Characterization of molecular and cellular functions of the cyclin-dependent kinase CDK9 using a novel specific inhibitor

Article abstract of DOI:10.1111/bph.12408

Background and Purpose The cyclin-dependent kinase CDK9 is an important therapeutic target but currently available inhibitors exhibit low specificity and/or narrow therapeutic windows. Here we have used a new highly specific CDK9 inhibitor, LDC000067 to interrogate gene control mechanisms mediated by CDK9. Experimental Approach The selectivity of LDC000067 was established in functional kinase assays. Functions of CDK9 in gene expression were assessed with in vitro transcription experiments, single gene analyses and genome-wide expression profiling. Cultures of mouse embryonic stem cells, HeLa cells, several cancer cell lines, along with cells from patients with acute myelogenous leukaemia were also used to investigate cellular responses to LDC000067. Key Results The selectivity of LDC000067 for CDK9 over other CDKs exceeded that of the known inhibitors flavopiridol and DRB. LDC000067 inhibited in vitro transcription in an ATP-competitive and dose-dependent manner. Gene expression profiling of cells treated with LDC000067 demonstrated a selective reduction of short-lived mRNAs, including important regulators of proliferation and apoptosis. Analysis of de novo RNA synthesis suggested a wide ranging positive role of CDK9. At the molecular and cellular level, LDC000067 reproduced effects characteristic of CDK9 inhibition such as enhanced pausing of RNA polymerase II on genes and, most importantly, induction of apoptosis in cancer cells. Conclusions and Implications Our study provides a framework for the mechanistic understanding of cellular responses to CDK9 inhibition. LDC000067 represents a promising lead for the development of clinically useful, highly specific CDK9 inhibitors.

Full text of DOI:10.1111/bph.12408

Novel CDK9 inhibitor
Characterisation of molecular and cellular CDK9 functions using a novel specific
inhibitor1
Running title: Novel CDK9 inhibitor
T K Albert1, C Rigault1,2, J Eickhoff3, K Baumgart1, C Antrecht1, B Klebl3, G Mittler4 and M
Meisterernst1,5
1Institute of Molecular Tumor Biology (IMTB), Faculty of Medicine, Westfalian Wilhelms
University Muenster (WWU), 48149 Muenster, Germany
2Present Address: Centre des Sciences du Goût et de l'Alimentation, CNRS 6265-INRA 1324-
Université de Bourgogne, 21000 Dijon, France
3LDC - Lead Discovery Center GmbH, 44227 Dortmund, Germany
4Max Planck Institute of Immunobiology and Epigenetics, 79108 Freiburg, Germany
5To whom correspondence should be addressed: Michael Meisterernst, Institute of Molecular
Tumor Biology, Faculty of Medicine, Westfalian Wilhelms University Muenster, Robert-
Koch-Strasse 43, 48149 Muenster, Germany, Email: meistere@uni-muenster.de
This article has been accepted for publication and undergone full peer review but has not been through the
copyediting, typesetting, pagination and proofreading process, which may lead to differences between this
version and the Version of Record. Please cite this article as doi: 10.1111/bph.12408
1
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Novel CDK9 inhibitor
Summary
Background and purpose
CDK9 has great potential as therapeutic target. Yet, currently available inhibitors suffer from
low specificity and/or narrow therapeutic windows. We present a novel high-specificity
CDK9 inhibitor termed 067 that serves to interrogate gene control mechanisms by CDK9 and
represents a promising lead for the development of clinically useful mono-specific CDK9
inhibitors.
Experimental approach
CDK9 selectivity of 067 was established in functional kinase assays. CDK9 functions in gene
expression were reinvestigated in in vitro transcription experiments, single gene analyses and
genome-wide expression profiling. Cell-based assays were used to investigate cellular
responses to the novel inhibitor.
Key results
The selectivity of 067 for CDK9 exceeded that of the established compounds flavopiridol and
DRB by far. 067 inhibited in vitro transcription in an ATP-competitive and dose-dependent
manner. Gene expression profiling of 067-treated cells revealed selective reduction of short-
lived mRNAs, including prominent regulators of proliferation and apoptosis. Analysis of de
novo RNA synthesis reasoned for a broad positive role of CDK9. At the molecular and
cellular level 067 recapitulated known hallmarks of CDK9 inhibition, i.e. enhanced pausing of
RNA polymerase II on genes and, most importantly, induction of apoptosis in cancer cells.
Conclusions and implications
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Novel CDK9 inhibitor
Our study provides a framework for the mechanistic understanding of cellular responses to
CDK9 inhibition. The data are predictably relevant for biomedical applications.
Keywords
Cyclin-dependent kinase, CDK9, gene expression, RNA polymerase II, transcription
Abbreviations
067: LDC000067
AML: acute myelogenous leukaemia
CDK: cyclin-dependent kinase
ChIP: chromatin immunoprecipitation
CLL: chronic lymphocytic leukaemia
CTD: carboxyterminal domain
DRB: 5,6-dichloro-1-beta-D-ribofuranosyl-benzimidazole
DSIF: DRB sensitivity-inducing factor
FRET: fluorescence resonance energy transfer
hnRNA: heterogeneous nuclear RNA
HPLC-MS: high performance liquid chromatography-mass spectometry
IC50: half-maximal inhibitory concentration
NELF: negative elongation factor
PIC: preinitiation complex
P-TEFb: positive transcription elongation factor b
RMA: robust multi-array average
RNAPII: RNA polymerase II
RT-qPCR: reverse transcription-quantitative real-time PCR
3
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Novel CDK9 inhibitor
Ser2/5/7-P: CTD phospho-serine 2/5/7
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Novel CDK9 inhibitor
Introduction
Cyclin-dependent kinases (CDKs) are a family of evolutionary conserved serine/threonine
kinases that form heterodimers with regulatory cyclin partner proteins (Malumbres and
Barbacid, 2005). CDKs can generally be classified into two major groups based on whether
they control cell cycle progression or regulate gene transcription by RNA polymerase II
(RNAPII). The first group includes CDK1 to CDK6, while CDK8, CDK9, CDK12 and
CDK19 are linked to transcription regulation (Loyer et al., 2005). CDK7 and CDK20 act in
both cell cycle control and transcription (Fisher, 2005, Wohlbold et al., 2006). Several CDKs
(such as CDK10, CDK11A, CDK11B, CDK13) are involved in RNA processing (Loyer et
al., 2005), while other CDKs have specialized roles in proliferation and other processes such
as cellular survival, homeostasis or development (Malumbres and Barbacid, 2005). Taken
together, CDKs present particularly promising drug targets for therapeutic interference in
human pathologies where these processes are affected (Malumbres and Barbacid, 2009,
Shapiro, 2006), and several CDK inhibitors are currently under evaluation in clinical trials
(http://clinicaltrials.gov/). In particular, inhibition of transcriptional CDKs such as CDK9
might present an effective strategy against proliferative diseases like cancer (Shapiro, 2006,
Wang and Fischer, 2008). This idea is supported by the observation that cancer cells often
rely on the production of short-lived antiapoptotic regulator proteins in order to resist
programmed cell death. Transcriptional down-regulation of such survival factors through
pharmacological CDK9 inhibition would result in antitumor activity due to reinstatement of
apoptosis. Indeed, down-regulation of the antiapoptotic MCL1 gene by the established CDK9
inhibitor flavopiridol (alvocidib) appears to be the primary mechanism underlying its
antitumor activity in chronic lymphocytic leukaemia (CLL) (Byrd et al., 2007, Chen et al.,
2005). While these results speak to the potential of CDK9 as a therapeutic target, flavopiridol
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Novel CDK9 inhibitor
has been shown to inhibit multiple CDKs (Liu et al., 2012) as well as other kinases such as
AKT (Caracciolo et al., 2012). Thus, inhibitors that exhibit higher specificity for CDK9 are
needed to establish final target validation and/or to investigate the underlying molecular
mechanisms of gene expression control by CDK9 in greater detail.
CDK9 and cyclin T form the P-TEFb complex that was originally identified as activity
controlling the early phase of transcription elongation via release of RNAPII from inherent
promoter-proximal pause sites (Li and Gilmour, 2011, Marshall and Price, 1995). This release
involves phosphorylation of the inhibitor DSIF, which consists of SPT4 and SPT5 subunits,
as well as subunits of negative elongation factor NELF (Fujinaga et al., 2004, Isel and Karn,
1999, Ivanov et al., 2000, Wada et al., 1998, Yamaguchi et al., 1999). DSIF and NELF are
thought to cooperate to cause promoter-proximal pausing of RNAPII (Peterlin and Price,
2006, Garriga and Grana, 2004). Subsequent phosphorylation of SPT5 and NELF subunits
leads to dissociation of NELF and conversion of DSIF into a positive processivity factor
(Martinez-Rucobo et al., 2011, Martinez-Rucobo and Cramer, 2013). CDK9 also
phosphorylates the carboxyterminal heptarepeat (consensus sequence Y1S2P3T4S5P6S7) within
the carboxyterminal domain (CTD) of the largest subunit of RNAPII. The CTD is modified
at various stages of transcription. RNAPII is recruited into the preinitiation complex (PIC)
with a hypophosphorylated CTD, and the CTD is phosphorylated on serine 5 (Ser5-P) during
initiation and then on serine 2 (Ser2-P) during elongation (Fuda et al., 2009). The latter task is
performed by CDK9 (Shim et al., 2002). The degree of phosphorylation at serine 2 increases
towards the 3'-ends of genes, which correlates to binding of termination and RNA processing
factors to the CTD (Bird et al., 2004). Ser2-P at promoter-proximal sites further helps to
release RNAPII from initiation and early elongation complexes (Zhou et al., 2012).
Proteomic studies identified two differently sized forms of P-TEFb, the larger one
representing an inactive form of P-TEFb in complex with HEXIM proteins and 7SK RNA
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Novel CDK9 inhibitor
(Nguyen et al., 2001, Yang et al., 2001). Targeting CDK9 with pharmacological inhibitors
causes its release from this pool, allowing it to bind to genes either on its own or in
combination with other proteins. Investigations of individual cellular and viral genes indicate
a widespread positive role for CDK9 in gene control (Chao and Price, 2001, Kanazawa et al.,
2000, Lis et al., 2000, Zhu et al., 1997). The requirement for CDK9 may quantitatively differ
from one gene to another and/or from one activator to another (Bottardi et al., 2011, Hou et
al., 2007, Wang et al., 2013).
Here we introduce a novel inhibitor based on a 2,4-aminopyrimidine scaffold, termed
LDC000067 (067), that originates from a small molecule screen of a chemical library that was
designed to bind to kinase ATP binding pockets. Functional kinase assays confirmed its
selectivity for CDKs, with a preference for CDK9-cyclin T1. 067 inhibited CDK9 in vitro
with an IC50 of 44 ± 10 nM. Its selectivity for CDK9 versus other CDKs was in the range of
55-fold (versus CDK2) to over 230-fold (versus CDK6 and CDK7) and exceeded that of the
widely used compounds 5,6-dichloro-1-beta-D-ribofuranosyl-benzimidazole (DRB) and
flavopiridol by far. This superior selectivity of 067 was confirmed in an ATP-competitive
kinase binding assay. 067 also inhibited in vitro transcription in an ATP-competitive and
dose-dependent manner. Furthermore, 067 decreased phosphorylation of residue serine 2
within the CTD of RNAPII, both in cells and nuclear extracts as well as in kinase assays using
recombinant GST-CTD as substrate. Cellular effects of 067 included induction of the tumor
suppressor protein p53 and apoptosis. Gene expression profiling of 067-treated cells revealed
selective reduction of short-lived mRNAs, including those that encode regulators of
proliferation and apoptosis such as MCL1 and MYC. Analysis of de novo RNA synthesis
reasoned for a broad positive role of CDK9. Finally, upon 067 treatment the previously
proposed forcing of pausing of RNAPII on MYC and other genes was observed, all of which
is consistent with specific inhibition of CDK9 by 067. In view of these specific properties,
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Novel CDK9 inhibitor
067 may be a valuable tool to further study CDK9 mechanisms of action in vitro and in vivo
as well as a potential drug to target CDK9 in diseases.
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Novel CDK9 inhibitor
Methods
Synthesis
of
LDC000067
(3-((6-(2-methoxyphenyl)pyrimidin-4-yl)amino)phenyl)
methanesulfonamide
Step 1: To a solution of 4,6-dichloropyrimidine (3.38 g; 22.7 mmol) in a mixture of
dimethoxyethane (30 mL) and water (6 ml) were successively added 2-methoxyphenylboronic
acid (3.45 g; 22.7 mmol), PdCl2(PPh3)2 (175 mg; 0.25 mmol), and potassium carbonate (1.69
g; 12.2 mmol). The mixture was stirred for 3 hours at 80°C and at room temperature over
night. It was concentrated under reduced pressure. The residue was dissolved in
dichloromethane (100 mL), the solution washed with water, dried over MgSO4, and
concentrated in vacuo. The intermediate 4-chloro-6-(2-methoxyphenyl)pyrimidine was
obtained as a pale yellow solid after chromatographic flash purification (silica gel;
dichloromethane/methanol gradient 100:0 to 90:10). Yield: 1.97 g (39 %).
Step 2: To a solution of the intermediate from Step 1 (100 mg; 0.453 mmol) in DMF (1 mL)
was added (3-aminophenyl) methanesulfonamide (84.3 mg; 0.453 mmol). The mixture was
heated for 1 hour at 80°C. It was diluted with water and the solvent removed by
lyophilization. The remaining yellow solid was passed through an SCX ion exchange
cartridge (eluent 7N ammonia in methanol). After another lyophilization step the pure title
1
compound was obtained as a white powder. Yield: 119.8 mg (71%). H NMR (400MHz, d6-
DMSO, 300K) δ 3.88 (s, 3H), 4.24 (s, 2H), 6.84 (bs, 2H), 7.00-7.09 (m, 2H), 7.17 (d, J = 8.4
Hz, 1H), 7.33 (t, J = 7.9 Hz, 1H), 7.42-7.47 (m, 2H), 7.62 (bs, 1H), 7.78-7.82 (m, 1H), 7.91
(dd, J = 1.7 Hz, J = 7.7 Hz, 1H), 8.68 (s, 1H), 9.76 (bs, 1H). MS (ES) C18H18N4O3S requires:
370, found: 371 (M+H)+. Purity of the compound was >95% as determined by HPLC-MS.
In vitro enzymatic kinase assay for CDKs
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Novel CDK9 inhibitor
The fluorescence resonance energy transfer (FRET)-based LANCE Ultra KinaSelect Ser/Thr
kit (Perkin Elmer) was used to determine IC50 values for various CDK inhibitors. Kinase
activity and inhibition in this assay was measured as recommended by the manufacturer.
Briefly, a specific ULight MBP peptide substrate (50 nM final concentration) was
phosphorylated by a CDK-cyclin pair in buffer (50 mM HEPES-KOH pH 7.5, 10 mM MgCl2,
1 mM EGTA, 2 mM dithiothreitol) containing ATP at the concentration of the KM values of
the individual kinases for 1 hour at room temperature. Subsequently, phosphorylation was
detected by addition of specific Europium (Eu)-labelled anti-phospho-antibodies (2 nM),
which upon binding to the phosphopeptide give rise to a FRET signal. FRET signals were
recorded in a time-resolved manner in a Perkin Elmer EnVision reader. Purified cyclin-kinase
pairs were obtained from the following suppliers: Carna Biosciences (CDK1-Cyclin B1,
CDK6-Cyclin D3, CDK7-Cyclin H-MAT1), ProQinase (CDK2-Cyclin A) and Invitrogen
(CDK9-Cyclin T1).
Competitive kinase binding/tracer displacement assay
The LanthaScreen Eu Kinase Binding Assay (Invitrogen) was performed for CDK-cyclin
pairs (suppliers: see above) to determine affinities of inhibitors binding to the ATP binding
pocket (Kd determination). It is a FRET assay based on binding and displacement of an ATP-
competitive tracer to the kinase of interest. Binding of an inhibitor to the kinase competes for
binding with the tracer, resulting in a loss of FRET. The assays were performed under the
conditions recommended by the supplier. Briefly, inhibitors were incubated for 1 hour with
kinase tracer 236 at the concentration corresponding to its individual Kd value for the kinase-
cyclin complex, 2 nM LanthaScreen Eu-Anti-His antibody and 5 nM of the purified kinase-
cyclin complex. Binding of the tracer to the kinase was measured at 340 nM excitation; 665
nm emission was used for the kinase tracer and 615 nM emission for normalisation.
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Novel CDK9 inhibitor
Kinase panel profiling
Kinase inhibition by 067 was measured in a radiometric assay by Reaction Biology (Malvern,
PA, USA). The assay directly measures kinase catalytic activity toward a specific substrate
(Anastassiadis et al., 2011). Briefly, 10 µM 067 or DMSO as solvent control were added to
Base Reaction Buffer (20 mM Hepes pH 7.5, 10 mM MgCl2, 1 mM EDTA, 0.02% Brij35,
0.02 mg/ml BSA, 0.1 mM Na3VO4, 2 mM DTT, 1% DMSO) containing substrates and
33
cofactors required by the individual kinase. P-ATP (specific activity 10 µCi/µl) was added
to the reaction mixture, and kinase reactions incubated for 120 min at room temperature.
Reactions were spotted on P81 ion exchange paper (Whatman), and filters extensively washed
in 0.75% phosphoric acid before radiometric quantification. Each protein kinase was
measured in duplicate and its catalytic activity expressed as residual kinase activity, i.e. the
percentage of average substrate phosphorylation compared to the solvent control reaction.
In vitro transcription
Transcription reactions were conducted with the adenovirus core promoter (template Gal-ML)
essentially as described previously (Boeing et al., 2010). For the kinetic elongation assay a
slightly modified immobilised Gal-ML template was used, which will be described elsewhere.
For immobilisation, templates were PCR amplified using a 5'-biotin-labelled primer that was
purified and coupled to paramagnetic streptavidin beads (Promega). Reactions were
conducted in 25 mM HEPES-KOH pH 8.2, 60 mM KCl, 5 mM MgCl2, 1 mM DTT, 0.01%
Igepal CA-630, 5-10% glycerol, 0.5 mg/ml BSA (Roche Applied Science) and 20 units of
RiboLock (Fermentas). Following transcription reactions, beads were washed, RNA eluted,
precipitated and analysed on denaturing gels.
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Novel CDK9 inhibitor
DNA microarray analysis
Gene expression profiling was conducted using high-density oligonucleotide arrays (Human
Gene 1.0 ST array, Affymetrix). Total RNA was isolated from three biological replicates of
067-treated (90 min) THP1 cells using the RNeasy RNA purification kit as recommended by
the supplier (Qiagen). Sample labeling, hybridisation, scanning, raw data extraction and
robust multi-array average (RMA) analysis of background-adjusted, normalized, and log-
transformed probe-set values were conducted by an authorised Affymetrix service provider
(KFB, Regensburg, Germany). Threshold settings for regulated genes were ≥1.4-fold
difference to DMSO-treated control cells, with p <0.05. Gene ontology analysis of regulated
genes was performed with DAVID (http://david.abcc.ncifcrf.gov/).
Reverse transcription quantitative PCR (RT-qPCR)
Total RNA was prepared using Trizol reagent (Invitrogen). Reverse transcription was
performed using either Superscript II reverse transcriptase (Invitrogen) or Quantitect reverse
transcription kit (Qiagen) with random hexamer primers. Quantitative real-time PCR (qPCR)
was carried out on the resulting cDNAs using SYBR Green PCR Master kit (Applied
Biosystems) according to manufacturers instructions on a Step One Plus PCR system
(Applied Biosystems). Relative transcripts levels were determined using the comparative
cycle threshold (CT) method. Exon-intron primers were designed with the program Primer3
(http://primer3.wi.mit.edu/). Sequences are available upon request.
Chromatin immunoprecipitation (ChIP) and antibodies
ChIP was performed as previously described (Albert et al., 2010). ChIP and input DNAs were
purified with the Qiaquick PCR kit (Qiagen), and qPCR was carried out as outlined above.
Antibodies used included IgG control (sc-2027), anti-RNAPII, subunit RPB1 (sc-899), anti-
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Novel CDK9 inhibitor
CDK9 (sc-484; all from Santa Cruz Biotechnology), and monoclonal rat antibodies anti-CTD
Ser2-P (clone 3E10), anti-Ser5-P (3E8), and anti-Ser7-P (4E12; all kind gifts from D. Eick).
Additional antibodies for Western blot analyses included anti-p53 phospho-serine 392 (sc-
56173) and anti-Tubulin (sc-8035; Santa Cruz Biotechnology).
Apoptosis measurement
Apoptosis was measured by flow cytometry using annexin-fluorescein and propidium iodide
staining according to standard protocols (BD Biosciences).
Results
LDC000067, a novel CDK inhibitor with high specificity for CDK9
Given the limited specificity of several widely used CDK9 inhibitors we set out to develop
highly specific ATP-competitive compounds based on a 2,4-aminopyrimidine scaffold.
Specificity was increased in iterative cycles of chemical modification and validated by
functional kinase assays. As an intermediate endpoint we present here the CDK9 inhibitor 067
(patent number WO 2008/129080; Fig. 1A), which shows promising biochemical and
pharmacological properties including high selectivity for CDK9, high stability as well as good
tolerability and low cytotoxicity in vivo.
The increased CDK9 selectivity of 067 in comparison to established CDK inhibitors
flavopiridol, DRB and SNS-032 (formerly BMS-387032) (Misra et al., 2004) was
demonstrated via substrate phosphorylation using a fluorescence resonance energy transfer
(FRET)-based assay. Half-maximal inhibitory concentrations (IC50) for the CDK9-cyclin T1
pair were 44 nM, 5.2 nM, 1.4 nM and 1.9 µM for 067, flavopiridol, SNS-032 and DRB,
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Novel CDK9 inhibitor
respectively (Table 1). Importantly, in this assay 067 displayed 55/125/210/>227/>227-fold
selectivity for CDK9 versus CDK2/1/4/6/7, while the other three compounds were much less
selective (e.g. 3/<1/7/59/20-fold for CDK9 versus CDK2/1/4/6/7 in the case of flavopiridol).
High CDK9 selectivity of 067 was confirmed via another FRET-based in vitro assay where
the ATP-competitive binding of 067, flavopiridol and DRB to CDK2-, CDK7- and CDK9-
cyclin complexes was compared (Table 2). The dissociation constant of 067 for CDK9-cyclin
T1 was 31-fold lower than for CDK2-cyclin A and nearly 500-fold lower than for the trimeric
CDK7-cyclin H-MAT1 complex; again, flavopiridol and DRB showed lower relative
differences in their dissociation constants for these CDKs. Since the IC50 of 067 for CDK2 is
55-fold and its Kd 31-fold higher than the corresponding values for CDK9, it seems unlikely
that 067 has a significant effect on CDK2 function when employed at the concentrations
required to block CDK9 function.
Further support for the selectivity of 067 was obtained by profiling a panel of 28
additional human recombinant non-CDK kinases for their response to 067. In this radiometric
assay, 10 µM 067 or DMSO as solvent control were added to a kinase reaction that directly
measures catalytic activity of the enzyme toward a specific substrate (Anastassiadis et al.,
2011). 22 out of the 28 kinases showed greater than 50% residual activity in the presence of
067, while CDK9-Cyclin T1 was severely affected (6% residual activity; Figure 1 B). In an
additional experiment, comparative IC50 values for several of the less affected kinases (e.g.
GSK3A, MAP4K4 or ABL2 with 9.5%, 17.5% and 27% residual activity, respectively) were
determined for 067 versus staurosporine, a prototypical ATP-competitive kinase inhibitor that
is known to bind to many kinases with high affinity but little selectivity (Karaman et al.,
2008). Strikingly, IC50 values were several hundred fold higher than the ones obtained with
the non-selective inhibitor staurosporine (Table 3).
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Novel CDK9 inhibitor
067 inhibits P-TEFb-dependent in vitro transcription in an ATP-competitive manner
We next asked whether 067 blocks transcription in a similar way to DRB and flavopiridol. In
vitro transcription experiments were conducted using either soluble or immobilised promoter
templates (Boeing et al., 2010) and nuclear extracts of HEK293T cells supplemented with
recombinant Gal4-VP16. Transcription assays revealed a half-maximal inhibition of 4 µM for
067 at 100 µM ATP (Fig. 1B). Using the same assay a roughly 20-fold higher potency of
flavopiridol and a 2.5-fold lower potency of DRB was revealed (Fig. 1C). Nuclear extracts
from four different cell lines were used to investigate the influence of ATP concentrations on
067-dependent inhibition of transcription activity (Fig. 1D). For these and most subsequent
experiments a final concentration of 10 µM was chosen which reduces transcription levels to
approximately 20-30% in vitro as well as in vivo (see below). High ATP levels (500 M)
markedly diminished the inhibitory effect of 067 on in vitro transcription when compared to
the routinely used 60 µM ATP. ATP competition was not unexpected, since (i) 067 emerged
from a chemical library containing scaffolds designed for binding to kinase ATP binding
sites, and (ii) 067 competed with/displaced the tracer from the ATP binding pocket in the in
vitro kinase binding assay (Table 2). We further investigated the effects of potassium or
magnesium chloride on the relative inhibitory potency of 067. While increasing the
concentration of KCl from the routinely used 50 mM up to 150 mM resulted in a strong
overall decrease of transcript levels, the relative inhibitory potency of 067 was largely
unaffected (KB, data not shown). In contrast, adding increasing amounts of MgCl2 during the
transcription elongation resulted in a significant Mg2+-dependent decrease of transcriptional
inhibition by 067 (Fig. 1E).
CDK9 inhibition by 067 reduces Ser2-P, induces p53 activation and leads to apoptosis
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Novel CDK9 inhibitor
Next the effects of CDK9 inhibition by 067 in living cells were investigated. One established
cellular target of CDK9 is serine 2 within the RNA polymerase CTD which becomes
phosphorylated during transcription elongation (Fuda et al., 2009). Previous experiments in
mouse embryonic stem cells (mESCs) had shown that flavopiridol at 1 µM significantly
reduced Ser2-P levels within 90 minutes (Rahl et al., 2010). We therefore compared the
effects of 067 versus flavopiridol treatment in mESCs. Cells were treated with either 10 µM
067 or 1 µM flavopiridol for 90 and 180 minutes and subsequently analysed by Western blot.
Treatment with the two compounds led to 1.6-fold and 3.4-fold reduction of Ser2-P after 90
minutes (Fig. 2A). While Ser2-P repression remained at the same level for 067 after 180
minutes, flavopiridol treatment nearly abolished the CTD modification at the later time point.
This quantitative difference might be attributed to the very high concentration of flavopiridol
which could result in non-specific effects especially at later time points (see discussion). We
also compared the effects of 067 treatment on CTD phosphorylation to the other widely used
CDK9 inhibitor DRB in HeLa cells (Fig. 2B). Again, a 60 min treatment with 10 µM 067
resulted in 1.6-fold reduction of Ser2-P, whereas Ser5-P and Ser7-P remained largely
unaffected; this reduction was similar to the one obtained with 50 µM DRB. Finally, we
confirmed these results in an in vitro kinase assay using recombinant GST-CTD as substrate
in HeLa nuclear extracts (Fig. 2C).
Blocking RNAPII transcription elicits a stress response that leads to activation of p53
(Ljungman et al., 1999). During this process p53 is stabilised and becomes activated by a
plethora of post-translational modifications, e.g. extensive phosphorylation. Next we
examined the effect of 067 on p53 signaling. Given that p53 is frequently mutated or inactive
in tumour cells (one example is HeLa) we used the breast carcinoma cell line MCF7, in which
p53 signaling is preserved. Indeed, after four hours treatment with 10 µM 067 (or 50 µM
DRB), the p53 protein was stabilised (data not shown) and activated as monitored by
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Novel CDK9 inhibitor
phosphorylation of serine 392 (Fig. 2D), a modification that is an integral event in the
induction of p53 by a diverse range of stimuli (Cox and Meek, 2010).
Various cell lines, in particular of haematopoietic origin, show pronounced sensitivity
to flavopiridol- and/or DRB-induced apoptosis (Konig et al., 1997, Parker et al., 1998, te
Poele et al., 1999). Due to its proapoptotic effects flavopiridol is currently under investigation
in phase II clinical trials as therapeutic agent in acute myelogenous leukaemia (AML) (Karp
et al., 2012). We tested the effects of 067 treatment on several cancer cell lines as well as on
primary patient-derived AML blasts using flow cytometry of annexin V-propidium iodide
double-stained cells (Fig. 2E-H). At concentrations >2 µM all cells showed a marked increase
in the percentage of apoptotic cells after 24 hours of exposure (Fig. 2E). Apoptosis induction
was most pronounced in cells of leukaemic origin such as THP1 (approximately 45%
apoptotic cells at 10 µM 067) and patient-derived blast cells (>60% at 10 µM). Titration of
067 in epithelial A549 lung carcinoma cells revealed near-saturation concentrations above 15
µM, with a 3-fold increase in apoptosis observed at 10 µM (Fig. 2F). 067 treatment of
HCT116 colon carcinoma cells which either contained or lacked wild-type p53 caused a
similar degree of apoptosis induction in both situations, suggesting a largely p53-independent
mechanism at least in this cellular background (Fig. 2G). Finally, comparison of cell death
rates in 067-treated (10 µM) or flavopiridol-treated (0.25 µM) A549 and MCF7 cells revealed
a similar degree of apoptosis induction (Fig. 2H). Taken together, these data indicate that 067
treatment of cells recapitulates several relevant cellular responses to CDK9 inhibition, most
importantly induction of programmed cell death in leukaemia cells.
Dose-dependent inhibition of de novo RNA synthesis by 067
To quantitatively determine the effects of 067 on gene transcription in vivo, a concentration-
dependent analysis of mRNA synthesis was conducted. Cells were treated with the inhibitor
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Novel CDK9 inhibitor
for 90 minutes, RNA was extracted and mRNA analysed via reverse transcription-quantitative
real-time PCR (RT-qPCR) at several representative cellular genes, including housekeeping
genes such as GAPDH and RPL3, cell cycle control genes such as p21, cyclin D1/CCND1,
PLK2, or the MYC gene (Fig. 3A). As newly synthesised RNA is rapidly spliced (within
minutes), and mature mRNA can be stable over days, we chose to analyse de novo
synthesised hnRNA using primer pairs located in neighbouring exon and intron regions.
Repression of mRNA synthesis was first detectable at a concentration of 1 µM, with 50% to
80% repression obtained at a concentration of 10 µM 067. Half-maximal repression by 067
was achieved in the range of 2 to 6 µM. While relative changes were comparable, the final
level of repression varied from gene to gene (Fig. 3A). Notably, housekeeping genes
(GAPDH, RPL3) were among the strongest responders. Similar results were obtained in
several human cancer cell lines (e.g. HeLa, THP1; data not shown) as well as in mESCs (Fig.
3B). Occasionally we observed moderate stimulatory effects, for instance on Myc in mESCs,
yet these were restricted to low concentrations of 067. The underlying reason remains unclear.
De novo RNA synthesis was also measured using a Tetracyclin-inducible luciferase
reporter gene system in HeLa cells (Boeing et al., 2010, Uhlmann et al., 2007). Treatment
with 10 µM 067 caused an 80% to 90% reduction of RNA synthesis (Fig. 3C). Luciferase
expression decreased in a concentration-dependent manner, with half-maximal repression
reached at a 067 concentration of 2 µM (Fig. 3D). Luciferase repression was stable over at
least three days, indicating that 067 was not metabolised to an inactive form but remained
fully active inside the cells (Fig. 3D). Together, these data classify 067 as an inhibitor of de
novo RNA synthesis by RNA polymerase II that is effective at low micromolar
concentrations.
P-TEFb broadly affects RNA synthesis of cellular genes
18
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Novel CDK9 inhibitor
Microarray gene expression profiling was conducted on RNA isolated from cells treated for
90 min with 067 (Suppl. Data Set 1). For this analysis we used the leukaemia cell line THP1
transformed by MLL-AF9, as it had shown high sensitivity for CDK9 inhibition by 067 (c.f.
Fig. 2E). Approximately 1,300 or 4.5% of all genes were reproducibly regulated by 067 (three
biological replicates; fold ≥1.4; p ≤0.05), out of which 94% were downregulated (Fig. 4A).
Within the top responders of the latter group were immediate early genes MYC (7-fold down-
regulated) and FOS (5-fold down). Other genes found were M-phase regulatory kinases
PLK2/3/4, chromosome segregation proteins CENPL/C1, anti-proliferative genes BTG1/2
and, consistent with the observed induction of apoptosis, MCL1, all of which were previously
identified as targets of flavopiridol and/or DRB (Lam et al., 2001, Garriga et al., 2010).
Interestingly, several miRNA genes, including miR-15a, miR-21, let-7f, the polycistronic
MYC-controlled miR-17-92 ONCOMIR cluster (Esquela-Kerscher and Slack, 2006), and at
least 106 non-coding RNA genes were shown to be downregulated. Approximately 80% of
the latter group represent small nucleolar RNAs (snoRNAs), many of which are located in the
introns of protein-coding genes that are subject to rapid splicing (Filipowicz and Pogacic,
2002). These data are generally consistent with and extend previous data from microarray
analyses using well-established CDK9 inhibitors such as flavopiridol or DRB.
Gene ontology analysis indicated an over-representation of transcription factors with fast-
decaying mRNAs in the group of downregulated genes (Yang et al., 2003) (Fig. 4B). We
hypothesised that genes with intrinsically stable mRNA might remain unaffected in the
microarrays. To investigate this possibility, de novo RNA synthesis was analysed at nine
genes that were shown not to be affected by 067 in the microarrays. This group of genes,
which included the housekeeping genes RPS15, RPL31 or MED1, showed a significant
decrease in their unspliced transcript levels (Fig. 4C). The projection of these and the previous
RT-qPCR analyses (Fig. 3) suggests that CDK9 positively controls mRNA synthesis of many
19
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Novel CDK9 inhibitor
more genes than was initially deducible from gene expression array data, in which RNA
stability appears to confer experimental bias.
CDK9 inhibition by 067 increases promoter-proximal RNAPII levels
CDK9 alleviates promoter-proximal pausing of RNAPII (Zhou et al., 2012). Release of the
polymerase from these positions is mediated through CTD phosphorylation as well as via
release of the inhibitory factors SPT5 and NELF (Yamaguchi et al., 1999). Correspondingly,
CDK9 inhibition by DRB or flavopiridol results in elevated RNAPII levels at promoter-
proximal pause sites (Glover-Cutter et al., 2008, Rahl et al., 2010). We next addressed
whether and how transcriptional inhibition by 067 would correlate to the distribution of
RNAPII at the prototypical paused MYC gene by conducting chromatin immunoprecipitation
(ChIP) analysis in 067-treated HeLa cells (Fig. 5). In line with the previous reports, 067
forced RNAPII to pause in a promoter-proximal position (Fig. 5A). Total RNAPII, and those
forms with a phosphorylated CTD at position serine 5 (Fig. 5D) and serine 7 (Fig. 5E)
moderately increased at promoter-proximal sites. Conversely, phosphorylation of the CTD at
serine 2 was low at the 5'-end of the MYC gene and steadily increased towards the 3'-end (Fig.
5C). The influence of 067 on serine 2 phosphorylation was surprisingly moderate under
conditions where MYC transcription was reduced to less than 50% (compare Fig. 3A). Of
further note, RNAPII levels in the 3’-region of the gene remained relatively high in the
presence of 067. Similar ChIP analyses of other genes and/or in other cells confirmed the
above above findings, i.e. increased RNAPII levels predominantly at the 5'-end of genes (data
not shown). Taken together, our data illustrate the situation of CDK9 inhibition by a dynamic,
ATP-competitive inhibitor at intermediate non-saturating conditions. It is likely relevant from
a mechanistic point of view as well as for potential applications as it illuminates the response
of RNAPII under limiting (but not eradicated) CDK9 activity.
20
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Novel CDK9 inhibitor
Discussion and Conclusions
In the current study we introduce a novel inhibitor and characterise it as a highly selective
binder of CDK9 that effectively interferes with the RNA synthesis of many genes. 067 is
significantly more specific for CDK9 than both flavopiridol and DRB (Tables 1 and 2), and it
has a particular advantage over the latter in that it can be used for biomedical studies in mice
(BK, unpublished observations). High CDK9 specificity is especially important in this context
since former studies performed with DRB or flavopiridol could have been compromised by
the known off-target effects for the other CTD kinases CDK1, CDK2 as well as CDK7.
Using this highly specific inhibitor we have confirmed a series of functions and
mechanisms that were previously associated with CDK9, namely (i) the general requirement
of CDK9 for efficient transcription in vitro (Fig. 1) as well as in vivo (Figs. 3 and 4), (ii) the
targeting of CTD residue serine 2 by CDK9 (Fig. 2), (iii) the activation of the tumour
suppressor p53 (Fig. 2), (iv) the induction of apoptosis (Fig. 2) and (v) the enhancement of
RNAPII pausing (Fig. 5) upon selective CDK9 inhibition by 067. In vivo RNA analysis
carried out in the present study uncovered a critical positive role for CDK9 in the de novo
RNA synthesis of all analysed genes. Relevant to therapeutic applications, 067 causes
apoptosis in several human cancer cell lines (THP1, A549, MCF7, HeLa), as has been
previously observed for other CDK9 inhibitors (Fig. 2). In addition, primary AML blasts
isolated from leukaemia patients undergo apoptosis upon treatment with 067. Cell death does
not depend on active p53 - a similar effect of 067 was observed in HCT116 cells lacking a
p53 gene. Instead, we assume that cell death relates to the repression of anti-apoptotic genes
such as MCL1, which was observed to be downregulated in our microarray studies and has
been reported previously in a different context (Lam et al., 2001, Ma et al., 2003). Beyond
this, our data generally imply that other genes like MCL1 that encode mRNAs and proteins
21
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Novel CDK9 inhibitor
with short half-lives could determine the initial biological response to CDK9 inhibitors. One
prominent example is MYC, whose mRNA and protein are subject to rapid turnover (e.g. Fig.
3, and data not shown). Our microarray analysis uncovered additional, novel CDK9 targets,
most notably several non-coding RNAs including cancer-associated miRNA genes such as
miR-15a, miR-21, let-7f (Esquela-Kerscher and Slack, 2006), and the polycystronic miR-17-
92 ONCOMIR cluster (He et al., 2005). Notably, the latter is a direct target of MYC,
suggesting that ONCOMIR downregulation (2.2-fold) was mediated through transcriptional
repression of MYC (O'Donnell et al., 2005).
Our in vitro transcription analysis clearly revealed competition between 067 and ATP
(Fig. 1D). Surprisingly, CDK9 inhibition by 067 was also strongly dependent on magnesium
(Fig. 1E). Currently, we can only speculate about the underlying mechanism. Recent
structure-function analyses of the closely related CDK2 demonstrated the requirement for
coordination of a second Mg2+ ion in the active site to stabilize ATP-binding and to
maximally enhance catalytic activity (Bao et al., 2011, Jacobsen et al., 2012). In analogy,
CDK9 active site chemistry might depend on this mechanism, too. In this scenario, increasing
the availability of magnesium might favour ATP- over 067-binding to the catalytic center of
CDK9, thereby diminishing the inhibitory activity of 067.
Reduction of RNAPII CTD phosphorylation at serine 2 by 067 was moderate in
comparison with the clinically evaluated flavopiridol, especially after longer drug exposure
(Fig. 2A). Of note, reduction in CTD phosphorylation by flavopiridol can not always be
directly linked with MCL1 ablation (Shapiro, 2006). On the other hand, abrogation of MCL1
expression and concurrent apoptosis induction is thought to be the critical mode of action for
flavopiridol in chronic lymphocytic leukemia cells (Chen et al., 2005), indicating that Ser2-P
is neither the sole (nor likely the most critical) target of CDK9 inhibitors. The more severe
effects of flavopiridol on Ser2-P might result from its lower specificity for CDK9. For
22
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Novel CDK9 inhibitor
example, the pan-specific flavopiridol could target additional CTD-Ser2 kinases such as
CDK12 and/or CDK13 (Bartkowiak et al., 2010, Blazek et al., 2011). Alternatively, Ser2-P
ablation by flavopiridol could simply reflect its higher affinity toward CDK9 (Table 2) (Chao
et al., 2000). Improvement of the comparably low affinity of 067 might help to illuminate this
issue in the future.
One important rationale for the development of 067 and related compounds is that
pan-CDK inhibitors such as flavopiridol exhibit a significant and dose-limiting level of
cytotoxicity (Shapiro, 2006), possibly due to their equipotent inhibition of multiple CDK
family members (see e.g. Table 1). This is predictably different for mono- or dual-selective
CDK inhibitors. Indeed, using 067 and analogous compounds from the series of
aminopyrimidines we could demonstrate that they exhibit a significant therapeutic window
(i.e. ratio of toxic-to-efficacious dose) in cells as well as in vivo (BK, unpublished
observations). Along the same line, a dual-selective CDK4/6 inhibitor (Fry et al., 2004) has
recently yielded very encouraging results in a breast cancer trial (Guha, 2013), demonstrating
that the selective inhibition of certain CDK family members is a valid therapeutic principle.
Taken together, our data provide a rationale for the development and optimization of
clinically useful, highly selective CDK9 inhibitors based on the 2,4-aminopyrimidine
scaffold, and 067 represents a promising lead in this context.
23
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Novel CDK9 inhibitor
Acknowledgements
We thank Dirk Eick for CTD antibodies. We are grateful to Tim Kelso for critical reading of
the manuscript and other members of the Meisterernst laboratory for helpful discussions. This
work was supported by the Ministry for Research and Technology (BMBF, grant number
0313860D to MM and 0313860B to GM) and by grants of the Westfalian Wilhelms
University Muenster to MM.
24
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Novel CDK9 inhibitor
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Novel CDK9 inhibitor
Table 1
Enzymatic kinase assay
IC50 [µM]
CDK4- CDK6-
CDK1-
CDK2-
CDK7-
CDK9-
Cyclin B1 Cyclin A Cyclin D1 Cyclin D3
Cyclin H-
MAT1
> 10
Cyclin T1
LDC000067
5.513
± 0.328
(n = 6)
> 10
2.441
± 0.227
(n = 6)
> 10
9.242
± 0.174
(n = 6)
> 10
> 10
0.044
± 0.010
(n = 6)
1.942
(n = 6)
> 10
(n = 6)
> 10
DRB
(n = 1)
< 0.005
(n = 1)
0.015
± 0.004
(n = 3)
0.006
± 0.001
(n = 3)
(n = 1)
0.038
± 0.008
(n = 3)
0.355
± 0.017
(n = 3)
(n = 1)
0.305
± 0.023
(n = 3)
3.404
± 0.139
(n = 3)
(n = 1)
0.103
± 0.023
(n = 3)
0.068
± 0.008
(n = 3)
(n = 1)
0.0052
± 0.0006
(n = 2)
0.0014
± 0.0005
(n = 3)
Flavopiridol
(n = 3)
0.052
± 0.009
(n = 3)
SNS-032
Half-maximal inhibition (IC50) of CDK inhibitors for the indicated CDK-cyclin pairs as
determined by an in vitro enzymatic kinase assay using time-resolved FRET analysis.
29
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Novel CDK9 inhibitor
Table 2
Kinase binding assay
Kd [µM]
CDK2-
CDK7-
CDK9-
Cyclin A
Cyclin H-MAT1
Cyclin T1
LDC000067
DRB
1.014
± 0.743
(n = 5)
n.d.
15.99
± 8.919
(n = 5)
> 10
0.0327
± 0.0151
(n = 35)
0.614
± 0.0269
(n = 2)
< 0.005
(n = 2)
0.0963
Flavopiridol
0.0302
± 0.0047
(n = 3)
± 0.0114
(n = 8)
(n = 8)
Dissociation constants (Kd) of CDK inhibitors 067, DRB and flavopiridol for the indicated
CDKs were determined by an in vitro kinase activity assay. All reactions contained ATP at
the concentration of the KM of the individual kinases (CDK2-Cyclin A: 3 µM; CDK7-Cyclin
H-MAT1: 25 µM; CDK9-Cyclin T1: 25 µM). N.d. = not determined.
30
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