Oncogene (2007) 26, 4179–4188

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ONCOGENOMICS

Identification and functional signature of genes regulated by structurally

different ABL kinase inhibitors

K Nunoda1, T Tauchi1, T Takaku1, S Okabe1, D Akahane1, G Sashida1, JH Ohyashiki2

and K Ohyashiki1
1
First Department of Internal Medicine, Tokyo Medical University, Shinjuku-ku, Tokyo, Japan and 2Intractable Disease Research
Center, Tokyo Medical University, Shinjuku-ku, Tokyo, Japan

ONCOGENOMICS
Dasatinib is an ATP-competitive, multi-targeted SRC and Introduction
ABL kinase inhibitor that can bind BCR-ABL in both the
active and inactive conformations. From a clinical SRC family kinases regulate multiple cellular events
standpoint, dasatinib is particularly attractive because it such as proliferation, differentiation, survival, cytoske-
has been shown to induce hematologic and cytogenetic letal organization, adhesion and migration as a con-
responses in imatinib-resistant chronic myeloid leukemia sequence of their ability to couple with many diverse
patients. The fact because the combination of imatinib and classes of cellular receptors and many distinct cellular
dasatinib shows the additive/synergistic growth inhibition targets (Thomas and Brugge, 1997). SRC kinases are
on wild-type p210 BCR-ABL-expressing cells, we rea- involved in BCR-ABL-mediated transformation and
soned that these ABL kinase inhibitors might induce the have been implicated in imatinib resistance (Donato
different molecular pathways. To address this question, we et al., 2003; Dai et al., 2004). Multiple domains of BCR-
used DNA microarrays to identify genes whose transcrip- ABL interact with and activate SRC kinases indepen-
tion was altered by imatinib and dasatinib. K562 cells dently of BCR-ABL kinase activity (Warmuth et al.,
were cultured with imatinib or dasatinib for 16 h, and gene 1997; Stanglmaier et al., 2003). The studies with
expression data were obtained from three independent dominant-negative mutants suggest that SRC kinases
microarray hybridizations. Almost all of the imatinib- and may contribute to the proliferation and survival of mye-
dasatinib-responsive genes appeared to be similarly loid cell lines expressing BCR-ABL in vitro (Lionberger
increased or decreased in K562 cells; however, small et al., 2000). HCK and LYN are expressed and activated
subsets of genes were identified as selectively altered in the acute phase of chronic myeloid leukemia (CML)
expression by either imatinib or dasatinib. The distinct patients, and their upregulation correlates with disease
genes that are selectively modulated by dasatinib are progression and resistance in patients with imatinib
cyclin-dependent kinase 2 (CDK2) and CDK8, which had (Hofmann et al., 2002; Donato et al., 2003; Dai et al.,
a maximal reduction of o5-fold in microarray screen. To 2004; Ptasznik et al., 2004). Therefore, dual SRC and
assess the functional importance of dasatinib regulated ABL kinase inhibitors are attractive for the treatment of
genes, we used RNA interference to determine whether imatinib-resistant CML.
reduction of CDK2 and CDK8 affected the growth Dasatinib is an orally available multitargeted SRC
inhibition. K562 and TF-1BCR-ABL cells, pretreated and ABL kinase inhibitor with two-log increased
with CDK2 or CDK8 small interfering RNA, showed potency relative to imatinib (Shah et al., 2004).
additive growth inhibition with imatinib, but not with Mutations in BCR-ABL that favor the adoption of an
dasatinib. These findings demonstrate that the additive/ active, imatinib-resistant conformation are effectively
synergistic growth inhibition by imatinib and dasatinib targeted by dasatinib, as shown in cell lines expressing
may be mediated in part by CDK2 and CDK8. 14 imatinib-resistant mutants (Shah et al., 2004; Burgess
Oncogene (2007) 26, 4179–4188; doi:10.1038/sj.onc.1210179; et al., 2005; O’Hare et al., 2005). Dasatinib prolonged
published online 8 January 2007 survival of mice with BCR-ABL-driven disease, and
inhibited proliferation of BCR-ABL-positive bone
Keywords: BCR-ABL; tyrosine kinase inhibitor; imatinib; marrow progenitor cells from patients with imatinib-
dasatinib sensitive or imatinib-resistant CML (Shah et al., 2004).
A recent saturation mutagenesis screening of BCR-ABL
also found that the spectrum of mutations that would
allow for dasatinib resistance is reduced compared
with that of imatinib, including L248R, Q252H,
Correspondence: Dr T Tauchi, First Department of Internal Medicine, E255K, V299L, T315I/A and F317V (Burgess et al.,
Tokyo Medical University, 6-7-1 Nishishinjuku, Shinjuku-ku, Tokyo 2005). Molecular docking studies also showed that
160-0023, Japan.
E-mail: [email protected]
dasatinib is likely to bind the inactive form of BCR-
Received 17 February 2006; revised 26 October 2006; accepted 27 October ABL, although requiring a lower conformational
2006; published online 8 January 2007 stringency, with the ability of binding more intermediate
 Microarray analysis of BCR-ABL tyrosine kinase inhibitors
K Nunoda et al
4180
conformations than imatinib (Gambacorti-Passerini imatinib would attenuate the downstream molecules of
et al., 2005). From a clinical standpoint, dasatinib is BCR-ABL (Figure 1b–d). As compared with treatment
particularly hopeful because it has been shown to induce with either agent alone, co-treatment with imatinib
hematologic and cytogenetic responses in imatinib- (0.5 mM) and dasatinib (5 nM) or imatinib (1.0 mM) and
resistant CML patients (Talpaz et al., 2006). The fact dasatinib (10 nM) for 24 h caused more inhibition of
that because the combination of imatinib and dasatinib BCR-ABL autophosphorylation (Figure 1b). Combined
shows additive/synergistic growth inhibition on wild- treatment with imatinib and dasatinib also caused more
type p210 BCR-ABL-expressing cells, we reasoned that attenuation of the levels of p-Akt, phosphor-nuclear
these ABL kinase inhibitors might induce different factor kappa B (NF-kB) and c-Myc, which are down-
molecular pathways. To address this question, we used stream of BCR-ABL (Figure 1c and d).
DNA microarrays to identify genes whose transcription
was altered by imatinib and dasatinib. The distinct genes
Dasatinib and imatinib-induced gene expression profiles
that are selectively modulated by dasatinib are cyclin-
K562 cells were cultured with dasatinib or imatinib for
dependent kinase 2 (CDK2) and CDK8. To assess the
16 h, and gene expression data from three independent
functional importance of dasatinib-regulated genes, we
microarray hybridizations were analysed (GPL 2523)
used RNA interference to determine whether reduction
(Figure 2a and b). The microarray experiment data were
of CDK2 and CDK8 affected the growth inhibition.
deposited in GEO (GSE 2810). Although the data in
K562 and TF-1BCR-ABL cells, pretreated with CDK2
this result demonstrated genuine differential expression,
or CDK8 small interfering RNA (siRNA), showed
most of these genes showed changes that were relatively
additive growth inhibition with imatinib but not with
small in magnitude. We, therefore, restricted to genes
dasatinib. These findings demonstrate that the additive
that showed at least a 1.5-fold change. Almost all of the
growth inhibition by imatinib and dasatinib may be
dasatinib or imatinib responsible genes appeared to be
mediated by CDK2 and CDK8.
similarly changed; however, small subsets of genes were
identified as selectively altered in expression by either
dasatinib or imatinib (Figures 3 and 4). In 16 h, 155 of
the 667 unique dasatinib genes and 144 of the 667
Results
unique imatinib genes were regulated >1.5-fold. This
finding suggests that these two structurally diverse BCR-
Analysis of combined drug effects
ABL tyrosine kinase inhibitors initially regulate highly
First, we investigated the combined use of dasatinib
overlapping common target genes and pathway. The
with the anti-leukemic agents imatinib, daunorubicin
common genes whose expression was affected by
(DNR), AraC and VP16 in vitro. Previous study
dasatinib and imatinib were categorized into different
demonstrated that imatinib showed additive or syner-
functional groups based on their biological function,
gistic effects in combination with some leukemic agents
and genes in the cell proliferation and apoptosis
(Nakajima et al., 2003). We therefore determined
categories were examined in greater detail (Figures 3
whether dasatinib could increase the effects of some
and 4).
anti-leukemic agents, including imatinib, in CML blastic
crisis cell line K562. The concentration of anti-leukemic
agents is plotted against percentage inhibition of Cell proliferation-related genes
proliferation, and each anti-leukemic agent alone is Dasatinib and imatinib affected the expression of several
compared with each anti-leukemic agent in combination cyclin-dependent kinases (CDK2, CDK4, CDC5R,
with 0.2 nM of dasatinib (Figure 1a). Regression lines CDK6 and CDK8), cell division cycle genes (CDC2L5,
generated by Microsoft Excel represent the best-fit CDC7, CDC25A and CDC25B) and cyclines (CCNA2,
relationship between drug concentration and the per- CCNC, CCND2, CCND3, CCNE1, CCNE2 and
centage inhibition of proliferation. R2 values are the CCNH) (Figure 3a and b). These regulators are known
square of the correlation coefficient. When imatinib was to be involved in G1/S and G2/M transition, and their
combined with 0.2 nM of dasatinib, the curve showed a decreased gene expression levels and thus reduced
substantial shift downward, consistent with increased activities are essential for cell cycle arrest at early stage.
antiproliferative activity of the drug combination Dasatinib and imatinib also affected the expression of
(Figure 1a). Similarly, with the combination of DNR mitotic inhibitors (CDKN1A, CDKN1B, CDKN1C,
or VP-16 and dasatinib, the curve also shifted down CDHN2C and CDK2AP1) (Figure 3b). Obviously,
(Figure 1a). When AraC was combined with dasatinib, downregulation/inactivation of such a large number of
the two curves crossed at 500 nM of AraC (Figure 1a). cell cycle-related genes may abolish the cell cycle
There was no additive effect of the combination of AraC machinery, probably a prerequisite for apoptosis in
and dasatinib. Although dasatinib and imatinib bind to dasatinib- or imatinib-treated CML cells. The distinct
overlapping sites within the BCR-ABL kinase domain, genes that are selectively modulated by dasatinib are
the clinically available concentrations of imatinib may CDK2 and CDK8, which had a maximal reduction
not exert an interfering effect by restricting the of o5-fold in microarray screen. CDK2 and CDK8
dasatinib’s access to its binding site. appeared to be candidates involved in the additive/
Next, we determined whether inhibition of BCR- synergistic growth inhibition by dasatinib and imatinib.
ABL-tyrosine kinase by co-treatment with dasatinib and Downregulation of these genes can be largely the result
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a Imatinib DNR b lP :
ABL
0 0

5nM Dasatinib
DNR only

10nM lmatinib
Imatinib only
20 20 R2 = 0.9068

% Inhibition
% Inhibition

2
R = 0.3741
40 40

60 2 60
R = 0.7343 R2 = 0.9343
with 0.2nM of 80 with 0.2nM of
80

10nM Dasatinib

1.0 M lmatinib
0.5 M lmatinib
1.0 M lmatinib
dasatinib

0.5 M lmatinib

5nM Dasatinib
dasatinib
0 0.1 0.2 0.3 0.4 0.5 0 20 40 60

Control
Imatinib (µM) DNR (nM)
AraC VP-16
0 0
VP-16 only BCR-ABL
2
20 20 R = 0.9625
% Inhibition

% Inhibition

AraC only
40 2 40
R = 0.3734 Image
60 60
R2 = 0.4726
Quantification 100 66 58 71 70 26 16 (%)
80 with 0.2nM of R2 = 0.4726 80 with 0.2nM of BLOT : P. Tyr
dasatinib dasatinib
0 200 400 600 0 200 400 600 BCR-ABL
AraC (nM) VP-16 (nM)
BLOT : ABL
c d
10nM Dasatinib
5nM Dasatinib

10nM Dasatinib
5nM Dasatinib
10nM Dasatinib
0.5 M lmatinib

1.0 M lmatinib
1.0 M lmatinib
0.5 M lmatinib

5nM Dasatinib
Control
10nM Dasatinib

0.5 M lmatinib
1.0 M lmatinib

1.0 M lmatinib
0.5 M lmatinib

5nM Dasatinib

Phospho-NF- B
Control

Image
Quantification 100 71 52 63 53 25 18 %
BLOT : Phospho-NFkB
Phospho-Akt NF- B
Image
Quantification 100 94 72 92 43 36 33 % BLOT : NF- B
BLOT : Phospho-Akt
c-Myc
Akt Image
Quantification 100 78 49 58 54 27 16 %
BLOT : Akt BLOT : c-Myc

Figure 1 Analysis of combined drug effects. (a) K562 cells were suspended to a final concentration of 1  105 cells/ml in fresh medium,
and incubated with anti-leukemic agents alone or in combination with 0.2 nM of dasatinib at 371C for 72 h. The number of cells in each
well was counted by flow cytometry. Regression lines generated by Microsoft Excel represent the best-fit relationship between drug
concentration and the percentage inhibition of proliferation. R2 values are the square of the correlation coefficient. Similar results were
obtained in each of three separate experiments. (b) K562 cells were cultured with indicated concentrations of dasatinib or imatinib for
24 h, and cell lysates were immunoprecipitated with ant-ABL mAb. The immunoprecipitates were immunoblotted with anti-
phosphotyrosine mAb (PY20) or anti-ABL Ab. (c) K562 cells were cultured with indicated concentrations of dasatinib or imatinib for
24 h, and the cell lysates were immunoblotted with anti-phospho-Akt or anti-Akt Ab. (d) K562 cells were cultured with indicated
concentrations of dasatinib or imatinib for 24 h, and the cell lysates were immunoblotted with anti-phospho-NF-kB, anti-NF-kB or
anti-c-Myc Ab.

of chain reactions of transcriptional inactivation. For Apoptosis-related genes

instance, CCD2 is known to be directly regulated by The intrinsic apoptotic pathway is important for
MYC at transcriptional sites, and its downregulation is dasatinib- or imatinib-mediated apoptosis. Both ABL
logically linked to downregulated MYC (Figure 3a). kinase inhibitors modulated the expression of many
genes that play a key role in this pathway, such as BAX,
BAK1, BID, BCL2, BCL6, BCL9, MCL1, CASP1,
Oncogenic signals genes CASP2, CASP3, CASP6, CASP7 CASP8, CASP9 and
Inactivation of BCR-ABL tyrosine kinase by dasatinib CASP10 (Figure 4a). Moreover, genes that regulate
or imatinib blocks the transcription factors, and thus the induction or prolongation of this pathway, includ-
represses expression of target genes such as those ing NF-kB-pathway genes (NF-kB1 and NF-kB1A),
encoding signal transduction molecules, as highlighted were also transcriptionally reduced (Figure 4b).
by members of STAT pathway (STAT1, STAT3, GADD45B, which induces cell cycle arrest at G2/M,
STAT4, STAT5A, STAT5B and STAT6) (Figure 3c). was also reduced by dasatinib and imatinib (Figure 4b).
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Figure 2 Hierarchical cluster analysis of gene expression profiles induced by dasatinib or imatinib in K562 cells. (a and b) K562 cells
were cultured with 100 nM of dasatinib or 5 mM of imatinib for 16 h, and gene expression data from three-independent microarray
hybridizations were analysed (GPL 2523). The microarray experiment data were deposited in GEO (GSE 2810). The scale in this figure
shows the level of expression, where red indicates increased gene expression, green indicates decreased expression, and the intensity of
color correlated to the magnitude changes. Black indicates no change.

Furthermore, genes coding tumor necrosis factor Validation of expression profiles of selected genes
(TNF) family receptors and ligands (TNF, TNFAIP3, To verify the changes of CDKs expression, which were
TNFRSR5 and TNFRSF12) were activated by dasatinib identified as selectively altered by either dasatinib or
or imatinib (Figure 4b). imatinib, we performed the immunoblot analysis focusing
on CDK2, CDK3, CDK4, CDK6, CDK8 and CDK9
(Figure 5a). K562 cells were cultured with indicated
DNA-damage repair genes concentrations of either dasatinib or imatinib for 48 h,
BCR-ABL-transformed cells appeared to be better and the cell lysates were immunoblotted with the
equipped to survive genotoxic damage because of indicated antibodies (Figure 5a). Protein expressions of
enhanced ability to repair DNA lesions, prolonged CDK2 and CDK8 were selectively reduced by dasatinib
activation of the G/2M checkpoint to provide more time treatment (Figure 5a). Quantitative real-time PCR
for repair, and inhibited apoptosis mechanisms. Dasa- analysis of these CDKs confirmed that protein expression
tinib and imatinib reduced the expression of several changes induced by dasatinib and imatinib correlated
DNA-damage repair pathway genes, such as RAD50, with changes in gene expression (data not shown). We
RAD51L3, RECQL, XRCC1, XRCC3 and XRCC4 also determined the protein expression of CDK2 and
(Figure 4c). Diverse modulation of these DNA-damage CDK8 in TF-1-BCR-ABL cells after dasatinib or
repair genes by both ABL kinase inhibitors may imatinib treatment (Figure 5b). Expressions of CDK2
participate in the reduced possibility of therapeutic drug and CDK8 was mainly reduced by dasatinib-treatment
resistance. (Figure 5b). As caspase-3 activity might affect the
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a Cell Proliferation-Related Genes-1 a Apoptosis-Related Genes-1
CDK2 BAX
Dasatinib Dasatinib
CDK4 BAK1
Imatinib Imatinib
CDK5R1 BID
CDK6 BCL2
CDK8 BCL6
CDK9 BCL9
CDC2L5 MCL1
CDC7 CASP1
CDC25A CASP2
CDC25B CASP3
CDC25C CASP6
MYC CASP7
E2F3 CASP8
E2F4 CASP9
E2F5 CASP10
-6 -4 -2 0 2 4 -6 -4 -2 0 2 4
Normalized ratio (log2) Normalized ratio (log2)
b Cell Proliferation-Related Genes-2
b Apoptosis-Related Genes-2
CCNA2
Dasatinib NFKB1
CCNC Dasatinib
Imatinib NFKB2
CCND2 Imatinib
NFKBIA
CCND3
IKBKB
CCNE1
IKBKE
CCNE2
IKBKG
CCNH
GADD45B
CDKN1A
CDKN1B TNF
CDKN1C TNFAIP3
CDKN2C TNFRSF5
CDK2AP1 TNFRSF12
DEDD
-6 -4 -2 0 2 4
-6 -4 -2 0 2 4 6
Normalized ratio (log2)
Normalized ratio (log2)
c Oncogenic Signals Genes
STAT1
c DNA Damage Repair Genes
Dasatinib
STAT3 RAD50
Imatinib Dasatinib
STAT4 RAD51
STAT5A Imatinib
RECQL
STAT5B XRCC1
STAT6 XRCC2
XRCC3
-6 -4 -2 0 2 4 6
XRCC4
Normalized ratio (log2) -6 -4 -2 0 2 4 6
Normalized ratio (log2)
Figure 3 Dasatinib- and imatinib-regulated cell proliferation
genes and STAT family genes. (a) Genes encode members of
cyclin-dependent kinases, cell division cycle genes, c-myc and E2F Figure 4 Dasatinib- and imatinib-regulated apoptosis genes and
family. (b) Genes encode members of cyclins, mitotic inhibitors. DNA-damage repair genes. (a and b) Genes encode members of
(c) Genes encode members of STAT family. apoptotic proteins. (c) Genes encode members of NF-kB pathways
and the death receptor pathway.

reduction of CDK2 or CDK8, therefore, we determined to determine whether reduction in CDK2 and CDK8
the time-dependent changes of CDK2/CDK8 expression affect the proliferation. K562 and TF-1BCR-ABL
and caspase-3 and poly(ADP-ribose)polymerase (PARP) cells were transfected with control siRNA or CDK2
activation in K562 cells (Figure 5c and d). K562 cells siRNA or CDK8 siRNA; then the CDK2 and CDK8
were cultured with either 100 nM of dasatinib or 10 mM of expression was analysed by immunoblotting after 48 h
imatinib for the indicated time, and the cell lysates were (Figure 6a). The siRNA to CDK2 or CDK8 specifically
immunoblotted with indicated antibodies (Figure 5c and reduced CDK2 or CDK8 expression (Figure 6a). At
d). The expression of CDK2 or CDK8 proteins was 48 h after transfection, K562 and TF-1BCR-ABL cells
reduced after 24 h of dasatinib treatment (Figure 5c); were treated with the indicated concentration of
however, the expression of CDK2 or CDK8 proteins was dasatinib or imatinib for 48 h, and viable cells were
nearly unchanged after imatinib treatment (Figure 5d). counted (Figure 6b and c). Increasing doses of imatinib,
As activation of caspase-3 or PARP was observed 12– in the presence of CDK2 siRNA or CDK8 siRNA,
24 h after dasatinib or imatinib treatment (Figure 5c and shifted the dose–response curve substantially down-
d), it is likely that caspase-3 activity may not be required ward, consistent with increased antiproliferative activity
for the reduction of CDK2/CDK8 protein level. (Figure 6b and c). When dasatinib was treated in the
presence of CDK2 siRNA or CDK8 siRNA, the two
curves crossed at 200 nM of dasatinib (Figure 6b and c).
siRNA-mediated knock down of CDK2 and CDK8 in These results demonstrated that transcriptional repres-
K562 cells and TF-1BCR-ABL cells sion of CDK2 or CDK8 can play an important role in
To assess the functional importance of dasatinib- and the combined growth inhibitory effects of dasatinib and
imatinib-regulated gene, we used RNA interference imatinib.
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Discussion and imatinib largely activated common apoptotic path-
ways, although there were differences in the activities
Imatinib and dasatinib are clinically active ABL tyrosine of the two compounds. Dasatinib is capable of bind-
kinase inhibitors that show the additive/synergistic ing BCR-ABL with less stringent conformational
growth inhibition on BCR-ABL-expressing cells requirements with respect to imatinib (Gambacorti-
(Figure 1a). Earlier studies demonstrated that dasatinib Passerini et al., 2005). Although both compounds inhibit

a Dasatinib nM Imatinib M Dasatinib nM Imatinib M b

0 10 50 100 1 2 5 10 0 10 50 100 1 2 5 10

CDK2
CDK3 TF-1 BCR-ABL
(%) 100 73 56 36 99 102 97 98 Image
Quantification 100 82 78 82 100 93 100 99 (%)
BLOT : CDK2 BLOT : Dasatinib nM Inatinib M
CDK3
0 10 50 100 1 2 5 10
CDK4
CDK2
100 51 33 32 86 85 46 37 (%)
CDK8 100 91 57 34 96 95 71 69 (%)
BLOT : CDK4
(%) 100 56 35 16 96 97 75 66 Image BLOT : CDK2
Quantification
BLOT : CDK8 CDK6
CDK8
100 53 44 38 94 99 62 51 (%)
BLOT : CDK6
100 87 39 17 91 90 83 77 (%)
BLOT : CDK8

actin CDK9
Cleaved PARP
BLOT : actin 100 86 50 41 91 94 66 63 (%)
BLOT : CDK9 BLOT : PARP

c Dasatinib d lmatinib
0 2 6 12 24 48 hrs 0 2 6 12 24 48 hrs
CDK2
CDK2
100 90 78 77 82 80 (%)
100 82 83 85 45 38 (%)
BLOT : CDK2
BLOT : CDK2
CDK8
CDK8
100 98 106 98 84 85 (%)
100 105 102 41 35 14 (%) BLOT : CDK8
BLOT : CDK8
cleaved
cleaved caspase-3
caspase-3
BLOT : cleaved-caspase-3
BLOT : cleaved caspase-3 cleaved
PARP
cleaved
PARP

BLOT : PARP BLOT : PARP

Figure 5 Validation of microarray data of cyclin-dependent kinase genes. (a) K562 cells were cultured with indicated concentrations
of either dasatinib or imatinib for 48 h, and the cell lysates were immunoblotted with the indicated antibodies. Quantitative analysis of
CDK2 and CDK8 expression was carried out by analysing hyper-ECL films of immunoblotting by using a densitometer. (b) TF-
1BCR-ABL cells were cultured with indicated concentrations of dasatinib or imatinib for 48 h, and the cell lysates were immunoblotted
with anti-CDK2 or anti-CDK8 or anti-PARP Ab. (c and d) K562 cells were cultured with either 100 nM of dasatinib (c) or 10 mM of
imatinib (d) for the indicated time, and the cell lysates were immunoblotted with anti-CDK2, anti-CDK8 or anti-cleaved caspase-3, or
anti-PARP Ab.

Figure 6 Knock down of CDK2 and CDK8 expression affected the proliferation in dasatinib- or imatinib-treated K562 cells and TF-
1BCR-ABL cells. (a) K562 cells and TF-1BCR-ABL cells transfected with 1.25 mM of control (GFP), or CDK2 siRNA or CDK8
siRNA were analysed 48 h after transfection for CDK2 or CDK8 expression by immunoblotting. (b and c) At 48 h after transfection,
K562 (b) and TF-1BCR-ABL cells (c) were treated with incubated concentrations of dasatinib or imatinib for 48 h; viable cells were
counted by using a Vi-cell XR automated cell viability analyzer (Beckman Coulter). The mean number of viable cells at different
concentrations of drug was normalized to the mean number of viable cells in the no-drug sample. Similar results were obtained in each
of three independent experiments.

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a K562 TF-1 BCR-ABL

CDK2 siRNA
CDK8 siRNA

CDK8 siRNA

CDK2 siRNA
CDK8 siRNA

CDK2 siRNA
CDK8 siRNA
CDK2 siRNA
Control

Control

Control

Control
CDK2 CDK8 CDK2 CDK8
(%) 100 32 98 100 102 45 100 33 82 100 106 21
BLOT : CDK2 CDK8 CDK2 CDK8

actin actin actin actin

BLOT : actin actin actin actin

b K562
100 lmatinib 100 lmatinib
lmatinib+CDK2siRNA lmatinib+CDK8 siRNA
80 80

60 60
%

%
R2 = 0.8183 R2 = 0.8183
40 40

20 20
R2 = 0.8548 R2 = 0.764
0 0
0 0.5 1.0 1.5 2.0 2.5 0 0.5 1.0 1.5 2.0 2.5
lmatinib ( M) lmatinib ( M)

100 Dasatinib 100 Dasatinib

Dasatinib+CDK2 siRNA Dasatinib+CDK8 siRNA
80 80
R2 = 0.5389 R2 = 0.5389
60 60
%

R2 = 0.8085 R2 = 0.7034
40 40

20 20

0 0
0 100 200 300 0 100 200 300
Dasatinib (nM) Dasatinib (nM)

c TF-1BCR-ABL

100 lmatinib 100 lmatinib

lmatinib+CDK2 siRNA lmatinib+CDK8 siRNA
80 80
R2 = 0.7819 R2 = 0.7819
60 60
%

40 40
R2 = 0.9071
R2 = 0.9924
20 20

0 0
0 0.5 1.0 1.5 2.0 2.5 0 0.5 1.0 1.5 2.0 2.5
lmatinib ( M) lmatinib ( M)

100 Dasatinib 100 Dasatinib

Dasatinib+CDK2 siRNA Dasatinib+CDK8 siRNA
80 80
R2 = 0.5086 R2 = 0.5086
60 60
%

R2 = 0.964
40 40
R2 = 0.7468
20 20

0 0
0 100 200 300 0 100 200 300
Dasatinib (nM) Dasatinib (nM)

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BCR-ABL tyrosine kinase, whether these two structu- S phase requires sequential activation of CDK2 and
rally diverse compounds mediate similar or disparate CDK4 (Sherr and Roberts, 1999). The role of CDK4 is
gene expression changes has not been addressed. In the well established; however, CDK2 may be regulated
present study, we used microarray gene expression differently in somatic cells and in cancer cells (Tetsu and
profiles to identify the genes commonly and selectively McCormick, 2003). In BCR-ABL-transformed cells,
regulated by dasatinib and imatinib. cyclin D activates CDK4/CDK6, which inturn transac-
On the basis of clustering, the transcriptional re- tivates cyclin E by releasing E2F. Cyclin E activates
sponses induced by dasatinib and imatinib were CDK2, and BCR-ABL causes relocation of p27 to the
remarkably similar (Figure 2a). Some degree of overlap cytoplasm, further stimulating CDK2 (Jiang et al.,
was expected because both compounds target BCR- 2000). Therefore, CDK2 seems to have a critical role
ABL kinase. Compatible with this phenotype, some in cell cycle progression in BCR-ABL-transformed
proapoptotic genes were upregulated, a number of cells. Downregulation of CDK2 or CDK8 by siRNA
antiapoptotic genes were downregulated by both ABL increased antiproliferative activity in imatinib-treated
kinase inhibitors, and a number of genes within the cells, but not in dasatinib-treated cells (Figure 6a–c).
same molecular pathway were coordinately regulated Our results clearly demonstrate that diverse regulation
(Figures 3 and 4). These include BAX, BCL-2, MCL-1, of CDKs by ABL kinase inhibitors may contribute at
CASP2 and CASP10 (Figure 4a). Although some of the least in part to additive growth inhibition in these
proapoptotic genes were repressed (i.e., BID, CASP3 compounds. The pleiotropic molecular sequel of SRC/
and CASP6), the overall response was one that would ABL inhibition does not allow us to conclude which
provide a strong proapoptotic signal (Figure 4a and b). pathways are most important for anti-tumor effect, but
Moreover, repression of the NF-kB pathway is also potentially offer a major therapeutic advantage, namely
associated with apoptotic stimulus (Figure 4b). Further- the simultaneous targeting of different proliferative/
more, the transcription of TNF superfamily genes and antiapoptotic pathways in tumor cells (Supplementary
genes involved in death-receptor signaling (DEDD) was Figure).
altered by dasatinib or imatinib (Figure 4b). One of the objectives of this study of the molecular
In addition to inducing apoptosis, ABL kinase profile of dasatinib-treated BCR-ABL-transformed cells
inhibitors can suppress cell cycle progression (Gesbert was to establish a framework for designing of combina-
et al., 2000; Parada et al., 2001). The transcriptional tion therapies with conventional anti-leukemia agents.
response was consistent with growth suppression, and a This study identified the enhanced antiproliferative
number of genes within key growth-regulatory pathways activity of combinations with dasatinib with anti-
were coordinately regulated (Figure 3a and b). For leukemia agents, such as imatinib, daunorubicine and
example, decreased expression of MYC, E2F3, E2F4 VP-16 (Figure 1a). The ability of dasatinib to suppress
and E2F5 is consistent with suppression of the MYC genes involved in cell proliferation-related genes (i.e.,
pathway (Figure 3a). The number of CDKs and CDK CDK2, CDK6, CDK8, CDC7, CCNA2, CCNC, CCNE1,
inhibitors, whose expression is highly coordinated to CCNE2 and MYC) and antiapoptotic genes (BCL2,
regulate appropriate cell cycle progression, was aber- MCL1 and NFkB) superior to imatinib constitute a
rantly expressed in dasatinib- or imatinib-treated cells molecular basis for the chemo-sensitizing effect of SRC/
(Figure 3b). CDK2 and CDK8 are selectively modulated ABL kinase inhibition.
by dasatinib, which had a maximal reduction of o5-fold This study provides comparative gene expression
in microarray screen (Figure 3a). We have shown that profiling analysis of the effect of dasatinib and imatinib,
dasatinib and imatinib and the combination of these two compounds that are clinically active for BCR-ABL-
compounds differently suppress the expression of c-Myc positive leukemia. Both agents commonly target a
protein (Figure 1d). Recently, Samanta et al. (2006) number of important molecular pathways that regulate
demonstrated that signal transduction by BCR-ABL/ cell growth and survival. A single proapoptotic or anti-
Jak2 network results in phosphorylation of Akt, which proliferative pathway may not be critical for the
leads to the stabilization of c-Myc and activates NF-kB therapeutic effects of ABL kinase inhibitors. The present
to cause elevation of c-Myc transcripts. As compared findings have important implications for the clinical use
with treatment with either agent alone, co-treatment of dasatinib and imatinib as an anti-leukemia agent,
with dasatinib and imatinib caused more attenuation of either alone or in combination with other agents.
the levels of phospho-Akt, phospho-NFkB and c-Myc
(Figure 1c and d).
As CDK2 is a c-Myc transcriptional target (Prathapam
et al., 2006), downregulation of a large number of cell Materials and methods
cycle-related genes, including CDK2 and CDK8, could
be largely the result of chain reactions of transcriptional Antibodies and reagents
Anti-CDK2 Ab (D-12), anti-CDK3 Ab (Y-20), anti-CDK4 Ab
inactivation by these ABL tyrosine kinase inhibitors. (H-22), anti-CDK6 Ab (C-21), anti-CDK8 Ab (D-9), anti-
Earlier study (Riley et al., 2001) demonstrated that CDK9 Ab (C-20), anti-actin Ab (C-2) and anti-ABL Ab (24-
v-SRC induces the expression of CDK2 in Rat-1 cells. 11) were purchased from Santa Cruz Biotechnology, Inc.
Therefore, inactivation of c-SRC and SRC-family (Santa Cruz, CA, USA). Antiphosphotyrosine mAb (PY20) was
kinase by dasatinib may decrease the expression of purchased from Becton Dickinson and Company (Franklin
CDK2. Progression through the cell cycle from G1 to Lakes, NJ, USA). Anti-Akt Ab, anti-phospho-Akt (Ser473)
Oncogene
 Microarray analysis of BCR-ABL tyrosine kinase inhibitors
K Nunoda et al
4187
Ab, anti-NF-kB p65 Ab and anti-phospho-NF-kB p65 USA) as raw data. The scanned data were normalized, verified
(Ser536) Ab were purchased from Cell Signaling (Beverly, and analysed using the Genomic Profiler software (Mitsui
MA, USA). Anti-c-Myc Ab was purchased from NOVUS Knowledge Industry, Tokyo, Japan) as described previously
(Littleton, CO, USA). Dasatinib was kindly provided by (Ohyashiki et al., 2005; Takaku et al., 2005; Zhang et al.,
Bristol-Myers Squibb (New York, NY, USA). DNR, cytosine 2006). First, the value was adjusted by subtraction of
arabinoside (Ara C) and etoposide (VP-16) were obtained background fluorescence of an equivalent area. Analysis was
from Sigma (St Louis, MO, USA). carried out by taking the median signal of the probe value for
each transcription set, and the 75% rank for the total
Cells and cell culture hybridization was calculated. Microarray data obtained from
K562 cells were obtained from the American Type Culture three independent experiments were then verified in a single
Collection (Rockville, MD, USA). TF-1BCR-ABL cells were file. The normalized log data of fluorescence ratio (Cy5–Cy3)
described previously (Komatsu et al., 2003). These cell lines which was quantified for each gene to reflect the relative
were cultured in RPMI1640 (Life Technology Inc., Rockville, abundance of gene in each experimental sample compared with
MD, USA), supplemented with 10% FCS (Hyclone Labora- reference sample, were deposited with GEO. For statistical
tories, Logan, UT, USA). analysis of host gene expression, we also utilized a GeneSifter
(VizXLabs, Seattle, WA, USA). Analysis of variance, and
Analysis of combined drug effects Student’s t-test were performed using GeneSifter. Values of
K562 cells were suspended to a final concentration of Po0.05 were considered to indicate a statistically significant
1  105 cells/ml in fresh medium, plated in 24-well dishes and difference, and the Benjamini–Hochberg algorithm was used
incubated with anti-leukemic agents alone or in combination for estimation of false discovery rates.
with 0.2 nM of dasatinib at 371C for 72 h. The anti-leukemic
agents used were imatinib, DNR, AraC or VP16. The number
Immunoblotting and immunoprecipitation
of cells in each well was counted by flow cytometry, and the
Immunoblotting and immunoprecipitation were performed
cell numbers were normalized by dividing the number of cells
as described previously (Tauchi et al., 1994). Quantitative
in the absence of anti-leukemic agents or with dasatinib alone.
analysis of CDK2 and CDK8 expression was carried out by
The data were plotted as the concentration of anti-leukemic
analysing hyper-enhanced chemiluminescence (ECL) films of
agents against the percentage inhibition of proliferation. To
immunoblotting by using a densitometer (Bio-Rad, Hercules,
determine the relationship between the percentage inhibition
CA, USA).
of proliferation and drug concentration, a best-fit regression
line was generated by Microsoft Excel.
Small interfering RNA experiments
Oligonucleotide DNA microarray hybridization siRNA oligonucleotides for CDK2 and CDK8 were purchased
We have designed two types of pathway focusing on low- from Santa Cruz Biotechnology Inc. (Santa Cruz, CA, USA),
density oligonucleotide microarrays (Novusgene Inc., Tokyo, and resuspended in RNase-free H2O at 20 mM. siRNA
Japan), which contain 667 selected genes related to cell growth, (1.25 mM) was added to prechilled 0.4 cm-gap electroporation
cell cycle, apoptosis, transcription, DNA repair and cell stress cuvettes (Bio-Rad). K562 (5  106) or TF-1BCR-ABL cells
responses (GEO accession number: GPL2523). This DNA chip (5  106) were washed twice in serum-free media and resus-
was made up of tetra-plicate spots of 60-mer highly specific pended to 5  106 cells per 250 ml of cold, serum-free RPMI
oligonucleotide probes. For DNA microarray analysis of 1640. Cells were added to the cuvettes, mixed, and further
genes, we used 1 mg of mRNA from K562 cells treated with mixed for 5 min on ice. Cells were then pulsed once at 250 mV,
dasatinib or imatinib for 16 h. Hybridization was carried out 960 mF and 200 O by using a Bio-Rad electroporator. At 48 h
automatically using Gene TAC Hybridization (Genomic after electroporation, protein knock down was determined by
Solution, Ann Arbor, MI, USA) according to the supplier’s immunoblotting, cells were treated with the indicated con-
instructions. The conditions of hybridization were 551C for centration of dasatinib or imatinib for 48 h, and viable cells
2 h, 501C for 2 h and 461C for 2 h, then 421C for 12 h. were counted by using a Vi-cell XR automated cell viability
analyzer (Beckman Coulter, Fullerton, CA, USA). The mean
Data analysis and statistic validation number of viable cells at different concentrations of drug was
The hybridization signals were scanned by GenePix 4000B normalized to the mean number of viable cells in the no-drug
Microarray Scanner (Axon Instruments, Union City, CA, sample.

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Supplementary Information accompanies the paper on the Oncogene website (http://www.nature.com/onc).

Oncogene