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

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Cyclin-Dependent Kinase 8: A New Hope in Targeted Cancer Therapy? Stephen Philip, Malika Kumarasiri, Theodosia Teo, Mingfeng Yu, and Shudong Wang J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.7b00901 • Publication Date (Web): 21 Dec 2017 Downloaded from http://pubs.acs.org on January 9, 2018

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Journal of Medicinal Chemistry

Cyclin-Dependent Kinase 8: A New Hope in Targeted Cancer Therapy? 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. § 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 1 ACS Paragon Plus Environment

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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 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 of 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 anti-cancer target. In this perspective, 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. § CDK8 OVERSEES DIVERSE BIOLOGICAL FUNCTIONS

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Among the many cellular functions of CDK8, the most notable is its involvement in transcription. CDK8 is part of the Mediator complex, a large multi-subunit protein complex that is central to the regulation of transcription in eukaryotes.24-26 The Mediator complex is a highly conserved transcriptional co-activator that acts as a molecular bridge to transfer regulatory signals from DNA-bound transcription factors (TFs) directly to the RNA polymerase II (RNAPII) pre-initiation complex (PIC). CDK8 reversibly associates with the Mediator complex as part of a four-subunit kinase module, which is additionally comprised of 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 pre-assembled 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, the positive regulatory role of CDK8 has been proposed33,34 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 with RNAPII-binding.24,27 Moreover it has been shown that binding of the kinase module and RNAPII to the core Mediator are mutually exclusive.36,37 Therefore, formation of the CDK8-Mediator complex may act as a molecular switch that prevents a second RNAPII from immediately reengaging the promoter, essentially terminating the reinitiation of transcription.



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Figure 1. The 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. 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 hypoxia4 ACS Paragon Plus Environment

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inducible 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 (RSmads) 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 phosphorylate R-Smads at their linker regions, permitting the binding to co-activators that are required for efficient transcription of target 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.



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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 co-activators. 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-γdependent genes. (IV) β-Catenin pathway: Binding of Wnt protein to a Frizzled family receptor activates dishevelled (Dsh) and subsequently deactivates a destruction complex that consist 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 nucleus to stimulate the transcription of TCF/LEF-dependent genes. CDK8 promotes β-catenin-dependent transcription by acting as a β-catenin co-activator 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. 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 7 ACS Paragon Plus Environment


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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 hetero-dimers 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 IFN-γ-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 anti-viral 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 phospho-acetylation 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 phospho-acetylation 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 translocate 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 8 ACS Paragon Plus Environment

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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 kinase domain, but differ considerably in the C-terminus.56 Consequently, they have some overlapping as well 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 9 ACS Paragon Plus Environment


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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 specific 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 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


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melanoma cells in a kinase-independent manner,17 while CDK19 regulates p53 responses independently of its kinase activity.61 § 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 contextspecific 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 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 cancerassociated 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 βcatenin, 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 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 11 ACS Paragon Plus Environment


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amplification.10,15,77 Furthermore, the kinase activity of CDK8 was required for both transcription of βcatenin target genes and proliferation of CRC cells. CDK8 was later identified to repress E2F1, a negative regulator of β-catenin/TCF-dependent 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 cell proliferation and anchorage-independent growth in normoxia and hypoxia. Inhibition of CDK8 caused downregulation of several genes e.g. SLC2A3, HK1, and ENO1 that involve in the glycolytic cascade and sensitized the HCT116 cells to glycolysis inhibitor 2-deoxy-D-glucose (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


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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 anti-leukemic 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 anti-cancer efficacy in AML xenograft models. These promising pre-clinical data obtained from two structurally distinct compounds suggest future development of CDK8/CDK19 inhibitors for AML therapy. Breast Cancer. Although its mortality rate has 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 13 ACS Paragon Plus Environment


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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 ER-induced 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 down-regulator (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 kinaseindependent manner.17 Loss of mH2A isoforms positively correlated with melanoma malignancy, and functional studies indicated that this loss drives melanoma progression through direct transcriptional 14 ACS Paragon Plus Environment

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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 cancer-associated 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 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 kappa-light-chain-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.



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Immuno-oncological Functions. Recent evidence indicate that phosphorylation of STAT1 at Ser727 by CDK8 suppressed NK cell-mediated cytotoxicity and tumor surveillance.70 NK cells spearhead 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, 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. § STRUCTURAL FEATURES OF CDK8 Structurally, CDK8 exhibits the typical bilobal kinase fold.89 The N-terminal domain contains mostly βsheets with two α-helices, 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-Met174-Gly175) 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.


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Figure 3. (A) Schematic representation of CDK8 bound to CycC (PDB ID: 4G6L). CDK8 is shown in yellow (C-terminus) and gray (N-terminus) 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 17 ACS Paragon Plus Environment


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consisting of nine residues are not fully resolved in the crystal structure. CycC is shown in green. (B) Inactive conformation of CDK8 (PDB ID: 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 ID: 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). 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 C-terminus, 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 Nterminal 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 (Figures 3B and 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 phospho-residue and interacts with the three well-conserved arginines, i.e., Arg65 (N-lobe), Arg150 (C-lobe) and Arg178 (T-loop). These interactions may drive the 18 ACS Paragon Plus Environment

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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 § 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 new-found interest in development of CDK8 inhibitors as potential anti-cancer agents, but some progress towards 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 local 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 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 inhibit potently several recombinant protein kinases including rho-associated 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 of 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 dose-dependent 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 anti-proliferative activity against several leukemia cell lines, and this activity was 19


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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 Table 1. Type I CDK8 inhibitors in pre-clinical development. Kinase inhibition: Inhibitor

Cellular activity

In vivo activity

Referencea

IC50 (nM) Anti-proliferative activity

Cortistatin A (1) CDK8 = 17 CDK19 = 10 ROCK1 = 250 ROCK2 = 220 Selective over 402 kinases at 10 µM

(GI50, nM):

Dose-dependent

SKNO-1 = 1

reduction in disease

RS4;11 = 3

progression and

SET-2 = 4

leukemia cell burden

MOLM14 = 5

in MV4;11 xenograft;

MV4;11 = 6

Suppressed tumor

UKE-1 = 7

growth (70% reduction

MEG-01 = 9

in tumor volume) in

TF-1 = 360

SET-2 xenograft

19 97

HCT116 > 1000 Inhibited tumor

SEL120-34A (2)

GI50 < 1000 nM against CDK8 = 4

leukemia cell lines: SKNO-

CDK19 = 10

1, KG-1, HEL-60, MOLM-

CDK9 = 1070

16, MV4;11, MOLM-6, Oci-

growth in KG-1 xenograft;

21

Reduced tumor volume in MV4;11 AML-2, and Oci-AML-3 xenograft


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Kinase inhibition: Inhibitor

Cellular activity

In vivo activity

Referencea

IC50 (nM) Senexin A (3)

Inhibition of p21-activated Inhibited tumortranscription (IC50, nM): 640

promoting paracrine

Inhibited β-catenin-

CDK8 = 280

80

effects of doxorubicin dependent transcription in in A549 xenograft HCT116 cells Senexin B (4)

Tumor growth CDK8 = 24-50 regression in LNCaPCDK19 = 4.2 LN3 xenograft; MAP4K2 and YSK4 (69

Inhibited β-catenin-

and 59 % inhibition at 2

dependent transcription in

µM, respectively)

HCT116 cells

Inhibited tumor

8,21,98,99

growth and potentiated the growth inhibition Selective over > 450 of fulvestrant in MCF7 kinases at 2 µM xenograft Wnt pathway inhibition (IC50, nM):

CDK8 = 5 CCT251545 (5)

7dF3 = 5

Tumor growth

LS174T = 23

inhibition in

SW480 = 190

xenografts of SW620

Colo205 = 35

(TGI = 36%), HCT116

PA-1 = 7

(TGI = 60%), and

Inhibition of p-STAT1-

LS513 (TGI = 81%)

CDK19 = 6 GSK3α = 462 GSK3β = 690

63,100,101

Selective over 291 kinases at 1 µM, and 55 receptors at 10 µM Ser727 in SW620 cells (IC50, nM): 23.3



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Cellular activity

In vivo activity

Referencea

IC50 (nM) Wnt pathway inhibition (IC50, nM): CCT251921 (6)

7dF3 = 11.8 CDK8 = 2.3

Tumor growth LS174T = 33

CDK19 = 2.6

inhibition in SW480 = 22

Selective over 279

xenografts of SW620

94,101

Colo205 = 15 kinases at 1 µM, and 55

(TGI = 44%) and PA-1 = 64

receptors at 10 µM

LS1034 (TGI = 55%) Inhibition of p-STAT1Ser727 in SW620 cells (IC50, nM): 25.5

3-methyl-1H-

CDK8 = 4.4

Wnt pathway inhibition

pyrazolo[3,4-

CDK19 = 3.8

(IC50, nM):

b]pyridine derivative

GSK3α = 691

7dF3 = 5.3

Tumor growth

(7)

Selective over 264

LS174T = 36.2

inhibition in

kinases at 1 µM, and 54

Colo205 = 9

xenografts of SW620

receptors at 10 µM with

PA-1 = 52

(TGI = 51%) and

an exception of


LS1034 (52%)

dopamine transporter

Ser727 in SW620 cells

(IC50 = 8.5 µM)

(IC50, nM): 8

96,101

1,6-Naphthyridine Wnt pathway inhibition

derivative (8) CDK8 = 5.1

(IC50, nM):

CDK19 = 5.6

7dF3 = 7.2

Selective over 264


kinases at 1 µM

Ser727 in SW620 cells

NAb

95

(IC50, nM): 17.9


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Cellular activity

In vivo activity

Referencea

NAb

95

NAb

102

NAb

64

NAb

103

IC50 (nM) Isoquinoline derivative (9)

Wnt pathway inhibition CDK8 = 32.7

(IC50, nM):

CDK19 = 18.8

7dF3 = 21.0

Selective over 307


kinases at 1 µM

Ser727 in SW620 cells (IC50, nM): 114 Wnt pathway inhibition

3-Benzylindazole derivative (10)

CDK8 = 53

(IC50, nM): 7dF3 = 65

CDK19 = 26 LS174T = 340 PASK = 260 PA-1 = 710 Selective over 307 Inhibition of p-STAT1kinases at 1 µM Ser727 in SW620 cells (IC50, nM): 49 6-Azabenzothiophene derivative (11) Anti-proliferative activity in CDK8 = 1.5 HCT116, EC50 = 6.7 µM

BRD6989 (12)

CDK8 ~500 CDK19 ~30000

IL-10 production

Selective over 414

EC50 = 1.35 µM

kinases at 1 µM a

Corresponding references for further information; bNA, not available.



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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 STAT1Ser727. 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 anti-tumor 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 down-stream 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 DNA-damaging 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 mitogen-activated 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-catenin-driven transcription in HCT116 cells. In mouse xenograft models, pre-treatment 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 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 24 ACS Paragon Plus Environment

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described above were reported in the form of patent application (i.e., WO2013116786) rather than peerreviewed 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 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 proteomics-based 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 (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. Pre-clinical testing of these compounds showed that they inhibited Wntdependent 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-β-catenin 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 25 ACS Paragon Plus Environment


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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 < 5% overlap of affected genes. Whereas treatment with compound 2 mostly affected the expression of STAT-dependent genes, only modest effects were seen with compound 6 despite the potent inhibition of STAT1-Ser727 phosphorylation in vivo. Although compounds 6 and 7 were claimed to be highly selective for CDK8/CDK19 in vitro at 1 µM, it remains unknown whether or not they acted specifically on targets at the doses used for xenograft studies. On the other hand, compound 5 was more potent than compounds 2 and 4 in inhibiting the growth of AML cell lines in spite of their similar inhibitory potency for CDK8/CDK19.21 Taken together, these findings warrant further investigation on the in vivo kinase selectivity of compounds 6 and 7 at the doses that inhibited the growth of CRC xenografts. Scaffold-hopping from 3,4,5-trisubstituted pyridines led to the discovery of 1,6-naphthyridine derivative 8 and isoquinoline derivative 9 (Table 1).95 Both compounds showed high selectivity for CDK8/CDK19 in kinase assays and potently inhibited the Wnt signaling in cell-based assays. The 3benzylindazole derivative 10102 and compound 6-Azabenzothiophene derivative 1164 are other inhibitors of CDK8/CDK19. Their chemical structures and kinase inhibitory profiles are summarized in Table 1. The pyridinyl tetrahydroquinoline BRD6989 (12, Table 1) was identified in a phenotypic screen for small molecules that enhance production of interleukin-10 (IL-10) from activated dendritic cells, and subsequent kinase profiling against a panel of 414 kinases identified CDK8 and CDK19 as its targets.103 Although showing a modest inhibition against CDK8 (IC50 500 nM), 12 demonstrated a high selectivity for CDK8 over CDK19. In bone-marrow-derived dendritic cells, 12 suppressed the phosphorylation of STAT1 at S727 and enhanced the production of IL-10, suggesting CDK8 as a negative regulator of IL10 production during innate immune activation. Like 12, other CDK8/CDK19 inhibitors, i.e., 1 and 5 were also capable of enhancing production of IL-10. It would be interesting to see whether the toxic


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effects associated with CDK8/CDK19 inhibitors on animal can be avoided with 12 due to its improved selectivity for CDK8. Aforementioned inhibitors bind to CDK8 in an ATP-competitive fashion (i.e., type I). Several type II inhibitors that were originally developed as tyrosine kinase inhibitors were also found to inhibit CDK8/CDK19. Sorafenib (12),89,104 ponatinib (13),105 and linifanib (14)63,106 inhibit CDK8/CDK19 potently in biochemical assays (Table 2).63 However, the biochemical potency of these compounds did not translate either to inhibition of the Wnt activity or of phosphorylation of STAT1-Ser727 in cells. Using compound 12 as a starting point, alternative type II inhibitors have been developed.107 Among them are pyrrolidine urea derivatives 15 and 16 (Table 2),107 both of which potently inhibit CDK8 in biochemical assays, but do not show biomarker activity against CDK8 in HCT116 cells. This relatively poor translation of in vitro biochemical potency into cellular activity with type II inhibitors is likely due to the fact that they target the inactive form of CDK8 that is poorly accessible in cells owing to CDK8 being locked in the active kinase conformation either in the Mediator complex or in the four subunit kinase module.63,107 Table 2. Type II inhibitors that inhibit CDK8. Main targets

Other targets

Cellular activity

Clinical

(IC50, nM)

(IC50, nM)

(IC50, µM)

development

Inhibitor Sorafenib (12)

RAF1 = 6

Approved for

BRAF= 22

advanced renal cell

BRAFV599E =

Wnt pathway

carcinoma,

38

CDK8 = 199

inhibition:

hepatocellular

VEGFR2 = 90

CDK19 = 206

7dF3 = 1.620

carcinoma and

LS174T = 4.520

radioactive iodine

FLK1 = 15 mutatedVEGR

resistant thyroid

3 = 20

carcinoma

Referencea

63,104,108



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Main targets

Other targets

Cellular activity

Clinical

(IC50, nM)

(IC50, nM)

(IC50, µM)

development

Inhibitor

Referencea

FLT3 = 58

Ponatinib (13)

Wnt pathway ABL = 9 ABL

T315I

= 56

CDK8 = 14

inhibition:

Approved for CML

CDK19 = 23

7dF3 = 0.041

and Ph+ALL

63,105,109

LS174T = 0.619

Phase III for KDR = 4 Linifanib (14)

advanced FLT1 = 3 Wnt pathway

hepatocellular

CDK8 = 14

inhibition:

carcinoma, and

CDK19 = 24

7dF3 = 1.29

phase II for

LS174T = 5.17

advanced non-

FLT4 = 190 PDGFRβ = 66

63,106,110,111

CSF1R = 3 KIT = 14 small cell lung FLT3 = 4 cancer Pyrrolidine urea derivative (15) CDK8 = 17.4 FLT3 (74% Selective over inhibition at 1

HCT116 >10

Pre-clinical

107

215 kinases at µM) 1 µM


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Main targets

Other targets

Cellular activity

Clinical

(IC50, nM)

(IC50, nM)

(IC50, µM)

development

CDK8 = 14.7

NAb

HCT116 > 4.8

Pre-clinical

107

CDK8 = 6.5

NAb

NAb

Pre-clinical

112

Inhibitor

Referencea

Pyrrolidine urea derivative (16)

W-34 (17)

a

Corresponding references for further information; bNA, not available.

§ DESIGNING INHIBITORS OF CDK8 Analyzing the chemical structures of inhibitors and their co-crystallized binding modes to CDK8 exposes common requirements for high binding affinities. The crystal structure of compounds 5 bound to CDK8 (Figure 4A) reveals a hydrogen bonding interaction between the pyridine nitrogen and backbone amino of Ala100 in the hinge region.63,94 The backbone carbonyls of Asp98 and Ala100 are also in close proximity (2.4 Å and 2.7 Å, respectively) to the C2 and C6 positions of the pyridine ring, strongly suggesting the existence of an interaction based on the observed electron density. In compound 6, the C2 position has an amino substituent, which facilitates a stronger interactions with Asp98.94 29 ACS Paragon Plus Environment


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Figure 4. The binding modes of compounds 5 (A, PDB ID: 5BNJ) and 12 (B, PDB ID: 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). Type I inhibitors are typically positioned in close proximity to the aromatic side chain of the gatekeeper residue, Phe97 (Figure 4A). Thus, lipophilic substituents that facilitate π-π interactions are favored at the pyridine-C3 position: the replacement of the chlorine in compound 6 with fluorine weakened the interaction with Phe97 and reduced inhibitory activity.94 In compounds 3 and 4, the electron rich cyano substituent appears to be preferred over halogens, at the C3 position. Given the orientation with respect to Phe97 phenyl ring, an edge-to-face π-π interaction is possible here. Type II inhibitors such as compound 12 bind to the inactive kinase conformation and occupy the ATPbinding site and an adjacent deep hydrophobic pocket (Figure 4B).113 CDK8 is the only CDK that has been crystallized in the inactive state. Typically, type II CDK8 inhibitors contain a hydrogen bond donoracceptor pair such as a urea or amide that is linked to a hydrophobic tail moiety on one side and a hinge binding moiety on the other side. This type of interaction is also seen in the case of compound 15. 30 ACS Paragon Plus Environment

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The uniquely extended CDK8 C-terminus lies in parallel and in close proximity (~5 Å) to the hinge, creating a potentially vital opportunity to increase inhibitor selectivity. Arg356 can play a critical role here. The high selectivity of compound 5 has been attributed to the formation of cation–π interactions between the pyridine-C5-branched phenyl group and the positively-charged guanidine side chain of Arg356.63 The addition of electron donating substituents such as a methyl group can enhance electron density of the phenyl ring, resulting in stronger cation-π interactions, and enhance selectivity and potency.114 Type II inhibitors also have a similar potential to benefit from Arg356 interactions. For example, compound W-34 (17, Table 2),112 a potent type II CDK8 inhibitor targets Arg356. Likewise, other residues may be targeted to enhance selectivity and potency. For example, Trp105, located on the αD helix adjacent to the hinge, is unique to CDK8 and CDK19, and no other CDK possesses an aromatic side chain at this position.19 In compound 1, a cation-p interaction between the protonated N,N-dimethylamino group and Trp105 can be observed in the co-crystal structure.19 This interaction is deemed to contribute to the high selectivity of compound 1 to CDK8 and CDK19. Since residues in the hydrophobic pocket are less conserved among the kinases, type II inhibitors may have a better chance to achieve higher selectivity when compared to type I analogs. For example, Phe176 in the deep pocket is unique to CDK8: other CDKs contain a leucine that is solvent-exposed in the active conformation.89 The 3-trifluoromethyl-4-chlorophenyl substituent of compound 12 is considered to interact hydrophobically with Phe176. The deep pocket also contains other hydrophobic residues such as Leu69, Leu70, Leu73, Val78, Val147, and Tyr32 that can also be targeted.112 § THE CHALLENGE AHEAD CDK8 has been shown to be involved in diverse signaling pathways that are crucial for the growth and survival of cancer cells. While suppression of CDK8 activity leads to the growth inhibition in many cancers, its contrary role in some other types of tumors has emerged. This cell-type specific nature of CDK8 may limit its application as a therapeutic target in certain types or sub-types of cancers. The 31 ACS Paragon Plus Environment


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breadth of such nature is currently not well understood, presenting a valuable opportunity for further studies. CDK8 was initially identified as an oncogene in CRC, and knockdown of CDK8 have been demonstrated to reduce both proliferation and the expression of β-catenin target genes (i.e., MYC, AXIN2, and LEF1) in CRC cell line Colo205.10 However, CDK8/CDK19 inhibitors, i.e., compounds 5, 6, and 7, showed negligible anti-proliferative activity in the above cell line, despite the potent inhibition of both Wnt-dependent reporter activity and the phosphorylation of STAT1 at Ser727.61,76 Similar results were also seen with other inhibitors, i.e., compounds 1 and 11, in HCT116 cells.19,62 Furthermore, changes in gene expression resulting from treatment of HCT116 and Colo205 cells with compounds 1 and 6, respectively, were significantly different from those arising from genetic knockdown of CDK8 in these cells.19,76 These discrepancies between kinase inhibition and genetic knockdown imply a noncatalytic role for CDK8, which may be an important determinant of oncogenic potential in CRC. However, beyond these narrow distinctions, much additional work is needed to clearly establish whether a non-catalytic function of CDK8 contributes to oncogenesis in CRC. An important question that has also arisen from pre-clinical studies of pharmacological inhibitors concerns the tumor types that would benefit most from targeting CDK8 and CDK19 kinase activity. Although CDK8/CDK19 inhibitors, i.e., compounds 5, 6, and 7, have shown negligible anti-proliferative activity in CRC cell lines, they have exhibited anti-tumor efficacy in xenografts bearing the same CRC cell lines, but it is uncertain whether any off-targets contribute to the anti-cancer effect observed.61,76 In AML cells, however, two distinct chemical classes of CDK8/CDK19 inhibitors, i.e., compounds 1 and 2, demonstrated anti-tumor activity in vitro and in vivo.19,21 Similarly, in ER-positive breast cancers, pharmacological inhibition of CDK8/CDK19 by compound 4 suppressed tumor growth and potentiated the anti-tumor activity of fulvestrant in vitro and in vivo.8 These divergent pre-clinical results suggest that targeting CDK8/CDK19 potentially offers an effective therapeutic strategy for AML and ER32 ACS Paragon Plus Environment

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positive breast cancers. Nevertheless, there is an urgent need to identify suitable biomarkers for the selection of appropriate patients that may respond to the treatment with CDK8/CDK19 inhibitors. A survey of literature suggests the use of phosphorylated STAT1-Ser727 and STAT5-Ser726 as markers in AML,21 but further validation of the experimental data is required. Clarke et al.101 has arisen a potential toxicology issue in developing CDK8/CDK19 inhibitor drug candidates. The authors were unable to establish a therapeutic window for their inhibitors, i.e., compounds 6 and 7 due to significant adverse effects in animals. However, no such toxicity in animal studies has been reported with other inhibitors, i.e., compounds 1, 2, and 4.8,19,21 Further investigation of pharmaceutical, pharmacokinetic, and pharmacodynamical properties of these compounds would provide a plausible explanation of the efficacy and toxicity observed. Another plausible strategy to better understand the toxicity of compounds 6 and 7 could be based on genetic models. In this regard, the generation of CDK8 knock-in mice, where CDK8 lacks the kinase function but retains the ability to interact with its cognate partner proteins, could aid in the prediction of pharmacodynamic effects of CDK8/CDK19 inhibition and help distinguish between on-target and off-target effects. The current understanding of the role of CDK8 in cancer therapy has been further complicated by the fact that all CDK8 inhibitors identified so far also target CDK19. The high sequence similarity between CDK8 and CDK19 in the kinase domain renders the development of mono-specific CDK8 inhibitors a challenging task. But the lack of CDK8-specificity confounds the ability in dissecting the kinase-targeted biology, mechanism of anti-cancer action, and associated physiology and toxicity. Finally, the role of CDK8 in the process of immune-evasion of cancer cells that likely derives from CDK8-mediated repression of the NK cell cytotoxicity may represent a unique opportunity for drug discovery. However, elucidating of the complex nature of CDK8 biology and its role in stimulating NK cells is a challenging task, requiring a significant advancement in establishing the appropriate in vitro 33 ACS Paragon Plus Environment


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and in vivo models. Nevertheless, identification of mono-specific inhibitors of CDK8 that possess favorable pharmacologic properties would be a major step towards addressing above issues so that development of CDK8-targeted cancer therapy can be progressed. § AUTHOR INFORMATION Corresponding Author *Phone: +61 8 83022372. Fax: +61 8 83021087. E-mail: [email protected]. ORCID Stephen Philip: 0000-0002-3945-8073 Mingfeng Yu: 0000-0002-0860-4816 Shudong Wang: 0000-0001-6225-5525 Author Contributions Conception and overall supervision: S.W. Wrote/revised/edited the manuscript: S.P., M.K., T.T., M.Y., and S.W. Notes The authors declare no competing financial interest. Biographies Stephen Philip completed his Master of Pharmacy in Pharmaceutical chemistry from JSS University, India in 2014. He then worked at Novartis as an associate scientist. He is currently pursuing his Ph.D. in medicinal chemistry from the University of South Australia under the supervision of Professor Shudong Wang. His work mainly focusses on the design and synthesis of novel protein kinase inhibitors for targeted cancer therapy. He is presently involved in the synthesis and optimization of inhibitors that target CDK8. Malika Kumarasiri received his Ph.D. in Chemistry from the Pennsylvania State University in 2009. He specialises in computer aided drug discovery and the dynamics of protein-drug interactions. After a 34 ACS Paragon Plus Environment

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successful four-year period as a postdoctoral fellow at the University of Notre Dame, he moved to University of South Australia to undertake computational investigations for the development of selective and potent kinase inhibitors. He is particularly interested in the selective inhibition of cyclin dependent kinases, and his current efforts are focused on the discovery of novel CDK8 inhibitors. Theodosia Teo received her Ph.D. in Cancer Biology from the University of South Australia in 2015. Her research is focussed on investigating the expression of proteins related to cancer growth, and development in normal and cancerous cells, upon treatment by protein kinase inhibitors. Upon graduation, she has continued her work, to dissect the effects of specific inhibition of CDK8 in various cancer types. Currently her research interests are centered on CDK8 and the mechanisms underlying its fundamental roles in colorectal cancer. Mingfeng Yu completed his Ph.D. in Organic Chemistry under the supervision of Associate Professors Matthew H. Todd and Peter J. Rutledge at the University of Sydney in 2013. Upon the completion of his Ph.D., he was appointed as a Post-doctoral Research Fellow at the University of South Australia. Under the guidance of Professor Shudong Wang, he conducts research to discover novel protein kinase inhibitors for targeted cancer therapy. He is currently interested in the development and optimization of small molecules that selectively and potently inhibit CDK8. Shudong Wang is Chair of Medicinal Chemistry at the University of South Australia. She began her research career in a British biotech company (CYCC) and then the School of pharmacy at University of Nottingham, UK. She is currently the Head of the Centre for Drug Discovery and Development where she leads a multi-disciplinary team with research spanning computational & medicinal chemistry, biochemistry, cell biology, pharmacology and pre-clinical drug evaluation. Her research interests focus on the discovery and development of novel classes of kinase targeted anti-cancer therapeutics. § ACKNOWLEDGMENTS 35 ACS Paragon Plus Environment


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S.P. acknowledges the support from the Australian Government Research Training Program Domestic Scholarship. § ABBREVIATIONS USED Cyc, cyclin; RNAPII, RNA polymerase II; PIC, pre-initiation complex; CTD, carboxy-terminal domain; SEC, super elongation complex; TF, transcription factor; HIF1A, hypoxia-inducible factor 1A; R-Smads, receptor-regulated Smad proteins; ER, estrogen receptor; Smad, mothers against decapentaplegic homolog; IEG, immediate early gene; BMP, bone morphogenetic protein; TGFβ, transforming growth factor β; NOTCH, neurogenic locus notch homolog protein; NICD, notch intracellular domain; STAT1, signal transducer and activator of transcription 1; IFN-γ, interferon gamma; JAK, Janus kinase; CycC, cyclin C; shRNA, short hairpin RNA; Skp2, S-phase kinase-associated protein 2; E2F1, E2 promoter binding factor 1; APC, adenomatous polyposis coli; NK, natural killer; mH2A1, macroH2A1; MEF, mouse embryonic fibroblast; SREBP-1C, sterol regulatory element-binding protein 1C; CRC, colorectal cancer; LEF1, lymphoid enhancer binding factor 1; AML, acute myeloid leukemia; TCF, T-cell factor; TNBC, triple-negative breast cancer; AXIN2, axis inhibition protein 2; SE, super enhancer; SERD, selective estrogen receptor down-regulator; EC50, Half maximal effective concentration; GREB1, growth regulation by estrogen in breast cancer 1; HER2, human epidermal growth factor receptor 2; ROCK, rhoassociated protein kinase; GSG2, haploid germ cell-specific nuclear protein kinase; IC50, half maximal inhibitory concentration; TGI, tumor growth inhibition; EC50, half maximal effective concentration; EGR1, early growth response 1; MAP4K2, mitogen-activated protein kinase kinase kinase kinase 2; YSK4, mitogen-activated protein kinase kinase kinase 19; GSK3α, glycogen synthase kinase 3α; PASK, PAS domain containing serine/threonine kinase; RAF1, RAF proto-oncogene serine/threonine-protein kinase; BRAF, serine/threonine-protein kinase B-Raf; VEGFR2, vascular endothelial growth factor receptor 2; FLT3, Fms related tyrosine kinase 3; ABL, Abelson tyrosine-protein kinase 1; KDR, kinase insert domain receptor; PDGFRβ, platelet derived growth factor receptor beta; CSF1R, colony 36 ACS Paragon Plus Environment

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stimulating factor 1 receptor; KIT, KIT proto-oncogene receptor tyrosine kinase; 2DG, 2-deoxy-Dglucose; NFκB, nuclear factor kappa-light-chain-enhancer of activated B cells; TNFα, tumor necrosis factor alpha, IL-10, interleukin-10; PI3KC2A, phosphatidylinositol-4,5-biphosphate 3-kinase C2A; AP1, activator protein 1. § REFERENCES (1)

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