The Journal of Clinical Investigation http://www.jci.org Volume 122 Number 8 August 2012
Targeting CDK1 promotes FLT3-activated
acute myeloid leukemia differentiation
Hanna S. Radomska,1 Meritxell Alberich-Jordà,1,2 Britta Will,1 David Gonzalez,1
Ruud Delwel,3 and Daniel G. Tenen2,4
1Beth Israel Deaconess Medical Center/Harvard Medical School, Boston, Massachusetts, USA. 2Harvard Stem Cell Institute,
Harvard Medical School, Boston, Massachusetts, USA. 3Erasmus University, Rotterdam, Netherlands.
4Cancer Science Institute, National University of Singapore, Singapore.
Mutations that activate the fms-like tyrosine kinase 3 (FLT3) receptor are among the most prevalent muta-
tions in acute myeloid leukemias. The oncogenic role of FLT3 mutants has been attributed to the abnormal
activation of several downstream signaling pathways, such as STAT3, STAT5, ERK1/2, and AKT. Here, we
discovered that the cyclin-dependent kinase 1 (CDK1) pathway is also affected by internal tandem duplication
mutations in FLT3. Moreover, we also identified C/EBPα, a granulopoiesis-promoting transcription factor,
as a substrate for CDK1. We further demonstrated that CDK1 phosphorylates C/EBPα on serine 21, which
inhibits its differentiation-inducing function. Importantly, we found that inhibition of CDK1 activity relieves
the differentiation block in cell lines with mutated FLT3 as well as in primary patient–derived peripheral
blood samples. Clinical trials with CDK1 inhibitors are currently under way for various malignancies. Our
data strongly suggest that targeting the CDK1 pathway might be applied in the treatment of FLT3ITD mutant
leukemias, especially those resistant to FLT3 inhibitor therapies.
In acute myeloid leukemia (AML), an immature cell can acquire
genetic changes, such as chromosomal translocations, insertions,
deletions, or point mutations, which lead to uncontrolled cell
growth, protection against cell death, and differentiation arrest.
Among the most common oncogenic mutations in AML are inter-
nal tandem duplications (ITD) or activating mutations in fms-
like tyrosine kinase 3 (FLT3). FLT3 is normally expressed in early
hematopoietic precursors and plays a role in their proliferation
and differentiation (1, 2), but its aberrant activation contributes
to the development of AML. FLT3ITD mutations occur in about
20%–30% of AML patients, and the majority of these mutations
(over 70%) are located in the juxtamembrane domain of FLT3.
A novel type of ITD mutation (over 28%) was recently identified
within the first kinase domain of the receptor (3). Several amino
acids in the kinase domain are also known to undergo activat-
ing point mutations, for example, mutations in aspartic acid 835,
which are seen in about 7% of AML cases (4). The consequences
of FLT3 mutations are self phosphorylation and ligand-indepen-
dent activation of the FLT3 receptor, followed by activation of
the downstream signaling pathways, mainly Stat5, Akt, ERK1/2,
Pim-1/2, and SHP-1 (5–11). Patients with activating FLT3 muta-
tions have a poor prognosis (1, 2, 4, 12–14); therefore, much effort
is being put forth to develop specific therapies. Small molecule
inhibitors that specifically inhibit the FLT3 activity are presently
undergoing clinical trials (1, 2, 4, 12–16). We have previously
demonstrated that one of the targets of the ERK1/2 kinase is
C/EBPα, a transcription factor playing a critical role in granu-
locytic differentiation (17) and often inactivated in various sub-
types of leukemia by multiple mechanisms, such as transcription-
al and translational silencing, as well as genetic mutations and
posttranslational modifications, which render C/EBPα protein
nonfunctional. The importance of C/EBPα as a molecular switch
is underscored by the fact that it is both necessary and sufficient
for granulocytic differentiation (18, 19). Activity of C/EBPα can
be modulated by phosphorylation, and a number of residues in
the C/EBPα protein that are subject to modifications have been
identified. However, until now, only phosphorylation of serine
21 has been shown to have clinical importance (20, 21). We have
shown that this single amino acid modification by the ERK1/2
pathway inhibits the function of C/EBPα and is responsible for
the differentiation block in FLT3ITD leukemic blasts (17, 21).
Pharmacological or genetic abrogation of this phosphoryla-
tion event in leukemic cells, for example, treatment with MEK1
inhibitor or substitution with a nonphosphorylatable mutant of
C/EBPα (S21A), permits granulopoiesis to proceed (17, 21). Phos-
phorylation of C/EBPα on serine 21 by p38 MAPK in hepatocytes,
on the other hand, increases its transactivation potential on the
phosphoenolpyruvate carboxykinase (PEPCK) gene promoter and
results in increased PEPCK expression (20). Thus, serine 21 phos-
phorylation in liver enhances gluconeogenesis and, therefore, may
play a role in diabetes.
Interestingly, among FLT3ITD patients, only 39% demonstrated
activation of MEK1, and thus the ERK1/2 pathway (22), yet C/EBPα
can still be inactivated by phosphorylation on serine 21 (this study).
Herein, we identified cyclin-dependent kinase 1 (CDK1, also known
as CDC2) as an FLT3ITD-activated kinase, which is responsible for
C/EBPα phosphorylation on serine 21 and the blocking of its func-
tion. Thus, we provide a molecular mechanism by which the consti-
Authorship note: Hanna S. Radomska and Meritxell Alberich-Jordà contributed
equally to this work.
Conflict of interest: The authors have declared that no conflict of interest exists.
Citation for this article: J Clin Invest. 2012;122(8):2955–2966. doi:10.1172/JCI43354.
2956 The Journal of Clinical Investigation http://www.jci.org Volume 122 Number 8 August 2012
tutively active FLT3 mutant receptor contributes to the pathogen-
esis of leukemia, and we propose the use of CDK1 inhibitors for the
treatment of FLT3ITD leukemia.
C/EBPα transcription factor can be phosphorylated on serine 21 by an
ERK1/2-independent kinase. We reported previously that the granu-
locytic differentiation-promoting function of C/EBPα could be
inhibited in FLT3ITD AML by ERK1/2-mediated phosphoryla-
tion of serine 21 (17, 21). It has also been reported that not every
FLT3ITD mutant can constitutively activate the ERK1/2 pathway
(22, 23). To test whether the differentiation block in FLT3ITD
AML can be mediated by an ERK-independent phosphorylation
of C/EBPα, we transiently coexpressed C/EBPα and FLT3ITD
mutant N51 (24), known not to activate the ERK1/2 pathway (ref.
23 and Radomska, unpublished observations), or empty vector
(MSCV-IRES-EGFP) in 293T cells and analyzed phosphorylation
of C/EBPα on serine 21 by Western blot. Figure 1 shows that while
activation of the ERK1/2 pathway by brief treatment with TPA
in the absence of FLT3ITD N51 resulted in a subtle increase in
phosphorylation of serine 21, a robust phosphorylation took place
when C/EBPα was coexpressed with FLT3ITD N51. Consistent
with the previous report (23), overexpression of FLT3ITD N51 in
the absence of TPA stimulation did not activate the ERK1/2 path-
way, and the treatment with MEK1/2 inhibitor PD98059 did not
affect the serine 21 phosphorylation levels. In contrast, treatments
with FLT3 inhibitors MLN518 and PKC412 led to a substantial
decrease in serine 21 phosphorylation. These results strongly sug-
gest that FLT3ITD mutant(s) can activate a novel pathway other
than ERK1/2, which is capable of phosphorylating C/EBPα on
serine 21 and blocking its function.
Phosphorylation of C/EBPα on serine 21 by CDK1 in vitro and in vivo.
To identify which kinase, in addition to ERK1/2, can phosphory-
late serine 21, we analyzed the amino acid sequence of C/EBPα in
the vicinity of serine 21 using Scansite software (http://scansite.
mit.edu/) and found that this residue lies within a motif likely to be
phosphorylated by CDK1. To verify this, we performed cell-free in
vitro kinase assay using purified active CDK1 kinase and GST-C/
EBPα proteins as substrates. As demonstrated in Figure 2A,
32P was specifically incorporated into the GST-C/EBPα WT pro-
tein only in the presence of the active enzyme, and mutating ser-
ine 21 to alanine abolished phosphorylation. Phenylalanine 31 is
located within the docking site for ERK1/2, and it was demon-
strated to be necessary for substrate recognition and phosphoryla-
tion of C/EBPα on serine 21 (17). In contrast to ERK1/2-mediated
phosphorylation, mutating this residue to alanine had no effect
on phosphorylation of C/EBPα by CDK1 (Figure 2A).
Furthermore, in transiently transfected 293T cells, overexpres-
sion of CDK1 led to an increase in serine 21 phosphorylation of
cotransfected C/EBPα without changing the activity of ERK1/2
(Figure 2B). It has been demonstrated that during mitosis CDK1
kinase reaches its highest activity level, while ERK1/2 activ-
ity subsides (25). Indeed, when C/EBPα-transfected 293T cells
were arrested at mitosis by nocodazole treatment, serine 21 was
phosphorylated despite the absence of detectable ERK1/2 activity,
and this phosphorylation process was abrogated by coexpression
of dominant negative mutant of CDK1 (DN CDK1; Figure 2B).
Mitotic arrest of U937 cells with nocodazole also showed increased
phosphorylation of endogenous C/EBPα, which decreased upon
release from arrest (Supplemental Figure 1A; supplemental mate-
rial available online with this article; doi:10.1172/JCI43354DS1).
Consistent with CDK1-mediated phosphorylation of C/EBPα,
treatment of mitotic U937 cells with MEK1 or CDK2/CDK5 inhib-
itors had no effect on phosphorylation of serine 21, while inhibi-
tion of CDK1 did (Supplemental Figure 1B). To further prove the
direct role of CDK1 in phosphorylating C/EBPα on serine 21, we
performed a knockdown experiment. shRNA specifically target-
ing CDK1 was expressed from a retroviral vector in MOLM-14
cells. Following sorting of GFP+ cells, the effectiveness of CDK1
knockdown and its effect on phosphorylation of C/EBPα were
measured by Western blot. As shown in Figure 2C, normalization
for the β-actin levels demonstrated that CDK1 protein expression
was decreased by about 60%. There was no change in C/EBPα total
protein expression, but there was an approximately 50% decrease
in phosphoserine 21 containing C/EBPα species. Notably, CDK1
knockdown had no effect on the levels of active ERK1/2 kinase.
Taken together, our results identify serine 21 of C/EBPα as a sub-
strate for CDK1 kinase.
FLT3ITD can induce phosphorylation of C/EBPα on serine 21 by a
non-ERK1/2 pathway. 293T cells were transiently cotransfected with a
C/EBPα expression vector together with an empty MSCV-IRES-EGFP
vector (lanes 1 and 2) or MSCV-IRES-EGFP–expressing FLT3ITD
mutant N51 (lanes 3–8). Whole-cell extracts were analyzed by West-
ern blot. The same membrane was stained sequentially with antibod-
ies indicated to the right. Samples shown in lanes 1 and 3 were left
untreated. Cells were also treated with TPA for 15 minutes to activate
the ERK1/2 pathway (lanes 2 and 4) in the absence or presence
(lane 6) of the MEK1/2 inhibitor PD98059. Lanes 7 and 8 contain sam-
ples of cells treated with FLT3 inhibitors, MLN518 and PKC412, respec-
tively. All samples shown were analyzed on the same blot.
The Journal of Clinical Investigation http://www.jci.org Volume 122 Number 8 August 2012
CDK1 pathway is activated in FLT3ITD AML. Constitutively active
FLT3ITD receptor kinase has been shown to stimulate a number of
downstream pathways. To determine whether CDK1 can be super-
activated by FLT3ITD as well, the CDK1 kinase complexes were
immunoprecipitated from asynchronously growing FLT3ITD AML
cell lines and used in in vitro kinase reaction with histone H1 as a
substrate. As shown in Figure 3A, all untreated and DMSO-treated
cell lines exhibited high CDK1 activity. In contrast, the kinase
activity was significantly repressed by the treatment with the FLT3
inhibitor MLN518 (Figure 3A). To ascertain whether the effect of
FLT3ITD on CDK1 is direct or indirect, we examined the cell-cycle
distribution of MOLM-14 cells treated with MLN518 or DMSO.
Figure 3B shows that FLT3 inhibitor treatment led to a significant
decrease in mitotic cells with enrichment of G0-arrested cells. Taken
together, these data indicate that CDK1 is a downstream pathway
activated by FLT3ITD mutant receptors in an indirect fashion.
Pharmacological and genetic inhibition of
CDK1 activity in FLT3ITD AML relieves
differentiation block. To determine the
biological effect of CDK1 inhibition
in FLT3ITD AML cells, we cultured
MV4;11, MOLM-13, and MOLM-14 cells
with small molecule inhibitors targeting
CDK1: flavopiridol, roscovitine (both
being presently tested in clinical trials;
http://clinicaltrials.gov), and NU6102.
For comparison, we also treated these
cells with the FLT3 inhibitor MLN518,
which we previously demonstrated as
decreasing ERK1/2 activity and phos-
phorylation of C/EBPα (21), and herein
we showed that it can also inhibit CDK1
activity (Figure 3). Figure 4A shows
the Western blot results obtained for
MOLM-14 cells; comparable results were
found for MOLM-13 and MV4;11 cells
(data not shown). The treatments with
all CDK1 inhibitors tested as briefly as 18
hours resulted in substantial hypophos-
phorylation of C/EBPα. While exposure
to 10 μM NU6102 decreased the levels of
phosphoserine 21-C/EBPα by 40%–60%
in all 3 cell lines (Figure 4A and data not
shown), a decrease in serine 21 phosphor-
ylation by 53%–86% was achieved with
100 nM flavopiridol (Figure 4A and data
not shown) and 77%–89% by 25 μM rosco-
vitine (Figure 4A and data not shown).
Neither of these CDK1 inhibitors down-
modulated the ERK1/2 activity (Figure
4A). In addition to affecting CDK1 activ-
ity, all 3 compounds are also known to
exert inhibitory effect against a kinase
highly homologous to CDK1, CDK2.
To eliminate the involvement of CDK2
in phosphorylation of C/EBPα, we cul-
tured the cells in the presence of anoth-
er compound, PNU 112455A, which
inhibits CDK2 and CDK5 (IC50 = 2 μM
for both) but does not display activity
against other kinases at concentrations as high as 100 μM. In con-
trast with NU6102, PNU 112455A had no effect on phosphoryla-
tion of C/EBPα in all cells (Figure 4A and data not shown).
We have previously reported that C/EBPα, when hypophos-
phorylated on serine 21, displays granulocytic differentiation-
promoting activity (17, 21). We cultured FLT3ITD cell lines in the
presence of CDK1 inhibitors for up to 3 days and monitored their
morphology as well as the changes in maturation marker expres-
sion. As shown in Figure 4B, MOLM-14 cells treated with 10 μM
NU6102 acquired granulocytic morphology as early as on day 2
of the culture, with more marked effect seen on day 3. Similar
changes in cell morphology were also noted after 2 days of treat-
ment with 12.5 μM roscovitine or 100 nM flavopiridol, although
a rapid onset of apoptosis was more pronounced in those cultures
(Figure 4B and data not shown). No morphological changes were
observed when the cells were treated with a vehicle control, DMSO,
C/EBPα phosphorylation on serine 21 by CDK1 in vitro and in vivo. (A) In vitro kinase assay.
Active CDK1 enzyme was incubated with GST (lane 1) or GST-C/EBPα fusion proteins (lanes
2–5) in the presence of 32P. Lanes 2 and 3 contain WT C/EBPα-GST as a substrate. Lane 4
contains Ser21Ala mutant, and lane 5 contains Phe31Ala mutant. Proteins were resolved on
acrylamide gel and blotted to nitrocellulose membrane. Upper panel: autoradiograph of the mem-
brane. Lower panel: staining with the N-terminal C/EBPα antibody. (B) Effect of CDK1 overex-
pression or mitotic arrest on C/EBPα phosphorylation. 293T cells transfected with empty vector,
or HA-tagged CDK1 expression vector (lanes 1 and 2). Cells expressing empty vector (lane 3) or
a dominant negative form of CDK1 (lane 4; DN CDK1) were arrested at mitosis by nocodazole
treatment. All samples were analyzed on the same gel, and the Western blot was stained with
the antibodies indicated. (C) CDK1 silencing results in decreased C/EBPα phosphorylation on
serine 21. MOLM-14 cells were transduced with a CDK1 shRNA, sorted for EGFP expression,
and cultured for 2 days (total of 4 days of viral transduction). Whole-cell extracts were analyzed by
Western blotting with the indicated antibodies. Signals were quantified and graphed. Black bars
indicate the relative expression levels of CDK1 and C/EBPα proteins or ERK1/2 activity following
the knockdown (compared with white bars for negative controls; NC). Gray bar shows the level
of phospho–serine 21 C/EBPα relative to the total C/EBPα protein in cells with downregulated
CDK1 expression. sh, short hairpin.
2958 The Journal of Clinical Investigation http://www.jci.org Volume 122 Number 8 August 2012
or CDK2/CDK5 inhibitor PNU 112455A (Figure 4B). Comparable
results were obtained for MOLM-13 cells (data not shown), while
for MV4;11 cells, morphological changes were less pronounced
(data not shown) and not seen with non-FLT3ITD AML cells
(U937, KG1a, and K562; data not shown). All 3 FLT3ITD cell lines
treated with NU6102 also demonstrated downregulation of c-myc,
which is indicative of myeloid maturation (Figure 4C and data not
shown). In addition, NU6102 treatment led to a time-dependent
increase in the number of surface CD11b–expressing MOLM-14
cells (up to 60% on days 3 and 4; Figure 4D), which is in accord
with granulocytic differentiation. The ERK1/2 pathway was origi-
nally discovered to be responsible for phosphorylation of serine 21
(17, 21). Previously, we reported that inhibition of this pathway in
FLT3ITD-expressing MV4;11 cells decreased phosphorylation of
C/EBPα and induced granulocytic differentiation (21). In MOLM-
14 cells, inhibition of the ERK1/2 pathway also led to differentia-
tion with similar kinetics, but the effect of the inhibition of CDK1
was more potent, as measured by the downregulation of c-myc,
upregulation of CD11b surface expression, increase in myeloper-
oxidase (MPO) and lysozyme mRNA expression, and morphologi-
cal changes (Supplemental Figure 2).
Next, we determined whether the differentiation-promoting
effect of CDK1 inhibitors was dependent on C/EBPα expression.
We designed an shRNA lentiviral construct specifically targeting
CEBPA and demonstrated C/EBPα downregulation at the protein
level (Supplemental Figure 3). As expected, MOLM-14 cells trans-
duced with a nonsilencing control shRNA showed upregulation
of CD11b expression upon treatment with the CDK1 inhibitor
NU6102 in comparison with the DMSO-treated cells, whereas
MOLM-14 cells transduced with the C/EBPα shRNA did not
respond to the treatment (Figure 5A). These changes observed by
flow cytometry nicely correlated with the morphological analysis of
cytospun cells (Figure 5B). These data indicate that the differenti-
ation-inducing effects of CDK1 inhibitors are C/EBPα dependent.
To test whether specific knockdown of CDK1 protein expres-
sion would have the same effect as treatments with small molecule
compounds, MOLM-14 cells were transduced with viral particles
expressing CDK1 shRNA and EGFP. We anticipated that rapid
inhibition of CDK1, which is necessary for cell-cycle progression
through mitosis, may lead to growth arrest and apoptosis. We
assumed that the expression level of CDK1 shRNA might paral-
lel the level of EGFP expression and thus be in inverse correlation
with the expression of the endogenous CDK1 protein. In order to
provide the gradient of CDK1 knockdown, EGFP+ cells were sorted
into 3 populations with low, medium, and high EGFP intensities.
Each population was maintained in complete culture medium,
and cell morphology was monitored daily. On day 4, we harvested
30,000 cells from each population, made lysates, and analyzed the
degree of knockdown by Western blot. Day 4 was selected based
on our previous knockdown experiment, showing that this was
the earliest time point demonstrating detectable and significant
decrease in CDK1 protein expression. Figure 6A shows that after
normalization for the β-actin protein, there was not much differ-
ence between the inhibition of CDK1 protein expression in cells
expressing medium and high levels of EGFP. Cells with low EGFP
had a modest, but detectable decrease in CDK1. Morphological
examination showed that cells sorted for high EGFP were enlarged
in size, but did not show clear signs of myeloid differentiation.
They also became growth arrested and died on day 7 (data not
shown). Cells sorted for medium and low EGFP levels, on the other
hand, acquired morphological changes consistent with granulo-
cytic maturation on day 10 (Figure 6B), and this effect was stron-
ger for low EGFP–expressing cells. Medium EGFP–expressing cells,
in addition to myeloid maturation, were accompanied by severe
cell death (data not shown).
Finally, primary FLT3ITD leukemic samples collected at diag-
nosis from the peripheral blood of patients were treated with
NU6102. As expected, CDK1 inhibition led to a remarkable hypo-
Inhibition of constitutive activity of FLT3 decreases CDK1 kinase activ-
ity in FLT3ITD AML cells and slows down the cell-cycle progression.
(A) CDK1 was immunoprecipitated from FLT3ITD-expressing cell
lines (MV4;11, MOLM-13, and MOLM-14) and used in kinase reac-
tions with histone H1 as a substrate and 32P. Proteins were separated
on acrylamide gels and blotted to nitrocellulose membranes. The top
panel shows the autoradiograph of the blot, and the bottom panel
shows staining with anti-CDK1 antibody to assure that equal amounts
of CDK1 were immunoprecipitated. The assay was done either in the
absence of treatment (–; lanes 1, 4, and 7), after 24-hour treatment
with 0.1% DMSO (DMSO; lanes 2, 5, and 8), or after 24-hour treat-
ment with FLT3 inhibitor (MLN518; lanes 3, 6, and 9). The percentages
indicate the CDK1 activities remaining after FLT3 inhibitor treatment
(DMSO-treated samples were set to 100%). (B) Cell-cycle distribu-
tion of MOLM-14 cells treated with DMSO (left panel) or FLT3 inhibitor
MLN518 (right panel). Inhibition of FLT3 arrests majority of MOLM-14
cells at G0. Numbers indicate the percentage of cells in each quadrant.
The Journal of Clinical Investigation http://www.jci.org Volume 122 Number 8 August 2012
phosphorylation of C/EBPα in the FLT3ITD patient cells in 3 out
of 4 FLT3ITD patient samples (Figure 7). Figure 7 shows the West-
ern blot analysis after 24 hours of treatment, although the effect
was already noticeable after 10 hours. Phosphorylation of serine
21 was also observed in leukemic samples with the WT FLT3 gene
(Figure 7), most likely due to constitutive activation of the ERK
pathway. This is in agreement with the findings that the enhanced
activity of this pathway was detected in over 80% of the AML sam-
ples tested, regardless of their FLT3 genotype status (26, 27). Nev-
ertheless, the samples not harboring FLT3ITD mutations showed
negligible or no effect upon treatment with the CDK1 inhibitor
(Figure 7). In addition, 8 patient samples carrying FLT3ITD and
2 patient samples with the WT FLT3 receptor were also examined
for the expression of mature (CD11b, CD11c, CD14, G-CSF-R,
CD15, and CD16) and immature (CD33, CD34, CD38, and
CD133) cell-surface markers following treatment with NU6102.
Although each sample demonstrated different specific profiles
of the response, all patient samples carrying FLT3ITD showed a
general tendency to increase the expression of maturation mark-
ers and decrease that of immature markers (Figure 8A). In con-
trast, samples with WT FLT3 (patients C and D), which did not
show a decrease in serine 21 phosphorylation, did not show any
signs of differentiation (Supplemental Figure 4). Whenever we had
enough material (patients A, F, and G), we also tested the mRNA
expression of granulocyte-specific genes, such as CEBPE, CSF3R
(coding for G-CSF receptor), neutrophil elastase, gelatinase A, and
lysozyme. All 3 patient samples showed increases in CSF3R and
CEBPE expression after treatment with CDK1 inhibitor; patient F
demonstrated an increase in expression of all 5 genes (Figure 8B).
Moreover, the treatment of FLT3ITD-carrying specimens with
NU6102 for 7 days was accompanied by morphological changes,
suggesting granulocytic differentiation (Supplemental Figure 5).
Since CDK1 inhibition was associated with substantial cell death,
we wanted to make sure that lobing of the nuclei seen on cytospin
Granulocytic differentiation of FLT3ITD cells after CDK1 inhibition. (A) Inhibition of CDK1 but not CDK2/CDK5 leads to hypophosphorylation of
C/EBPα on serine 21. MOLM-14 cells were treated with CDK1 inhibitors NU6102 (lane 3), flavopiridol (flavo; lanes 6–7), or roscovitine (rosco;
lanes 8–9) for 18 hours at the concentrations indicated. For control, cells were treated with DMSO (lanes 1 and 5). Treatment with the FLT3 inhibi-
tor MLN518 (lane 2) was used for a positive control. Lane 4 shows cells cultured in the presence of CDK2/CDK5 inhibitor PNU 112455A (PNU).
Shown is a Western blot stained with antibodies indicated. (B) Morphological differentiation of MOLM-14 cells treated with CDK1 inhibitors. Cells
were either untreated or treated with 0.1% DMSO for 3 days (DMSO), 10 mM PNU112455A for 3 days (PNU112455A), 10 mM NU6102 for 3 days
(NU6102), 12.5 mM roscovitine for 2 days, or 100 nM flavopiridol for 2 days (flavo). Cytospin was stained with Wright-Giemsa. Red arrowheads
point to the cells displaying granulocytic maturation. Original magnification, ×40. (C) Downregulation of c-myc expression in response to CDK1
inhibition. MOLM-14 cells were grown in the presence of the indicated drugs for 18 hours and analyzed by Western blot. (D) Increase in CD11b
surface expression on MOLM-14 cells treated with CDK1 inhibitor. Cells were treated with CDK1 inhibitor NU6102 or vehicle control (0.1% DMSO)
for up to 4 days and analyzed daily for the expression of CD11b by flow cytometry. Left panel: histograms obtained on day 3. Right panel: y axis
indicates the percentages of CD11b+ cells during the time course.
2960 The Journal of Clinical Investigation http://www.jci.org Volume 122 Number 8 August 2012
preparations was caused by true differentiation, rather than apop-
tosis. Therefore, FLT3ITD leukemic samples treated with NU6102
were sorted for CD15+ cells and tested by the Wright-Giemsa meth-
od. As shown in Figure 8C, the same morphological forms were
observed and importantly, CD15+ cells were nearly 90% viable (Fig-
ure 8C). In summary, inhibition of CDK1 in FLT3ITD cells led to
an increase in C/EBPα function by its hypophosphorylation on
serine 21 and the relief of the differentiation arrest.
Activity of CDK1 is controlled by a multiple-step process, but ulti-
mately, CDK1 can be activated by binding to cyclin B1 (28, 29). To
determine how the constitutively active FLT3ITD receptor affects
CDK1 activity, MOLM-14 cells were untreated or treated with FLT3
inhibitor MLN518 or vehicle control DMSO and analyzed by West-
ern blot staining with anti–cyclin B1 antibody. As shown in Fig-
ure 9A, treatment of MOLM-14 cells with FLT3 inhibitor led to a
decreased expression of cyclin B1 protein. Conversely, introduction
of FLT3ITD into Ba/F3 cells led to about a 2-fold increase in total
cyclin B1 protein levels (Figure 9B). These data suggest a possible
involvement of cyclin B1 in the activation of CDK1.
Activating mutations in FLT3 receptor tyrosine kinase are among
the most common mutations in AML and indicate poor prognoses
(1, 2, 4, 12–14). Thus, development of small molecule inhibitors
specifically targeting FLT3 seemed to be a promising approach
for treating a large number of AML cases (15). Although different
activating mutations in FLT3 exhibit divergent sensitivities toward
different FLT3 inhibitors (30), the preclinical and clinical trials
involving the first generation of drugs (PKC-412, MLN518, CEP-
701, SU11248, and AC220) showed biologic activity and favorable
toxicity (15, 16, 31–36). However, in several cases, patients dem-
onstrated primary resistance to the drugs (3, 37). Alternatively,
an initial response was soon followed by emergence of second-
ary mutations, for example, mutations N676K and D835Y in
the kinase domain of FLT3 (38, 39). To bypass this problem, a
search for a new generation of FLT3 inhibitors is under way. In the
meantime, combination therapies with selective FLT3 inhibitors
and conventional cytotoxic chemotherapy are being investigated,
but those have been characterized by unacceptable toxicity and
poor tolerance (40). Alternative treatments of FLT3 mutant AML
may involve inhibitors of the downstream pathways activated by
FLT3ITD mutations. For example, we demonstrated before that
inhibition of the ERK1/2 pathway in FLT3ITD-expressing AML
cells leads to their differentiation (21). The multitude of down-
stream signaling pathways activated by mutant FLT3 receptors
(5–11) may increase the repertoire of possible drug combinations.
A majority of the FLT3ITD downstream pathways control sur-
vival and apoptosis, while ERK1/2 signaling plays a role in the dif-
ferentiation block by phosphorylating the C/EBPα transcription
factor on serine 21 and inhibiting its function (17, 21). Interest-
ingly, only a fraction of FLT3ITD patients exhibited activation of
the ERK1/2 pathway (22). Of note, due to technical difficulties
in examining the ERK activity in FLT3ITD leukemias, additional
studies are needed to determine whether activation of ERK can
serve as a specific biomarker of FLT3 signaling in primary leuke-
mias (22). Moreover, we observed serine 21 phosphorylation on
C/EBPα in cells with an FLT3ITD mutant receptor, which is dis-
abled in ERK1/2 activation (mutant N51; refs. 23, 24). We hypoth-
esized that a kinase other than ERK1/2 may be responsible for
C/EBPα phosphorylation and differentiation block. In this report,
we identified CDK1 (also known as CDC2) as the kinase specifically
modifying C/EBPα on serine 21 in AML with FLT3ITD mutations.
Thus, our data provide a potential molecular mechanism explain-
ing the maturation block in FLT3ITD cases without activation of
ERK1/2. However, in addition to ERK1/2, CDK1 is another modu-
lator of C/EBPα differentiation function, and we cannot discard
the contribution of other mediators to the differentiation block
seen in FLT3ITD AML. Further, our observations do not rule
out a potential interplay between ERK1/2 and CDK1 activity on
C/EBPα function in certain FLT3ITD AML cases. The FLT3ITD
and CDK1 connection was previously reported by Odgerel et al.
(41). While they reported that CDK1 is partially inactivated in
FLT3ITD AML cell lines, our work concludes that CDK1 can be
activated by FLT3ITD mutations. This apparent contradiction
could be explained by the use of different FLT3 inhibitors (PKC412
and MLN518, respectively) and the concentrations used, resulting
in either effects in apoptosis (41) or differentiation (this study).
Several studies described modulation of C/EBPα activity by
phosphorylation on various residues (17, 42–44). However, phos-
Downregulation of C/EBPα expression prevents the granulocytic dif-
ferentiation induced by CDK1 inhibitors. (A) MOLM-14 cells were trans-
duced with a nonsilencing control shRNA (NSC) or an shRNA-targeting
C/EBPα (C/EBPα sh) lentivirus. Cells were treated with either DMSO
control (D) or 5 mM NU6102 (NU). The y axis indicates the percent-
age of CD11b-positive cells after 4 days of culture. (B) Morphological
analysis of MOLM-14 cells upon C/EBPα silencing and CDK1 inhibitor
treatment. Cells infected with the NSC or the C/EBPα shRNA lentivirus
were treated for 4 days in the presence of 5 mM NU6102. Cytospins
were stained with Wright-Giemsa. Original magnification, ×40.
The Journal of Clinical Investigation http://www.jci.org Volume 122 Number 8 August 2012
phorylation of a single amino acid, serine 21, seems to have the
most remarkable effect by shifting the activity of C/EBPα from
a granulocytic differentiation-promoting factor (unphosphory-
lated) to a dominant negative form (phosphorylated) (17). Serine
21 can be phosphorylated by ERK1/2 (17, 21), p38 MAPK (20, 45),
and CDK1 (this report). It is the only phosphorylation site on
C/EBPα with clinical significance identified so far. Hyperphos-
phorylation of serine 21 in leukemic blasts blocks their matura-
tion (21). P38MAPK-mediated phosphorylation of serine 21-
C/EBPα acts as a switch inhibiting neutrophilic differentiation of
CD34+ progenitors while permitting their eosinophilic matura-
tion and may be responsible for disturbed neutrophilic develop-
ment in severe congenital neutropenia (45). In liver cells, however,
phosphorylation of serine 21 by p38 MAPK increases the activity
of C/EBPα on promoters of genes involved in gluconeogenesis,
thus possibly contributing to diabetes (20, 46).
CDK1 belongs to a family of cyclin-dependent kinases, which are
critical regulators of cell division. While individual CDK proteins
may substitute for each other’s function, gene-targeting experi-
ments demonstrated that CDK1 is the only family member whose
role in promoting mitosis cannot be substituted by any other CDK
(47). Misregulation of CDK1 expression/activity in solid tumors
is well documented, and several clinical trials with CDK1 inhibi-
tors are currently under way. In contrast, there is very little known
about the role of CDK1 during leukemogenesis. Higher expression
levels of CDK1 were detected in leukemic cells with del(5q) (48).
Also, a recent report described upregulation of CDK1 in leukemia
with translocation liposarcoma/ETS-related gene (TLS-ERG) and
attributed increased expression of CDK1 to the differentiation
block (49). Similarly, we found that in AML with constitutively
active FLT3 receptor, CDK1 can contribute to the maturation
block by inhibiting function of the transcription factor C/EBPα,
which is required for granulopoietic development. Our findings
open the door to the possibilities of using pharmacological inhibi-
tors of CDK1 in leukemia as well.
Studies described herein demonstrated that various CDK1
inhibitors affect C/EBPα phosphorylation and promote the dif-
ferentiation process to variable degrees. This might be due to
their broad spectrum of action; by inhibiting multiple pathways
at the same time, they can rapidly induce apoptosis. While we
believe that much of the activity of these inhibitors is through
their effects on C/EBPα phosphorylation, our experiments do not
rule out effects on other pathways as well. We also found that acti-
vation of the CDK1 pathway by FLT3ITD involves upregulation
of cyclin B1 rather than upregulation of CDK1 itself (49). While
these data suggest that cyclin B1 is involved in the activation
of CDK1, we cannot rule out that other CDK1 regulators, such
as Myt1 and cdc25c, could also be involved (41). NU6102 dem-
onstrated the best differentiation-promoting activity, perhaps
because of the higher specificity against CDK1 versus other CDKs.
The knockdown experiments are in accord with this hypothesis.
Cells expressing lower levels of EGFP and presumably lower levels
of shRNA showed more pronounced maturation, while the cells
with higher levels of EGFP (and presumably the highest levels of
shRNA) exhibited mainly apoptosis (this study).
In summary, we demonstrate that constitutive activation of
the FLT3 receptor can lead to abnormal activation of multiple
downstream signaling pathways (Figure 10), which are capable
of inhibiting the function of C/EBPα, contributing to the differ-
entiation block. Inhibiting either FLT3 receptor, MEK1 kinase,
or CDK1 can restore the activity of C/EBPα and induce myeloid
maturation of leukemic blasts.
Cell lines. Human AML lines carrying FLT3ITD mutations were kindly
donated by Yoshinobu Matsuo (MOLM-13, Fujisaki Cell Center, Hayas-
hibara Biochemical Labs, and the Kurashiki Medical Center, Kurashiki,
Okayama, Japan), Neill Giese (MOLM-14; Calistoga Pharmaceuticals,
Inc.), Stefan Heinrichs (MV4;11, Department of Pediatric Oncology, Far-
ber Cancer Institute, Boston, Massachusetts, USA), and David Sternberg
(Ba/F3-FLT3ITD, OSI Pharmaceuticals). MOLM-13, MOLM-14 (50), and
MV4;11 (CRL 9591; ATCC) as well as U937 (CRL 1593; ATCC) were grown
Induction of granulocytic differentiation following genetic downregula-
tion of CDK1 expression. (A) shRNA-mediated decreased expression
of CDK1 in MOLM-14. MOLM-14 cells were virally transduced with
CDK1-targetting shRNA (CDK1 sh) or negative control. Two days later,
populations expressing low (Lo), medium (Med), or high (Hi) GFP lev-
els were sorted. Following an additional 2 days of culture, an aliquot of
each cell population was lysed and analyzed by Western blot with anti-
CDK1 antibody (top panel). β-actin antibody was used for loading con-
trol. All samples were loaded on the same gel but were noncontiguous.
Quantifications of the band intensities are shown below. Neg, negative
for GFP. (B) Downregulation of CDK1 expression leads to granulocytic
maturation. Following sorting, cells were cultured for up to 8 more days
and their morphology was monitored daily on Wright-Giemsa–stained
slides. Original magnification, ×40.
2962 The Journal of Clinical Investigation http://www.jci.org Volume 122 Number 8 August 2012
in RPMI 1640 with 10% FBS. The murine bone marrow–derived IL-3–
dependent Ba/F3 cell line (51) was cultured in RPMI with 10% FBS and
10% WEHI-3B conditioned medium. A Ba/F3 stable line expressing the
FLT3ITD mutant N51 (24) was maintained in RPMI/10% FBS/10% WEHI-
3B conditioned medium and 750 μg/ml active G418. Human Embryonal
Carcinoma 293T (HEK 293T; CRL 11268, ATCC) and Phoenix-Ampho
Packaging (Orbigen) cell lines were cultured in DMEM with 10% FBS.
Patient samples. After informed consent was obtained, peripheral blood
samples of AML patients were collected at the time of diagnosis before ini-
tiation of treatment. Blasts and mononuclear cells were purified by Ficoll-
Hypaque (Nygaard) centrifugation and cryopreserved. Cells were thawed at
37°C, incubated on ice for 10 minutes, and washed twice in ice-cold HBSS.
Cells were precultured in EX-VIVO (BioWhittaker) medium supplemented
with 10 ng/ml human IL-3 (hIL-3), 10 ng/ml hIL-6, and 25 ng/ml hSCF
at 37°C for 45 minutes on 150 mm Petri dishes to remove adherent cells.
Suspension cells were collected and incubated at 37°C in the presence of
5 or 10 μM NU6102 or 0.1% DMSO (vehicle control).
Reagents. All inhibitors were prepared in DMSO. FLT3 inhibitors
MLN518 (CT53518; Millenium Pharmaceuticals) and PKC-412 (Biomol)
as well as CDK1 inhibitors NU6102 (Calbiochem/EMD Biosciences) and
flavopiridol (Sigma-Aldrich) were reconstituted at 10 mM. Roscovitine
was reconstituted at 25 mM. The CDK2/CDK5 inhibitor PNU112455A
(Calbiochem) was dissolved at 10 mM, and the MEK1 inhibitor PD98059
was reconstituted at 50 mM. TPA stock solution was prepared at a 1-mM
concentration. All reagents except MLN518 (kept at 4°C) were stored at
–20°C. A stock solution of nocodazole (Sigma-Aldrich) was prepared in
DMSO at 10 mg/ml.
Plasmids. MSCV-IRES-EGFP-FLT3ITD (N51) was described previously
(24). The GST-C/EBPα fusion expression vectors (containing 139 of the
N-terminal amino acids of either WT C/EBPα, S21A, or F31A mutations)
were described previously (21). WT and dominant negative (DN-CDK1)
isoforms of human CDK1 cDNAs were HA tagged at the C termini and
cloned in pCMV-neo-Bam expression vector (52). Liu Yang (Departments
of Orthopedics and Medicine/Hematology, University of Washington,
Seattle, Washington, USA) provided CDK1 siRNA lentiviral constructs.
The shRNA oligonucleotide (GGATTCCAGGTTATATCTCATCTCGA-
GATGAGATATAACCTGGAATCCTTTTTTT; targeting nt 350–370 of
the human CDK1 coding sequence) was cloned under the control of the
human H1 promoter in pNantx retroviral vector, which contains a GFP
reporter (described in ref. 53). The human CEBPA and a nonsilencing con-
trol shRNA sequences were cloned into the lentiviral vector pGhU6 con-
taining an EGFP reporter. The shRNA oligonucleotide sequence targeting
CEBPA was ACCCCGCCAAGAAGTCGGTGGACAAGAACATCAAGAGT-
GTTCTTGTCCACCGACTTCTTGGCTTTTTGGAA (930–954 nt), and the
nonsilencing control sequence was ACCCCATCTCGCTTGGGCGAGAG-
In vitro kinase assays. C/EBPα-GST fusion proteins were isolated from
BL21(DE3)pLys cells stimulated with 1 mM isopropyl-β-D-thio galacto-
pyranoside (IPTG) for 1 hour. Bacteria were then centrifuged at 1,500 g for
10 minutes, and bacterial pellets were subjected to a single cycle of freeze-
thawing. Lysed bacteria were suspended in 1 ml BugBuster Protein Extrac-
tion Agent (Novagen) and supplemented with 25 U of Benzonase Nuclease
(Novagen). Following a 5-minute incubation at room temperature and cen-
trifugation at 13,000 g for 20 minutes, the supernatant was collected and
incubated with 0.2 ml of Glutathione Sepharose 4FF beads (Amersham) for
1 hour at room temperature. Beads were pelleted and washed 4 times with
PBS and immediately suspended in in vitro kinase reaction mixture con-
taining [γ32P]ATP (10 Ci/mmol), purified CDK1 kinase (cat. P6020S; NEB),
and supplied reaction buffer. The reactions were carried out at 30°C for 10
minutes, then stopped by adding 4× Laemmli buffer and boiling samples at
100°C for 10 minutes. The products were resolved on SDS-PAGE, blotted
to nitrocellulose membrane, and analyzed by autoradiography. To control
for the amount of C/EBPα-GST protein, the same membrane was stained
with the N-terminal anti-C/EBPα antibody.
In vivo kinase assay. FLT3ITD AML cells were treated with 10 μM MLN518
or 0.1% DMSO for 24 hours. Equal numbers of cells were harvested and
Decreased C/EBPα phosphorylation upon CDK1 inhibi-
tion in FLT3ITD AML patients. Four patient samples with
FLT3ITD mutations (patients A, B, F, and H) and 4 patient
samples with WT FLT3 receptor (patients C, D, K, and L)
were cultured in vitro for 24 hours in the presence of 10 mM
NU6102 or 0.1% DMSO. Whole-cell lysates were tested
by Western blot. Signals for serine 21–phosphorylated
proteins were normalized for the total C/EBPα protein and
plotted (shown below Western blot; D, DMSO, N, NU6102).
The numbers above bars indicate the percentage of
phosphorylated C/EBPα species compared with samples
treated with DMSO (set to 100%).
The Journal of Clinical Investigation http://www.jci.org Volume 122 Number 8 August 2012
lysed in RIPA lysis buffer. Active CDK1 kinase complexes were immunopre-
cipitated with anti-CDK1 antibody and captured on Immobilized Protein A
beads (IPA-300; Repligen). Following 3 washes with ice-cold PBS and
1 wash with ADBI CDK1 kinase reaction buffer (Upstate Biotechnology),
the CDK1-containing beads were used in kinase reactions in ADBI con-
taining purified histone H1 as a substrate and [γ32P]ATP (10 Ci/mmol).
The reactions were carried out at 30°C for 10 minutes, stopped by boiling
in Laemmli buffer for 10 minutes, subjected to SDS-PAGE electrophore-
sis, and transferred onto nitrocellulose membranes. The incorporation of
32P into the substrate was examined by autoradiography, and the amounts
of immunoprecipitated CDK1 kinase were compared by staining the same
membrane with anti-CDK1 antibody.
Mitotic arrest. U937 cells were arrested at mitosis by incubation in the
presence of 100 μg/ml nocodazole for 16 hours. Cells were released
from the block by washing twice and by subsequent culture in complete
medium without nocodazole.
Granulocytic differentiation of FLT3ITD cells after inhibition of CDK1 activity. (A) Inhibition of CDK1 in patient samples with FLT3ITD induces
granulocytic differentiation. Blood specimens from FLT3ITD AML patients (patient A and patient B; same as shown in Figure 7) were cultured in
the presence of 0.1% DMSO or 10 mM NU6102 for 7 days. Cell aliquots were stained with anti-CD11c, anti-CD15, anti-CD14, anti-CD11b, anti-
CD16, anti–G-CSF-R, anti-CD33, anti-CD133, anti-CD34, and anti-CD38 antibodies and analyzed by flow cytometry. The y axes indicate the
percentage of positive cells. (B) Patient samples were treated as in A for 5 days and analyzed for mRNA expression of granulocytic cell-surface
markers by quantitative RT-PCR. The y axes indicate relative expression to GAPDH. (C) CD15+ cells with granulocytic-like morphology are viable.
CD15 expression on FLT3ITD AML patient A sample treated with DMSO (gray) or 5 mM NU6102 (white) during 10 days (left panel). Number
indicates the percentage of CD15+ cells upon NU6102 treatment. CD15+ cells were sorted, cytocentrifuged, and stained with Wright-Giemsa
method (middle panel). Original magnification, ×40. CD15+ cells were also stained with annexin V and PI to determine the extent of their viability
(right panel). The numbers in each quadrant indicate percentages of viable (lower left), early apoptotic (lower right), late apoptotic (upper right),
and necrotic cells (upper left).
2964 The Journal of Clinical Investigation http://www.jci.org Volume 122 Number 8 August 2012
hours later, FLT3 inhibitors (0.5 μM MLN518 and 0.2 μM PKC-412) were
added and the cells were cultured for an additional 16 hours. PD98059 (at
100 μM) was added 1.5 hours before the cell harvest, and TPA (at 10 nM) was
added for the last 15 minutes of the culture. At the end of the treatments,
cells were collected and lysed in 600 μl of 1× Laemmli Sample Buffer. Then
30 μl per lane were loaded on PAGE/SDS gels (7.5%).
Retroviral transductions. Retroviruses were produced by transfecting Phoe-
nix A cells. Virus-containing supernatants were collected at 48 and 72
hours after transfection, filtered through 0.45-μm filter, and concentrated
using a Centricon Plus-70 100000 MWCO column (Millipore).
Retroviral transduction was performed in culture dishes (Falcon 1008;
BD) coated with 12 μg/ml RetroNectin during 2 consecutive days using a
MOI between 2.5 and 5. The EGFP-expressing cells were enriched by sort-
ing on day 3 and cultured for up to 8 additional days.
Lentiviral transductions. 293T cells were cotransfected using Lipofectamine
2000 with C/EBPα shRNA in pGhU6 vector or the shRNA control and len-
tiviral constructs Gag-Pol and Env. Virus was harvested and concentrated
using a Centricon Plus-70 100000 MWCO column (Millipore). A single len-
tiviral transduction was performed in the presence of polybrene (8 μg/ml)
(Sigma-Aldrich). MOLM-14 cells were infected with an MOI of 5. One day
after transduction, cells were treated with either 0.01% DMSO control or 5 μM
NU6102. Infected cells were determined by EGFP flow cytometry analysis.
Morphological examination. About 104 cells were spun at 500 g for 5 min-
utes onto glass slides and Wright-Giemsa stained with Diff-Quik solu-
tions (Dade Behring).
Western blot. Typically, 5 × 106 cells were spun (1 K, 5 minutes), washed
in PBS, lysed in 400 μl of 1× Laemmli Sample Buffer, and boiled at 100°C
for 10 minutes. From 30 to 40 μl of each lysate was loaded on 7.5% SDS-
PAGE gels and proteins transferred to nitrocellulose membranes. Following
blocking in 5% milk/TBST (TBST: 25 mM Tris-HCl pH 7.4, 137 mM NaCl,
2.7 mM KCl, 0.1% Tween 20), membranes were stained with primary anti-
bodies diluted in 5% BSA/TBST/0.1% sodium azide overnight at 4°C and
then with HRP-conjugated secondary antibodies at room temperature for
1 hour. Signals were detected by enhanced chemiluminescence and quan-
tified by ImageQuant software (Molecular Dynamics). The primary anti-
bodies were goat N-terminal C/EBPα (N-19; 1:1,000; sc-9315, Santa Cruz
Biotechnology Inc.), rabbit phospho–Ser21-C/EBPα (1:1,000; #2841, Cell
Signaling Technology), goat C-terminal C/EBPα (C-18, 1:1,000; sc-9314,
Santa Cruz Biotechnology Inc.), rabbit C/EBPα (14AA, 1:1,000; sc-61, Santa
Cruz Biotechnology Inc.), rabbit CDK1 (1:1,000; PC25, Calbiochem), rab-
bit phospho-(T202/Y204)-ERK1/2 (1:1,000; #9101, Cell Signaling Technol-
ogy), panERK (1: 1,000; #610123, BD Transduction Laboratories), cyclin A
(C-19; sc-596) β-actin (1:10,000; A5441, Sigma-Aldrich), and β-tubulin
(1:4,000 clone 2-28-33; T5293, Sigma-Aldrich). All secondary antibodies
were HRP conjugated (Santa Cruz Biotechnology Inc.) and diluted 1:5,000
for rabbit-HRP, 1:3,000 for mouse-HRP, and 1:2,000 for goat-HRP.
Transfections. For transient expression, 293T cells were plated out at 2 × 105
cells per well on 6-well plates and transfected by 5 μl of TransFectin (Bio-
Rad) complexed with 2 μg of MSCV-IRES-EGFP or MSCV-FLT3ITD-IRES-
EGFP (mutant N51; ref. 24) and 0.5 μg of pcDNA3-WT C/EBPα (21). Five
The activity of CDK1 in FLT3ITD-expressing MOLM-14 cells is cor-
related with cyclin B1 protein levels. MOLM-14 cells were left untreated
(U) or treated for 24 hours with either 0.05% DMSO or 5 mM MLN518
FLT3 inhibitor (MLN), and whole-cell lysates were analyzed by West-
ern blot with antibodies indicated on the right. (A) Cyclin B1 protein
levels decrease upon inhibition of FLT3 receptor in MOLM-14 cells.
(B) Forced expression of FLT3ITD mutant (N51) in BaF3 cells leads to
2.1-fold increase in cyclin B1.
Effect of constitutive activation of the FLT3 receptor in leukemo-
genesis. Activation of FLT3 receptor by ITD mutations (stars)
promotes cell survival and inhibits apoptosis by activation of
STAT3, STAT5, and AKT. Differentiation of myeloid precursors
is mediated by the C/EBPα transcription factor, which exhibits
full maturation-promoting activity when hypophosphorylated
on serine 21. Activation of ERK1/2 and/or CDK1 (via increased
expression of cyclin B [Cycl B]) leads to hyperphosphoryla-
tion of C/EBPα, which abolishes its function and results in a
differentiation block. Broken arrows indicate a likely cascade
of downstream pathways from the activated FLT3 leading to
upregulation of c-myc and cyclin B. Pharmacological inhi-
bition of either the FLT3, MEK1,or CDK1 pathway results in
decreased phosphorylation of C/EBPα on serine 21, increase
in its activity, and induction of granulopoiesis.
The Journal of Clinical Investigation http://www.jci.org Volume 122 Number 8 August 2012
Apoptosis assay. The analysis of apoptotic cells was performed using
annexin V–FLUOS kit (Roche) according to the manufacturer’s protocol.
Simultaneous labeling with propidium iodide (PI) was used for exclusion
of the necrotic cells.
Cell-cycle analysis. Cells were suspended in phosphate-citrate buffer
solution with 0.02% saponin for permeabilization and then incubated
with 20 μg/ml Hoechst 33342 (Invitrogen) and 1 μg/ml Pyronin Y
(Sigma-Aldrich). Incorporation of Hoechst 33342 and Pyronin Y were
measured by flow cytometry.
Study approval. Patients’ informed consent was obtained in accordance
with the Declaration of Helsinki. The study was approved by the Institu-
tional Review Board: Committee on Clinical Investigations of Beth Israel
Deaconess Medical Center.
We thank Karen O’Brien for useful suggestions and Christopher
Hetherington for technical assistance. We also thank Yoshinobu
Matsuo for MOLM-13, Neill Giese for MOLM-14, Stefan Hein-
richs for MV4;11, and David Sternberg for Ba/F3-flt3ITD cell
lines. Liu Yang provided CDK1 siRNA lentiviral constructs. We
thank members of the Tenen and Gary Gilliland laboratories for
many useful discussions and Mary Singleton and Toya Dessesaure
for help in preparation of the manuscript. This research was sup-
ported by grants to D.G. Tenen from the NIH (P01 CA66996, P01
DK080665, and R01 CA 118316).
Received for publication April 15, 2010, and accepted in revised
form June 7, 2012.
Address correspondence to: Daniel G. Tenen, Center for Life Sci-
ences, 3 Blackfan Circle, Room 437, Boston, Massachusetts 02115,
USA. Phone: 617.735.2205; Fax: 617.735.2222; E-mail: dtenen@
Britta Will’s present address is: Albert Einstein College of Medi-
cine, New York, New York, USA.
Flow cytometry. Cells were washed once in PBS and blocked in 2% FBS/
PBS on ice for 15 minutes. Surface staining was performed on ice for
30–40 minutes followed by 2 washes with PBS. Antibodies used were as
follows: PE-conjugated anti-human CD11b (#555388; BD Biosciences —
Pharmingen), PE-Cy5–conjugated anti-human CD11b (used in MOLM-
14 cells transduced with pGhU6; #301308, Biolegend), FITC-conjugated
anti-human CD11c (#11-0116-73; eBioscience), FITC-conjugated anti-
human G-CSF–R/CD114 (# FAB381F; R&D Systems), APC-conjugated
anti-human CD14 (# 561383; BD Biosciences), Pacific Blue–conjugated
anti-human CD15 (#57-0159-73; eBioscience), eFluor605NC-conjugated
anti-human CD16 (#93-0168-41, eBioscience), PE Cy7–conjugated anti-
human CD33 (#25-0338-42, eBioscience), APC-conjugated anti-human
CD34 (#343510, Biolegend), PE-Cy7–conjugated anti-human CD38 (#25-
0389-41, eBioscience), and biotin-conjugated anti-human CD133 (#13-
1338, eBioscience). Exclusion of dead cells was done by addition of DAPI.
Cell sorting was performed using a FACSAria cell sorter, and immuno-
phenotyping was done on an LSRII flow cytometer (BD Biosciences). Data
were analyzed with FlowJo software (Treestar Inc.).
Quantitative RT-PCR. RNA was isolated by TRI Reagent (MRC Inc.),
treated with DNaseI, and reverse-transcribed into cDNA (Invitrogen).
Quantitative RT-PCR was performed using iQ Sybr Green Supermix (Bio-
Rad). Amplification was done with a Corbett Rotor Gene 6000 (QIAGEN)
using the following parameters: 95°C (10 minutes), 45 cycles of 95°C (15 s)
and 60°C (1 minute). Primer sequences were as follows: human GAPDH
F: 5′-CCACATCGCTCAGACACCAT-3′; human GAPDH R: 5′-CCAG-
GCGCCCAATACG-3′; human G-CSF-R F: 5′-TTTCAGGAACTTCTCTT-
GACGAGAA-3′; human G-CSF-R R: 5′-CGAGCCGAGCCTCAGTTTC-3′;
human C/EBPε F: 5′-CTCCGATCTCTTTGCCGTGAA-3′; human
C/EBPε R: 5′-TGGGCCGAAGGTATGTGGA-3′; human gelatinase A
F: 5′-GTGGGACAAGAACCAGATCACAT-3′; human gelatinase A
R: 5′-GTCTGCCTCTCCATCATGGATT-3′; human neutrophil elastase
F: 5′-CCACCCGGCAGGTGTTC-3′; human neutrophil elastase R:
5′-GTGGCCGACCCGTTGAG-3′; human MPO F: 5′-AGACCTGCTG-
GAGAGGAA-3′; human MPO R: 5′-CGCAGCCGCTTGACTTG-3′;
human lysozyme F: 5′-GCTGCAAGATAACATCGCT-3′; human lysozyme
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