The Journal of Experimental Medicine
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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.
on December 26, 2015
Published January 30, 2006
Supplemental Material can be found at:
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.
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