The Journal of Experimental Medicine
JEM © The Rockefeller University Press $8.00
Vol. 203, No. 2, February 20, 2006 371–381 www.jem.org/cgi/doi/10.1084/jem.20052242
Acute myelogenous leukemia (AML) can be
defi ned as an accumulation of immature mye-
loid cells in the bone marrow and blood result-
ing from dysregulation of normal proliferation,
diff erentiation, and apoptosis. AML is the most
common type of leukemia in adults and occurs
in approximately one third of newly diagnosed
patients. Multiple genetic defects have been
implicated in the pathogenesis of AML (1),
such as chromosomal deletions or additions,
and chromosomal translocations resulting in
production of in-frame fusion proteins. Based
on current detection techniques, up to 45% of
AML cases show normal karyotype (2); thus, in
those cases, point mutations or small rearrange-
ments may aff ect critical genes. One such gene,
which is mutated in up to 30% AML cases, is
the FLT3 receptor tyrosine kinase gene (3).
The most common (20–25% AML patients)
form of mutations in FLT3 are small in-frame
internal tandem duplications (ITDs) in the jux-
tamembrane domain (3–5). In ?7% of AML
cases, point mutations in aspartic acid 835 in
the kinase domain have been reported as well
(6, 7). Both types of mutations result in the
constitutive activation of the FLT3 receptor
and abnormal activation of the downstream
pathways: Stat5, Stat3, Akt, and extracellular
signal–regulated kinase (ERK)1/2 (8–11). Be-
cause FLT3 is normally expressed in early pre-
cursors and plays role in proliferation and
diff erentiation of hematopoietic progenitors
<doi>10.1084/jem.20052242</doi><aid>20052242</aid>Block of C/EBP훂 function by
phosphorylation in acute myeloid
leukemia with FLT3 activating mutations
Hanna S. Radomska,1 Daniela S. Bassères,1 Rui Zheng,3 Pu Zhang,1
Tajhal Dayaram,1 Yukiya Yamamoto,1 David W. Sternberg,2
Nathalie Lokker,4 Neill A. Giese,4 Stefan K. Bohlander,5,6
Susanne Schnittger,5 Marie-Hélène Delmotte,7 Roger J. Davis,7
Donald Small,2 Wolfgang Hiddemann,5,6 D. Gary Gilliland,2
and Daniel G. Tenen1
1Beth Israel Deaconess Medical Center/Harvard Medical School and 2Howard Hughes Medical Institute/Brigham and Women’s
Hospital, Boston, MA 02115
3Johns Hopkins University School of Medicine, Baltimore, MD 21231
4Millennium Pharmaceuticals, Inc., San Francisco, CA 94080
5Laboratory of Leukemia Diagnostics, Department of Internal Medicine III and 6CCG Acute Leukemias, GSF National Research
Center of Environment and Health, D-81377 Munich, Germany
7Howard Hughes Medical Institute and Program in Molecular Medicine, University of Massachusetts Medical School,
Worcester, MA 01655
Mutations constitutively activating FLT3 kinase are detected in ? ?30% of acute myelog-
enous leukemia (AML) patients and affect downstream pathways such as extracellular
signal–regulated kinase (ERK)1/2. We found that activation of FLT3 in human AML inhibits
CCAAT/enhancer binding protein 훂 (C/EBP훂) function by ERK1/2-mediated phosphoryla-
tion, which may explain the differentiation block of leukemic blasts. In MV4;11 cells,
pharmacological inhibition of either FLT3 or MEK1 leads to granulocytic differentiation.
Differentiation of MV4;11 cells was also observed when C/EBP훂 mutated at serine 21 to
alanine (S21A) was stably expressed. In contrast, there was no effect when serine 21 was
mutated to aspartate (S21D), which mimics phosphorylation of C/EBP훂. Thus, our results
suggest that therapies targeting the MEK/ERK cascade or development of protein therapies
based on transduction of constitutively active C/EBP훂 may prove effective in treatment of
FLT3 mutant leukemias resistant to the FLT3 inhibitor therapies.
Daniel G. Tenen:
Abbreviations used: AML, acute
binding protein α; ER, estrogen
receptor; ERK, extracellular
signal–regulated kinase; ITD,
internal tandem duplication;
MAP, mitogen-activated pro-
tein; NBT, Nitro blue tetrazo-
lium; PDGFR, platelet-derived
growth factor receptor.
H.S. Radomska and D.S. Bassères contributed equally to
The online version of this article contains supplemental material.
372 INHIBITION OF C/EBPα FUNCTION IN FLT3 MUTANT AML | Radomska et al.
(12, 13), it is not surprising that constitutive activation of
FLT3 contributes to development of AML. AML patients
with FLT3 mutations have poor prognosis (14–19). There-
fore, small molecule inhibitors that specifi cally target FLT3
activity are undergoing clinical trials (20–23), but so far they
have produced rather disappointing results. Because FLT3
regulates an intricate signaling network consisting of multiple
downstream eff ectors, identifi cation of the critical FLT3 tar-
gets involved in mediating the leukemic phenotype will pos-
sibly lead to the identifi cation of novel alternative therapeutical
targets for treatment of activated FLT3 leukemias.
Another critical gene involved in the pathogenesis of
AML is the CCAAT/enhancer binding protein α (C/EBPα).
C/EBPα is a leucine zipper transcription factor that is impor-
tant for normal myeloid cell diff erentiation. Within the he-
matopoietic system, expression of C/EBPα is detectable in
early myeloid precursors and is up-regulated as they commit
to granulocytic diff erentiation pathway and mature (24, 25).
Consistent with this expression pattern, mice lacking C/
EBPα have no mature neutrophils, but rather accumulation
of myeloblasts in the bone marrow (26). Conversely, overex-
pression of C/EBPα in precursor cell lines triggers neutro-
philic diff erentiation (24, 27–29). Several studies from our
group and others’ showed that expression or function of C/
EBPα is inactivated in various types of leukemia (AML and
CML) by diverse molecular mechanisms (30–40). Impor-
tantly, provision of fully functional C/EBPα into leukemic
cells could restore their diff erentiation program (24, 28, 31).
Recently, we have found that C/EBPα can be directly
phosphorylated by ERK1/2 on S21, which aff ects the ability
of C/EBPα to induce diff erentiation (28). Ectopic expression
of the phosphomimetic C/EBPα mutant (S21D) inhibited
granulocytic diff erentiation (28). In the present work, we
provide evidence that the activating mutations in FLT3 in
AML patients and cell lines inactivate C/EBPα function by
ERK1/2-mediated phosphorylation on S21. Either allevia-
tion of ERK1/2 activity or ectopic expression of a function-
ally active mutant of C/EBPα (S21A) in FLT3 ITD-expressing
cells rescues myeloid diff erentiation. Thus, we provide a new
molecular mechanism by which constitutively active FLT3
contributes to the pathogenesis of leukemia.
Activation of FLT3 leads to hyperphosphorylation
of C/EBP훂 on serine 21
We hypothesized that the diff erentiation block in AML
with FLT3 activating mutations is mediated by compro-
mised expression and/or function of C/EBPα. Therefore,
we analyzed C/EBPα mRNA and protein expression in fi ve
human FLT3 mutant AML cell lines. Three lines (MOLM-
13, MOLM-14 [reference 41], and MV4;11 [references 42,
43]) carry ITD mutations (44) and two (MonoMac1 and
MonoMac6 [reference 45]) have an activating point muta-
tion (V592A) in the juxtamembrane domain (46). All cell
lines expressed easily detectable C/EBPα mRNA (Fig. 1)
and protein (Fig. 2, C and F, and not depicted). During
the treatment with a specifi c FLT3 inhibitor, MLN518 (47),
C/EBPα mRNA levels did not change signifi cantly (Fig. 1).
Considering that all fi ve cell lines expressed relatively high
levels of C/EBPα, which were not up-regulated by MLN518
treatments (Figs. 1 and 2; not depicted), it appears that the
suppression of C/EBPα expression by mutated FLT3 is not
a general mechanism of the diff erentiation block in human
AML cell lines.
We have recently reported that the function of C/EBPα
is inhibited by phosphorylation on S21 by ERK1/2 (28). In-
terestingly, ERK1/2 is one of several downstream signaling
pathways activated by FLT3 (9, 11). Therefore, we sought
to determine if C/EBPα protein is phosphorylated upon ac-
tivation of FLT3. As shown in Fig. 2 A, the NH2-terminal
region of C/EBPα (amino acids 1–119) is specifi cally phos-
phorylated by ERK1/2 and not by other mitogen-activated
protein (MAP) kinases. To determine if activation of FLT3
increases phosphorylation of endogenous C/EBPα, THP-1
cells, which express C/EBPα and wild-type FLT3 receptor,
were serum starved and stimulated with FLT3 ligand. West-
ern blot with phospho-specifi c antibodies (Fig. 2 B) showed
that stimulation as short as 5 min resulted in a rapid increase
Figure 1. Human AML cell lines with activating mutations in FLT3
express C/EBP훂 mRNA. (A) Cell lines (top) were not treated (0) or
treated with 1 μM MLN518 for 8 or 24 h (indicated above the lanes).
(top) Hybridization of the Northern blot to a human C/EBPα probe.
(bottom) A control hybridization to 18S ribosomal RNA. RNA from KG1a
and U937 cells served as negative and positive controls, respectively.
(B) The same RNA samples were analyzed in triplicate by TaqMan
Real-Time PCR and normalized to 18S RNA.
JEM VOL. 203, February 20, 2006
Figure 2. Activation of FLT3 in human AML cells leads to C/EBP훂
protein phosphorylation on S21 by the ERK1/2 pathway. (A) COS7
cells were untreated or treated with UV-C (60 J/m2; UV) or 100 nM PMA
and incubated for 60 min. Endogenous c-Jun NH2-terminal protein ki-
nase (JNK), p38, and ERK were immunoprecipitated and used for in vitro
kinase assays. Control tests showed that ERK phosphorylated Elk1,
JNK-phosphorylated c-Jun, and p38 MAP kinase phosphorylated ATF2.
(B) THP-1 cells expressing wild-type FLT3 were serum starved for 6 h (0),
or starved and stimulated with 100 ng/ml FLT3 ligand (FL) for 5 and
10 min before harvest. Western blot of whole cell extracts was sequen-
tially stained with pS21-C/EBPα, C/EBPα, pThr202/Tyr204-ERK1/2, and
β-tubulin antibodies. (C) Human AML cell lines with mutant FLT3 have
increased phosphorylation of C/EBPα on S21. All cell lines were serum
starved for 7 h in the absence (−) or presence (+) of 1 μM MLN518.
Western blot was analyzed as in B. (D) MV4;11 cells were serum starved
in the absence (−) or presence of the FLT3 inhibitors AG1296 and
MLN518 at concentrations marked above the lanes, or with vehicle con-
trol (DMSO). Western blot analyses were done as in B. (E) Phosphoryla-
tion status of C/EBPα in MV4;11 and MOLM-13 cells as a dose response
to MLN518. Cells were serum starved for 7 h in the presence of MLN518
at concentrations indicated above the lanes. Western blot was stained as
in B. (F) Time course of FLT3 inhibition and its effect on C/EBPα phos-
phorylation. At each time point, MV4;11, MOLM-13, or MOLM-14 cells
were serum starved for a total period of 8 h. MLN518 (1 μM) was added
for the fi nal hours (indicated above the lanes) of the treatment. For ex-
ample, “1 hr” means that the cells were starved for 7 h in the absence of
the inhibitor, and for 1 fi nal hour in its presence. Western blot staining
was as described previously. (G) Decrease of pS21-C/EBPα by inhibition
of FLT3 activity in AML patient samples. Leukemic blasts from bone mar-
row (patient no. 592), or peripheral blood (patient no. 667) were cul-
tured in the absence (0), or presence (1) of MLN518 for 6 h. Western
blot of whole cell extracts was analyzed with antibodies recognizing
activated FLT3 (P-FLT3), phosphorylated C/EBPα (P-Ser21-C/EBPα), or
total C/EBPα protein. MV4;11 cells were included for control. Quantifi ca-
tion of C/EBPα phosphorylation normalized to the total C/EBPα protein
is shown (bottom).
374 INHIBITION OF C/EBPα FUNCTION IN FLT3 MUTANT AML | Radomska et al.
in levels of phosphorylated (activated) ERK1/2 as well as C/
EBPα on S21. We also tested whether constitutive activation
of mutant FLT3 in AML also results in phosphorylation of
C/EBPα. All fi ve cell lines tested (Fig. 2 C and not depicted)
demonstrated phosphorylation of C/EBPα on S21, which
correlated with activation of ERK1/2. Treatment of these
cell lines with MLN518 led to a dramatic reduction in levels
of pS21C/EBPα, coincident with a decrease in pERK1/2
levels (Fig. 2 C) and pFLT3 (not depicted). Similarly, treat-
ment of MV4;11 cells with another FLT3 inhibitor, AG1296
(48), showed a dose-dependent decrease in phosphorylation
of C/EBPα and ERK1/2 (Fig. 2 D). The absolute levels of
C/EBPα protein in all fi ve lines were not signifi cantly al-
tered by the treatments with any of the inhibitors. Fig. 2 E
shows that MLN518 reduces pS21-C/EBPα in MV4;11
and MOLM-13 cells in a dose-dependent manner. When
normalized for the levels of C/EBPα protein, as little as 0.1
μM MLN518 led to a threefold decline in pS21-C/EBPα
levels in both lines, and as great as a 50-fold decrease was
noted for treatment of MOLM-13 cells with 1 μM inhibi-
tor. A time course experiment using FLT3 ITD cell lines
was performed to test the kinetics of the MLN518 eff ect
on C/EBPα phosphorylation (Fig. 2 F). The inhibition of
S21 phosphorylation was apparent within the fi rst 2 h and
reached maximum between 4 and 6 h. The treatments had
no eff ect on the overall levels of C/EBPα protein, but corre-
lated with activation of ERK1/2. Activation of ERK1/2 and
hyperphosphorylation of C/EBPα were also seen in 293T
cells transiently cotransfected with FLT3 ITD and wild-type
C/EBPα expression vectors; these eff ects were abrogated by
MLN518 (Fig. S1, available at http://www.jem.org/cgi/
content/full/jem.20052242/DC1), indicating a causal role of
Next, we sought to determine whether constitutive ac-
tivation of FLT3 aff ects phosphorylation of C/EBPα in
AML patients with FLT3 ITD. Primary cells from BM (pa-
tient no. 592) or PBMCs (patient no. 667) were incubated
in the absence or presence of MLN518, and whole cell ly-
sates were analyzed by Western blot shown in Fig. 2 G.
Staining with pY591-FLT3 antibody, which detects one of
the tyrosine residues phosphorylated as a result of activation
(unpublished data), showed that treatments with MLN518
led to an ?50–60% decrease in FLT3 activation. Inhibition
of FLT3 in patient samples was also paralleled by a 40–60%
decrease in pS21-C/EBPα. Control treatment with DMSO
had no eff ect on phosphorylation of FLT3 or C/EBPα (un-
Pharmacological inhibition of FLT3 activation by MLN518
triggers myeloid differentiation of MV4;11 cells
We showed previously that mutating S21 in C/EBPα to D
mimics constitutive phosphorylation of S21, inhibits the dif-
ferentiation-promoting activity of C/EBPα, and can inhibit
retinoic acid-mediated granulocytic diff erentiation of U937
cells (28). Therefore, we hypothesized that constitutive acti-
vation of FLT3 may result in diff erentiation block through
phosphorylation and thus, inactivation of C/EBPα. Because
MLN518 decreases the level of FLT3 activation and increases
the pool of C/EBPα protein not phosphorylated on S21, we
expected that the treatment with this inhibitor might result
in the onset of diff erentiation. As shown in Fig. 3 A, the 3-d
course of culture of MV4;11 cells with MLN518 in the pres-
ence of serum led to a dramatic decrease in phosphorylation
of C/EBPα, noted as early as on day 1. In contrast, up to 3 d
of DMSO treatment had no eff ect on C/EBPα phosphory-
lation. After 2–3 d of MLN518 treatment, MV4;11 cells ex-
hibited indentation and bending of the nuclei and an increase
in cytoplasmic/nuclear ratio characteristic of band and meta-
myelocyte stages of granulocytic diff erentiation (Fig. 3 B).
Cells treated with 0.05% DMSO for the same period of time
remained blastic. Morphological changes were accompanied
by respiratory burst activity determined by Nitro blue tetra-
zolium (NBT) reduction assay (Fig. 3 B), another indicator of
granulocytic diff erentiation. Although growth tests indicated
that the IC50 of MLN518 for this cell line is between 0.3
and 1 μM (unpublished data), we noted diff erentiation when
MV4;11 cells were cultured in 5 or 10 μM MLN518, but not
in 1 μM (unpublished data). To eliminate the possibility that
these higher concentrations of MLN518 aff ects other FLT3-
related receptor kinases, such as c-kit or platelet- derived
growth factor receptor (PDGFR), we treated MV4;11 and
MOLM-13 cells with STI571 (49), and found no eff ect on
phosphorylation of C/EBPα (Fig. S2, available at http://
One of the early events essential for granulocytic diff er-
entiation is down-regulation of c-myc expression through
C/EBPα-mediated inhibition of E2F function (50, 51). Very
rapid down-regulation of c-myc protein was seen in all tested
FLT3 mutant cell lines in response to MLN518 (Fig. 3 C; not
depicted). However, because c-myc protein stability is re-
gulated by ERK1/2-mediated phosphorylation (52), we also
investigated c-myc mRNA levels to eliminate the possibility
that the decrease in c-myc protein is a result of its dephos-
phorylation and decrease in its stability. Fig. 3 C shows that
the c-myc down-regulation occurs also at the mRNA level in
all fi ve cell lines studied. In all cases, the down-regulation of
c-myc closely correlated with decrease in pS21-C/EBPα
(Figs. 2 F and 3 C show the same blot). In support of the diff er-
entiation onset triggered by MLN518 treatment, we also ob-
served up-regulation of elastase, lysozyme, myeloperoxidase,
and lactoferrin gene expression in MV4;11 cells (Fig. 3 D).
In accordance with earlier published data (47), the MLN518-
induced diff erentiation of MV4;11 cells was accompanied by
a notable apoptosis (Table S1, available at http://www.jem.
Inhibition of MEK1 activity decreases phosphorylation
of C/EBP훂 and induces granulocytic differentiation
of MV4;11 cells
In addition to the ERK1/2 pathway, activation of FLT3 af-
fects other pathways, such as Stat3/5, or Akt, which are also
involved in leukemogenesis (8, 9, 11). To determine each
JEM VOL. 203, February 20, 2006
pathway’s contribution to the diff erentiation block in FLT3
mutant leukemia, we treated MV4;11 cells with a MEK1 in-
hibitor, PD98059, as well as a PI3 kinase inhibitor, LY294002,
and the JAK-2 protein tyrosine kinase inhibitor, AG490.
Cells were monitored daily for morphological changes, NBT
reduction activity, cell growth, and apoptosis. LY294002 and
AG490 retarded growth and induced apoptosis (Table S1),
but did not induce diff erentiation. In contrast, the MEK1
inhibitor decreased pS21-C/EBPα levels in as early as 9 h
(Fig. 4 A) and relieved diff erentiation block of MV4;11 cells
(Fig. 4 B). The fi rst morphological and functional (NBT)
signs of diff erentiation were noted 2 d later (Fig. 4 B). Further
Figure 3. Inhibition of FLT3 induces granulocytic differentiation
of MV4;11 cells. (A) MV4;11 cells were treated with 5 μM MLN518 for
3 d. Samples were withdrawn daily and phosphorylation of C/EBPα on
S21 was analyzed by Western blot with phospho-specifi c (top) and
C/EBPα (bottom) antibodies. Quantifi cation of signals with respect to
total cellular protein levels (as determined by Ponceau S staining) is
shown (bottom). (B) MV4;11 cells were cultured in the presence of
5 μM MLN518 or vehicle control (0.05% DMSO). Cell aliquots were
examined daily for morphological changes (top) and for NBT reduction
activity (bottom). Differential counts are summarized in the chart.
(C) Inhibition of FLT3 leads to down-regulation of c-myc. (top) FLT3
mutant AML cell lines were serum starved for a total of 8 h in the
absence or presence of 1 μM MLN518 for the indicated number of
hours (at the end of starvation period). Western blot was stained with
c-myc or β-tubulin antibodies. (bottom) FLT3 mutant lines, MV4;11,
MOLM-13, MOLM-14, MonoMac1 (MM1), and MonoMac6 (MM6) were
grown in the absence (0) or presence of 1 μM MLN518 for the indicated
times (hours). Total RNA was examined by Northern blot with probes
specifi c for c-myc and GAPDH (to control for RNA integrity). (D) Up-
regulation of granulocyte-specifi c gene mRNA in MLN518-treated cells.
Same RNA samples as in C, were examined by Real Time-PCR with
primer sets specifi c for the elastase, lysozyme, myeloperoxidase, and
lactoferrin genes. Data were normalized for GAPDH RNA and presented
as fold change.
376 INHIBITION OF C/EBPα FUNCTION IN FLT3 MUTANT AML | Radomska et al.
incubation of cells with MEK1 inhibitor for up to 7 d re-
sulted in even more pronounced granulocytic morphology
(Fig. 4 B). Thus, we conclude that activation of ERK1/2
pathway by FLT3 mutations is the main event responsible for
mediating the diff erentiation block. Because apoptosis, but
not diff erentiation, was noted when cells were treated with
inhibitors blocking Stat3/5 or Akt pathways, it appears that
activation of those pathways plays a role in cell survival rather
than diff erentiation.
Expression of C/EBP훂 mutant lacking Ser21
phosphorylation relieves the differentiation block
in FLT3-ITD human AML cells
We have demonstrated that C/EBPα with S21 mutated to
alanine (S21A), such that it is no longer a substrate for
ERK1/2 kinases, retains full ability to induce of granulocytic
diff erentiation of U937 and K562 cells. In contrast, mutation
of S21 to aspartate (S21D), which mimics constitutive phos-
phorylation of S21 (S21D) inhibits granulocytic diff erentia-
tion (28). We predicted that expressing a C/EBPα protein
lacking the ERK1/2 phosphorylation site (S21A mutant)
would restore the diff erentiation of FLT3 mutant AML cells.
To test this hypothesis, S21A and S21D C/EBPα mutants
were fused to the estrogen receptor (ER) ligand-binding do-
main and stably introduced into MV4;11 and MOLM-14
cells. In this system, the expression of C/EBPα-ER proteins
is constitutive with cytoplasmic localization and their nuclear
translocation is induced by treatment with β-estradiol (28,
31, 53). Several independent stable lines of MV4;11 cells were
established with high expression of C/EBPα-ER (Fig. 5 A;
not depicted). As assessed by examination of Wright-Giemsa
stained cells (Fig. 5 B), a 24-h treatment of the MV4;11 stable
lines expressing S21A-C/EBPα (MV-S21A) with β-estradiol
resulted in morphological changes typical of granulocytic dif-
ferentiation. These cells also exhibited oxidative burst activity
(NBT; Fig. 5 B). In contrast, MV4;11 clones expressing the
S21D mutant of C/EBPα (MV-S21D) did not show any of
these characteristics and remained immature (Fig. 5 C). Simi-
lar results were obtained in MOLM-14 cells (unpublished
data). We also tested the eff ect of induction of the C/EBPα
mutant proteins on the expression of c-myc. We found that,
whereas β-estradiol–treated MV-S21A lines down-regulated
c-myc protein (not depicted) and mRNA (Fig. 5 D), the
MV-S21D lines maintained c-myc expression (Fig. 5 D). As
down-regulation of c-myc is a critical event in C/EBPα-
induced diff erentiation (50, 51), these results further demon-
strate the importance of nonphosphorylated C/EBPα for
FLT3 is overexpressed or coexpressed with FLT3 ligand in
>90% of AML cases (3, 5), providing another mechanism of
constitutive receptor activation. Therefore, specifi c targeting
of activated FLT3 receptor by small molecule inhibitors is an
attractive therapeutic approach for AML. Although several
chemical compounds have been developed to inhibit FLT3
activity (46–48, 54–56), none of these inhibitors is directed
solely to the FLT3 receptor; they can aff ect activities of other
kinases, such as PKC, TrkA, VEGFR, KIT, or PDGFR, thus
increasing the possibility of nonspecifi c toxicity (3, 5). The
eff ectiveness of a given inhibitor may also vary depending on
the actual mutation in the FLT3 receptor, raising a possibility
of drug resistance. Therefore, elucidating the pathways
downstream of FLT3 might lead to the development of bet-
ter therapeutic approaches in AML.
The major known eff ect of all FLT3 inhibitors is the
induction of apoptosis (47). Ours is the fi rst report demon-
strating that treatment of FLT3 ITD cells with MLN518,
in addition to apoptosis, can also trigger diff erentiation.
We also showed that inhibition of the ERK1/2 pathway
in FLT3 mutant AML line could achieve the same eff ect,
whereas inhibition of other downstream pathways (Akt,
Stat3/5) resulted only in apoptosis. Thus, activation of FLT3
aff ects signaling pathways controlling both diff erentiation
Several clinical trials with various FLT3 inhibitors are
currently in progress (21–23). In one of them, oral admin-
istration of CEP-701 to 14 AML patients expressing FLT3-
activating mutations demonstrated sustained FLT3 inhibition
Figure 4. MV4;11 cells differentiate upon inhibition of the
ERK1/2 pathway. (A) Western blot showing decreased pS21-C/EBPα
(top) in response to continuous treatment with 100 μM of the MEK1
inhibitor PD98059 or vehicle control (DMSO). For DMSO-treated cells, a
72-h incubation is shown. (bottom) The same blot stained with C/EBPα
antibody. (B) (top) MV4;11 cell differentiation was monitored by morpho-
logical examination of Wright-Giemsa stained cytospin preparations. Red
arrows point to cells with granulocytic morphology. (bottom) Respiratory
burst activity as assessed by the NBT assay.
JEM VOL. 203, February 20, 2006
and clinical evidence of biologic activity in only 5 patients
(22). The same study also showed that two out of eight pa-
tients exhibited >90% inhibition in FLT3 receptor acti-
vation, but remained resistant to the clinical eff ect of this
compound. The molecular eff ects of another FLT3 inhibi-
tor, SU11248, were evaluated on patient samples and showed
transient or sustained decrease in ERK1/2 activation in 80%
of patients. Interestingly, activation of the upstream kinase,
MEK1, was seen in only 39% patients. Thus, components
of the FLT3–MEK1–ERK1/2 cascade can be dissociated in
Previously, we have identifi ed C/EBPα as a transcription
factor necessary and suffi cient for neutrophilic diff erentiation
(24, 26–29). It was logical to assume that such important
molecule might be a target in pathogenesis of leukemia. In
fact, the expression or function of C/EBPα are disturbed in
various subtypes of leukemia (30–40), providing an explana-
tion for the block in diff erentiation.
In the course of this work, we show that, in mutant FLT3
AML, the diff erentiation-promoting function of C/EBPα is
inhibited at yet another level: posttranslational modifi cation
by phosphorylation. We were able to show, using myeloid
and 293T cells, that FLT3 activation induces phosphorylation
of C/EBPα on S21, which inactivates C/EBPα function.
Consistent with the known role of C/EBPα in promoting
granulocytic diff erentiation, inhibition of FLT3 or introduc-
tion into the cells of S21A C/EBPα mutant rescued the dif-
ferentiation block in AML cells. Furthermore, inhibition of
FLT3 activity decreased the levels of S21 phosphorylation in
FLT3 mutant cell lines and AML patients. Nonetheless, inhi-
bition of FLT3 was less eff ective in decreasing the C/EBPα
phosphorylation in patient samples when compared with cell
lines. One possible explanation for this diff erence is that the
activities of serine/threonine phosphatases, such as protein
phosphatase 1 (PP1), or MAP kinase phosphatase-1 (MKP-1)
may be lower in patients (57, 58).
Several groups previously generated 32Dcl3 stable lines
expressing mutants of FLT3 (59, 60). In those cells, C/EBPα,
was shown to be down-regulated at the mRNA level (60).
Also, two out of three FLT3 ITD-positive patients had very
low levels of C/EBPα mRNA, which increased approxi-
mately twofold following the FLT3 inhibition therapy (60).
In our own studies with murine 32Dcl3 and 503 (PU.1−/−
Figure 5. The dephosphorylated form of C/EBP훂 is suffi cient to
mediate granulocytic differentiation of MV4;11 cells. (A) MV4;11
cells were stably transfected with inducible C/EBPα expression vectors.
Independent clones were analyzed by Western blot. Parental MV4;11
cells expressing only the endogenous C/EBPα protein are shown (left).
Clone nos. 7, 25, and 29 express an ectopic C/EBPα-ER fusion protein in
which S21 of C/EBPα was mutated to alanine (S21A). Clones C, D, and N
express C/EBPα-ER protein with S21 mutated to aspartate (S21D) to
mimic phosphorylation. (B) MV4;11 cells with induced expression of
S21A-C/EBPα protein undergo granulocytic differentiation. Granulocyte-
specifi c morphological changes in Wright-Giemsa stained cytospins
(indicated by red arrows) were seen (top) as well as an increase in NBT
reduction activity (bottom). (C) Induction of S21D-C/EBPα did not
induce granulocytic maturation as shown by morphological examina-
tions (top) or NBT reduction assay (bottom). The inductions shown are
for 24 h; however, no changes were observed after longer treatments
with β-estradiol (not depicted). (D) Parental MV4;11 cells and C/EBP-
ER–expressing stable lines (S21A, and S21D) were left untreated (0) or
treated with 1 μM β-estradiol for 4, 8, and 24 h. RNA was collected and
analyzed by Northern blot with probes specifi c for c-myc (top) and
GAPDH (bottom). For positive controls, K562 and U937 cells were used.
The U937 stable line with Zn-inducible expression of ectopic C/EBPα
(reference 24) was included as negative control, as they were shown
to down-regulate c-myc expression upon induction of C/EBPα expres-
sion (reference 51).
378 INHIBITION OF C/EBPα FUNCTION IN FLT3 MUTANT AML | Radomska et al.
line expressing endogenous C/EBPα; unpublished data) stable
lines, we found that FLT3 ITD mutation did not lead to a
strong activation of ERK1/2 and we did not observe an in-
crease in C/EBPα phosphorylation on S21, which is located
in a protein region highly conserved among mammalian spe-
cies. Furthermore, in a mouse transplantation model, FLT3
ITD mutations resulted in development of myeloproliferative
disease rather than AML (61). These fi ndings suggest that the
signaling pathways activated by FLT3 are diff erent in mice
and humans. In fact, intrinsic diff erences have been reported
between mouse and human control of hematopoiesis medi-
ated by C/EBPα (62). We cannot rule out the possibility
that both C/EBPα-inactivating mechanisms (transcriptional
repression and functional inhibition) may be operational in
human FLT3 mutant AML, possibly depending on the dif-
ferentiation stage of the transformed cell.
Overexpression or constitutive activation of the ERK
pathway has been shown to play an important role in the
pathogenesis and progression of various cancers (63). Cur-
rently, preclinical trials for pancreas, colon, breast cancers,
and leukemia are ongoing with compounds specifi cally inhib-
iting MEK1/2 component of this pathway (64, 65). Abnor-
mal activation of the ERK pathway also occurs in leukemia
because of the activating mutations in FLT3, Ras, as well as
genes in other pathways (PI3K, PTEN, Akt) (65). Thus, tar-
geting the Ras–Raf–MEK–ERK pathway in leukemia may
off er a potential alternative to standard chemotherapy. It has
been shown that the primary eff ect of down-modulation of
MEK–ERK pathway activation in AML primary blasts by
a selective inhibitor of MEK1 (PD98059) was a cell cycle
arrest followed by apoptosis (66). Our results demonstrate
that PD98059 could decrease pS21-C/EBPα levels in the
FLT3/ITD AML lines and induce granulocytic diff erentia-
tion. Our data provide strong indication that inhibitors of
MEK–ERK cascade could have signifi cant clinical benefi t in
the treatment of FLT3 mutant AML, especially in cases of
resistance to FLT3 inhibitors. Furthermore, development of
protein therapies based on transduction of constitutively ac-
tive C/EBPα (such as the S21A mutant) may prove eff ective
in treatment of subtypes of leukemia with inadequate expres-
sion/function of C/EBPα.
MATERIALS AND METHODS
Cell lines. MV4;11 (CRL 9591; American Type Culture Collection [refer-
ences 42, 43]), MOLM13, MOLM14 (67), MonoMac1 (68), U937 (CRL
1593; American Type Culture Collection), and KG1a (CCL 246.1; Ameri-
can Type Culture Collection) were grown in RPMI 1640 with 10% FBS.
MonoMac6 (45) were cultured in RPMI 1640/10% FBS supplemented with
MEM Non-Essential Amino Acid Solution and OPI Media Supplement
(Sigma-Aldrich). THP-1 (TIB 202; American Type Culture Collection)
were grown in RPMI 1640/10% FBS with 0.05 mM β-mercaptoethanol.
For MV4;11 stable lines, phenol red-free RPMI 1640/10% charcoal dextran
stripped FBS and 0.25 μg/ml puromycin were used.
Patient samples. Patient samples were obtained from bone marrow (pa-
tient no. 592) or peripheral blood (patient no. 667) at the Laboratory of
Leukemia Diagnostic, Grosshadern. Samples were collected at time of diag-
nosis before initiation of treatment. The percentage of blasts in the samples
was 70% (patient no. 592; male, 71 yr, FAB M2, 46, XY , MLL-PTD−,
FLT3-LM+, FLT3-TKD−, KIT−, NRAS−) and 95% (patient no. 667; fe-
male, 74 yr, FAB M1, 47, XX, +8 , MLL-PTD+, FLT3-LM+, FLT3-
TKD−, KIT−, NRAS−). Blast cells were collected after patient consent,
purifi ed by Ficoll-Hypaque density centrifugation, and frozen in 40%
IMDM/50% FCS/10% DMSO. Cells were thawed in IMDM/20% FCS,
400 IE/ml Heparin, 100 U/ml DNase, washed once in IMDM (without se-
rum), and incubated at 37°C for 6 h in IMDM with or without MLN518,
or with DMSO (vehicle control).
Reagents. FLT3 inhibitor MLN518 (CT53518) (47) and FLT3/PDGFR
inhibitor AG1296 were reconstituted in DMSO at 10 mM. PD98059
(MEK1 inhibitor), LY294002 (PI3 kinase inhibitor), and tyrphostin AG490
(JAK2 inhibitor) were dissolved in DMSO to 50 mM. The fi nal concentra-
tions were 25 μM or 50 μM for LY294002, and 50 μM or 100 μM for
AG490. Recombinant human FLT3 ligand was reconstituted at 10 μg/ml.
All reagents, except MLN518 (kept at 4°C) were stored at −20°C.
In vitro kinase assays. Protein kinase assays were performed as described
previously (69). The details are provided in the supplemental Materials
and methods section (available at http://www.jem.org/cgi/content/full/
Western blot. 5 × 106 cells were spun (1 K, 5 min), washed in PBS, lysed
in 400 μl of 1× Laemmli sample buff er (70), and boiled at 100°C for 10 min.
30–40 μl were loaded on 7.5% SDS-PAGE gel. After 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 antibodies in 5%
BSA/TBST/0.1% sodium azide overnight at 4°C and with horseradish per-
oxidase (HRP)–conjugated secondary antibodies at room temperature for
1 h. Signals were detected by enhanced chemiluminescence and quantifi ed
by ImageQuant software (Molecular Dynamics). The primary antibodies
were rabbit pS21-C/EBPα (1:1,000; Cell Signaling), goat C/EBPα (1:1,000;
Santa Cruz Biotechnology, Inc.), pY591-FLT3 (1:1,000 54H1; Cell Signal-
ing), rabbit p(T202/Y204)-ERK1/2 (1:1,000; Cell Signaling), and β- tubulin
(1:1,000 clone 2–28-33; 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.
Plasmids. pBabe-(S21A)C/EBPαER and pBabe-(S21D)C/EBPαER plas-
mids were described previously (28). They contained coding regions of mu-
rine C/EBPα mutated in S21 and fused to the human ER ligand-binding
domain. Puromycin gene was included for stable line selection.
Transfections. Stable lines were made by Nucleofection (Amaxa). Cells
were grown in phenol red-free RPMI 1640/10% charcoal-stripped FBS for
at least 48 h before transfection. 107 cells were mixed with 100 μl of Nucleo-
fector Solution T and 5 μg of ScaI linearized plasmid. Pulses were delivered
with Nucleofector device and program T-05. Cells were resuspended in
10 ml of phenol red-free RPMI 1640/20% charcoal-stripped FBS supple-
mented with 10% of MV4;11 cells-conditioned medium and plated on 96-well
plates. Selection with 0.25 μg/ml puromycin was performed 72 h later.
RNA isolation and analysis. Total RNA was isolated with TriReagent.
For Northern blots, 20 μg of RNA was separated on agarose gels and trans-
ferred to Biotrans Plus membranes. The blots were hybridized to the 700-bp
EcoRI–HindIII fragment of the human C/EBPα 3′ UTR (24) and a 305-bp
XbaI–EcoRI cDNA fragment of human c-myc (50). For loading control,
the blots were stripped and rehybridized to GAPDH or 18S ribosomal RNA
probes. Quantitation was performed with Image Quant. Details on SYBR
green and TaqMan Real-Time PCR assays are provided in supplemental
Materials and methods.
Morphological examination. Approximately 104 cells were spun at 500
revolutions/min for 5 min onto glass slides and Wright-Giemsa stained with
JEM VOL. 203, February 20, 2006
Diff Quick solutions (Dade Behring). Diff erential counts were performed on
at least 10 fi elds from each slide.
Nitroblue tetrazolium reduction assay. 5 × 105 cells were incubated in
0.5 ml solution containing PBS, NBT (1 tablet in 10 ml PBS; Sigma-
Aldrich), and 0.33 μM PMA for 20–30 min at 37°C. Cytospin slides were
prepared and counterstained with 0.5% safranin in 20% ethanol.
Online supplemental material. In Fig. S1, FLT3 ITD mutant induces
activation of ERK1/2 pathway and phosphorylation of C/EBPα on S21
in transiently transfected 293T cells. Fig. S2 shows that a c-kit and PDGFR
inhibitor, STI571, do not aff ect levels of pS21-C/EBPα in FLT3 ITD cell
lines. Table S1 depicts FLT3, PI3K, and Jak2 inhibitors that induce apopto-
sis of MV4;11 cells. Supplemental Materials and methods contains descrip-
tion of procedures for an in vitro kinase assay and real-time PCR. Online
supplemental material is available at http://www.jem.org/cgi/content/full/
We thank C. Sullivan for help with experiments. We are grateful to B. Neel,
S. Koschmieder, F. Rosenbauer, G. Huang, and E. Weisberg for useful suggestions;
B. Scheijen, and S. Whitman for help in obtaining patient material. We thank
Drs. Y. Matsuo for MOLM-13, and S. Heinrichs for MV4;11 and MonoMac1 cell lines.
Members of the Tenen and Gilliland laboratories are acknowledged for many useful
discussions. We thank K. O’Brien, M. Singleton, and A. Lugay for help in preparation
of the manuscript.
This research was supported by grants to D.G. Tenen from the National
Institutes of Health (no. P01 CA72009), and to H.S. Radomska from the National
Institutes of Health (no. DK62064).
The authors have no confl icting fi nancial interests.
Submitted: 7 November 2005
Accepted: 22 December 2005
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