Cyclin-Dependent Kinase 8: A New Hope in Targeted Cancer Therapy

Dec 21, 2017 - It is deemed to contribute to the exceptionally high affinity of CDK8 for CycC due to larger interaction surface area. Structural Aspec...
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Cyclin-Dependent Kinase 8: A New Hope in Targeted Cancer Therapy? Miniperspective Stephen Philip, Malika Kumarasiri, Theodosia Teo, Mingfeng Yu, and Shudong Wang* Centre for Drug Discovery and Development, Sansom Institute for Health Research and School of Pharmacy and Medical Sciences, University of South Australia, Adelaide, South Australia 5001, Australia

ABSTRACT: Cyclin-dependent kinase 8 (CDK8) plays a vital role in regulating transcription either through its association with the Mediator complex or by phosphorylating transcription factors. Myriads of genetic and biochemical studies have established CDK8 as a key oncogenic driver in many cancers. Specifically, CDK8-mediated activation of oncogenic Wnt-β-catenin signaling, transcription of estrogen-inducible genes, and suppression of super enhancer-associated genes contributes to oncogenesis in colorectal, breast, and hematological malignancies, respectively. However, while most research supports the role of CDK8 as an oncogene, other work has raised the possibility of its contrary function. The diverse biological functions of CDK8 and its seemingly context-specific roles in different types of cancers have spurred a great amount of interest and perhaps an even greater amount of controversy in the development of CDK8 inhibitors as potential cancer therapeutic agents. Herein, we review the latest landscape of CDK8 biology and its involvement in carcinogenesis. We dissect current efforts in discovering CDK8 inhibitors and attempt to provide an outlook at the future of CDK8-targeted cancer therapies.

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INTRODUCTION Cyclin-dependent kinases (CDKs) are a family of serine/ threonine protein kinases that coordinate critical regulatory events during cell cycle and transcription.1 Their namesake is derived from the functional dependence on binding various cyclins (Cycs), and to date, at least 20 CDKs and 30 Cycs have been identified. CDKs are engaged in diverse biological processes such as metabolism, neuronal differentiation, hematopoiesis, angiogenesis, stem cell self-renewal, and spermatogenesis.2 Given these fundamental roles, it is unsurprising that deregulation of CDKs is a common feature of many cancers. In particular, CDK8, a ubiquitously expressed, primarily transcriptional member of the CDK family, has come under focus owing to investigations of its central roles in transcription and oncogenesis. CDK8 is also one of the most eccentric CDK family members for exerting both positive and negative impacts on transcription and executing context-specific roles in oncogenic pathways.3 CDK8 (initially termed K35) was initially discovered as a putative kinase partner of cyclin C (CycC).4,5 It was originally reported as a negative regulator of transcription; however, later © XXXX American Chemical Society

studies have revealed its positive roles in gene-specific transcription.6−12 CDK8 gene (located on human chromosome 13q12) is translated into a 53 kDa protein comprising 464 amino acids,5 where its kinase activity is regulated by the association with CycC.4 The 13q12 chromosome region is often amplified in colorectal cancer (CRC).10,13−15 In fact, it was later demonstrated that CDK8 is overexpressed in a large fraction of CRC.10 Emerging evidence also indicates that CDK8 is involved in the development of melanoma, acute myeloid leukemia (AML), prostate cancer, and breast cancer.8,16−21 While CDK8 deficiency induces the developmental arrest of embryos through transcriptional deregulation of developmentally critical genes and prevents the generation of viable mice,22 knockdown of CDK8 in Drosophila S2 cells and cultured mammalian 293FT cells does not affect their viability, and conditional deletion of CDK8 in adult mice has no major effect on normal cellular viability.23 Taken together, these findings suggest CDK8 as a potential anticancer target. In this review, Received: June 19, 2017 Published: December 21, 2017 A

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Figure 1. Role of CDK8 in regulation of RNAPII transcription. (I) In the initiation phase, (1) yeast CDK8 negatively regulates transcription by phosphorylating RNAPII CTD in vitro, precluding its incorporation into PIC. (2) Human CDK8 phosphorylates CycH in vitro, thereby preventing the initiation of transcription via deactivating CDK7, although contrary to this, a positive role of CDK8 in regulation of transcription has been suggested. (II) After RNAPII promoter escape, the kinase module can bind to the core Mediator, and this facilitates pause release and elongation at certain genes via recruitment of SEC. This binding also induces a change in the conformation of the core Mediator, rendering it incapable of binding to another RNAPII. (III) As RNAPII cannot bind to the CDK8-Mediator complex, this terminates the reinitiation of transcription.

with RNAPII-binding.24,27 Moreover it has been shown that binding of the kinase module and RNAPII to the core Mediator is mutually exclusive.36,37 Therefore, formation of the CDK8Mediator complex may act as a molecular switch that prevents a second RNAPII from immediately reengaging the promoter, essentially terminating the reinitiation of transcription. Subsequent studies revealed that CDK8 potentiates the expression of stimulus-specific genes through regulating transcription elongation. 6−8 Knockdown of CDK8 in HCT116 cells showed a clear decrease in phosphorylation of RNAPII CTD at Ser2 and Ser5 with concomitant impairment of transcription elongation at immediate early genes (IEGs) that are activated in response to serum stimulation.6 This knockdown did not affect RNAPII recruitment but instead prevented the recruitment of super elongation complex (SEC) to the gene loci of IEGs (Figure 1, II). Likewise, another study demonstrated that in response to hypoxia, hypoxia-inducible factor 1A (HIF1A) dependent transactivation of hypoxiainducible genes requires the CDK8-Mediator complex to facilitate RNAPII pause release via recruitment of SEC (Figure 2, VII).7 A more recent report suggested that CDK8 potentiates estrogen receptors (ERs) in breast cancers by promoting estrogen-induced phosphorylation of RNAPII CTD at Ser2, assisting in the completion of the transcription of ER-inducible genes more effectively.8 Phosphorylation of Transcription Factors. CDK8 can regulate gene expression by phosphorylation of TFs including mothers against decapentaplegic homolog (Smad),9 neurogenic locus notch homolog protein (NOTCH),38 signal transducer and activator of transcription 1 (STAT1),12 and sterol regulatory element-binding protein 1C (SREBP-1C).39 Phosphorylation can directly affect TF activity or prime them for ubiquitin-mediated degradation. CDK8 enhances the transactivation potential and turnover of receptor-regulated Smad proteins (R-Smads) in the bone morphogenetic protein (BMP)/transforming growth factor β (TGFβ) pathways (Figure 2, I).9 The TGFβ family of cytokines are important regulators of cell cycle, differentiation, and apoptosis.40 Nucleoplasmic CDK8/CDK9 phosphorylates RSmads at their linker regions, permitting the binding to coactivators that are required for efficient transcription of target

following a brief overview of the biological functions of CDK8, we analyze the key roles of CDK8 in carcinogenesis. Then we focus on recent advancement in the development of pharmacological CDK8 inhibitors. Finally, we provide an outlook of the rational design of potential CDK8 inhibitors.

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CDK8 OVERSEES DIVERSE BIOLOGICAL FUNCTIONS Among the many cellular functions of CDK8, the most notable is its involvement in transcription. CDK8 is part of the Mediator complex, a large multisubunit protein complex that is central to the regulation of transcription in eukaryotes.24−26 The Mediator complex is a highly conserved transcriptional coactivator that acts as a molecular bridge to transfer regulatory signals from DNA-bound transcription factors (TFs) directly to the RNA polymerase II (RNAPII) preinitiation complex (PIC). CDK8 reversibly associates with the Mediator complex as part of a four-subunit kinase module, which additionally comprises MED12, MED13, and CycC.24,27−29 While MED12 and CycC are required for the kinase activity of CDK8, MED13 is involved in recruiting the preassembled kinase module to the core Mediator. 24,29 Upon binding, the kinase module dramatically alters the structure and function of the core Mediator.24,30 Early in vitro biochemical studies indicate both human CDK8 and its homolog in yeast as transcriptional repressors (Figure 1). Yeast CDK8 was shown to repress transcription by phosphorylating the carboxy-terminal domain (CTD) of RNAPII, precluding its incorporation into a functional PIC.31 Human CDK8/CycC seems inhibiting transcription initiation by phosphorylating CycH at its Ser5 and Ser304, resulting in a loss of the kinase activity of CDK7.29,32 However, this mode of transcriptional repression has not been recapitulated in vivo, and, instead, a positive regulatory role for CDK8 has been proposed.33,34 Human CDK8-Mediator complex has also been shown to repress reinitiation of transcription.24 Following the initiation of transcription, RNAPII escapes from PIC to leave behind a scaffold complex which facilitates reinitiation of transcription.35 The kinase module can bind to core Mediator after transcription initiation.24 This binding induces a change in the conformation of the core Mediator that is incompatible B

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Figure 2. Biological functions of CDK8. (I) Smad transactivation: C-terminal domain-phosphorylated R-Smads complex with Smad4 and translocate to the nucleus. CDK8/CDK9-mediated phosphorylation of R-Smads at the linker region enhances the transactivation of TGFβ/BMP-regulated genes by facilitating their interaction with coactivators. This phosphorylation also leads to turnover of R-Smads, shutting down target gene expression. (II) NOTCH signaling: Binding of a ligand to NOTCH receptor induces proteolytic cleavage to release NICD, which stimulates target gene expression in the nucleus. CDK8 phosphorylates NICD, priming it for degradation and ultimately terminating target gene expression. (III) STAT1 signaling: Upon IFN-γ stimulation, JAK-phosphorylated STAT1 translocates to the nucleus. CDK8 phosphorylates STAT1 at Ser727 in the nucleus to regulate the expression of IFN-γ-dependent genes. (IV) β-Catenin pathway: Binding of Wnt protein to a Frizzled family receptor activates dishevelled (Dsh) and subsequently deactivates a destruction complex that consists of GSK3β, AXIN, and APC. In the absence of Wnt signaling, this destruction complex phosphorylates β-catenin for degradation within the cytoplasm. In the presence of Wnt signaling, β-catenin translocates to the C

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Figure 2. continued nucleus to stimulate the transcription of TCF/LEF-dependent genes. CDK8 promotes β-catenin-dependent transcription by acting as a β-catenin coactivator and by phosphorylating E2F1, a β-catenin antagonist. (V) Histone phosphorylation: CDK8-Mediator complex phosphorylates Ser10 of histone H3, an event associated with the activation of gene expression. (VI) Cell cycle: Skp2 promotes mH2A1 ubiquitination and CDK8 expression to regulate G2/M transition. (VII) Hypoxia-induced genes: CDK8-Mediator complex regulates the expression of hypoxia-induced genes via recruitment of SEC. (VIII) Lipogenesis: CDK8 inhibits lipogenesis by phosphorylating SREBP-1C, priming it for degradation.

genes.9 This phosphorylation also primes R-Smads for eventual degradation. Hence, CDK8/CDK9-mediated phosphorylation directs the transcriptional activity and turnover of activated Smad proteins. CDK8 regulates the NOTCH signaling pathway (Figure 2, II), which is vital for cell-to-cell communication, neuronal development, and T-cell differentiation.38,41−43 Binding of a ligand to the extracellular domain of NOTCH receptors induces proteolytic cleavage to release NOTCH intracellular domain (NICD), which enters the cell nucleus to activate the transcription of NOTCH target genes.44,45 CDK8 controls the function of NICD by phosphorylation, resulting in its ubiquitination and subsequent termination of target gene expression.38 This termination is thought to be important in resetting target genes for the subsequent round of NOTCH signaling activation and in establishing a NOTCH signaling gradient.46 The STAT family of proteins are important mediators of cytokine responses and their deregulation is often associated with increased vulnerability to viral infections, immune disorders, and hematopoietic malignancies.47 Upon interferon-γ (IFN-γ) signaling, phosphorylation of STAT1 at Tyr701 by the Janus kinases (JAKs) causes STAT1 homo- or heterodimers to translocate to the nucleus, where they bind to DNA and regulate transcription (Figure 2, III).48 However, studies have shown that an additional phosphorylation at STAT1-Ser727 is required for the complete expression of IFNγ-dependent genes.49 CDK8 was found to phosphorylate STAT1 at Ser727 to fully activate STAT1-dependent transcription on IFN-γ signaling.12 This phosphorylation is also required for full-blown antiviral responses. For example, CDK8 knockdown resulted in increased sensitivity to vesicular stomatitis virus. Covalent modification of histones by phosphorylation and acetylation has traditionally been associated with transcriptional activation.50 A special CDK8-Mediator complex containing GCN5L acetyltransferase can catalyze the phosphoacetylation of histone H3 at Ser10/Lys14.51 CDK8 phosphorylates Ser10 on histone H3 (Figure 2, V), which in turn stimulates the acetylation of Lys14 by GCN5L. This phosphoacetylation of histones is frequently associated with transcriptional activation of IEGs.29,52 Regulation of lipogenesis is primarily mediated by the SREBP family of TFs (Figure 2, VIII).53 Upon insulin signaling, SREBP-1C undergoes proteolytic maturation at the Golgi apparatus and translocates to the nucleus to activate the transcription of lipogenic genes. CDK8/CycC negatively regulates de novo lipogenesis by phosphorylating SREBP-1C at Thr402, resulting in increased SREBP-1C ubiquitination and protein degradation.39 Consistent with this finding, knockdown of CDK8 in Drosophila, mammalian hepatocytes, and mouse liver resulted in enhanced expression of lipogenic genes with increased lipid accumulation. CDK8 in Cell Cycle. Prior work implied that the regulation of p27 by S-phase kinase-associated protein 2 (Skp2), the

receptor component of an Skp2, cullin, F-box (SCF) ubiquitin ligase complex, might in part contribute to the modulation of G2/M transition.54 Recently, Skp2-macroH2A1 (mH2A1)CDK8 axis was found to regulate G2/M transition (Figure 2, VI).16 In this regard, the histone variant mH2A1 was identified as a new substrate of Skp2, and its degradation by Skp2 led to the enhanced expression of CDK8. Ectopic expression of mH2A1 phenocopied Skp2-deficient mouse embryonic fibroblasts (MEFs) G2/M arrest, whereas restoration of CDK8 partially rescued the phenotypes of Skp2-deficient MEFs including the defect in G2/M transition. Paralogs of Kinase Module Subunits and Their Functions. Importantly, three subunits of the kinase module, i.e., CDK8, MED12, and MED13, have undergone independent gene duplications in vertebrates to produce paralogs CDK19, MED12L, and MED13L, respectively.55−57 Little is known about the biological functions of these paralogs. The original subunits and their respective paralogs appear to assemble in a mutually exclusive fashion in the four-subunit kinase module.58 This raises the possibility that multiple variants of the kinase module may exist, thus allowing further functional specialization of the Mediator complex. CDK8 and CDK19 are highly homologous proteins with 97% sequence identity in the catalytic site but differ considerably in the C-terminus.56 Consequently, they have some overlapping as well as diverse biological functions. For example, both kinases phosphorylate RNAPII CTD and TFs such as NICD and STAT1 to regulate their functions.12,19,59,60 Similarly, expression of dominant drug-resistant alleles of either CDK8 or CDK19 was able to restore the growth of AML cells that had been inhibited by a CDK8/CDK19 inhibitor, i.e., cortistatin A (1, Table 1).19 Studies also indicated their different roles in development and gene expression programs. CDK8 knockout in mice leads to early embryonic lethality, irrespective of the presence of CDK19.22 Knockdown of CDK8 in HCT116 cells downregulated 65% of genes induced in response to hypoxia, whereas only 13% was affected by CDK19 knockdown.7 In contrast, CDK19 depletion in HCT116 cells showed a more substantial effect on gene expression elicited in response to genotoxic stress and glucose deprivation than did CDK8 depletion. CDK8 inhibition by shRNA knockdown or by CRISPR/CAS9 knockout blocked ER signaling in BT474 cells, in spite of the presence of CDK19.8 The two kinases also show different patterns of expression in human tissues.60 Northern blot analysis of various human tissues indicated that CDK8 was ubiquitously expressed whereas CDK19 expression was tissue-specific. A recent study also showed that CDK19 was highly expressed in SJSA cells, whereas CDK8 was almost undetected.61 Collectively, these studies indicate that CDK8 and CDK19 have both overlapping and distinct biological functions. Pharmacological Inhibition and Knockdown of CDK8/ CDK19. Studies have shown that the effects of pharmacological inhibition of CDK8/CDK19 are distinct from their gene knockdown. For example, while CDK8 knockdown suppressed D

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Table 1. Type I CDK8 Inhibitors in Preclinical Development

E

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Table 1. continued

F

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Table 1. continued

G

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Table 1. continued

a

Corresponding references for further information. bNA, not available.

oncogene status.10,66 It is also overexpressed in melanoma, breast, prostate, and pancreatic cancers.16−18,20,67 Inhibition of CDK8 by microRNA, small interfering RNA, or short hairpin RNA (shRNA) suppressed the proliferation in melanoma, prostate, CRC, and breast cancers.10,17,20,68,69 Stimulatingly, CDK8 inhibition has been shown to augment the cytotoxicity of NK cells.70 Consequently, CDK8 represents a promising target for therapeutic validation. This section details the different mechanisms by which CDK8 contributes to tumorigenesis and how pharmacological inhibition of CDK8 could be relevant to the treatment of such cancers. Colorectal Cancer. CRC is the third most common type of cancer and fifth in terms of cancer-associated deaths, accounting for 694 000 deaths and 1.36 million new cases every year.71,72 CRC arises in colon epithelial cells as a result of accumulated genetic and epigenetic changes. The most frequent genetic alteration is the deactivation of the APC tumor suppressor gene, leading to aberrant activation of the Wnt signaling pathway.73 This aberrance results in the stabilization and nuclear translocation of β-catenin, which binds to T-cell factor (TCF) and enhances the transactivation of genes (e.g., MYC, AXIN2, and LEF1) that are implicated in CRC.74−76 The therapeutic value of targeting CDK8 started with the report by Hahn and co-workers that proposed CDK8 as an

the phosphorylation of E2 promoter binding factor 1 (E2F1) at Ser375,62 CDK8/CDK19 inhibitor CCT251545 (5, Table 1)63 did not affect this phosphorylation. Furthermore, CDK8/ CDK19 kinase inhibitors do not produce the same phenotype as CDK8 knockdown.10,19,64 For example, while CDK8 knockdown reduced the proliferation of HCT116 cells, CDK8/CDK19 inhibitors had little effect on these cells.19,64 Furthermore, the transcriptomic effects of CDK8 or CDK19 knockdown and their pharmacological inhibition in HCT116 were also different.65 Taken together, these results demonstrate that the effects of CDK8 and/or CDK19 knockdown are distinct from those caused by inhibition of their kinase activities. In fact, kinase-independent functions of CDK8 and CDK19 have been documented in the literature. CDK8 was shown to stimulate the proliferation of melanoma cells in a kinase-independent manner,17 while CDK19 regulates p53 responses independently of its kinase activity.61

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CLINICAL RELEVANCE OF CDK8 CDK8 is involved in several key developmental and cellular pathways, and hence it is unsurprising that dysregulation of CDK8 is often implicated in cancers. Nonetheless, CDK8 appears to play context-specific roles in different cancers: while it acts as a promoter in many cancers,8,10,19,21 cases to the contrary are emerging.23 In CRC, CDK8 has been conferred an H

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oncogene in CRC.10 CDK8 was shown to be overexpressed in 60% of CRC and that CDK8 knockdown reduced the proliferation in CRC cells and xenograft models harboring CDK8 amplification.10,15,77 Furthermore, the kinase activity of CDK8 was required for both transcription of β-catenin target genes and proliferation of CRC cells. CDK8 was later identified to repress E2F1, a negative regulator of β-catenin/TCFdependent transcription, by phosphorylating it at Ser375.11,62 Thus, CDK8 promotes CRC by facilitating the transcription of β-catenin target genes and suppressing E2F1 (Figure 2, IV). CDK8 has also been reported to be required to maintain CRC cells in an undifferentiated state. Adler et al.77 revealed that loss of CDK8 promoted differentiation and inhibited tumor growth in CRC xenograft models. In addition, CDK8 has been shown to play a critical role in allowing cancer cells to use glucose as an energy source.78 Cancer cells often exibit upregulated glycolysis to meet the high demand for proliferation and survival even under aerobic conditions, a phenomenon known as Warburg effect.79 HCT116 cells engineered to carry a kinase-dead CDK8 (CDK8as/as) reduced glucose transporter (GLUT3) expression and glucose import, as well as cell proliferation and anchorageindependent growth in normoxia and hypoxia. Inhibition of CDK8 caused downregulation of several genes, e.g., SLC2A3, HK1, and ENO1, that are involved in the glycolytic cascade and sensitized HCT116 cells to glycolysis inhibitor 2-deoxy-Dglucose (2DG). These results were also reproduced with a CDK8/CDK19 inhibitor, senexin A (3, Table 1).80 As upregulation of glycolysis is a common feature of cancer, CDK8 inhibitor and its combination with drugs that block glycolysis may enable specific targeting of cancer cells without harmful effects on normal cells. The oncogenic role of CDK8 in CRC was further validated by two cohort studies of CRC cases. Among 372 colon cancer patients, the 5-year survival rate was significantly lower in association with higher expression level of CDK8.66 However, this was not the case in rectal cancer, where no correlation between CDK8 and the overall mortality rate was observed. Another study attested that in CRC, levels of CDK8 positively correlated with stage of the disease.15 Its overexpression was higher in stages III and IV than in stage I, raising the possibility that CDK8 could be involved in the transformation of colorectal adenoma to carcinoma. While significant data support the role of CDK8 as an oncogene, other work has raised the possibility of its contrary function. McCleland et al.23 reported that deletion of CDK8 in APCMin intestinal tumor model shortened the survival and increased tumor burden compared to CDK8 wild-type mice. In human CRC, mutation in Wnt-β-catenin signaling is one of the earliest events of tumorigenesis and progression. Additional mutations, such as CDK8 amplification, would accumulate over time, providing selective advantage for tumor growth or metastasis. However, in the ApcMin mice, CDK8 is deleted prior to tumor initiation; in the absence of CDK8, the tumors may exploit alternative and more aggressive transcriptional and signaling pathways for growth. Further studies, for example, utilizing transgenic CDK8 overexpression, would be valuable to assess pro-oncogenic role of CDK8. Hematological Malignancies. Studies with CDK8/ CDK19 inhibitors, i.e., 1 and SEL120-34A (2, Table 1),21 have focused on the inhibition of CDK8/CDK19 kinase activity in AML.19 CDK8 and CDK19 were identified as negative regulators of super-enhancer (SE)-associated genes in AML.19

Inhibition of CDK8/CDK19 by compound 1 led to robust antileukemic activity in AML cells by a mechanism involving upregulation of SE-associated genes with tumor suppressing and lineage-controlling functions. On the other hand, compound 2 inhibited the growth of AML cells with high levels of phosphorylated STAT1-Ser727 and STAT5-Ser726.21 The in vitro effectiveness of the two compounds has also translated to anticancer efficacy in AML xenograft models. These promising preclinical data obtained from two structurally distinct compounds suggest future development of CDK8/ CDK19 inhibitors for AML therapy. Breast Cancer. Although its mortality rate has been reduced owing to favorable prognosis, breast cancer remains the second common cause of female cancer-associated deaths.71 Xu et al.16 reported that CDK8 exerted its oncogenic role in breast cancer via the Skp2-mH2A1-CDK8 regulatory axis. Skp2-deficient breast cancer cells showed a reduction in proliferation characterized by upregulation of mH2A1 and downregulation of CDK8. Notably, this defect in proliferation could be rescued upon knockdown of mH2A1 or by restoration of CDK8. Mechanistically, breast tumor suppression upon Skp2 loss was mediated by mH2A1-mediated repression of CDK8. Furthermore, expression levels of CDK8 and Skp2 in 189 human breast cancer samples positively correlated with primary tumor status, nodal metastasis, and stage in breast cancers.16 A more recent study showed that targeting CDK8 may have a positive impact on hormone therapy of ER-positive breast cancers.8 A vast majority of breast cancer patients express ERα and are designated as ER-positive.81 The binding of estrogen to ER triggers the transcription of genes, which eventually drives the proliferation and survival of ER-positive breast cancers. Although several ER signaling inhibitors have been developed, many patients develop resistance, particularly in the metastatic setting where tumors modify their ER, making them less reliant on ER, or utilize alternate signal transduction pathways to substitute ER. McDermott et al.8 showed that CDK8 exerted its effect downstream of ER as its pharmacological inhibition suppressed estrogen-induced phosphorylation of RNAPII CTD at Ser2 and thus inhibited the transcriptional elongation of ERinduced gene, GREB1. Furthermore, it was demonstrated that senexin B (4, Table 1)8 suppressed the development of estrogen independence, inhibited tumor growth in vitro and in vivo, and synergized with fulvestrant, a selective estrogen receptor downregulator (SERD). Melanoma. In spite of being the least common and being curable if detected early, melanoma accounts for the majority of skin cancer deaths.82,83 CDK8 has been implicated in melanomagenesis, where it has been shown to be regulated by the histone variant mH2A and drives proliferation in a kinase-independent manner.17 Loss of mH2A isoforms positively correlated with melanoma malignancy, and functional studies indicated that this loss drives melanoma progression through direct transcriptional upregulation of CDK8. This study has important implications for therapeutic approaches relying on agents that inhibit CDK8 kinase activity as the kinase-independent functions may offer alternative routes for the survival of cancer cells. Prostate Cancer. Prostate cancer is the second most frequently diagnosed cancer and sixth leading cause of cancerassociated death in males.84 Although being curative in the early stages, few treatment options are currently available for advanced-stage disease. CDK8 and CDK19 have been linked to oncogenic potential in advanced prostate cancers.20 I

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Figure 3. (A) Schematic representation of CDK8 bound to CycC (PDB code 4G6L). CDK8 is shown in yellow (C-terminus) and gray (Nterminus) with the αC helix in cyan, DMG motif in violet, C-terminal tail in blue, insertion in pink, and hinge in orange. The extended C-terminal tail and the unique insertion consisting of nine residues are not fully resolved in the crystal structure. CycC is shown in green. (B) Inactive conformation of CDK8 (PDB code 3RGF). In the inactive state, Glu99CycC cannot interact with Arg65, Arg150, and Arg178, and the salt bridge between Lys52 and Glu66 cannot be established as they are further apart. (C) Active conformation of CDK8 (PDB code 4G6L). In the active conformation, Glu99CycC interacts with Arg65, Arg150, and Arg178, and the salt bridge between Lys52 and Glu66 is established. For (B) and (C), CDK8 is shown in yellow (C-terminus) and gray (N-terminus) with selected residues in red. CycC is shown in green with selected residues in magenta. The figure was prepared using PyMOL1.3 (Schrödinger Inc., 2013).

Although no mechanistic details have been elucidated, the two kinases were found to be specifically overexpressed in advanced-stage prostate cancer, and knockdown or pharmacological inhibition of both decreased invasion and migration in prostate cancer cells, highlighting their potential as novel therapeutic targets in prostate cancer. Transcription of Proinflammatory Cytokines. CDK8 and CDK19 have been shown to promote the transcription of tumor-promoting cytokines regulated by nuclear factor κ-lightchain-enhancer of activated B cells (NFκB).85 The NFκB family of transcription factors has been implicated in several major diseases including inflammatory disorders and cancer.86 Activation of NFκB in cancers has been linked to proliferation, angiogenesis, metastasis, and resistance to apoptosis. Majority of NFκB inhibitors show significant side effects, likely due to sustained NFκB suppression, and thus drugs affecting induced, but not basal, NFκB activity may provide some therapeutic benefit. Chen et al.85 reported that inhibition of CDK8/CDK19 by small molecule inhibitors, i.e., 3 and 4 (Table 1), or by shRNA knockdown downregulated the NFκB-initiated transcription of tumor-promoting proinflammatory cytokines including IL8, CXCL1, and CXCl2. Notably, the CDK8/ CDK19 inhibition had little effect on the basal level of NFκB

regulated genes. The IL8, CXCL1, and CXCl2 cytokines play a major role in chemotherapy-induced tumor-promoting paracrine activities, suggesting the potential therapeutical opportunity for pharmacological inhibitors of CDK8/CDK19. Immuno-Oncological Functions. Recent evidence indicated that phosphorylation of STAT1 at Ser727 by CDK8 suppressed NK cell-mediated cytotoxicity and tumor surveillance.70 NK cells spearhead the body’s natural defense against viral infections and malignant cells.87 Putz et al.70 showed that CDK8 phosphorylates STAT1 at Ser727 in resting NK cells in the absence of cytokine stimulation, to suppress their cytotoxic action. Consistently, depletion of CDK8 from NK cells with shRNA reduced this phosphorylation and enhanced their cytotoxicity. This notion was further substantiated by STAT1S727A mice which were significantly resilient to the development of B16F10 melanoma and ν-Abl+ leukemia and entirely resistant to 4T1 breast cancer metastasis. The increased cytotoxicity of NK cells in STAT1-S727A mice was attributed to the increased expression of perforin and granzyme B. Moreover, NK cells have been implicated in eradicating leukemic stem cells which often cause the relapse of the disease.88 Consequently, CDK8 inhibition may enhance cancer prognosis by boosting NK-cell mediated defenses. J

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STRUCTURAL FEATURES OF CDK8 Structurally, CDK8 exhibits the typical bilobal kinase fold.89 The N-terminal domain contains mostly β-sheets with two αhelices, while the C-terminal domain is mostly α-helical (Figure 3A). The two domains are connected by a region known as the hinge. The catalytic cleft is bordered by the hinge region and rests between the two domains with active site residues contributed by both. The C-terminal domain portion of the ATP-binding site contains a unique DMG (Asp173-Met174Gly175) motif, which replaces the DFG motif that is highly conserved among CDKs. The conformation of the DMG motif and the αC helix are critical for its activation. CDK8 possesses certain characteristic secondary structural features, i.e., an extended C-terminal tail that lies adjacent to the hinge and a unique insertion consisting of nine residues (240EDIKTSNPY248) in front of the αG helix (Figure 3A).89 CDK7 is the only other CDK to have such an extended Cterminus, but the composition of the two extensions is different.90,91 The αGH1-αGH3 helices are solvent-exposed and exist between αG and αH. The αD helix is uniquely extended and contains a cluster of positively charged amino acids that are pointing to the loop region between αD and αE.89 The αC helix is in the N-terminal domain and can interface with CycC when in the correct conformation. CDK8 also has an additional helix, αB, in the N-terminus. It is deemed to contribute to the exceptionally high affinity of CDK8 for CycC due to larger interaction surface area. Structural Aspects of CDK8 Activation. The active conformation of CDK8 is achieved with its binding to CycC, resulting in a movement of the αC helix and subsequently the DMG motif, relieving the steric block at the catalytic site (Figure 3B and Figure 3C). The formation of a conserved salt bridge between Lys52 and Glu66 assists in stabilizing this active conformation.89 In contrast to other CDKs, the active conformation of CDK8 lacks a phosphorylated residue in the T-loop. Instead, it is believed that Glu99 in CycC mimics the effect of a phosphorylated residue and interacts with the three well-conserved arginines, i.e., Arg65 (N-lobe), Arg150 (Clobe), and Arg178 (T-loop). These interactions may drive the conformational change, opening up the ATP-binding site.92 The binding of MED12/MED13 to CDK8/CycC may also facilitate further conformational changes. Studies on the recombinant human kinase module have revealed that MED12 is required for kinase activity,29 and structural studies have shown that the binding of kinase module to the core Mediator occurs via MED13.24

for the successful therapeutic application of these CDK8/ CDK19 dual inhibitors. The steroidal alkaloid 1 (Table 1), isolated from the marine sponge Corticium simplex, is the first compound to demonstrate a high affinity for CDK8.97 Compound 1 was found to potently inhibit several recombinant protein kinases including rhoassociated protein kinases (ROCKs) and CDK19 by biochemical assays when testing against a panel of 402 kinases at 10 μM. The selectivity of 1 was also evaluated in MOLM-14 cell lysate utilizing a KiNativ platform, but no binding to ROCK1 or ROCK2 was detected up to 2.5 μM 1, suggesting its high specificity for CDK8 in the cells.19 In MOLM-14 cells, 1 inhibited the phosphorylation of CDK8 substrates including STAT1-Ser727, Smad2-Thr220, and Smad3-Thr179 in a dosedependent manner. X-ray crystallographic analysis of 1 in complex with CDK8/CycC revealed a type I binding mode. Despite not inhibiting the growth of HCT116 cells, 1 exhibited impressive antiproliferative activity against several leukemia cell lines, and this activity was deprived upon a point mutation of Trp to Met at position 105 in CDK8/CDK19. To the best of our knowledge, 1 is the only CDK8/CDK19 inhibitor validated with this dominant drug-resistant allele approach. The cellular effectiveness of 1 has translated into tumor growth inhibition in two xenograft models (MV4;11 and SET-2) with no apparent toxicity to the host.19 The tricyclic benzimidazole 2 (Table 1) developed by Selvita is another potent ATP-competitive inhibitor of CDK8/ CDK19.21 This molecule displayed good selectivity over CDKs 1, 2, 4, 5, 6, and 7 but weakly inhibited CDK9. However, it should be noted that its kinome-wide selectivity profile has not been disclosed. Compound 2 has been shown to inhibit the phosphorylation of STAT1 at Ser727 and of STAT5 at Ser726 in vitro and to induce an arrest in the growth of AML cells where such phosphorylation is especially high. In two AML xenograft models (i.e., KG-1 and MV4;11), this compound suppressed tumor growth with no detectable signs of toxicity and inhibited the phosphorylation of STAT5 at Ser726 in a dose-dependent manner without affecting the phosphorylation of known CDK8 substrate STAT1-Ser727. Surprisingly, compound 2 was inactive on MOLM-14 cell line,21 which was sensitive to compound 1.19 These findings warrant further investigation with regard to its antitumor activity. The 4-aminoquinazoline derivative 3 (Table 1) is a potent ATP-competitive inhibitor of CDK8/CDK19.80 It was initially identified in a high-throughput screen for downstream inhibitors of p21-activated transcription. Compound 3 inhibited β-catenin-driven transcription in HCT116 cells and the induction of TF early growth response 1 (EGR1) upon serum stimulation in HT1080 cells, consistent with the biological functions of CDK8.6 In addition, compound 3 suppressed tumor-promoting paracrine activities of DNAdamaging drug doxorubicin both in vitro and in vivo.80 Further optimization of compound 3 led to the discovery of compound 4 (Table 1), which showed increased potency against CDK8/ CDK19.98 Besides CDK8/CDK19, it also inhibited mitogenactivated protein kinase kinase kinase kinase 2 (MAP4K2) and mitogen-activated protein kinase kinase kinase 19 (YSK4) to a lesser extent. Like compound 3, analog 4 inhibits β-catenindriven transcription in HCT116 cells. In mouse xenograft models, pretreatment of mice with compound 4 delayed engraftment of human A549 lung cancer and MDA-MB-468 triple-negative breast cancer (TNBC) cells. It was also shown

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CDK8 INHIBITORS FOR CANCER THERAPY A large body of evidence suggests the oncogenic role of CDK8 in several types of cancers and substantiates that its inhibition can impede tumor growth. This has spurred a deep and newfound interest in development of CDK8 inhibitors as potential anticancer agents, but some progress toward clinic has only been made recently. BCD-115, a CDK8/CDK19 inhibitor (structure not disclosed),93 has advanced into phase I clinical trials for the treatment of ER-positive human epidermal growth factor 2 (HER2)-negative locally advanced and metastatic breast cancer. All of the CDK8 inhibitors discovered so far also inhibit CDK19,19,21,80,94−96 due to the high degree of structural similarity between the two kinases.57 The biology of CDK19, particularly its role in cancer, is less explored and could be vital K

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Table 2. Type II Inhibitors That Inhibit CDK8

L

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Table 2. continued

a

Corresponding references for further information. bNA, not available.

to sensitize TNBC xenografts to doxorubicin therapy, and the combined treatment displayed significant antimetastatic potential compared to monotherapy with doxorubicin. Biological activities of compound 4 described above were reported in the form of patent application (i.e., WO2013116786) rather than peer-reviewed publication.98 In addition, compound 4 inhibited the growth in AML and ER-positive breast cancer cell lines.8,21 In an in vivo model of ER-positive breast cancer (MCF7), compound 4 suppressed tumor growth and enhanced

the growth-inhibitory effect of SERD fulvestrant without apparent toxicity to the animal. The 3,4,5-trisubstituted pyridine derivative 5 (Table 1) was initially identified as a potent inhibitor of the Wnt signaling pathway.100 A follow-on study using a chemical proteomicsbased approach identified CDK8/CDK19 as its targets.63 In addition to CDK8/CDK19, this molecule also weakly targets glycogen synthase kinases 3α (GSK3α) and GSK3β in vitro. Subsequently reported CCT251921 (6, Table 1),78 an analog of 5, and 3-methyl-1H-pyrazolo[3,4-b]pyridine derivative 7 M

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Figure 4. Binding modes of compounds 5 (A, PDB code 5BNJ) and 12 (B, PDB code 3RGF). CDK8 is shown in gray with selected residues colored magenta. Compounds 5 and 12 are shown with green carbons. Interactions between CDK8 and each inhibitor are depicted in black dashed lines. The figure was prepared using PyMOL1.3 (Schrödinger Inc., 2013).

(Table 1)79 were more potent inhibitors of CDK8/CDK19. While compounds 5, 6, and 7 were claimed to be selective over a panel of 291, 279, and 264 kinases, respectively, at 1 μM, CDK8 itself was not included in any of these kinase panels as a positive control. Preclinical testing of these compounds showed that they inhibited Wnt-dependent activity in a number of CRC cell lines and the phosphorylation of STAT1 at Ser727. Despite the potent inhibition of the Wnt signaling in cell-based reporter assays, both series of compounds did not inhibit the proliferation of CRC cell lines in vitro but were able to modestly suppress tumor growth in CRC xenografts.101 However, compounds 6 and 7 resulted in a loss of body weight in mice and further toxicological profiling in rats and dogs revealed toxicity to key organs including bone marrow, heart, liver, and lungs. This toxicity might be attributed to altered gene expression patterns that extend beyond the Wnt-βcatenin pathway. In fact, compounds 6 and 7 were found to affect the expression of genes involved in bone development, immunology, and inflammation. In contrast to the toxicity observed for compounds 6 and 7, the aforementioned compounds 1 and 2 were well tolerated without any apparent deleterious effects in mice, although detailed toxicity studies have not been carried out.19,21 This discrepancy might, at least in part, be explained by the findings from a recent study by Rzymski et al. 21 In specific, transcriptomic effects of compounds 2 and 6 in Colo205 xenograft models were compared, revealing only a