Single-agent Chk1 inhibition is anti-proliferative in leukemia cells in vitro and in vivo
Kurtis D. Davies, Michael J. Humphries, Francis Sullivan, Ira von Carlowitz, Yvan Le Huerou, Peter J. Mohr, Bin Wang, James F. Blake,
Michael A. Lyon, Indrani Gunawardana, Mark Chicarelli, Eli Wallace, Stefan Gross
Abstract # 3874
Array BioPharma Inc., Boulder, CO
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Chk1Chk1-A
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40nM Chk1-A
Kinase
IC50(nM)
Chk1
0.9
Chk2
654
Rsk3
110
Mylk
51
1uM Camptothecin
Vehicle Control
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FITC-A
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Figure 4: Chk1-A induces S-phase accumulation and an eventual subG1 population in HEL92.1.7 cells
R
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Selectivity Screen In a panel of 257 kinases, Chk1-A [0.1μM] is highly selective for Chk1,
with no significant Chk2 inhibition and no activity against other kinases involved in checkpoint
control, cell cycle progression, or apoptosis.
Cell Cycle Analysis HEL92.1.7 cells
were treated with a dose range of Chk1A for 24, 48, or 72hr then fixed with
EtOH and stained with propidium iodide
(BD Pharmingen). Cell cycle profile was
determined by flow cytometry.
Figure 2: Chk1-A exhibits low nanomolar cellular activity
25
%<G1
300nM Chk1-A
vehicle
3, 1, 3
%G1
%S
%G2
1, 2, 2
100nM Chk1-A
40nM Chk1-A
40nM Chk1-A
3, 2, 2
54, 49, 62 26, 31, 25
15, 15, 8
2, 4, 4
100nM Chk1-A
2, 3, 3
57, 48, 54 27, 34, 29 12, 13, 11
3, 4, 4
200nM Chk1-A
4, 7, 10
50, 41, 32 31, 39, 51
12, 9, 7
3, 4, 4
300nM Chk1-A
7, 13, 15
46, 38, 28 36, 42, 53
9, 5, 2
3, 3, 3
Vehicle Control
PI
% of vehicle control
20
4000
2000
30
0
Chk1-A (nM)
AnnexinV Apoptosis Assay HEL92.1.7 cells
were treated with a dose range of Chk1-A or
1uM camptothecin for 48 or 72hr. Cells were
then stained using Annexin-V-FLOUS kit
(Roche). Stained cells were analyzed by flow
cytometry. Graphed values represent the
percentage of cells positive for AnnexinV
staining but negative for propidium iodide
staining (n=1). Inset is an example
demonstrating the analyzed population.
60
0
1u
M
C
pt
Chk1-A (nM)
15
0
19
pt
C
60
0
1u
M
30
0
15
0
75
ve
hi
cl
e
0
38
AnnexinV+/ PI(% of total cells)
600
75
300
38
150
19
600
75
Figure 7: Chk1-A treatment does not lead to pre-mature mitosis in
HEL92.1.7 cells
G2
vehicle control
<G1 G1
G2
40nM Chk1-A
<G1 G1
G2
%>G2
51, 53, 57 26, 27, 29 20, 18, 10
200nM Chk1-A
Cellular Activity Assays For Chk1 serine-296 autophosphorylation inhibition assay, HT-29
colon carcinoma cells were treated for 30min with a dose range of Chk1-A and then treated
with 100nM camptothecin for 2.5hr. Cells were then harvested and lysates were analyzed by
Western blot. For checkpoint abrogation assay, HT-29 cells were treated with 100nM
camptothecin for 16hr and then treated with a dose range of Chk1-A for 5hr. Induction of
phosphorylation on serine-10 on histone H3 (mitotic marker) was then analyzed via an indirect
immunofluorescence -based method using a LI-COR Aerius imager.
300
Immunofluorescent Analysis of H2A.X Phosphorylation HEL92.1.7 cells were
synchronized with 300nM nocodazol for 16hr then released into media containing Chk1-A or
1uM camptothecin. Cells were harvested 14hr later (time corresponding to late S-phase),
spun onto microscope slides with a Cytospin, and then fixed with 4% paraformaldehyde
followed by MetOH. Cells were then stained with an antibody to H2A.X phospho-serine-139
(Millipore) and Hoechst 33342 DNA stain (Invitrogen). Images were acquired by fluorescent
microscopy using a 60X objective. Experimenter was blinded to conditions during scoring
(graph at left). For the representative images (right) p-H2A.X is green and DNA is blue.
Phospho-HH3 S10
Checkpoint abrogation (pHH3)
48hr
72hr
Caspase 3/7 Assay HEL92.1.7 cells were
seeded at 4,000 cells/100μL in 96-well plates
and then treated with a dose range of Chk1-A or
1uM camptothecin. Caspase 3/7 activity was
determined at 24, 48, and 72hr using the
Caspase-Glo 3/7 assay (Promega). Raw assay
output was normalized to an approximation of
cell number as determined by CellTiter-Blue
cellular viability assay (Promega) (n=1).
Figure 9: Chk1-A inhibits growth of HEL92.1.7 tumor xenografts in vivo
24hr, 48hr, 72hr
48hr
40
24hr
48hr
72hr
6000
0
200nM Chk1-A
20
<G1 G1
24hr
AnnexinV
40
300nM nocodazol
11
38
60
0
IC50 (nM)
Auto-pChk1 (S296) inhibition
150
80
8000
60
100nM Chk1-A
<G1 G1
G2
200nM Chk1-A
<G1 G1
G2
72hr
DNA content
400nM Chk1-A
<G1
G1
G2
pHH3 and PI Staining
HEL92.1.7 cells were
treated with a dose range
of Chk1-A or 300nM
nocodazol (positive control
for pHH3) for 24 hours and
then fixed in EtOH. After
fixation, cells were stained
with an antibody to histone
H3 phospho-serine-10
(Millipore) and then
propidium iodide (BD
Pharmingen). pHH3
positivity and DNA content
were then determined by
flow cytometry.
1400
Mean Tumor Volume (mm3)
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K
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M
A
K
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3
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O
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%cells p-H2A.X foci
%cells pan-nuclear p-H2A.X
CFSE Cell Division Assay
HEL92.1.7 cells were synchronized
with 300nM nocodazol for 16hr and
then released and immediately
stained with carboxyfluorescein
diacetate succinimidyl ester (CFSE,
Invitrogen). Cells were then treated
with a dose range of Chk1-A or 1uM
camptothecin for 72hr, at which point
they were analyzed by flow
cytometry. Each successive peak on
the histogram (from right to left)
represents a division of the signal by
2 and thus 1 round of cell division.
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# cells
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Figure 6: Chk1-A induces a primarily pan-nuclear distribution as
opposed to nuclear foci of p-H2A.X in HEL92.1.7 cells
1
Chk1-A (uM)
M
0.1
8hr
C
hk
1A
0
4hr
Western Analysis
HEL92.1.7 cells were
treated with a dose
range of Chk1-A or
1uM camptothecin for
4, 8, or 24hr.
Harvested cells were
then lysed with RIPA
buffer and cleared
lysates were analyzed
by Western blot using
commercially available
antibodies.
20
0n
400
GAPDH
M
800
Rad51
10
0n
1200
Cdc2 p-T14/Y15
hk
1A
1600
Cdk2 p-Y15
C
2000
H2A.X p-S139
40
nM
2400
Figure 1: Chk1-A is selective against a broad kinase panel
Chk1
Chk1
Proliferation Assay HEL92.1.7
cells were seeded at 4,000
cells/100μL in 96-well plates and
then immediately treated with a dose
range of Chk1-A. Proliferation was
determined at 24, 48, and 72hr using
the CellTiter-Blue cellular viability
assay (Promega). Values represent
the mean ± S.E. (n=4 for 24 and
48hrs, n=5 for 72hrs).
24hr
48hr
72hr
successive divisions
Cdk’s
Chk1 p-S345
ve
hi
cl
e
2800
0.01
A
K
T
1
Chk1-A (nM)
Chk2 p-T68
Results
Chk2
Chk1-A (nM)
19
230
Vehicle
K562
600
39
300
126
150
MV411
Molm13
Chk1-A (nM)
38
27
75
72hr Proliferation IC50 (nM)
HEL92.1.7
19
Leukemia Cell Line
1uM Cpt
Figure 8: Chk1-A induces apoptosis in HEL92.1.7 cells
Vehicle
Figure 5: Chk1-A induces activating phosphorylation of DNA damage
response / cell-cycle checkpoint proteins and reduces inhibitory
phosphorylation on cyclin-dependent kinases in HEL92.1.7 cells
1uM Cpt
Figure 3: Chk1-A is anti-proliferative as a single-agent in leukemia cells
in vitro
1uM Cpt
Results
3200
RFU
Here we show that single-agent treatment with Chk1-A, a potent and selective
small molecule inhibitor of Chk1, alone is anti-proliferative in human leukemia cell
lines. Employing HEL92.1.7 cells, a line particularly sensitive to Chk1-A, we sought to
understand the mechanisms by which Chk1 inhibition derives the anti-proliferative
effect. Chk1-A treatment resulted in S-phase accumulation and induction of several
biochemical markers of DNA damage and cell-cycle checkpoint activation.
Furthermore, the anti-proliferative effect correlated with the induction of apoptosis but
was not associated with pre-mature entry into mitosis. In vivo, we found that
HEL92.1.7 tumor xenografts were sensitive to oral administration of Chk1-A at a dose
that was well tolerated. Together, these studies suggest that inhibition of Chk1
results in DNA damage that induces apoptosis and that use of a Chk1 inhibitor as a
single-agent could be an effective strategy to treat certain types of human cancers.
Results
Vehicle
Chk1 is a serine/threonine kinase that plays important roles in the cellular
response to genotoxic stress. For this reason, there is a great deal of interest in using
inhibitors of Chk1 to potentiate the effects of DNA-damaging chemotherapeutics. In
addition, multiple studies have demonstrated that Chk1 activity is essential during an
unperturbed cell cycle to ensure proper DNA replication and maintain genomic
integrity. Therefore, it is plausible that a Chk1 inhibitor could also be efficacious as a
single-agent therapeutic for human cancer.
Results
%cells p-H2A.X +
Introduction
75
Available at www.arraybiopharma.com
In Vivo TGI Female SCID-Beige
mice bearing growth-staged
HEL92.1.7 tumors were
administered Chk1-A (25 mg/kg
PO, BID) for 13 days as indicated.
This dosing regimen was well
tolerated with minimal body
weight loss over the course of the
study.
1200
1000
800
600
400
Vehicle
200
0
25mg/kg Chk1-A BID
0
5
10
15
20
25
Dosing
Day of Study
Treatment
% Mean
Regression
Max % Body
Weight Loss
% TGI
Vehicle
0
3.7, day 8
-
-
25 mg/kg
Chk1-A
4.6, day 4
5.7, day 13
68.6
12.7
Growth Delay (Days)
All in vivo studies were performed in accordance with IACUC guidelines and in harmony with
the Guide for Laboratory Animal Care and Use.
Conclusions
-Chk1-A, a potent and selective inhibitor of Chk1, is anti-proliferative as a single-agent
in leukemia cells in vitro and inhibits the growth of HEL92.1.7 tumor xenografts in vivo
-Inhibition of proliferation correlates with S-phase accumulation, induction of
biochemical markers of DNA damage, and apoptosis - suggests that Chk1 inhibition
produces lethal DNA lesions during replication
-Although Chk1-A treatment reduces inhibitory phosphorylation of Cdc2, it does not
result in histone H3 phosphorylation in cells with <4N DNA content - suggests that
apoptosis is not due to pre-mature mitotic entry
-Single-agent Chk1 inhibition may be an effective treatment strategy for certain cancers