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Leukemia (2005) 19, 329–334
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REVIEW
CEBPA point mutations in hematological malignancies
H Leroy1, C Roumier1, P Huyghe2, V Biggio1, P Fenaux3 and C Preudhomme1
Laboratoire d’Hématologie A, CHRU Lille, U524 INSERM Lille, France; 2Service des Maladies du sang, CHRU Lille, France; and
Service d’Hématologie Clinique, Hôpital Avicenne-Paris 13 University, France
1
3
The CCAAT/enhancer-binding protein-alpha (CEBPA) is a
transcription factor strongly implicated in myelopoiesis
through control of proliferation and differentiation of myeloid
progenitors. Recently, several works have reported the presence of CEBPA-acquired mutations in hematological malignancies. In this work, we analyzed characteristics of mutations
and their correlation with disease characteristics described
in previous studies. In the 1175 patients reported, 146 CEBPA
mutations were identified in 96 patients. Mutations were found
in the whole gene sequence, but cluster regions were clearly
identified. Furthermore, two categories of mutations were
reported: out-of-frame ins/del often in the N-terminal region,
and in-frame ins/del often in the C-terminal region. CEBPA
mutations were reported exclusively in acute myeloid
leukemia (AML) (according to WHO classification criteria)
and mutated patients preferentially belonged to M1, M2 and
M4 FAB subtypes. All but one case belonged to the ‘intermediate’ prognostic subgroup of MRC classification. In the
absence of poor prognostic factors, patients with CEBPA
mutation had favorable outcome, very similar to that of the
t(8;21), inv(16), t(15;17) subgroup. Systematic analysis of
CEBPA mutations, in addition to that of alterations in master
genes of hematopoiesis, may be useful to assess the prognosis
of AML particularly in patients belonging to the ‘intermediate’
prognostic subgroup.
Leukemia (2005) 19, 329–334. doi:10.1038/sj.leu.2403614
Published online 13 January 2005
Keywords: CEBPA; mutations; AML; prognosis; transcription factor;
CCAAT/enhancer-binding protein
Introduction
Among the many oncogenes affecting proliferation and cell
death, anomalies of the genes implicated in the closely
regulated pathways of hematopoietic differentiation are key
oncogenic events.1 In acute myeloid leukemia (AML), cell
differentiation arrest can occur at different levels by alteration
of specific genes like those of the CBF complex.2 The CEBPA
gene (located on chromosome 19q13.1 band) belongs to the
CCAAT/enhancer-binding protein family, which is involved
in the balance between cell proliferation and terminal
differentiation. CEBPA gene mRNA can be translated from
the first AUG encoding the 42-kDa normal isoform and also
from the second AUG (nt 508–510) encoding the 30-kDa
normal isoform, which has lost the 119 first AA including
the TAD1 functional domain. The functions of CEBPA 30-kDa
protein are not well known. Nevertheless, Pabst et al3
demonstrated that this shorter CEBPA protein had lost normal
CEBPA functions and had dominant negative effect on 42-kDa
Correspondence: Dr C Preudhomme, Laboratoire d’Hématologie A –
hôpital Calmette, CHRU, Place de Verdun, 59000 Lille, France; Fax:
þ 33 3 20 44 55 10; E-mail: [email protected]
Received 19 July 2004; accepted 4 November 2004; Published online
13 January 2005
wild-type (wt) CEBPA. The 42-kDa CEBPA protein has four
principal domains: the C-terminal part containing a leucine
zipper domain mediating homo- or heterodimerization; the
DNA-binding domain (DBD), a basic positively charged domain
able to interact with specific DNA sequences; and two
regulatory and transactivating domains TAD1 and TAD2
(Figure 1).
The 42-kDa normal protein acts as a transcription factor with
a crucial role during differentiation of various cell types
including hepatocytes, adipocytes, enterocytes, keratinocytes,
lung, mammary gland cells and hematopoietic cells. In
hematopoiesis, CEBPA plays a pivotal role in early stages of
myeloid differentiation and is particularly expressed in myelomonocytic cells.4–8 CEBPA has multiple actions such as downregulation of C-MYC expression allowing differentiation, direct
upregulation of the expression of granulocytic lineage-specific
genes and synergistic action with other key genes in myeloid
development including CBF complex genes and PU.1.5,9–11 In
addition to specific DNA binding, CEBPA could act by protein–
protein interaction. The principal partners of those interactions
are p21, CDK2, CDK4 and E2F. Repression of E2F-dependent
transcription genes by CEBPA had previously been shown to be
a critical event in suppressing cellular proliferation and inducing
granulocytic or adipocyte differentiation. CEBPA also inhibits
cell proliferation by activating transcription of p21/WAF1, by
stabilizing p21 and inhibiting CDK2 and CDK4.12–14
CEBPA expression begins with the commitment of myeloid
lineage precursors and is upregulated during granulocytic differentiation.7 CEBPA-defective mice have no mature
granulocytes, whereas cells of the other lineages are not
affected.15 In addition, CEBPA expression could block monocytic differentiation.16
The strong implication of CEBPA in granulocytic differentiation points to this gene as a key target in leukemogenesis, as
shown now in many studies. Pabst et al3 found that events
leading to the loss of CEBPA function observed in AML
contribute to leukemogenesis by blocking granulocytic differentiation. Moreover, myeloid blasts observed in those cases
were committed myeloid cells generally classified in the M1 or
M2 FAB AML subtypes.17 Recently, three mechanisms of CEBPA
inactivation have been reported. One is downregulation of
CEBPA expression by the AML1-ETO fusion transcript in t(8,21)
leukemia cells. In this model, conditional expression of CEBPA
in those cells is sufficient to trigger granulocytic differentiation.18 The second mechanism is inhibition of the translation of
CEBPA mRNA by interaction with hnRNPE2, induced by
BCR-ABL fusion protein.19 This mechanism could contribute
to the transition from chronic phase to myeloid blast crisis in
CML, by blocking myeloid differentiation. Finally, inactivating
CEBPA mutations have been reported in hematological
malignancies, especially in AML.3,20–26 In this review, we
focused on CEBPA mutations and how, through inactivation of
CEBPA mutations in hematological malignancies
H Leroy et al
330
120
2nd ATG
AA :
99
104
183
189
286
nt :
445
462
697
714
1006
Clustering regions
R1
nt
1
445
R2
462
306
317
345
1068 1099
1183
R3
732
855
R4
1059 1062
R5
1137
1152
1196
%
60
50
40
Single
alteration
30
20
10
0
R1
R2
R3
R4
R5
R1
R2
R3
R4
R5
%
60
50
Multiple
alterations
40
30
20
10
0
Legend:
: Transactivating Domain 1 (Poly-Gly)
: Transactivating Domain 2 (Poly-Pro)
: Out of frame ins / del mutations
: In frame ins / del mutations
: DNA Binding Domain (DBD)
: Other type mutations
: Leucine Zipper Domain
Figure 1
Schematic representation of CEBPA functional domain; incidence of the different types of mutation and localization of mutation hot
spot regions.
transcriptional properties of the CEBPA protein, they could lead
to leukemogenesis.
Correlation of CEBPA mutations with hematological
parameters and prognosis
CEBPA mutations and morphological classification
Overview of CEBPA gene mutations reported
In the last 3 years, 1175 patients were screened for CEBPA
mutations in seven studies.3,21–25,27.Those patients included 962
AML (Table 1), 156 myelodysplastic syndromes, 23 acute
lymphoblastic leukemia and 34 non-Hodgkin’s lymphoma.
Among those 1175 patients, 96 (8.2%) had CEBPA mutations
including nine with silent mutations and 87 with acquired
nonsilent mutations (described in online additional data,
supplementary data). CEBPA mutations were only observed in
myeloid malignancies.
Leukemia
All but two of the 87 reported patients with CEBPA mutation had
AML, including 30 M1 AML, 35 M2 AML, 14 M4 AML, three M5
AML, two unclassified AML and one therapy-related AML
(incidence in AML: 85 on 962 patients (8.8 %) ; see Table 1).
The two remaining patients had RAEB-t (a disorder now
included in AML in the new WHO classification of myeloid
disorders).
FAB classification of AML and MDS was available in 712
patients studied for CEBPA mutations. Incidences of mutations
were 14.5 % of M1, 6.4% of M2, 4% of M4, 2% of M5, one of
CEBPA mutations in hematological malignancies
H Leroy et al
331
the seven therapy-related AML and one of the 12 RAEB-t. No
mutations were found in M0, M3, M6 and M7-AML.
In these four studies, the complete remission rate was not
significantly different in patients with or without CEBPA
mutations, but all studies except that of Snaddon et al21 showed
better relapse-free survival (RFS) or overall survival (OS) in
mutated cases. Discrepancies between Snaddon et al21 and
other studies could have been due to some characteristics of
their mutated cases, including higher peripheral blood blast
count (median of 93 109/l vs o24 109/l in the mutated cases of
other studies), the localization of mutations (five of eight
mutated patients had a single alteration of the C-terminal part
of CEBPA protein vs zero of 27 in the other studies), and,
although this is more speculative, the fact that five of their eight
mutated patients received standard dose AraC instead of highdose AraC in the other studies.
An analysis of cooperating mutations, made in two studies,
showed no difference in the incidence of FLT3 and MLL
alterations in CEBPA-mutated and -nonmutated patients.24,27
However, in patients with CEBPA mutation, Preudhomme
et al,24 showed that the presence of FLT3 internal tandem
duplication (FLT3-ITD) had a negative prognostic influence,
whereas, in the study of Fröhling et al,27 the presence of FLT3ITD or FLT3 mutations had no prognostic impact. Nevertheless,
in the two studies, the size of the population studied was too
small for definite conclusions (15 patients in Preudhomme et al,
study24 and 36 patients in Fröhling et al, study27).
Cytogenetic findings
Cytogenetic data was available in 83 of the 87 mutated patients
(see online additional data). Only one had a complex karyotype
with more than three abnormalities; 58 cases (70%) had a
normal karyotype and eight (10%) had only one abnormality. In
the remaining 15 mutated cases, the karyotype was classified in
the ‘intermediate’ prognostic subgroup without further details;
no structural or numerical alteration of chromosome 19 and no
‘favorable’ karyotype (t(8;21), inv(16), t(16;16), t(15;17)) was
reported in those patients. Therefore, all except one of the
mutated cases belonged to the ‘intermediate’ subgroup of the
MRC classification.28 This may suggest that CEBPA loss of
function induced by balanced translocations and CEBPA loss of
function by point mutation are mutually exclusive.
Other clinical and biological correlations (Table 2)
When they were studied, no correlation was observed between
CEBPA mutations and age, sex, WBC count and bone marrow
blast percentage.
Prognostic value of CEBPA mutations
CEBPA mutation profile in mutated cases (Table 3)
Four studies have evaluated the prognostic impact of CEBPA
mutations in 71 mutated patients, by comparison to 670 patients
without CEBPA mutation.21,22,24,27
A total of 45 patients had a single CEBPA alteration, whereas 42
cases had multiple alterations resulting from biallelic mutations
or from several mutations on the same allele. There was no
Table 1
Prevalence of nonsilent CEBPA mutations in acute myeloid leukemia (AML)
Study
No of patients studied
1
236
M0 AML
M1 AML
M2 AML
M3 AML
M4 AML
M5 AML
M6 AML
M7 AML
Sec, AML
Uncl, AML
Incidence of mutations
2
78
FAB subtypes not available
36/236
6/78
3
137
4
99
5
135
6
277
Cumulative incidence (%)
0/1
2/8
5/62
0/22
0/32
0/5
NC
NC
1/7
NC
8/137
NC
6/56
2/43
NC
NC
NC
NC
NC
NC
NC
8/99
0/1
8/33
2/34
NC
3/19
2/32
0/5
0/4
NC
0/7
15/135
0/10
7/62
4/64
0/22
1/50
0/61
0/3
NC
NC
0/5
12/277
0
14.5
6.4
0
4
2
0
0
14.3
0
8.8 (85/962)
Sec. AML ¼ secondary AML; Uncl. AML ¼ unclassified AML; NC ¼ no case.
Study 1 ¼ Fröhling et al; Study 2 ¼ Gombart et al; Study 3 ¼ Pabst et al; Study 4 ¼ Snaddon et al; Study 5 ¼ Preudhomme et al; Study
6 ¼ Khosrovani et al.
Table 2
Clinical and biological characteristics of AML patients with and without CEBPA mutation (median values)
WT
Fröhling et al
Preudhomme et al
Snaddon et al
Khosrovani et al
47
45
46.5
NA
Age
Mutated
47
45
55.5
44
% Female
WT
Mutated
WBC (giga/l)
WT
Mutated
55
49
36
NA
19.9
13
NA
NA
50
33
62
33
28.9
20
NA
34.5
PB blasts
WT
Mutated
40%
NA
9.2 G/l
NA
62%
NA
9.2 G/l
NA
BM blasts (%)
WT
Mutated
Platelets (giga/l)
WT
Mutated
80
NA
75
NA
93
NA
NA
NA
80
NA
80
64.5
45
NA
NA
41
WT ¼wild-type; WBC ¼ white blood count; PB ¼ peripherical blood; Na ¼ not available.
Leukemia
CEBPA mutations in hematological malignancies
H Leroy et al
332
significant difference in location and type of mutations between
patients with single or multiple alterations (Figure 1).
multiple mutations, patients had preferentially M1-AML (45%),
whereas, in case of single mutation, patients had preferentially
M2-AML (44%) (Table 3).
Characteristics of CEBPA mutations
Pathogenetic role of CEBPA mutations
Until now, 146 CEBPA mutations have been described in 96
patients,3,21–25,27 including nine isolated silent mutations without transcriptional effect in nine patients and 137 nonsilent
CEBPA mutations in 87 patients (five missense mutations, 54 inframe ins/del mutations, 78 nonsense mutations including five
STOP codon point mutations and 73 out-of-frame ins/del
mutations inducing a STOP codon downstream). Reported
mutations were nonrandomly distributed all over the CEBPA
gene sequence and occurred in five regions of the protein
(regions R1 to R5) (Figure 1): R1 corresponding to the AA before
the Poly-Gly domain, R2 between the Poly-Pro and the Poly-Gly
domain, R3 corresponding to the N-terminal part of the DBD
and the region just before the DBD domain, R4 corresponding to
the C-terminal part of the DBD and to the N-terminal part of the
b-Zip domain (fork region), R5 corresponding to the C-terminal
part of the B-zip domain.
R1 and R4 were the two main cluster regions as 76% of the
137 nonsilent mutations belonged to these regions. Moreover,
the distribution of the type of mutations between these two
regions was heterogeneous. Nearly all out-of-frame ins/del
nonsense mutations occurred in R1 region, whereas most 54 inframe ins/del mutations occurred in R4 region. Functional study
of the mutated protein showed important loss of transactivation
properties of CEBPA in almost all cases of mutation25
Interestingly, among the 78 nonsense mutations, 65 occurred
at the 50 of the second ATG codon of CEBPA (R1 region) leading
to increased translation of the alternative 30-kDa form of the
protein. As demonstrated by Pabst et al, this shorter protein has a
dominant negative effect on 42-kDa wt CEBPA. Conversely,
none of the 54 in-frame ins/del mutations lead to increased
synthesis of the dominant negative 30-kDa isoform.
A minor cluster region (R2 region) has been reported by
Fröhling et al,27 who found 14 recurring mutations in the TAD2
domain. Those alterations were 6 bp duplications or 3 bp
deletions inducing the addition of H-P AA in a motif of three
H-P repeats, or deletion of one P in a motif of seven P repeats.
Of the nine cases with TAD2 mutation, seven had no 30-kDa
dominant negative form. The significance of such recurrent
alterations (ie repeats of GCC nucleotide triplets) remains to be
understood.
Overall, in 55 of the 87 mutated cases, mutations lead to the
synthesis of the dominant negative 30-kDa form, which inhibits
CEBPA normal function. No significant correlation between
localization, number of mutations and FAB subtypes could be
established (Figure 1). Of note, however, is the fact thatin case of
Table 3
Expression of 30-kDa dominant negative isoform according to FAB subtype and number of CEBPA mutations
Single alteration
30-kDa
isoform
Leukemia
M1 AML
M2 AML
M3 AML
Other
5
11
8
2
(45%)
(55%)
(89%)
(40%)
Total
26 (75%)
30-kDa
isoform+
6
9
1
3
(55%)
(45%)
(11%)
(60%)
19 (25%)
Multiple alteration
30-kDa
isoform
30-kDa
isoform+
5
2
1
1
14
13
4
2
(26%)
(13%)
(20%)
(33%)
9 (21%)
(74%)
(87%)
(80%)
(66%)
33 (%)
How CEBPA mutations can contribute to leukemogenesis in AML
and to prognosis remains uncertain. It has been shown that
CEBPA mutations induced modifications in the gene expression
profile obtained from blast cells, and that, if AML patients were
classified according to the gene expression profile, CEBPA
mutated patients segregated in two homogeneous clusters.29 A
review of the currently published CEBPA mutations suggests two
main types of mutated cases. The first group contains patients
with at least one mutation in R1 or R2 clustering regions leading
to increased translation of the 30-kDa isoform from the second
AUG downstream of the mutation, inducing the indirect loss of
CEBPA main function in a dominant negative manner. The other
group contains patients where CEBPA gene mutations result in
loss of the main transregulatory function of CEBPA, mainly due to
multiple alterations of the gene; absence of wt CEBPA protein in
blast cells could explain the differentiation arrest of these cells.
How can mutations contributing to the loss of function of a
master gene in early myeloid differentiation result in a myeloid
type of leukemia? It has previously been reported that biallelic
mutations of another early myeloid differentiation gene (AML1/
RUNX1) could induce M0 AML subtype.2 Furthermore, mice
models of CML blast crisis with CEBPA knockout result in
immature erythroid leukemia30,31 and expression of a dominant
negative 30-kDa CEBPA protein inhibits differentiation of myeloid
and erythroid progenitors.32 On the other hand, all CEBPAmutated patients reviewed in this work had M1, M2, M4 or M5
AML subtypes, clearly showing persistence of myeloid differentiation in blast cells. However, in mutated cases with multiple
alterations of CEBPA, preventing expression of wt protein, or in
single alterations with expression of p30 dominant negative form,
the persistence of CEBPA residual cellular functions has been
demonstrated.31,33,34 Therefore, those cases are not identical to
KO models since the loss of transactivating properties is
dependent of on cis-regulating elements of the CEBPA target
genes. Moreover, other members of the CEBPA family, including
CEBPB and CEBPE, could partially prevent the effects of the loss of
function of mutated CEBPA and allow incomplete myeloid
differentiation resulting in M1, M2, M4 and M5 blasts.
Pabst et al18 demonstrated that, when CEBPA nonsense
mutations occurred before the second AUG, the 30-kDa CEBPA
isoform was increased by 4.2–8.6-fold compared with unmutated cases. This shorter form of CEBPA lacks antiproliferative
activity35 and most of the transactivation properties of the
normal protein,36,37 and acts dominantly on wt CEBPA by
inhibiting DNA binding of CEBPA on targeted genes.3 Indeed, in
the presence of equal amounts of 42-kDa and 30-kDa in
vitro, the 30-kDa isoform totally abolished transactivation of
a plasmid reporter by the full-length CEBPA protein.18 The
30-kDa protein binds DNA with only 14% affinity compared
with the full-length protein and can inhibit DNA binding of wt
CEBPA.3 However, the 30-kDa isoform does not inhibit 42-kDa
normal function on each CEBPA-transregulated genes. For
example, MPO gene expression is not abrogated by the
30-kDa form.31,34 The predominance of M1 and M2 FAB
subtypes in CEBPA-mutated AML, with partial myeloid differentiation block in blast cells, is compatible with this effect and
the higher incidence of CEBPA mutations in M1, M2 and M4
FAB subtypes supports a critical role of CEBPA gene function in
CEBPA mutations in hematological malignancies
H Leroy et al
333
the intermediate stages of granulocytic differentiation. One may
hypothesize that events inducing loss of CEBPA gene function in
transformed myeloid precursor cells could lead to M1, M2 or
M4 FAB AML.
In mutated cases where no 30-kDa is synthetized, other
mechanisms are required to explain the contribution of CEBPA
mutations to leukemogenesis. Since as few as three-fold increases
in CEBPA 42-kDa protein are sufficient to induce differentiation
of myeloid cell lines,8 it is possible that only moderate decreases
of CEBPA protein in vivo are sufficient to explain the differentiation block in those AML. This could be achieved by haploinsufficiency resulting from single C-terminal mutations.
It has been shown that patients with CEBPA mutations had
similar prognosis as patients belonging to the ‘favorable’ group
according to MRC classification (including t(8;21), inv(16) and
t(15;17) AML). It is difficult to speculate on the reasons of this
favorable prognosis of AML with CEBPA mutations. However,
there are some similarities between the cellular effects of the
chimeric oncoproteins observed in AML with ‘favorable’
karyotype and the cellular effects of CEBPA mutations found
in AML with normal karyotype (belonging to the ‘intermediate’
karyotype subgroup of MRC classification). In both situations,
CEBPA function is repressed. Indeed, chimeric proteins induce
the disruption of CEBPA normal functions: in t(8;21), AML1-ETO
is able to suppress CEBPA protein expression by decreasing the
level of CEBPA mRNA18,38; in inv(16), CBFb-MYH11 is also able
to disturb CEBPA functions38 and in t(15;17), Truong et al,39
showed that PML-RARa blocked CEBPA activity. Those similarities could contribute to explain why AML with CEBPA
mutations have favorable prognosis in AML.
This favorable prognostic value and the predominance of
CEBPA mutations in M1, M2 and M4 AML justifies, in our opinion,
systematic screening for CEBPA mutations in those AML groups,
especially in patients belonging to the intermediate prognosis
cytogenetic group. Analysis of the currently reported CEBPA
mutations has shown a nonrandom distribution of mutations in the
coding sequence of the gene, with two principal mutation cluster
regions (R1 and R4). Mutation types are not equally distributed
between the two regions as more than 87% of in-frame ins/del
mutations occurred in the R4 region (if we exclude the TAD2 inframe ins/del) and nonsense out-of-frame mutation localized in the
R1 region in 82% of the cases. As the size of ins/del mutations is
shorter in out-of-frame ins/del mutations, the best screening
methods may differ for each region: gene scan method after
amplification may be preferable for the C-terminal region (R3 and
R4 regions) and direct sequencing for the N-terminal region
(genomic sequence coding for AA 1–200, including R1 and R2
region). By these methods, 92% of the 137 CEBPA mutations and
100% of the mutations inducing the synthesis of dominant
negative 30-kDa isoform would have been identified.
Systematic screening of CEBPA mutations in association with
the search of other alterations in master genes implicated in
leukemogenesis such as FLT3, MLL or RAS should probably be
performed systematically in AML, particularly in patients
belonging to the ‘intermediate’ prognostic group, to determine
more precisely the impact of the association between genetic
events on overall survival and therapeutic response in large
cohorts of patients.
Acknowledgements
This work was supported by the ‘Fondation de France’ and by the
‘Ligue contre le cancer, Comité Nord’. We thank Dan Tenen for
critical reading of this manuscript.
Supplementary Information
Supplementary Information accompanies the paper on the
Leukemia website (http://www.nature.com/leu).
References
1 Tenen DG. Disruption of differentiation in human cancer: AML
shows the way. Nat Rev Cancer 2003; 3: 89–101.
2 Roumier C, Fenaux P, Lafage M, Imbert M, Eclache V,
Preudhomme C. New mechanisms of AML1 gene alteration in
hematological malignancies. Leukemia 2003; 17: 9–16.
3 Pabst T, Mueller BU, Zhang P, Radomska HS, Narravula S,
Schnittger S et al. Dominant-negative mutations of CEBPA,
encoding CCAAT/enhancer binding protein-alpha (C/EBPalpha),
in acute myeloid leukemia. Nat Genet 2001; 27: 263–270.
4 Tenen DG, Hromas R, Licht JD, Zhang DE. Transcription factors,
normal myeloid development, and leukemia. Blood 1997; 90:
489–519.
5 Reddy VA, Iwama A, Iotzova G, Schulz M, Elsasser A, Vangala RK
et al. Granulocyte inducer C/EBPalpha inactivates the myeloid
master regulator PU.1: possible role in lineage commitment
decisions. Blood 2002; 100: 483–490.
6 Zhang P, Nelson E, Radomska HS, Iwasaki-Arai J, Akashi K,
Friedman AD et al Induction of granulocytic differentiation by 2
pathways. Blood 2002; 99: 4406–4412.
7 Keeshan K, Santilli G, Corradini F, Perrotti D, Calabretta B.
Transcription activation function of C/EBPalpha is required
for induction of granulocytic differentiation. Blood 2003; 102:
1267–1275.
8 Radomska HS, Huettner CS, Zhang P, Cheng T, Scadden DT,
Tenen DG. CCAAT/enhancer binding protein alpha is a regulatory
switch sufficient for induction of granulocytic development
from bipotential myeloid progenitors. Mol Cell Biol 1998; 18:
4301–4314.
9 Johansen LM, Iwama A, Lodie TA, Sasaki K, Felsher DW, Golub TR
et al. c-Myc is a critical target for c/EBPalpha in granulopoiesis.
Mol Cell Biol 2001; 21: 3789–3806.
10 Smith LT, Hohaus S, Gonzalez DA, Dziennis SE, Tenen DG. PU.1
(Spi-1) and C/EBP alpha regulate the granulocyte colony-stimulating factor receptor promoter in myeloid cells. Blood 1996; 88:
1234–1247.
11 Klempt M, Melkonyan H, Hofmann HA, Eue I, Sorg C. The
transcription factors c-myb and C/EBP alpha regulate the monocytic/myeloic gene MRP14. Immunobiology 1998; 199: 148–151.
12 D’Alo F, Johansen LM, Nelson EA, Radomska HS, Evans EK, Zhang
P et al. The amino terminal and E2F interaction domains are
critical for C/EBP alpha-mediated induction of granulopoietic
development of hematopoietic cells. Blood 2003; 102: 3163–
3171.
13 Wang QF, Cleaves R, Kummalue T, Nerlov C, Friedman AD. Cell
cycle inhibition mediated by the outer surface of the C/EBPalpha
basic region is required but not sufficient for granulopoiesis.
Oncogene 2003; 22: 2548–2557.
14 Timchenko NA, Wilde M, Nakanishi M, Smith JR, Darlington GJ.
CCAAT/enhancer-binding protein alpha (C/EBP alpha) inhibits cell
proliferation through the p21 (WAF-1/CIP-1/SDI-1) protein. Genes
Dev 1996; 10: 804–815.
15 Zhang DE, Zhang P, Wang ND, Hetherington CJ, Darlington GJ,
Tenen DG. Absence of granulocyte colony-stimulating factor
signaling and neutrophil development in CCAAT enhancer binding
protein alpha-deficient mice. Proc Natl Acad Sci USA 1997; 94:
569–574.
16 Friedman AD. Transcriptional regulation of granulocyte and
monocyte development. Oncogene 2002; 21: 3377–3390.
17 Bennett JM, Catovsky D, Daniel MT, Flandrin G, Galton DA,
Gralnick HR et al. Proposals for the classification of the acute
leukaemias. French–American–British (FAB) co-operative group.
Br J Haematol 1976; 33: 451–458.
18 Pabst T, Mueller BU, Harakawa N, Schoch C, Haferlach T, Behre
G et al. AML1-ETO downregulates the granulocytic differentiation
factor C/EBPalpha in t(8;21) myeloid leukemia. Nat Med 2001; 7:
444–451.
Leukemia
CEBPA mutations in hematological malignancies
H Leroy et al
334
19 Perrotti D, Cesi V, Trotta R, Guerzoni C, Santilli G, Campbell K
et al. BCR-ABL suppresses C/EBPalpha expression through
inhibitory action of hnRNP E2. Nat Genet 2002; 30: 48–58.
20 Nerlov C. C/EBPalpha mutations in acute myeloid leukaemias. Nat
Rev Cancer 2004; 4: 394–400.
21 Snaddon J, Smith ML, Neat M, Cambal-Parrales M, Dixon-McIver
A, Arch R et al. Mutations of CEBPA in acute myeloid leukemia
FAB types M1 and M2. Genes Chromosomes Cancer 2003; 37:
72–78.
22 van Waalwijk van Doorn-Khosrovani SB, Erpelinck C, Meijer J,
van Oosterhoud S, van Putten WL, Valk PJ et al. Biallelic mutations
in the CEBPA gene and low CEBPA expression levels as prognostic
markers in intermediate-risk AML. Hematol J 2003; 4: 31–40.
23 Kaeferstein A, Krug U, Tiesmeier J, Aivado M, Faulhaber M, Stadler
M et al. The emergence of a C/EBPalpha mutation in the clonal
evolution of MDS towards secondary AML. Leukemia 2003; 17:
343–349.
24 Preudhomme C, Sagot C, Boissel N, Cayuela JM, Tigaud I, de
Botton S, et al., ALFA Group. Favorable prognostic significance of
CEBPA mutations in patients with de novo acute myeloid
leukemia: a study from the Acute Leukemia French Association
(ALFA). Blood 2002; 100: 2717–2723.
25 Gombart AF, Hofmann WK, Kawano S, Takeuchi S, Krug U, Kwok
SH et al. Mutations in the gene encoding the transcription factor
CCAAT/enhancer binding protein alpha in myelodysplastic
syndromes and acute myeloid leukemias. Blood 2002; 99:
1332–1340.
26 Chim CS, Wong AS, Kwong YL. Infrequent hypermethylation of
CEBPA promotor in acute myeloid leukaemia. Br J Haematol 2002;
119: 988–990.
27 Fröhling S, Schlenk RF, Stolze I, Bihlmayr J, Benner A, Kreitmeier S
et al. Mutations in younger adults with acute myeloid leukemia
and normal cytogenetics: prognostic relevance and analysis of
cooperating mutations. J Clin Oncol 2004; 22: 624–633.
28 Grimwade D, Walker H, Oliver F, Wheatley K, Harrison C,
Harrison G et al. The importance of diagnostic cytogenetics
on outcome in AML: analysis of 1612 patients entered into
the MRC AML 10 trial. The Medical Research Council Adult
and Children’s Leukaemia Working Parties. Blood 1998; 92:
2322–2333.
29 Valk PJ, Verhaak RG, Beijen MA, Erpelinck CA, Barjesteh van
Waalwijk van Doorn-Khosrovani S, Boer JM et al. Prognostically
Leukemia
30
31
32
33
34
35
36
37
38
39
useful gene-expression profiles in acute myeloid leukemia. N Engl
J Med 2004; 350: 1617–1628.
Iwama A, Osawa M, Hirasawa R, Uchiyama N, Kaneko S,
Onodera M et al. Reciprocal roles for CCAAT/enhancer binding
protein (C/EBP) and PU.1 transcription factors in Langerhans cell
commitment. J Exp Med 2002; 195: 547–558.
Wang QF, Friedman AD. CCAAT/enhancer-binding proteins are
required for granulopoiesis independent of their induction of the
granulocyte colony-stimulating factor receptor. Blood 2002; 99:
2776–2785.
Schwieger M, Lohler J, Fischer M, Herwig U, Tenen DG, Stocking
C. A dominant-negative mutant of C/EBPalpha, associated with
acute myeloid leukemias, inhibits differentiation of myeloid and
erythroid progenitors of man but not mouse. Blood 2004; 103:
2744–2752.
Cleaves R, Wang QF, Friedman AD. C/EBPalphap30, a myeloid
leukemia oncoprotein, limits G-CSF receptor expression but not
terminal granulopoiesis via site-selective inhibition of C/EBP DNA
binding. Oncogene 2004; 23: 716–725.
Friedman AD, Keefer JR, Kummalue T, Liu H, Wang QF, Cleaves
R. Regulation of granulocyte and monocyte differentiation by
CCAAT/enhancer binding protein alpha. Blood Cells Mol Dis
2003; 31: 338–341.
Lin FT, MacDougald OA, Diehl AM, Lane MD. A 30-kDa
alternative translation product of the CCAAT/enhancer binding
protein alpha message: transcriptional activator lacking antimitotic
activity. Proc Natl Acad Sci USA 1993; 90: 9606–9610.
Calkhoven CF, Bouwman PR, Snippe L, Ab G. Translation start site
multiplicity of the CCAAT/enhancer binding protein alpha mRNA
is dictated by a small 50 open reading frame. Nucleic Acids Res
1994; 22: 5540–5547.
Ossipow V, Descombes P, Schibler U. CCAAT/enhancer-binding
protein mRNA is translated into multiple proteins with different
transcription activation potentials. Proc Natl Acad Sci USA 1993;
90: 8219–8223.
Cilloni D, Carturan S, Gottardi E, Messa F, Messa E, Fava M et al.
Down-modulation of the C/EBPalpha transcription factor in
core binding factor acute myeloid leukemias. Blood 2003; 102:
2705–2706.
Truong BT, Lee YJ, Lodie TA, Park DJ, Perrotti D, Watanabe N et al.
CCAAT/enhancer binding proteins repress the leukemic phenotype
of acute myeloid leukemia. Blood 2003; 101: 1141–1148.