Development of a Potent, Specific CDK8 Kinase Inhibitor Which Phenocopies CDK8/19 Knockout Cells

Article abstract of DOI:10.1021/acsmedchemlett.5b00278

Beginning with promiscuous COT inhibitors, which were found to inhibit CDK8, a series of 6-aza-benzothiophene containing compounds were developed into potent, selective CDK8 inhibitors. When cocrystallized with CDK8 and cyclin C, these compounds exhibit an unusual binding mode, making a single hydrogen bond to the hinge residue A100, a second to K252, and a key cation-πinteraction with R356. Structure-based drug design resulted in tool compounds 13 and 32, which are highly potent, kinase selective, permeable compounds with a free fraction >2% and no measurable efflux. Despite these attractive properties, these compounds exhibit weak antiproliferative activity in the HCT-116 colon cancer cell line. Further examination of the activity of 32 in this cell line revealed that the compound reduced phosphorylation of the known CDK8 substrate STAT1 in a manner identical to a CDK8 knockout clone, illustrating the complex effects of inhibition of CDK8 kinase activity in proliferation in these cells.

Full text of DOI:10.1021/acsmedchemlett.5b00278

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Letter
Potent, Specific CDK8 Kinase Inhibitors Lack CDK8-dependent
Antiproliferative Activity in HCT-116 Colon Cancer Cell Line
Michael F.T. Koehler, Philippe Bergeron, Elizabeth M. Blackwood, Krista Bowman, Kevin R
Clark, Ron Firestein, James R Kiefer, Klaus Maslos, Mark L. McCleland, Linda Orren, Laurent
Salphati, Steve Schmidt, Elisabeth V Schneider, Jiansheng Wu, and Maureen H. Beresini
ACS Med. Chem. Lett., Just Accepted Manuscript • DOI: 10.1021/acsmedchemlett.5b00278 • Publication Date (Web): 06 Jan 2016
Downloaded from http://pubs.acs.org on January 7, 2016
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Potent, Specific CDK8 Kinase Inhibitors Lack CDK8-
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Cell Line
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Michael F.T. Koehler†*, Philippe Bergeron†, Elizabeth M. Blackwood‡, Krista Bowman§, Kevin R.
Clarkǁ, Ron Firestein#, James R. Kiefer§, Klaus Maskos∥, Mark L. McCleland#, Linda Orrenǁ, Laurent
Salphati⊥, Steve Schmidtǁ, Elisabeth V. Schneider∥○, Jiansheng Wu◊ and Maureen H. Beresiniǁ
†Department of Discovery Chemistry, ‡Department of Translational Oncology, §Department of Structural Biology,
ǁDepartment of Biochemical and Cellular Pharmacology, #Department of Pathology, ⊥Department of Drug Metabolism and
Pharmacokinetics and ◊Department of Structural Biology, Genentech, Inc., 1 DNA Way, South San Francisco, CA 94080,
United States
∥Proteros Biostructures GmbH, Bunsenstr. 7a, Dꢀ82152 Martinsried, Germany
○MaxꢀPlanckꢀInstitut für Biochemie, Am Klopferspitz 18a, Dꢀ82152 Martinsried, Germany
KEYWORDS CDK8, Cyclin C, kinase inhibitor
ABSTRACT: Beginning with promiscuous COT inhibitors, which were found to inhibit CDK8, a series of 6ꢀazaꢀbenzothiophene
containing compounds were developed into potent, selective CDK8 inhibitors. When coꢀcrystallized with CDK8 and cyclin C, these
compounds exhibit an unusual binding mode, making a single hydrogen bond to the hinge residue A100, a second to K252 and a
key cationꢀπ interaction with R356. Structure based drug design resulted in tool compounds 13 and 32, which are highly potent,
kinase selective, permeable compounds with a free fraction >2% and no measurable efflux. Despite these attractive properties, these
compounds exhibit weak antiproliferative activity in the HCTꢀ116 colon cancer cell line. Further examination of the activity of 32
in this cell line revealed that the compound reduced phosphorylation of the known CDK8 substrate STAT1 in a manner identical to
a CDK8 knockout clone, illustrating the complex effects of inhibition of CDK8 kinase activity in proliferation in these cells.
CDK8 is a cyclinꢀdependent kinase that forms part of the
Mediator complex, which itself regulates the transcriptional
activity of RNA polymerase II. A number of studies have
shown that CDK8 modulates the transcriptional output
from distinct transcription factors involved in oncogenic
control. 1 These factors include the Wnt/βꢀcatenin pathway,
Notch, p53 and TGFꢀβ.2,3
Additionally, the conditional knockout of Cdk8 in young
adult mice produced no gross abnormalities, while Cdk8
deletion in the ApcMin tumor model reduced survival and
increased tumor burden.10
To date, the investigations of the role of CDK8 in both celꢀ
lular signaling and colon cancer have relied upon RNAi
mediated suppression of CDK85 and on the use of a kinase
dead mutant CDK84. In order to more fully
While the means by which CDK8 activity accomplishes the
regulation of these various pathways remains an active area
of investigation, CDK8 has been found to be amplified and
overexpressed in colon cancer. In this context, CDK8 has
been reported to act as a colon cancer oncogene4, driving
Wnt pathway activity by mediating βꢀcatenin transcriptionꢀ
al output.5 Suppression of CDK8 expression in colon canꢀ
cer cell lines expressing high levels of both CDK8 and βꢀ
catenin led to reduced proliferation of these cell lines and
6
induced differentiation. Furthermore, in a survey of coloꢀ
rectal cancer tissue samples, CDK8 was associated with βꢀ
catenin activation and with reduced rates of survival.7 More
recently, it has been shown that suppression of CDK8 exꢀ
pression in melanoma cells reduces proliferation. 8 Interestꢀ
ingly, the reverse has also been reported; mutations in
MED12 which disrupt the association of CDK8 with the
mediator complex are associated with uterine leiomyomas.9
Figure 1. Kinase selectivity for compounds 1 and 2 measured
at a concentration of 0.1 ꢀM in a panel of 52 kinases. Each
vertical line represents a single kinase and is colored by the
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Table 2. Alternatives to the tetrazolea
percent inhibition seen. Selected potently inhibited kinases are
highlighted. Complete data is in the supplementary material.
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Table 1. Potency, permeability and plasma protein binding
data for 2-tetrazolyl-thieno[2,3-c]pyridines 1-10
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a PPB figures in human, rat and mouse plasma (H/R/M)
investigate the role of CDK8 in colon cancer, we aimed to
develop a potent and selective small molecule inhibitor of
CDK8. In order for such a molecule to serve as a useful
tool compound in understanding CDK8 activity in colon
cancer cell lines, we required that it also be cell permeable
with minimal efflux, and have a high free fraction in plasꢀ
ma.
lower than approximately 100Å2 were permeable as measꢀ
ured in a standard MDCK assay. None of the compounds
examined exhibited any significant level of efflux.
As might be expected from a set of lipophilic compounds
containing a tetrazole, which would be negatively charged
at physiological pH, the compounds were found to be highꢀ
ly protein bound, with all compounds >99.7% bound in
human, rat and mouse serum. In an effort to reduce plasma
protein binding, polar functionality was introduced at the 4ꢀ
position of our aryl ether. These modifications did not adꢀ
versely affect the potency of these compounds for CDK8,
but as the TPSA went above 100Å2, the MDCK permeabilꢀ
ity dropped dramatically, making them unlikely to be active
in a cellular context. We therefore intiated an examination
of what functional groups would be tolerated in place of the
tetrazole.
The two small molecule inhibitors of CDK8 reported at the
start of our investigations, sorafenib11 and Senexin A12
exhibited insufficient kinase selectivity and potency, reꢀ
spectively, to serve our purposes. We therefore examined
our inꢀhouse database of kinase inhibition data and looked
for compounds which might have been prepared for a difꢀ
ferent target, but serendipitously inhibited CDK8.
We were pleased to find that compound 2, which has been
reported to be a COT inhibitor13,14, potently inhibited
CDK8. As part of our standard followꢀup for a lead, we
resynthesized the material and assayed intermediates in the
synthesis in a fluorescence polarization binding assay. We
were surprised to discover that compound 1 was a still
more potent inhibitor of CDK8 than our initial lead, and
exhibited superior selectivity as well (Figure 1 & Table 1).
We began our exploration of substitution at the 2ꢀposition
of the thieno[2,3ꢀc]pyridines by examining the carboxylate
11 (Table 2).15 As expected, the activity of the compound
was only slightly diminished, and the plasma protein bindꢀ
ing was reduced only very slightly. To determine if an aniꢀ
on was required at physiological pH, we prepared inactive
ester 12 and amides 13ꢀ18. With the smallest amide subꢀ
stituents, the inhibition of CDK8 remained potent, but alkyl
groups larger than methyl and particularly disubstitution of
the amide
Having determined that the second aryl ring in the biphenyl
ether provided no advantage, we prepared and evaluated
compounds 2ꢀ10 in an effort to understand the limitations
of this chemotype. Despite the presence of the highly polar
tetrazole group, compounds with a topological polar surꢀ
face
area
(TPSA)
Table 3. Incorporation of polar functional groups on aryl
ether.a
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a PPB figures in human, rat and mouse plasma (H/R/M)
a PPB figures in human, rat and mouse plasma (H/R/M)
complex with 22 bound to facilitate design. The structure
contains a single monomer of the CDK8/cyclin C complex
in the asymmetric unit, with CDK8 stabilized in the “acꢀ
tive” conformation (helix C “in” and the sidechain of
Glu66 of the helix coordinated to the active site Lys252).
Prior structures of sorafenib and other inhibitors bound the
kinase with both DMGꢀin and DMGꢀout binding
modes.11,16 The complex with 22 elicited the DMGꢀin conꢀ
formation in the enzyme and interactions between the proꢀ
tein and 22 resemble those proposed for molecules of this
type binding to COT (Figure 2). A cationꢀΠ interaction is
seen in the close approach of Arg356 to the iodophenyl
ring, stabilizing a 65° rotation of the iodophenyl ring out of
the plane of the heterocyle. Hydrogen bonds are formed
between the pyridyl nitrogen of 22 and the backbone nitroꢀ
gen of Ala100 of the hinge as well as from the sidechain of
Lys52 to the amide carbonyl. While the binding mode of
the compound is clear from the electron density (Table S1),
the temperature factors of the ligand suggest its presence at
50ꢀ75% occupancy; however, the intermediate resolution of
the data precluded definitive refinement of that parameter.
Interestingly, a formate ion is observed at partial occupancy
in the region underneath the P loop, adjacent to the inhibiꢀ
tor, raising the possibility of building into that region to
increase the buried surface area. Examination of the strucꢀ
ture prompted the design of a few targeted analogs in an
effort to maximize potency.
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resulted in 10ꢀ to 100ꢀfold losses in activity. More promisꢀ
ingly, the plasma protein binding was seen to diminish in
both the caboxamide 13 and Nꢀmethyl carboxamide 14.
Finally, we examined the necessity of having a hydrogen
bond donor as part of the 2ꢀsubstituent and prepared comꢀ
pounds 19ꢀ21. As with the amides, the smaller substituents
were more potent, but we observed a 5ꢀfold or greater loss
in affinity relative to the carboxamide, and plasma protein
binding was greater than 99% for all the ketones examined.
With the knowledge that carboxamides combined the best
potency and the lowest PPB yet obtained, we prepared anꢀ
other series of analogs to determine whether we could conꢀ
tinue to reduce PPB while maintaining potency and permeꢀ
ability by incorporating polar functional groups at the 4ꢀ
position of our aryl ether (Table 3).
In preparing these analogs we discovered that alkyl ethers
at this position led to decreases in potency, as seen in comꢀ
pounds 24 and 25, perhaps due to the incorporation of the
polar oxygen atom, which can be partially compensated for
by the increased lipophilicity of the trifluoromethyl group
in 25. Addition of a further heteroatom in amide 27 and
reverse amide 28 led to potency very similar to that of the
methyl ether. Nitrile 26 pushes its heteroatom further away
from the
ring and offered good potency, reduced PPB and moderate
kinetic solubility.
Our first analogs were targeted at probing the region in the
vicinity of the hinge binding nitrogen, which accepts a hyꢀ
drogen bond from the NH of Ala100. We noted that there
was space available to make a contact with the main chain
of Asp98 from the 7ꢀposition of our heterocycle, and we
made a series of analogs to access this region (Table 4).
Interestingly, hydrophobic analogs 29ꢀ31 retained much of
their CDK8 binding
Having generated SAR data at both of the two most easily
modified regions of our molecules, we determined the 2.2Å
resolution crystal structure (PDB: 5CEI and Supporting
Information) of the CDK8/cyclin C
Table 4. 7-substituted thieno[2,3-c]pyridines.a
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Table 6. Antiproliferative activity of compounds 13, 31 and
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2
3
32 on HCTꢀ116 cells. Compounds 5-8, 11 and 25 were also
evaluated, but had EC50 values >10 ꢀM under both high and
low serum conditions.
4
5
6
HCT116 EC50
10% FBS (ꢀM)
HCT116 EC50
0.625% FBS (ꢀM)
compound
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8
9
13
>10
>10
8.8
8.7
31
32
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5.4
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flavopiridol
0.034
0.059
Compound 13 had an IC50 value of 2.2 ꢀM for
CDK9/cyclin T1, and therefore a selectivity of 350ꢀfold for
CDK8/cyclin C. Compound 32 had an IC50 value of 1.1
ꢀM, and a selectivity of 730ꢀfold. The overall profile of
these two molecules now met our goal of producing potent,
selective CDK8 inhibitors with solubility, plasma protein
binding, permeability and low efflux (Table S2).We thereꢀ
fore evaluated the antiproliferative effects of our comꢀ
pounds in the colorectal carcinoma HCTꢀ116 cell line,
which had previously been shown to exhibit sharply reꢀ
duced proliferation in response to CDK8 protein depletion
using targeted shRNA.4 Cells were treated with compounds
for 72h in media containing fetal bovine serum (FBS) at a
concentration of either 0.625% or 10%. The lower concenꢀ
tration of FBS was used to mitigate the effects of the low
free fraction of some of our compounds. Throughout the
course of the experiment, the cells were in growth phase,
and yet we did not observe significant growth inhibition
(Table 6). Flavopiridol, a potent inhibitor of multiple
CDKs17with known antiproliferative properties18 was used
as a positive control. In each case, the activity of the comꢀ
pounds appeared at approximately 1000ꢀfold higher conꢀ
centration than the IC50 value for the purified enzyme.
Concerned that the compounds examined were failing to
act on CDK8 in the cellular context, we examined phosꢀ
phorylation of Ser727 of STAT1, a substrate of CDK819 as
a biomarker.
Figure 2. Structure of compound 22. Hydrogen bonding interꢀ
actions with Ala100 and Lys52 are indicated with dashed
lines.
activity, with 29 losing less than 3ꢀfold, and the others less
than 2ꢀfold. Compound 32 was found to bind 4 fold more
potently than its parent compound 13, which confirmed our
modeling of the compound which indicates that the NH2
can readily make a hydrogen bond to the backbone carbonꢀ
yl of Asp98.
Having generated extremely potent binders of CDK8, it
was important to examine the selectivity of these comꢀ
pounds relative to other kinases. We focused especially on
ensuring that there was minimal activity against CDKs 1
and 4, as these would be expected to have antiproliferative
effects via disruption of the cell cycle, confounding our
ability to assess the effects of inhibiting CDK8. We thereꢀ
fore submitted compounds 13 and 32 for profiling against a
panel of 209 kinases. We were pleased to find that both
compounds exhibit excellent selectivity for CDK8 over
every other kinase examined (Figure 3) even when tested at
concentrations 160ꢀ and 670ꢀfold greater than the measured
IC50, respectively.
In order to validate STAT1 Ser727 as a substrate in
HCT116 cells, CRISPR guide RNAs were created to target
CDK8 for genomic deletion.20-23 Three separate clones
showed complete ablation of CDK8 protein while its close
homolog CDK1924 and actin expression remained unꢀ
changed (Figure 4A). Clone 1 was then used to generate
three CDK8/CDK19 double knockouts, for which all three
generated clones showed the complete absence of both proꢀ
teins. Interestingly, cyclin C levels were greatly reduced in
the absence of CDK8 alone, and the protein was undetectaꢀ
ble in the double knockout, likely due to instability when
unable to associate with the other components of the meꢀ
diator complex, and highlighting the possibility of secondꢀ
ary effects when protein expression is altered using siRNA
or CRISPR.
Having identified CDK9/cyclin T1 as our most active offꢀ
target, we therefore determined the IC50 values for 13 and
32 in a fluorescence polarization activity assay
Figure 3. Kinase selectivity for compounds 13 and 32 measꢀ
ured at a concentration of 1 µM in a panel of 209 kinases.
Each vertical line represents a single kinase and is colored by
the percent inhibition seen. Selected potently inhibited kinases
are highlighted. Complete data is in the supplementary materiꢀ
al.
HCTꢀ116 cells along with their respective CDK8 knockout
and CDK8/CDK19 double knockout clones were then
probed with an antibody specific for phosphorylated
Ser727 on STAT1. As had been previously reported in
bone marrow derived macrophages19 and peripheral blood
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mononuclear cells25, stimulation of HCTꢀ116 cells with
interferonꢀγ resulted in increased expression of STAT1 and
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4
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7
8
a large increase in the amount of phosphorylated Ser727
(Figure 4B). This phosphorylation was reduced in the
CDK8 knockout, and further reduced in the CDK8/CDK19
double knockout clones stimulated in the same manner
while the total amount of STAT1 expressed remained relaꢀ
tively constant. Encouragingly, cells treated with 2 ꢀM
compound 32 showed levels of phosphorylated Ser727
nearly identical to that of the double knockout cell line,
clearly demonstrating that this molecule is able to downꢀ
regulate signaling in cultured cells.
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Having demonstrated that 32 was in fact inhibiting CDK8
function in HCTꢀ116 cells, we used the CDK8 and
CDK8/19 knockout cell lines created to demonstrate that
the observed antiproliferative effects of 32 were independꢀ
ent of their inhibition of the targeted protein. Wild type
HCTꢀ116 cells, along with the CDK8 and CDK8/19 deleꢀ
tion clones derived from them, were incubated with varying
concentrations of 32. As can be seen in Figure 4C, each of
these cell lines showed identical growth inhibition curves,
indicating that growth inhibition in these cells is an offꢀ
target effect.
During the preparation of this manuscript, additional selecꢀ
tive CDK8 inhibitors have been reported, both of which
have been shown to sharply reduce the presence in cells of
STAT1ꢀpS727 analogously to 32. Cortistatin A is reportꢀ
ed26 to have mixed antiproliferative effects on cell lines
with sensitivity driven by the set of genes upregulated upon
treatment, with HCTꢀ116 cells among the insensitive lines.
The compound CCT251545, identified through its inhibiꢀ
tion of the WNT pathway in cells, binds to and inhibits
CDK8 and CDK19,27 and inhibits proliferation of SW620
colon cancer cells after 96h in culture.
Figure 4. A. Immunoblot analysis of Cdk8 knockout and
Cdk/Cdk19 double knockout cells. Three independent clones
of each mutant are shown. B. Pharmacological inhibition of
Cdk8 kinase activity reduces STAT1 Ser727 phosphorylation
following stimulation with IFNγ. Cells were treated for 24h in
the presence or absence of 2 µM 32. C. Cell viability in the
presence of 32 at 72h.
Our development of potent, highly specific CDK8 inhibiꢀ
tors and their lack of CDK8ꢀdependent antiproliferative
activity on the HCTꢀ116 colon cancer cell linehas therefore
served to substantiate the emerging, complex picture of the
role of CDK8 in cancer. STAT1 pSer727 has been potently
downregulated by inhibition of CDK8/19 by different comꢀ
pounds, and appears to be a useful biomarker of inhibition.
The impact of CDK8 kinase inhibition in proliferation is
cell line dependent, and possibly condition dependent, and
appears to diverge from the scaffolding functions of CDK8
as evidenced by the differing antiproliferative effects of
CDK8 knockdown and kinase inhibition in HCTꢀ116 cells.
Further investigation of these differences is clearly imꢀ
portant to understanding the roles of CDK8.
ASSOCIATED CONTENT
Supporting Information. A description of the preparation of 1ꢀ
32 are provided along with kinase selectivity and experimental
procedures used in the determination of the coꢀcrystal structure of
22 with CDK8 (PDB: 5CEI). Additionally, the preparation and
characterization of our genetically altered HCTꢀ116 cell line is
presented. This material is available free of charge via the Internet
at http://pubs.acs.org.
AUTHOR INFORMATION
Corresponding Author
* mkoehler@gene.com, (650) 225ꢀ8135
ACKNOWLEDGMENT
We thank members of the DMPK and purification groups within
Genentech Small Molecule Drug Discovery for purification and
analytical support.
The crystallographic experiments were performed on the X06SA
beamline at the Swiss Light Source, Paul Scherrer Institut, Villiꢀ
gen, Switzerland.
REFERENCES
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(1)
Conaway, R. C.; Sato, S.; TomomoriꢀSato, C.; Yao, T.;
TNFꢀAlpha Production. Bioorg. Med. Chem. Lett. 2009, 19, 1722–
1725.
(15) Zhu, G.ꢀD.; Gunawardana, I. W.; Boyd, S. A.; Melcher, L.
Conaway, J. W. The Mammalian Mediator Complex and Its Role in
Transcriptional Regulation. Trends Biochem. Sci. 2005, 30, 250–255.
1
2
3
4
5
6
7
8
9
(2)
Nemet, J.; Jelicic, B.; Rubelj, I.; Sopta, M. The Two Faces
M. A Facile and General Synthesis of 2,4ꢀDiꢀ and 2,4,7ꢀTrisubstituted
Thieno[2,3ꢀC]pyridines. J. Heterocycl. Chem. 2008, 45, 91–96.
(16) Schneider, E. V.; Böttcher, J.; Huber, R.; Maskos, K.;
Neumann, L. StructureꢀKinetic Relationship Study of CDK8/CycC
Specific Compounds. Proc. Natl. Acad. Sci. USA 2013, 110, 8081ꢀ
8086.
(17) Karaman, M. W.; Herrgard, S.; Treiber, D. K.; Gallant, P.;
Atteridge, C. E.; Campbell, B. T.; Chan, K. W.; Ciceri, P.; Davis, M.
I.; Edeen, P. T.; et al. A Quantitative Analysis of Kinase Inhibitor
Selectivity. Nat. Biotechnol. 2008, 26, 127–132.
(18) Jung, C.; Motwani, M.; Kortmansky, J.; Sirotnak, F. M.;
She, Y.; Gonen, M.; HaimovitzꢀFriedman, A.; Schwartz, G. K. The
CyclinꢀDependent Kinase Inhibitor Flavopiridol Potentiates Gammaꢀ
IrradiationꢀInduced Apoptosis in Colon and Gastric Cancer Cells.
Clin. Cancer Res., 2003, 9, 6052–6061.
(19) Bancerek, J.; Poss, Z. C.; Steinparzer, I.; Sedlyarov, V.;
Pfaffenwimmer, T.; Mikulic, I.; Dölken, L.; Strobl, B.; Müller, M.;
Taatjes, D. J.; et al. CDK8 Kinase Phosphorylates Transcription Facꢀ
tor STAT1 to Selectively Regulate the Interferon Response. Immunity
2013, 38, 250–262.
(20) Cong, L.; Ran, F. A.; Cox, D.; Lin, S.; Barretto, R.; Habib,
N.; Hsu, P. D.; Wu, X.; Jiang, W.; Marraffini, L. A.; et al. Multiplex
Genome Engineering Using CRISPR/Cas Systems. Science, 2013,
339, 819–823.
(21) Jinek, M.; East, A.; Cheng, A.; Lin, S.; Ma, E.; Doudna, J.
RNAꢀProgrammed Genome Editing in Human Cells. eLife. 2013, 2,
e00471.
(22) Mali, P.; Yang, L.; Esvelt, K. M.; Aach, J.; Guell, M.; Diꢀ
Carlo, J. E.; Norville, J. E.; Church, G. M. RNAꢀGuided Human Geꢀ
nome Engineering via Cas9. Science, 2013, 339, 823–826.
(23) Canver, M. C.; Bauer, D. E.; Dass, A.; Yien, Y. Y. Characꢀ
terization of Genomic Deletion Efficiency Mediated by Clustered
Regularly Interspaced Palindromic Repeats (CRISPR)/Cas9 Nuclease
System in Mammalian Cells. J. Biol. Chem. 2014, 289, 21312ꢀ21324.
(24) Tsutsui, T.; Fukasawa, R.; Tanaka, A.; Hirose, Y.; Ohkuma,
Y. Identification of target genes for the CDK subunits of the mediator
complex. Genes to Cells 2011, 16, 1208ꢀ1218.
of Cdk8, a Positive/Negative Regulator of Transcription. Biochimie.
2014, 97, 22–27.
(3)
M. R.; Liu, L.; Wang, H.; King, B.; Shaik, S.; et al. Cyclin C Is a
Haploinsufficient Tumour Suppressor. Nat.Cell Biol. 2014, 16, 1080–
1091.
Li, N.; Fassl, A.; Chick, J.; Inuzuka, H.; Li, X.; Mansour,
(4)
Firestein, R.; Bass, A. J.; Kim, S. Y.; Dunn, I. F.; Silver, S.
J.; Guney, I.; Freed, E.; Ligon, A. H.; Vena, N.; Ogino, S.; et al.
CDK8 Is a Colorectal Cancer Oncogene That Regulates BetaꢀCatenin
Activity. Nature. 2008, 455, 547–551.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
(5)
Seo, JꢀO.; Han, S. I.; Lim, SꢀC. Role of CDK8 and βꢀ
Catenin in Colorectal Adenocarcinoma. Oncol. Rep., 2010, 24, 285ꢀ
291.
(6)
drusan, Z.; Soukup, T. M.; RooseꢀGirma, M.; Blackwood, E. M.;
Firestein, R. CDK8 Maintains Tumor DeꢀDifferentiation and Embryꢀ
onic Stem Cell Pluripotency. Cancer Res. 2012, 72, 2129–2139.
Adler, A. S.; McCleland, M. L.; Truong, T.; Lau, S.; Moꢀ
(7)
Firestein, R.; Shima, K.; Nosho, K.; Irahara, N.; Baba, Y.;
Bojarski, E.; Giovannucci, E. L.; Hahn, W. C.; Fuchs, C. S.; Ogino, S.
CDK8 Expression in 470 Colorectal Cancers in Relation to ΒꢀCatenin
Activation, Other Molecular Alterations and Patient Survival. Int. J.
Cancer. 2010, 126, 2863ꢀ2873.
(8)
Kapoor, A.; Goldberg, M. S.; Cumberland, L. K.;
Ratnakumar, K.; Segura, M. F.; Emanuel, P. O.; Menendez, S.;
Vardabasso, C.; Leroy, G.; Vidal, C. I.; Polsky, D.; Osman, I.; Garcia,
B.A.; Hernando, E.; Bernstein, E. The Histone Variant macroH2A
Suppresses Melanoma Progression Through Regulation of CDK8.
Nature. 2010, 468, 1105–1109.
(9)
Turunen, M.; Spaeth, J. M.; Keskitalo, S.; Park, M. J.;
Kivioja, T.; Clark, A. D.; Mäkinen, N.; Gao, F.; Palin, K.; Nurkkala,
H.; et al. Uterine LeiomyomaꢀLinked MED12 Mutations Disrupt
MediatorꢀAssociated CDK Activity. CellReports. , 2014, 7, 654–660.
(10) McCleland, M.L.; Soukup, T.M.; Liu, S.D.; Esensten, J.H.; de
Sousa, E.; Melo, F.; Yaylaoglu, M; Warming, S.; RooseꢀGirma, M.;
Firestein, R. Cdk8 deletion in the Apc(Min) murine tumour model
represses EZH2 activity and accelerates tumourigenesis. J Pathol.
2015, 237, 508ꢀ519.
(11) Schneider, E. V.; Böttcher, J.; Blaesse, M.; Neumann, L.;
Huber, R.; Maskos, K. The Structure of CDK8/CycC Implicates Specꢀ
ificity in the CDK/Cyclin Family and Reveals Interaction with a Deep
Pocket Binder. J. Mol. Biol. 2011, 412, 251–266.
(12) Porter, D. C.; Farmaki, E.; Altilia, S.; Schools, G. P.; West,
D. K.; Chen, M.; Chang, B. D.; Puzyrev, A. T.; Lim, C.; Rokowꢀ
Kittell, R. CyclinꢀDependent Kinase 8 Mediates Chemotherapyꢀ
Induced TumorꢀPromoting Paracrine Activities. Proc. Natl. Acad. Sci.
USA 2012, 109, 13799–13804.
(13) George, D.; Friedman, M.; Allen, H.; Argiriadi, M.; Barꢀ
beris, C.; Clabbers, A.; Cusack, K.; Dixon, R.; FixꢀStenzel, S.; Gorꢀ
don, T.; Janssen, B.; Jia, Y.; Moskey, M.; Quinn, C.; Salmeron, JꢀA.;
Wishart, N.; Woller, K.; Yu, Z. Discovery of Thieno[2,3ꢀC]Pyridines
as Potent COT Inhibitors. Bioorg. Med. Chem. Lett. 2008, 18, 4952ꢀ
4955.
(25) Lehtonen, A.; Matikainen, S.; Julkunen, I. Interferons Upꢀ
Regulate STAT1, STAT2, and IRF Family Transcription Factor Gene
Expression in Human Peripheral Blood Mononuclear Cells and Macꢀ
rophages. J. Immunol. 1997, 159, 794–803.
(26) Pelish, H.E.; Liau, B.B.; Nitulescu, I.I; Tanpeerachaikul, A.;
Poss, Z.C.; Da Silva, D.H.; Caruso, B.T.; Arefolov, A.; Fadeyi, O.;
Christie, A.L.; Du, K.; Banks, D.; Schneider, E.V.; Jestel, A.; Zou, G.;
Si, C.; Ebmeier, C.C.; Bronson, R.T.; Krivtsov, A.V.; Myers, A.G.;
Kohl, N.E.; Kung, A.L.; Armstrong, S.A.; Lemieux, M.E.; Taatjes,
D.J.; Shair, M.D. Mediator kinase inhibition further activates superꢀ
enhancerꢀassociated genes in AML. Nature. 2015, 526, 273ꢀ276.
(27) Dale, T.; Clarke, P.A.; Esdar, C.; Waalboer, D.; Adenijiꢀ
Popoola, O.; OrtizꢀRuiz, MꢀJ; Mallinger, A.; Samant, R.S.; Czoꢀ
drowski, P.; Musil, D; Schwarz, D.; Schneider, K.; Stubbs, M.; Ewan,
K.; Fraser, E.; TePoele, R.; Court, W.; Box, G.; Valenti, M.; Brandon,
A. d.; Gowan, S.; Rohdich, F.; Raynaud, F.; Schneider, R.; Poeschke,
O.; Blaukat, A.; Workman, P.; Schiemann, K.; Eccles, S.A.; Wienke,
D.; Blagg, J. A selective chemical probe for exploring the role of
CDK8 and CDK19 in human disease. Nat Chem. Biol. 2015, 11, 973ꢀ
980.
(14) Cusack, K.; Allen, H.; Bischoff, A.; Clabbers, A.; Dixon,
R.; FixꢀStenzel, S.; Friedman, M.; Gaumont, Y.; George, D.; Gordon,
T.; Gronsgard, P.; Janssen, B.; Jia, Y.; Moskey, M.; Quinn, C.; Salmꢀ
eron, A.; Thomas, C.; Wallace, G; Wishart, N.; Yu, Z. Identification
of a Selective Thieno[2,3ꢀC]Pyridine Inhibitor of COT Kinase and
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