time (N15 min). PDB ID

DMG conformation

Compound name

CDK8, Kd (μM)

Binding kinetics

Resolution (Å)

Release date

3RGF 4F6S 4F7J 4F70 4F6U 4F7N 4F7L 4F6W 4F7S 4G6L

Out Out Out Out Out Out Out Out In In

Sorafenib SKR-7 SKR-3 SKR-4 SKR-5 SKR-11 SKR-2 SKR-1 Type I compound –

0.31 [78] 3.24 [85] 5.82 [85] 1.82 [85] 0.70 [85] 0.08 [85] 0.01 [85] 0.03 [85] 2.44 [85] –

LRT [85] SRT [85] SRT [85] SRT [85] DRT [85] DRT [85] LRT [85] LRT [85] LRT [85] –

2.20 2.60 2.60 3.00 2.10 2.65 2.90 2.39 2.20 2.70

2011-08-10 2013-05-01 2013-05-01 2013-05-01 2013-05-01 2013-05-01 2013-05-01 2013-05-01 2013-05-01 2013-05-01

compensate the lack of the phosphoresidue [86,87]. The partially active and non-phosphorylated CDK2/CycA showed Glu162CDK2 mimicking pThr160CDK2 by means of forming interactions with conserved Arg126CDK2 and Arg150CDK2 (Fig. 4, middle panel). However, even though it is binding ATP according to the expected binding mode, the nonphosphorylated CDK2/CycA structure was reported to show only 1% of kinase activity compared to the fully activated form [86]. Phosphorylation of CDK2 appears as crucial for the activity, however this not necessarily has to be true for CDK8 in every context. CDK8/CycC complex as a member of multiprotein transcription regulatory complex (part of CDK-module of mediator [88]) might undergo further structural rearrangements upon association with its CDK-module partners (MED12, MED13) leading to an open form of a kinase. In summary, cyclin C binding is sufficient for the first step of CDK8 activation (“αC helix pushed-in” conformation) but the second step (T-loop open conformation) is not clear. Therefore, there is a missing piece in the puzzle to reveal the complete mechanism of CDK8 activation. Studies focused on the regulation of CDK8 [11,31,39] provided valuable evidences that kinase activity was related not only to the kinase itself but also to the substrate binding, enabled by other members of mediator complex. For example it was found that MED12 – but not MED13 – was required for kinase activity toward RNAPII CTD [11]. In turn, association of the CDK-module with the core of mediator, resulted in kinase activity toward histone H3 on chromatin [11]. As a conclusion, in order to fully understand structural aspects of CDK8, it needs to be regarded within the specific context of its already deciphered biological function. Currently available structures of CDK8/CycC complexes give a detailed insight into flexibility of the ATP binding site and the surrounding residues. CDK8 is the only member of the CDK family which was shown to bind type II inhibitors and adopt DMG-out conformation [70]. As an example is the structure of CDK8/CycC complexed with sorafenib (Fig. 7). It displays deep pocket binding, disrupting salt bridge between Glu66CDK8 and Lys52CDK8 and forms two hydrogen bonds with Glu66CDK8 and one hydrogen bond with Asp173CDK8 (backbone amide) in the DMG motif (Fig. 5, left panel). Additionally, sorafenib forms two hydrogen bonds with the hinge region (Ala100CDK8) and many van der Waals interactions as its buried surface is 92.1%. There are seven other structures

of CDK8/CycC complexed with type II inhibitors with deep pocket binding moiety grafted from BIRB-796 (Fig. 2). They also display DMG-out conformation and together with kinetic studies [85] provide a concrete picture of structure-kinetic relationship (Table 2). It was shown that elongation of the residence time which is regarded as a key success factor for compound optimization in drug discovery [77], is attributed to a conserved interactions with the hinge and hydrophobic contacts within CDK8 front pocket [85] including Arg356, His 106, Trp 105, Asp103 and Ala155 (Fig. 5, right panel). The latter type of contacts is present in another CDK8/CycC structure with type I inhibitor (Figs. 2, 6). Additionally, it forms one hydrogen bond with the hinge (Ala100CDK8), one water mediated hydrogen bond with Asp173CDK8, π–π stacking interaction with the gatekeeper residue (Phe97CDK8) and van der Waals interactions with side chains that constitute “the ceiling” and “the floor” of the ATP binding pocket (Ala50CDK8, Val27CDK8, Gly28CDK8, Val35CDK8, Tyr99CDK8, Ile79CDK8, Leu158CDK8, Ala172CDK8). Interestingly, the structure of this compound is a sub-structure of known CDK8 inhibitors referred as SNX2-class compounds (described below). Additionally, CDK8 structure was studied in the context of CDKmodule from yeast [89,90,91] and human [11,31] by means of macromolecular electron microscopy. It was noted that the shape of yeast CDK-module [90] is strikingly similar to the human CDK-module [31]. Additionally, it was found that Med13 was mediating interaction between CDK-module and mediator core [31,90]. However, the subunit organization of CDK-module was delineated only in the case of yeast [90]. According to these studies, CDK8/CycC complex forms the head domain (“Head”) of the CDK-module, Med12 subunit corresponds to the central density of the CDK-module, termed “Body” and MED13 subunit forms the mobile “Leg” [90]. This architecture suggests that Med12 contributes to regulation of CDK8 kinase activity by providing an access to its substrates [90] which is in accordance with aforementioned experiments [11]. At the same time, both Med12 and Med13 mediate assembly and stabilization of the CDK-module, while CDK8/CycC forms a stable sub-complex [90]. In summary, CDK8/CycC is an extensively studied target in respect of 3D structure as well as quaternary structure as part of multiprotein complexes.

Fig. 2. Ligands complexed with CDK8/cyclin C structure (sorafenib presented in Fig. 7).

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Fig. 3. Peculiarities of CDK8 structure. Left panel: CDK8/CycC complex (4G6L) with DMG motif as spheres. Atypical fragments in CDK family found in CDK8 structure colored red. Undetermined structural fragments shown in dotted line. Cyclin C—purple, N lobe—blue, C lobe—orange. Middle panel: CDK9/CycT complex (3BLQ) with DFG motif as spheres. Cyclin T—gray, CDK9—cyan. Right panel: CDK2/CycA (1P5E) with DFG motif as spheres. Cyclin A—yellow, CDK2—green. All protein complexes shown in ribbon representation. Pictures prepared with PyMOL.

11. Development of CDK8 inhibitors Although CDK8 is a promising novel target in oncology, the search of CDK8 inhibitors seems to have just started as most of CDK8-specific inhibitors are found in patent applications published in 2013 and 2014. Several known kinase inhibitors demonstrated binding affinity to CDK8 when evaluated against a panel of human protein kinases [78,79]. In this way following CDK8 binders/inhibitors were found: BMS-387032/ SNS-032, CP-724714, EXEL-2880/GSK-1363089, Flavopyridol, PLX-4720, Staurosporine and type II inhibitors ABT-869, AST-487, BIRB-796, Sorafenib (Fig. 7). Most of the primary targets for these kinase inhibitors are members of a tyrosine kinase group (TK) or containing CDK, MAPK, GSK3 and CLK (CMGC, Table 3). Selectivity of these compounds ranges from low (Staurosporine, AST-487, EXEL-2880/GSK-1363089), through medium (Flavopyridol, ABT-869, Sorafenib, BMS-387032/SNS-032, PLX-4720, BIRB-796) to high (CP-724714) [78,79]. Noteworthy, CP-724714 is binding to only five other kinases with Kd b 3 μM, including CDK19 (referred to as CDK11 in the original paper [78]) (Kd = 1100 nM), EGFR (Kd = 42 nM), ERBB2 (Kd = 43 nM), ERBB4 (Kd = 240 nM) and MKNK2 (Kd = 840 nM) [78]. Noteworthy, CDK19 is a CDK8 paralog with a high degree of amino acid sequence conservation [92]

(sporadically referred as CDK11). CDK19 associates with mediator complex [93], but its function is not completely understood [30]. It was proposed that CDK8 and CDK19 play opposite roles [94,95,96], however these observations may well be result of compensatory overexpression of the paralog in specific knockdown cells. Mutual exclusivity of CDK8 and CDK19 within the CDK–mediator complex and in the context of expression patterns in cells and tissues raised important questions related to the required selectivity of preferred inhibitors [80]. There are few compounds that appeared as CDK8 binders/inhibitors by compounds profiling on kinase panels. This set includes compound 4 [81], compound 3 [82] and compounds with an urea moiety 3b, 3e [83], 13c [84] (Fig. 8). The binding/inhibition data for these compounds were determined in different screening systems therefore comparison of these compounds is difficult (Table 3). In spite of that, it can be noted that potency of majority of these compounds is comparable to their primary targets. Interestingly, primary targets for compound 13c are phosphoinositide-3 kinase (PI3K) and an atypical kinase mTOR. Moreover compound 13c is reported to be highly selective toward other kinases except CDK8 and CDK19. In 2009, steroidal alkaloid, cortistatin A (Fig. 9) isolated from marine sponge Corticium simplex was reported as a high affinity binder to CDK8

Fig. 4. T-loop stabilization in phosphorylated CDK2/CycA (left panel), non-phosphorylated CDK2/CycA (middle panel) and non-phosphorylated CDK8/CycC (right panel). Fragment of CDK2/CycA structure (1P5E) that contains pThr160 forming interactions with side chains of Arg50, Arg126 and Arg150; Fragment of CDK2/CycA structure (1FIN) that contains Glu162 forming interactions with side chains of Arg126 and Arg150; Fragment of CDK8/CycC structure (4G6L) that contains Glu99CycC which interacts with side chains of Arg65, Arg150 and backbone of Arg178; Selected residues are presented as ball-and-sticks. CDK2—green ribbon, green carbon atoms; cyclin A—yellow ribbon, yellow carbon atoms; CDK8—orange ribbon, gray carbon atoms; cyclin C—purple ribbon, pink carbon atoms. Pictures prepared with Maestro (Schrödinger).

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Fig. 5. Binding mode of sorafenib (left panel, 3RGF) and compound SKR-2 (right panel, 4F7L) in CDK8/Cyc structures. CDK8—orange ribbon, protein carbon atoms—grey ball-and-stick, ligand carbon atoms—green ball-and-stick. Pictures prepared with Maestro (Schrödinger).

(Kd = 17 nM [74]). In addition to its strong binding affinity, it showed high level of selectivity (only 4.5% of 359 non mutant kinases inhibited at 35 percent of control). Cortistatin A was also described as a high affinity ligand for CDK19 (Kd = 10 nM) and Rho-associated, coiledcoil containing protein kinase (ROCK I, Kd = 250 nM, ROCK II Kd = 220 nM) [74]. Cortistatin A displays impressive differential antiproliferative activity against human umbilical vein endothelial cells (HUVECs, IC50 = 1.8 nM) versus normal human dermal fibroblasts (NHDF, IC50 = 6.0 μM) [74]. The cortistatins appeared as attractive lead compounds for drug discovery in antiangiogenic cancer chemotherapy and their mechanism of action was sought to be revealed. In 2012, SNX2-class compounds were revealed as selective inhibitors of CDK8 and its isoform CDK19 [53]. This group of compounds, based on the 4-aminoquinazoline scaffold, was discovered in a highthroughput screening (HTS) of over 100 000 compounds for downstream inhibitors of p21-activated transcription [58]. Exemplary hits from HTS are SNX2 (IC50 = 6.1 μM) and SNX14 (IC50 = 3.8 μM) (Fig. 9). Subsequent structure optimization of hit compounds resulted in more efficacious carbonitrile derivatives with SNX2-1-108 (IC50 = 0.74 μM) and Senexin A (IC50 = 0.64 μM) as examples 9 (Fig. 9). Further characterization using a kinome panel revealed high selectivity

of SNX2-1-108 for two closely related kinases CDK8 and CDK19 [85]. In turn, Senexin A was characterized as an inhibitor with Kd = 0.83 μM and Kd = 0.31 μM for CDK8 and CDK19, respectively, and CDK8 kinase activity was inhibited with IC50 = 0.28 μM [85]. Cellular effects of Senexin A related to CDK8 activity were studied as well. It was shown that Senexin A inhibited β-catenin-dependent transcription in HCT116 colon carcinoma cells, and the induction of transcription factor EGR1 upon serum treatment in quiescent HT1080 cells [85]. Additionally, the effect of Senexin A on p21-activated transcription was corroborated with cortistatin A. Further development of SNX2-class compounds aimed at improvement of solubility and potency (WO2013116786A1). The most potent compound of new generation is Senexin B (SNX-1-165, Fig. 9) which is more active than any other previously discovered SNX2-class compounds. Senexin B, similarly to Senexin A, inhibits p21-activated transcription (HT1080 cells) and the oncogenic β-catenin activity (HCT116 colon carcinoma cells). Senexin B strongly binds to CDK19 and CDK8 with Kd values equal to 80 nM and 140 nM, respectively. Inhibition of CDK8 kinase activity by Senexin B expressed as IC50 values is ranging from 24 to 50 nM in different assays. Kinase profile where activity of a single concentration (2 μM) of the compound to inhibit

Fig. 6. Binding mode of type I inhibitor in CDK8/CycC structure (4F7S). CDK8—orange ribbon, ball-and-stick, grey carbon atoms; inhibitor—ball-and-stick, green carbon atoms; glycerol—ball-and-stick, cyan carbon atoms. Picture prepared with Maestro (Schrödinger).

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Fig. 7. Known kinase inhibitors identified as CDK8 binders.

ATP pocket binding of over 450 kinases is measured showed high selectivity of Senexin B. The strongest inhibition was observed for CDK19 (98.6% inhibition) and CDK8 (97.8% inhibition). There were only two other kinases inhibited at the level N 50%, MAP4K2 (69% inhibition) and YSK4 (59% inhibition). Senexin B was investigated in pharmacokinetic studies (mice and rats), A549 lung cancer and triple-negative breast cancer (TNBC) xenograft studies (WO2013116786A1) with positive outcome. In 2014, three companies (Hoffmann-La Roche, Selvita and Nimbus) reported novel CDK8 inhibitors. Four series of CDK8 inhibitors described in patent applications were published by Hoffman La Roche, namely (i) phenyl-pyridine/pyrazine amides (WO2014029726A1), (ii) pyridine/ pyrazine series substituted by hydroxyethylamino (WO2014106606A1) (iii) bi-ring phenyl-pyridines (WO2014090692A1), and (iv) pyrrole derivatives (WO2014154723A1). These novel compounds were tested in the CDK8/CycC LANCE TR-FRET kinase assay and in vitro cell

proliferation assay on cancer cells including colorectal cancer HCT116 and gastric cancer AGS cell line. Selvita reported substituted tricyclic benzimidazoles as CDK8 inhibitors (WO2014072435A1). Representative compounds were screened for activity against CDK8, CDK9, CDK1, CDK2, CDK5 and CDK7 at 1 μM concentration of a compound using kinase activity test procedures and reported as percent of inhibition. The compounds of the invention appeared as highly selective CDK8 inhibitors within the set of tested CDKs and within large commercial kinase panel (personal communication). Additionally, several compounds tested in mitochondrial reduction of a tetrazolium component (MTS) viability assay on colon cancer HCT116 and SW480 cells showed cytotoxic effect at the level below 1 μM concentration. Capability of inhibiting oncogenic tumor growth in vivo after oral administration was demonstrated for two exemplary compounds. According to company information, Selvita identified a clinical candidate molecule, SEL120-34 that is currently in preclinical

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Table 3 Binding/inhibition data for known kinase inhibitors identified as CDK8 binders and literature compounds with CDK8 as an off-target. CDK8 and primary targets are reported (when more than one target is listed, given values/kinase group refer to the first one in the list). B-POC (binding percentage of control), Ambit score, ctr (%)—lower numbers indicate stronger hits. Compound name

CDK8 binding/inhibition parameter

Primary targets

Primary target kinase group

Primary target binding/inhibition parameter

References

BMS-387032/SNS-032 CP-724714 EXEL-2880/GSK-1363089 Flavopiridol PLX-4720 Staurosporine ABT-869 AST-487 BIRB-796 Sorafenib Compound 3 Compound 4 Compound 3b Compound 3e Compound 13c

Kd = 1200.0 nM Kd = 2300.0 nM Kd = 130.0 nM Kd = 120 nM Kd = 1900 nM Kd = 510 nM Kd = 95.0 nM Kd = 1.4 nM Kd = 220 nM Kd = 310 nM Inhibition (10 μM) = 82% B-POC (10 μM) = 3.4 Ambit score (5 μM) = 19 Ambit score (5 μM) = 2.8 ctr (%) 10 μM = 13

CDK2 ERBB2 MET,AXL,VEGFR2 CDK2, CDK4 BRAF PRKCH, Pan-Inhibitor FLT3, CSF1R, VEGFR2 FLT3, KIT p38-alpha VEGFR2, FLT3, BRAF JNK1, JNK2, JNK3 PDGFR, CK1, RAF SRC SRC PI3K, mTOR

CMGC TK TK CMGC TKL AGC TK TK CMGC TK CMGC TK TK TK

Kd = 69 nM Kd = 43 nM Kd = 1.4 nM Kd = 550 nM Kd = 440 nM Kd = 4.8 nM Kd = 0.63 nM Kd = 0.79 nM Kd = 0.45 nM Kd = 59 nM Inhibition (10 μM) = 99% B-POC (10 μM) = 4.1 Ambit score (5 μM) = 11 Ambit score (5 μM) = 12 ctr (%) 10 μM = 11

[78,79] [78] [79] [78,79] [79] [78,79] [78,79] [78,79] [78,79] [78,79] [82] [81] [83] [83] [84]

development and anticipated initiation of clinical development in 2016. This is most likely the most advanced therapeutic program, according to publicly available information, selectively targeting CDK8 kinase, that can deliver clinical proof of concept in the near future. Nimbus reported CDK8 inhibitors (WO2014194201A2, WO2014194245A2) providing exemplary compounds with given IC50 values measured by the CDK8 FRET assay. Additionally one compound was further studied by means of mass spectrometry CDK8 inhibition assay which confirmed its inhibitory potency toward CDK8. Concluding remarks It is now evident that CDK8 is a remarkably flexible transcriptional regulator capable of playing diverse, usually cancer promoting roles, however ultimate effects of CDK8 depend on the context. These findings warrant continued development of selective CDK8

inhibitors in oncology supported by expanding knowledge about structural features of this target. First selective compounds have already been identified by several biotech and pharmaceutical companies and are in preclinical development and hopefully a clinical proof of concept of targeting CDK8 in cancer can be delivered in the near future. Evolving understanding of very specific CDK8 activity might allow development of a robust responder hypothesis and personalized clinical approach.

Transparency document The Transparencydocument associated with this article can be found, in the online version.

Fig. 8. Literature compounds with CDK8 as an off-target.

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Fig. 9. Cortistatin A and examples of SNX2-class compounds.

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