Download myeloid leukemia in acute microRNA-181a up

From bloodjournal.hematologylibrary.org by RAUL RIBEIRO on February 19, 2013. For personal use only.
2013 121: 159-169
Prepublished online October 25, 2012;
doi:10.1182/blood-2012-05-428573
Lenalidomide-mediated enhanced translation of C/EBPα-p30 protein
up-regulates expression of the antileukemic microRNA-181a in acute
myeloid leukemia
Christopher J. Hickey, Sebastian Schwind, Hanna S. Radomska, Adrienne M. Dorrance, Ramasamy
Santhanam, Anjali Mishra, Yue-Zhong Wu, Houda Alachkar, Kati Maharry, Deedra Nicolet, Krzysztof
Mrózek, Alison Walker, Anna M. Eiring, Susan P. Whitman, Heiko Becker, Danilo Perrotti, Lai-Chu
Wu, Xi Zhao, Todd A. Fehniger, Ravi Vij, John C. Byrd, William Blum, L. James Lee, Michael A.
Caligiuri, Clara D. Bloomfield, Ramiro Garzon and Guido Marcucci
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Regular Article
MYELOID NEOPLASIA
Lenalidomide-mediated enhanced translation of C/EBP␣-p30 protein
up-regulates expression of the antileukemic microRNA-181a in acute myeloid
leukemia
Christopher J. Hickey,1 Sebastian Schwind,1 Hanna S. Radomska,1 Adrienne M. Dorrance,1 Ramasamy Santhanam,1
Anjali Mishra,2 Yue-Zhong Wu,1 Houda Alachkar,1 Kati Maharry,1,3 Deedra Nicolet,1,3 Krzysztof Mrózek,1 Alison Walker,1
Anna M. Eiring,2 Susan P. Whitman,2 Heiko Becker,1 Danilo Perrotti,2 Lai-Chu Wu,4 Xi Zhao,5 Todd A. Fehniger,6 Ravi Vij,6
John C. Byrd,1 William Blum,1 L. James Lee,5 Michael A. Caligiuri,1 Clara D. Bloomfield,1 Ramiro Garzon,1 and
Guido Marcucci1
1Division
of Hematology, Department of Internal Medicine, and 2Department of Microbiology, Virology, Immunology, and Medical Genetics, Comprehensive
Cancer Center, The Ohio State University, Columbus, OH; 3Alliance for Clinical Trials in Oncology Statistics and Data Center, Mayo Clinic, Rochester, MN;
Departments of 4Molecular and Cellular Biochemistry, and 5Chemical and Biomolecular Engineering, The Ohio State University, Columbus, OH; and 6Division of
Oncology, Siteman Cancer Center, Washington University School of Medicine, St Louis, MO
Recently, we showed that increased miR-181a expression was associated with improved
outcomes in cytogenetically normal acute myeloid leukemia (CN-AML). Interestingly,
• High miR-181a levels associmiR-181a expression was increased in CN-AML patients harboring CEBPA mutations,
ate with CEBPA mutations and which are usually biallelic and associate with better prognosis. CEBPA encodes the
C/EBP␣ transcription factor. We demonstrate here that the presence of N-terminal
likely contribute to the favorCEBPA mutations and miR-181a expression are linked. Indeed, the truncated C/EBP␣able prognostic impact of
p30
isoform, which is produced from the N-terminal mutant CEBPA gene or from the
these mutations in AML.
differential
translation of wild-type CEBPA mRNA and is commonly believed to have no
• Lenalidomide induces
transactivation activity, binds to the miR-181a-1 promoter and up-regulates the miCEBPA-dependent miR-181a
croRNA expression. Furthermore, we show that lenalidomide, a drug approved for
expression and in turn inmyelodysplastic syndromes and multiple myeloma, enhances translation of the C/EBP␣creases sensitivity to chemop30 isoform, resulting in higher miR-181a levels. In xenograft mouse models, ectopic
therapy in AML blasts.
miR-181a expression inhibits tumor growth. Similarly, lenalidomide exhibits antitumorigenic activity paralleled by increased miR-181a expression. This regulatory pathway
may explain an increased sensitivity to apoptosis-inducing chemotherapy in subsets of AML patients. Altogether, our data provide a
potential explanation for the improved clinical outcomes observed in CEBPA-mutated CN-AML patients, and suggest that
lenalidomide treatment enhancing the C/EBP␣-p30 protein levels and in turn miR-181a may sensitize AML blasts to chemotherapy.
(Blood. 2013;121(1):159-169)
Key Points
Introduction
Cytogenetically normal acute myeloid leukemia (CN-AML) is the
largest subset of AML, comprising up to 45% of cases.1 In
CN-AML patients, several molecular markers have been identified
as predictors of clinical outcome, including the mutational status of
the CEBPA gene.2-5 CEBPA encodes the CCAAT enhancer-binding
protein ␣ (C/EBP␣), a transcription factor critical for normal
myeloid cell differentiation.6 In the hematopoietic system, C/EBP␣
is expressed early in myeloid precursors and up-regulated during
their commitment to granulocytic pathway and maturation.6-8
Several reports demonstrated that diverse molecular mechanisms
are responsible for C/EBP␣ inactivation of expression or function
in various types of leukemia (AML and chronic myelogenous
leukemia).9,10
The C/EBP␣ protein contains multiple N-terminal transactivation domains and 1 C-terminal basic-leucine zipper region, responsible for DNA binding and protein dimerization.11,12 As a result of
differential translation initiation from a single CEBPA mRNA
molecule, 2 protein isoforms are produced (full-length p42 and
N-terminally truncated p30),13 which play different functions in
gene regulation and proliferation.14-16 Approximately 15% of CN-AML
patients carry mutations in the CEBPA gene that are clustered in
2 regions. The N-terminal frame-shift mutations, which occur in
approximately 90% of CEBPA mutant patients, prevent expression
of the full-length transcriptionally active C/EBP␣-p42 protein, but
allow translation of the truncated C/EBP␣-p30 isoform.2,14,17,18 In
contrast, the C-terminal mutations disrupt the basic-leucine zipper
Submitted May 7, 2012; accepted October 5, 2012. Prepublished online as
Blood First Edition paper, October 25, 2012; DOI 10.1182/blood-2012-05-428573.
The publication costs of this article were defrayed in part by page charge
payment. Therefore, and solely to indicate this fact, this article is hereby
marked ‘‘advertisement’’ in accordance with 18 USC section 1734.
The online version of this article contains a data supplement.
© 2013 by The American Society of Hematology
BLOOD, 3 JANUARY 2013 䡠 VOLUME 121, NUMBER 1
159
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160
BLOOD, 3 JANUARY 2013 䡠 VOLUME 121, NUMBER 1
HICKEY et al
domain, and abolish C/EBP␣ DNA-binding.14 In most cases, the
N-terminal mutations occur concurrently with a C-terminal mutation and are present on different alleles. Paradoxically, those
biallelic double CEBPA mutations have a more favorable prognostic impact.3,19 However, the mechanisms through which CEBPA
mutations lead to better response to treatment and improved
outcomes have not been described.
MicroRNAs (miRNAs) are small noncoding RNAs that repress
translation and/or initiate degradation of specific target messenger
RNAs (mRNA) and have recently been shown to play a role in
carcinogenesis.20 Genome-wide expression profiling of AML has
revealed miRNA signatures associated with distinct cytogenetic or
molecular subtypes of the disease5,21 and have proven useful to
predict clinical outcome in AML patients.22
Recently, we and others reported an association of higher
miR-181a expression and favorable outcomes especially in a
molecular subset of high-risk CN-AML patients (FLT3-ITD positive and/or NPM1 wild-type) and in AML patients with cytogenetic
abnormalities.22-24 In healthy cells, miR-181a regulates B-cell
development, influences T-cell sensitivity to antigens by modulating T-cell receptor signaling intensity, and is involved in early steps
of hematopoiesis.25 A tumor suppressor activity of miR-181a was
reported in chronic lymphocytic leukemia,26 gliomas,27 and astrocytomas,28 and was attributed to direct targeting of BCL2 family
members.28 Recently, it was shown that ectopic expression of
miR-181a sensitizes AML cell lines to chemotherapy.29 In addition,
we reported that higher miR-181a expression was associated with
the presence of CEBPA mutations in CN-AML patients.5
In this study, we show that higher miR-181a expression in
CN-AML patients is predominantly present in the patients harboring CEBPA N-terminal mutations, as opposed to those having
single C-terminal mutations or wild-type CEBPA. We also demonstrate that miR-181a expression is directly modulated by the
C/EBP␣-p30 isoform. Furthermore, we show that the immunomodulatory agent lenalidomide enhances the expression of the N-truncated
C/EBP␣-p30 that is followed by increased miR-181a expression
and augmented sensitivity of leukemia cells to chemotherapy.
Finally, using murine models engrafted with human AML cells
either expressing ectopic miR-181a, or treated in vivo with
lenalidomide, we observed a strong inhibition of tumor growth.
Thus, our findings support future investigations seeking novel
pharmacologic therapies for AML patients with inadequate expression and/or function of the full-length C/EBP␣-p42, with the intent
to enhance the expression of the N-truncated C/EBP␣-p30 isoform
and thus, the expression of miR-181a. The therapeutic induction of
C/EBP␣-p30 and in turn of miR-181a are intended to recapitulate
the favorable outcomes of AML patients with double CEBPA
mutations after treatment with intensive chemotherapy.
Methods
Cell lines and AML patient samples
The cell lines THP-1 (TIB-202), MV4-11 (CRL-9591), HL60 (CCL-240),
KG1a (CCL-246.1), and HEK-293T were from ATCC. CEBPA mutations
were analyzed in adults ⬍ 60 years of age with untreated, primary
molecular high-risk CN-AML enrolled onto the cancer and leukemia
Group B (CALGB) treatment protocols 9621 and 19 808.5 RNA samples
were analyzed for miRNA expression using a previously reported Ohio
State University (OSU) customized miRNA chip.5 Images of the miRNA
microarrays (E-MTAB-1320) were acquired, and calculations, normalizations, and filtering of signal intensity for each microarray spot were
performed as previously reported.5 Expression levels of miR-181a were
compared according to CEBPA mutation status using the Wilcoxon-Rank
sum test. Estimated probabilities of survival were calculated using the
Kaplan-Meier method, and the log-rank test evaluated differences between
survival distributions. Written informed consent for these studies was
obtained from all patients in accordance with the Declaration of Helsinki.
The AML patient blasts used for ex vivo experiments were obtained from
apheresis blood samples collected from patients treated at OSU and stored
in the OSU Leukemia Tissue Bank. After thawing, the cells were placed in
RPMI medium supplemented with 20% FBS and cytokine cocktail
supporting proliferation of hematopoietic progenitors (StemSpan CC100,
StemCell Technologies) and the adherent cells were removed. Informed
consent to use the tissue for laboratory studies was obtained from each
patient according to the OSU institutional guidelines.
Plasmids transfection and miRNA detection by
nanoelectroporation
Nanoelectroporation (NEP) is a microchannel-nanochannel array with the
2 separate microchannels for cell and plasmid loading, that is capable of
delivering the precise amount of transfection reagents into individual
cells.30 C/EBP␣ expression vectors containing either wild-type, N-terminal
(H24Afs), or C-terminal (R300L) mutants were loaded in one side of
microchannels, whereas KG1a cell suspension (⬍ 103/mL) was loaded into
the other side of microchannels with a single cell in each microchannel
being moved to the tip of the nanochannel by optical tweezers. Two electric
pulses across the nanochannels (240V, 5ms each) were applied through
platinum electrodes to conduct the transfection. Transfected cells were
cultured in situ after the removal of the plasmid solution. To detect the
miR-181a expression level inside individual cells, a locked nucleic acid
(LNA) molecular beacon (MB; miR-181a LNA-MB sequence: 5⬘-FAMaccgcg-ActCacCgaCagCgtTgaAtgtt-cgcggt-BHQ1-3⬘ [upper cases represent LNA]; Sigma-Aldrich) was transfected 24 hours later through the same
nanochannel. The images were acquired 4 in 4% PFA solution, at 20°C by
Nicon Eclipse Ti microscope (Nikon Instruments) using Nikon Plan Apo
VC 100X, N.A. ⫽ 1 and Evolve 512 EMCCD camera (Photometrics).
NIS-Elements AR 3.2 software (Nikon Instruments) was used for image
acquisition and processing. The average fluorescence of each cell (with
background correction) was measured 45 minutes after the MB transfection. Three or 4 cells were transfected by NEP for each plasmid.
Xenograft models in NOD/SCID mice
THP-1 cells were injected (10 ⫻ 106 cells/mouse) subcutaneously (1 tumor
per mouse) into 4- to 6-week-old NOD/SCID mice obtained from The
Jackson Laboratory. Lenalidomide (50 mg/kg) or vehicle control (1 ⫻ PBS)
were directly injected into the tumors twice a week for 2 weeks. Five tumors
were treated with lenalidomide and 3 tumors were injected with vehicle.
After 6 weeks the mice were killed and tumor sizes were assessed using
caliper and by weight. To test the effect of miR-181a overexpression on
tumor growth, THP-1 cells were transiently transfected with the Amaxa
system (Solution V, Lonza) with pSUPERIOR-retro-puro vector overexpressing miR-181a, or empty vector. After a 6-hour culture, the cells were
injected, and 6 weeks later tumor sizes were determined as described above.
The in vivo animal studies were conducted with the approval of the OSU
Institutional Lab Animal Care and Use Committee, and in accordance with
the National Institutes of Health Guidelines for animal care.
Additional methods are provided in supplemental Methods (available
on the Blood Web site; see the Supplemental Materials link at the top of the
online article).
Results
N-terminal C/EBP␣ mutations are associated with higher
miR-181a levels in CN-AML
We demonstrated that CN-AML patients with either high miR-181a
expression,22,24 or harboring CEBPA mutations5 have a more
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BLOOD, 3 JANUARY 2013 䡠 VOLUME 121, NUMBER 1
LENALIDOMIDE INDUCES C/EBP␣-p30 AND miR-181a IN AML
161
Figure 1. C/EBP␣-p30 expression is correlated with
increased miR-181a expression. (A) Overall Survival of
younger (18-59 years) CN-AML patients with CEBPA
wild-type, single mutations (N or C-terminal), or double
mutations (N and C-terminal mutations together).
(B) miR-181a expression in younger (18-59 years)
CN-AML patients with either CEBPA wild-type (CEBPA
wt), monoallelic C-terminal mutations (C-term. mut.),
monoallelic N-terminal mutations (N-term. mut.), or concurrent N and C-terminal mutations (N⫹C term. mut.).
(C) Representative patient bone marrow samples with
characterized CEBPA mutations effecting either the
N-terminus or C-terminus of the C/EBP␣ protein. Quantitative real-time RT-PCR data showing the association of
the CEBPA mutation status and expression of the precursor (pre) miR-181a-1 (encoded on chromosome 1 locus)
or miR-181a-2 (encoded on chromosome 9 locus). Each
bar represents the average of triplicate measurements
and error bars denote SEM.
favorable prognosis. Mutations in CEBPA tend to cluster to
2 regions, N or C-terminal and are present mainly in CN-AML
patients.5,13 The majority of these patients have mutations in both
alleles (double mutants), which have better outcomes compared
with the patients with monoallelic mutations or wild-type gene
(Figure 1A). We also showed that patients with CEBPA mutations
had higher miR-181a expression.5
We sought to determine whether the higher miR-181a expression was associated with a particular type of CEBPA mutation
(N vs C-terminal). We analyzed the miR-181a expression among
younger (⬍ 60 years) CN-AML patients (n ⫽ 171) with wild-type
CEBPA, monoallelic C-terminal, monoallelic N-terminal, or biallelic (concurrent N and C-terminal) mutated CEBPA (see supplemental Methods). Expectedly, very few patients presented with single
N-mutations (n ⫽ 4). Patients with N-terminal mutations (single or
double) tended to have a higher miR-181a expression than patients
with a single C-terminal mutation or wild-type CEBPA (Figure 1B).
These differences were statistically significant when patients with
biallelic (N and C-terminal) mutations compared with patients with
single C-terminal mutations (P ⫽ .03) or with wild-type alleles
(P ⬍ .001). Given the very small number, single N-terminal mutation
patients could not be compared with those in the remaining subgroups.
These results were confirmed by quantitative RT-PCR in 3 patients
(Figure 1C), each representing a different CEBPA genotype (wildtype, N-terminal I68Lfs, and C-terminal E167GfsX3 mutants). The
relative expression of C/EBP␣-p42 and p30 polypeptides were
determined (supplemental Figure 1). miR-181a is coded by 2 separate
genes, miR-181a-1 and miR-181a-2, located on chromosomes
1q32.1 and 9q33.3, respectively. Thus, we tested if the expression
of both genes were associated with the distinct C/EBP␣ mutants by
measuring the levels of miR-181a precursors, chromosome 1-derived
pre-miR-181a-1 and chromosome 9-derived pre-miR-181a-2. A
12-fold higher expression of pre-miR-181a-1 was observed in the
patient harboring an N-terminal C/EBP␣ mutation compared with
the patient expressing the wild-type C/EBP␣ (Figure 1C). Only a
slight increase of pre-miR-181a-1 expression was seen in the
sample containing C-terminal C/EBP␣ mutation. In contrast, the
expression of precursor miR-181a-2 was not affected in the AML
patient with C-terminal mutation, and up-regulated only 3.73-fold
in the case with N-terminal mutation, compared with the patient
with wild-type CEBP〈 (Figure 1C). These data suggest that
miR-181a-1 gene expression derived from chromosome 1 is
predominantly affected in patients harboring N-terminal CEBPA
mutations. To further validate these results functionally and to
avoid the interplay of various C/EBP␣ protein isoforms produced
simultaneously from the wild-type and mutant alleles in individual
AML specimens, we constructed vectors expressing either wildtype or mutant CEBPA genes and introduced them individually into
the K562 cell line, which does not express endogenous C/EBP␣.6,31
Schematic diagrams for the various C/EBP␣ wild-type and mutant
proteins are shown in Figure 2A. Two N-terminal mutations
(P23Qfs and H24Afs) led to frame-shifts precluding expression of
the full-length p42 protein, but allowing for reinitiation of translation at the downstream in-frame translational start site; this resulted
in the expression of N-terminally truncated C/EBP␣-p30 isoform.9,14 The C-terminal mutation, R300L, has an amino acid
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162
HICKEY et al
BLOOD, 3 JANUARY 2013 䡠 VOLUME 121, NUMBER 1
Figure 2. Forced expression of patient-derived
N-terminal mutated CEBPA into K562 cells induces
up-regulation of the endogenous miR-181a. (A) Schematic diagram of C/EBP␣ expressed from vectors containing the CEBPA gene isolated from AML patients with
wild-type CEBPA or identified CEBPA mutations.
N-terminal mutations locations, P23Qfs and H24Afs, are
depicted by inverted triangles on the wild-type protein.
N-terminal mutations produce the N-truncated C/EBP␣p30 isoform. C-terminal mutation contains an amino acid
substitution R300L located within the nuclear localization
sequence domain (V-K; underlined amino acids are
believed to be required for nuclear localization). DNAbinding domain (DBD) and leucine-zipper (L-ZIP) domain
are represented by white and black boxes, respectively.
(B) Confocal microscopy images for K562 cells transiently expressing those C/EBP␣ isoforms described in
panel A. All C/EBP␣ isoforms are identified with green
labeling and DAPI staining performed as described,50
indicates location of nucleus shown in blue. The wild-type
C/EBP␣ and N-terminal mutant C/EBP␣ were localized to
the nucleus. In contrast, the C-terminal mutant C/EBP␣
(R300L) was localized in the cytoplasm. (C) Western blot
analysis of cellular fractions after ectopic expression of
C/EBP␣ isoforms described in panel A and expressed in
the leukemia cell line K562. Notably, all isoforms are
localized in the nucleus except for R300L which is
predominately cytoplasmic. UBC9 serves as a positivecontrol for C/EBP␣-p30 expression.33 Ku-70 and Actin
serve as internal loading controls. (D) Quantitative realtime RT-PCR analysis for miR-181a expression in the
K562 transiently transfected with the expression constructs described in panel A. Notably, miR-181-1 expression is highest in those cells transiently expressing the
C/EBP␣-p30 isoform. Each bar represents average of
triplicate measurements and error bars denote SEM.
(E) FAM fluorescence images of KG1a cells transfected
with C/EBP␣ expression plasmids (N-terminal or
C-terminal mutants, wild-type C/EBPA, or empty vector)
and 1 day later with a fluorescent miR-181a LNA-MB
(left), assessed for relative expression of endogenous
miR-181a levels (right). FAM is released from its quencher
and emitted fluorescence when MBs unfold and bind to
miR-181a. Average of 3 to 9 (n) measurements is shown and
error bars denote SEM.
substitution within the highly conserved nuclear localization sequence (NLS).32 Confocal microscopy of transiently transfected
K562 cells (Figure 2B, supplemental Figure 2A) showed that the
wild-type C/EBP␣ proteins were localized in the nuclei, as were
the products from the N-terminally mutated genes (P23Q and
H24A). In contrast, the C-terminal mutant protein (R300L) was
found mainly in the cytoplasm. These results were confirmed by
Western blot analyses using nuclear and cytoplasmic fractions
prepared from the same cells (Figure 2C). In accord with the
published data,14 the N-terminal mutants produced the C/EBP␣p30 protein isoform, which correlated with the increased expression of
nuclear protein UBC9, previously described as being indirectly regulated by C/EBP␣-p30 and not by the full-length p42 isoform.33
Next, we measured the expression of miR-181a precursors
(pre-miR-181a-1 and pre-miR-181a-2) in K562 cells expressing
ectopic C/EBP␣ mutant or wild-type proteins (Figure 2D). Consistent with our previous data, the highest expression of pre-miR181a-1 was observed in the cells expressing the N-terminal
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BLOOD, 3 JANUARY 2013 䡠 VOLUME 121, NUMBER 1
mutants (3- to 4.5-fold), however only a slight increase in
pre-miR-181a-1 expression was observed for the wild-type and
C-terminally mutated isoforms, compared with the empty vector
control. The slight increase of pre-miR-181a-1 expression seen
among the C-terminally mutated isoform was probably because of
the small but noticeable presence of C/EBP␣ protein in the nuclear
fraction. The expression of H24Afs N-terminal mutant resulted in
the highest levels of pre-miR-181a-1 (3.2-fold more than the levels
induced by the wild-type C/EBP␣), whereas the expression of the
P23Qfs N-terminal mutant was associated with 2.2-fold higher
pre-miR-181a-1 expression (Figure 2D). In general, the increase in
miR-181a-1 expression was associated with the expression and
function (as determined by UBC9 expression; Figure 2C) of the
C/EBP␣-p30. The expression of miR-181a-1 was also up-regulated
in cells coexpressing N and C-terminal mutants of C/EBP␣
similarly to what we observed in patients with double CEBPA
mutations (supplemental Figure 2). In agreement with the results
obtained from AML patient samples (Figure 1B), the effect of each
C/EBP␣ isoform on the chromosome 9-derived pre-miR-181a-2 expression was not as prominent (Figure 2D).
To demonstrate the differential impact of CEBPA mutants and
wild-type on miR-181a expression, we transfected plasmids encoding wild-type, N-terminal and C-terminal mutated CEBPA in AML
KG1a cells that do not express CEBPA (C.J.H., unpublished data,
February 2013; also Radomska et al37) using a nanochannel
electroporation (NEP) technique, which was developed by our
group to deliver precise amounts of charged molecules or particles
into individual cells with minimal cell damage.30 NEP relies on the
selection of applied electric voltage, pulse duration and pulse number to
achieve plasmid dosage control. Using the same poration conditions, a
similar amount of plasmids was delivered to each transfected cell.30
A fluorescent LNA-MB was used to measure miR-181a at a single cell
resolution. Figure 2E shows a significant increase in miR-181a expression in cells receiving the N-terminal mutated CEBPA compared with
those receiving C-terminal mutated or wide-type CEBPA.
Taken together, our data suggest that N-terminal mutants are
functionally more active than wild-type and C-terminal mutants in
enhancing the expression of miR-181a-1.
The expression of the pre-miR-181a-1 is regulated by the
N-terminally truncated C/EBP␣-p30 protein
As noted in the introduction and diagrammed in Figure 2A, CEBPA
mRNA can be translated from at least 2 in-frame translation start
sites, leading to production of full-length C/EBP␣-p42 and
N-terminally truncated C/EBP␣-p30 proteins.34 Because we observed a correlation between the expression of patient-derived
N-terminal frame-shift mutants and miR-181a levels, we investigated the C/EBP␣-p30/miR-181a interplay using inducible K562 stable
cell lines expressing either C/EBP␣-p42 or C/EBP␣-p30 isoforms
fused to human estrogen receptor (ER) ligand binding domain.31
On ␤-estradiol treatment, C/EBP␣-ER fusion proteins translocate
into nuclei, where they can exert their effects. Figure 3A shows
miR-181a precursor levels assayed in K562 stable lines after a
48-hour treatment with ␤-estradiol and compared with the levels in the
control cell line that was transduced with the vector expressing the
ER portion of the fusion protein. In the presence of C/EBP␣-p30-ER,
pre-miR-181a-1 expression was increased 6.3-fold (Figure 3A).
A somewhat lesser effect (2.42-fold) was observed in the cell line
expressing C/EBP␣-p42-ER (Figure 3A), similar to the data observed
among cells expressing the full-length C/EBP␣ isoform (Figure 2D).
Similar data were also obtained for THP-1 cells stably transduced with
constitutively active HA-tagged C/EBP␣-p3035 expression construct
LENALIDOMIDE INDUCES C/EBP␣-p30 AND miR-181a IN AML
163
Figure 3. Truncated C/EBP␣-p30 isoform induces expression of pre-miR181a-1. (A) K562 cells were stably transfected with ␤-estradiol inducible C/EBP␣-p30
or p42 ER fusion constructs31,37 enabling nuclear translocation of ectopically
expressed C/EBP␣ proteins. For negative control, vector expressing the ER domain
alone was included. Total RNA was analyzed for the expression of miR-181a-1 (black
bars) and miR-181a-2 (white bars) precursors by quantitative real-time PCR. (B) Total
protein lysates from K562 stable lines described in panel A were analyzed for the
relative expression of C/EBP␣-ER fusion proteins by Western blot stained with
C/EBP␣ antibody. To control for loading, staining with ␤-tubulin antibody was used.
(C) Quantitative real-time RT-PCR data and (D) Northern blot data of THP-1 cells
stably expressing HA-tagged C/EBP␣-p30 isoforms or empty vector.35 Mature
miR-181a expression was found to be highest in those THP-1 cells expressing the
HA-tagged C/EBP␣-p30 isoform. Northern blot shows an increase for both pre-miR181a-1 and mature miR-181a expression for those cells expressing the HA-tagged
C/EBP␣-p30 (snRNA U6 was used as an internal loading control). (E) Western blot
data showing the expression of HA-tagged C/EBP␣-p30 and empty vector for those
cells described in panels C and D. In panels A and C, average of triplicate
measurements is shown and error bars denote SEM.
(Figures 3C-E). Viable THP-1 cells constitutively expressing HA-C/
EBP␣-p42 could not be established because of differentiation-inducing
activity of C/EBP␣-p42 protein (unpublished observations). However,
compared with the empty vector control line, the cells expressing
C/EBP␣-p30 demonstrated a 3.5-fold increase in expression of pre-miR181a-1 (Figure 3C). Increases in precursor miR-181a-1, as well as
mature species were also apparent in Northern blot (Figure 3D).
In summary, using different in vitro systems we demonstrated
that C/EBP␣-p30, and to a lesser extent C/EBP␣-p42, can induce
chromosome 1-derived miR-181a-1 expression in AML cell line
models. Because the N-terminal CEBP〈 mutations in AML are not
capable of producing the wild-type full-length C/EBP␣-p42 isoform,
but allow for translation of the C/EBP␣-p30 protein (Figure 2C),14 we
conclude that C/EBP␣-p30 is the main isoform responsible for upregulation of miR-181a-1 in CEBPA mutated CN-AML patients.
C/EBP␣-p30 physically interacts with the promoter of
miR-181a-1 and regulates its expression
Given that our previous5 and current data showed a correlation
between the expression of C/EBP␣-p30 and increased expression
of miR-181a-1 in AML patients, next we sought to identify the
putative C/EBP␣-responsive element within the miR-181a-1 promoter.
It was recently reported that a region of approximately 600 base pairs
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BLOOD, 3 JANUARY 2013 䡠 VOLUME 121, NUMBER 1
mutation of the putative site abrogated the binding of both C/EBP␣
proteins. The addition of C/EBP␣ antibody (which recognizes both
p42 and p30 isoforms) to the binding reactions led to formation of
super-shifted complexes. Western blot data revealed that C/EBP␣p42 and C/EBP␣-p30 isoforms were expressed at similar levels
(Figure 4B). In contrast, the binding of C/EBP␣ proteins to the
distal predicted C/EBP site (nts ⫺159/⫺154) was not observed
(data not shown).
To determine the transactivation potential of each C/EBP␣
isoform on the miR181a-1 promoter, we inserted a 192 bp proximal
promoter fragment upstream of the firefly luciferase gene and
tested the resulting luciferase activity in the absence and presence
of each C/EBP␣ isoform. As demonstrated in HEK-293T cells and
shown in Figure 4C, both isoforms transactivated the reporter gene,
although C/EBP␣-p30 exhibited a stronger activity than the
C/EBP␣-p42 isoform (6.5-fold vs 4.2-fold, respectively). Part of
the promoter activity in C/EBP␣-p42–transfected cells may be
caused by the expression of the p30 isoform produced from the
p42 expression vector (Figure 4A-C). The reporter construct
containing a C/EBP␣ site mutation (which abrogated C/EBP␣
binding, Figure 4A) showed a decrease in transactivation by both
C/EBP␣-p42 and p30 (22% and 32%, respectively) but did not
completely inhibit it. These data suggest that in addition to the
direct binding to the miR181a-1 promoter, both C/EBP␣ isoforms,
albeit p30 more efficiently than p42, may contribute to miR181a-1
regulation by an indirect mechanism via protein-protein interaction
with other miR-181a-1 promoter-binding factors.
Figure 4. Human miR-181a-1 promoter is regulated by C/EBP␣. (A) C/EBP␣
binds specifically to a site within the human miR-181a-1 proximal promoter. Nuclear
extracts from HEK-293T cells transiently transfected with pcDNA3-FLAG (vect.),
pcDNA3-C/EBP␣-p30-FLAG (␣-p30), or pcDNA3-C/EBP␣-p42-FLAG (␣-p42) were
used in electrophoretic mobility shift assay (EMSA). The radiolabeled probes
contained either wild-type predicted C/EBP-binding site, or mutant (mut.) sequences
(shown below). Lane labeled “probe” contains binding reaction in the absence of
nuclear extract. Where indicated by “⫹” above the lanes, C/EBP␣ specific antibody
was added to the binding reactions. Solid arrowhead shows protein/DNA complex,
whereas open arrowhead indicates binding complex super-shifted with the antibody.
Unbound probe is shown on the bottom of the gel (free probe). (B) Relative
expression of C/EBP␣ proteins in nuclear extracts used in EMSA in panel A is
demonstrated in Western blot stained with C/EBP␣-specific antibody. The p42 and
p30 C/EBP␣ polypeptides are indicated to the right. (C) C/EBP␣-dependent transactivation of the miR-181a-1 promoter. Human miR-181a-1 192 bp promoter fragment
containing wild-type C/EBP-binding site (boxed sequence) or mutated sequence
(indicated below the box) were linked to firefly luciferase gene (black box; diagrammed on top) and transiently transfected to HEK-293T cells with either empty
expression vector pcDNA3-FLAG (⫺), or pcDNA3-C/EBP␣-FLAG vectors (p42 or
p30). Cell lysates were analyzed for luciferase activity and normalized to cotransfected Renilla luciferase activity. For control, C/EBP␣ expression vectors were also
tested with promoter-less luciferase vector, pGL4-11 (vector). Each bar represents
average of 3 transfection experiments and SEM bars are shown. On the right,
representative aliquots of cell lysates used for luciferase assay were also analyzed by
Western blot to demonstrate comparable levels of C/EBP␣ protein.
(bp) upstream of the miR-181a-1 gene exhibits a promoter activity
in immortalized human oral keratinocytes, but the molecular
mechanism(s) regulating this activity were not investigated.36
Using several transcription factor binding site prediction engines
(TFSearch, TESS, MatInspector), we identified 2 potential C/EBPbinding sites located between nucleotides ⫺101/⫺96 and ⫺159/
⫺154 of the miR-181a-1 promoter. To test the binding of C/EBP␣
to those sites, we prepared nuclear extracts from HEK-293T cells
transiently transfected with CEBPA expression vectors coding for
either the full-length or N-truncated C/EBP␣ isoforms. As shown
in Figure 4A, the more proximal site (nts ⫺101/⫺96) bound both
C/EBP␣-p42 and p30 isoforms in a sequence-specific manner:
The immunomodulatory compound lenalidomide induces both
C/EBP␣-p30 and miR-181a expression
Thus far, our data showed that N-terminal CEBPA mutations
(expressing C/EBP␣-p30 protein) associated with higher miR-181a
levels, which in turn predict better outcomes in CN-AML patients,22-24
and that C/EBP␣-p30 was more potent in the up-regulation of miR181a-1 than C/EBP␣-p42. Therefore, we reasoned that the pharmacologic up-regulation of C/EBP␣-p30 and in turn of miR-181a
would be an attractive therapeutic option to improve the clinical
outcomes of AML patients carrying wild-type CEBPA, but with the
expression of a nonfunctional C/EBP␣-p42 (such as C-terminal
mutant, or serine 21 phosphorylated form).31,37 Thus, we focused
our attention on compounds that could act as modulators of
C/EBP␣ protein expression. The immunomodulatory compound,
lenalidomide (Revlimid, Celgene) was selected based on its ability
to up-regulate erythroid-specific genes and its clinical activity in
AML.38-40 We also reported that a gene profile for CEBPA mutant
AML was consistent with partial erythroid differentiation.5 Based
on these observations, we hypothesized that lenalidomide was a
probable candidate for inducing expression of miR-181a via
modulation of C/EBP␣ isoforms.
AML patient-derived blasts were cultured in vitro in the
presence of 3.0␮M lenalidomide and cells were collected hourly
for Western blot analyses. The 3.0␮M concentration of lenalidomide was previously shown to be clinically achievable in vivo.39
We observed an induction of both C/EBP␣ isoforms and an
increase of C/EBP␣-p30 expression compared with C/EBP␣-p42
(Figure 5A). The induction of the C/EBP␣-p30 isoform expression
reached maximum between 5 and 8 hours of the treatment. As
expected the miR-181a expression increased 2.5-fold after 12 hours
and 3.0-fold after 24 hours of lenalidomide treatment (Figure 5B;
same patient sample is shown in Figure 5A-B; also see supplemental Figure 3). Furthermore, AML primary samples treated with
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165
based on the availability of material for the analysis. As shown in
Figure 5C, C/EBP␣ protein expression was induced by day 5 of
lenalidomide treatment (before initiation of chemotherapy) compared with the pretreatment baseline and paralleled by the increase
in miR-181a levels. Although an increase of both C/EBP␣ isoforms
was observed in these samples, it was probable that C/EBP␣-p30
induced miR-181a expression more effectively than C/EBP␣-p42,
as we demonstrated in our previous experiments (Figures 3 and
4). Consistently, the expression levels of miR-181a and C/EBP␣
expression were higher after lenalidomide treatment in bone
marrow samples from 3 AML patients (Nos. 4-6) with available
material and treated on a Washington University clinical trial
(supplemental Figure 4).40 We conclude that lenalidomide treatment in vitro and in vivo leads to increase of C/EBP␣ expression
and induction of miR-181a expression in AML patient blasts. Of
note, the lenalidomide-mediated induction of miR-181a expression
was found only in C/EBP␣-expressing cells (THP-1 and HL60;
Figure 6B and supplemental Figure 5, respectively), and not in cells
lacking the C/EBP␣ expression (K562; supplemental Figure 5),
supporting that C/EBP␣ mediates the lenalidomide-dependent
mechanisms of miR-181a up-regulation.
Lenalidomide treatment sensitizes leukemic cells to cytarabine
chemotherapy
Figure 5. Increased C/EBP␣-p30 expression after treatment with the immunomodulatory compound, lenalidomide. (A) Western blot analysis of AML patient
blasts treated in vitro with 3.0␮M lenalidomide followed by hourly collections. The
expression of C/EBP␣ (p30 and p42) was detected by C/EBP␣ antibody. The signal
intensities were assessed and the p30/p42 ratios were calculated (shown below
C/EBP␣ stained blot). Staining with Actin antibody served as an internal loading
control. The data shown are representative for 3 patient samples. (B) Quantitative
real-time RT-PCR data for miR-181a expression in the same AML blasts shown in
panel A. The expression of miR-181a was increased at 12 hours and 24 hours after
3.0␮M lenalidomide treatment (black bars) compared with the vehicle control (white
bars) for the same time point. Average of triplicate measurements from a single
patient sample is shown and error bars denote SEM. Similar results shown in panels
A and B were observed in a total of 3 separate AML patient blasts used in the same
experiment (not shown). Additional data from the long-term in vitro treatment with
lenalidomide are shown in supplemental Figure 3. (C) Quantitative real-time RT-PCR
data from 3 bone marrow samples from patients treated with lenalidomide induction
therapy (top). Samples were analyzed before treatment (white bars) and 5 days after
lenalidomide induction therapy (black bars). Data are shown as average of triplicate
measurements and error bars denote SEM. Increased expression of miR-181a was
observed on day 5 after lenalidomide induction therapy. Corresponding whole cell
lysates for those patients were analyzed using Western blot (bottom). The expression
of C/EBP␣ (p42 and p30) was increased on day 5 after lenalidomide induction
therapy. Actin served as an internal loading control. Patients’ cytogenetic and clinical
characteristics before the therapy are summarized in supplemental Table 1.
lenalidomide in vitro for 5 days showed a decrease in cell viability
(supplemental Figure 3). Next, we performed analyses of bone
marrow samples from relapse/refractory AML patient who participated in the OSU clinical trial (NCT01132586) receiving lenalidomide before induction chemotherapy. Bone marrow samples (Nos.
1-3) from 3 younger AML patients (⬍ 60 years) were selected
Cytarabine (Ara-C) is a pyrimidine antagonist, which interferes
with DNA synthesis and is used in upfront and salvage regimens
for AML.41,42 To improve the cytotoxic activity of Ara-C treatment,
various novel drug combinations have been explored.43,44 Recently,
it was demonstrated that miR-181a can sensitize a chemotherapyresistant HL60 cell line to Ara-C treatment.29 Having found that
miR-181a is up-regulated in response to lenalidomide, we asked
whether pretreatment of AML cells with this compound can
similarly sensitize the cells to Ara-C.
When THP-1 cells were treated with either 3␮M lenalidomide,
1␮M Ara-C, or both compounds simultaneously, an additive
cytotoxic effect was observed (Figure 6A). Using THP-1 cells
transiently transfected with antagomiR-181a, or a nonsilencing
control oligoribonucleotide, we asked whether the lenalidomide
effect was mediated by miR-181a expression. As shown in Figure
6B, antagomiR-181a efficiently down-regulated levels of the
endogenous miR-181a and the lenalidomide-induced up-regulation
of miR-181a was effectively blocked by antagomiR-181a. Regardless of the levels of miR-181a, lenalidomide treatment led to an
increase in C/EBP␣-p30 protein (Figure 6C), which indicates that
the up-regulation of miR-181a is a downstream event with respect
to regulation of C/EBP␣.
After the transfection with antagomiR-181a or nontargeting
control, THP-1 cells were treated with 3.0␮M lenalidomide or
vehicle every 24 hours for 3 days, followed by a single application
of Ara-C at concentrations ranging between 0␮M and 5.0␮M and
allowed to incubate for 72 hours. Cellular proliferation was
determined by MTS assay. As shown in Figure 6D, Ara-C alone
inhibited cell proliferation in a dose-dependent fashion; however,
the addition of lenalidomide had a stronger inhibitory effect.
Furthermore, down-regulation of miR-181a levels by the transfected antagomiR-181a prevented the antiproliferation effect associated with the lenalidomide treatment (Figure 6D solid blue),
whereas antagomiR-181a had no effect on the cell response to
Ara-C alone (Figure 6D). From these data, we concluded that
lenalidomide sensitizes leukemic cells to conventional Ara-C
therapy via miR-181a.
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Figure 6. Lenalidomide induced miR-181a expression sensitizes leukemia cells to conventional chemotherapy. (A) Untransfected THP-1 cells were cultured for 6 days
in the presence of 3␮M lenalidomide alone, 1␮M ara-C alone, both drugs together (as indicated by “⫹” or “⫺” below the graph), or vehicle control (PBS; the left-most bar) and
MTS proliferation assay was performed. The bars represent averages from 3 to 4 readings. SEM and relative percentages of proliferation rate are shown. (B) Quantitative
real-time RT-PCR analysis for the expression of miR-181a in THP-1 cells transiently transfected with nontargeting control (negative control) or antagomiR-181a. Endogenous
expression of miR-181a was found to be lower in those cells transfected with antagomiR-181a before drug treatments (black bars). After the treatment with 3.0␮M
lenalidomide, miR-181a expression was increased among those cells previously transfected with nontargeting control (red bars). In contrast, the expression of miR-181a was
relatively unchanged among those cells transfected with antagomiR-181a followed by 3.0␮M lenalidomide treatment (blue bars). Data are shown as an average of
measurements and error bars denote SEM. (C) Western blot for those cells described in panel A with the additional treatment with either 3.0␮M lenalidomide or vehicle control
for 3 days. The expression of C/EBP␣ (p42 and p30) was found to be higher in those cells treated with lenalidomide, regardless of earlier transfection status described in panel
A. Actin served as an internal loading control. (D) THP-1 cells transfected with nontargeting control, or antagomiR-181a were cultured with various concentrations (0-5␮M) of
cytarabine (Ara-C) in the presence of 3.0␮M lenalidomide (lenalid.; solid lines), or vehicle (broken lines) for 72 hours and cellular proliferation was measured by MTS assay.
Each datapoint represents an average of 3 measurements.
Lenalidomide treatment in vivo causes a decrease in xenograft
tumor size
Patients responding to lenalidomide were found to have increased
miR-181a expression levels (Figure 5). Moreover, forced expression of miR-181a resulted in reduced cell growth (supplemental
Figure 6A) and colony-forming ability (supplemental Figure 6B),
as well as increased spontaneous death (increased number of cells
in sub-G1 phase; supplemental Figure 6C). In addition, transient
expression of miR-181a in AML patient blasts led to a 2-fold
increase of annexin V labeling, suggesting that blasts expressing
increased levels of miR-181a had a lower apoptosis threshold
(supplemental Figure 6D). To further prove the causal role of
miR-181a in in vivo tumor growth inhibition, mice were injected
with THP-1 cells transiently transfected with miR-181a expression
vector (n ⫽ 5) or with empty control vector (n ⫽ 5). After 6 weeks,
mice were killed and tumors measured. A tumor-suppressing
activity of miR-181a was revealed by decreased tumor sizes and
weights, in mice engrafted with cells forced to express miR-181a
(P ⬍ .01; Figure 7A).
Having established that lenalidomide increases miR-181a expression, we asked whether, similar to miR-181a, this compound could
also suppress tumor growth in vivo. NOD/SCID mice were injected
subcutaneously with untransfected THP-1 cells (10 ⫻ 106 cells).
Four weeks after engraftment, lenalidomide (50 mg/kg; n ⫽ 5) or
vehicle (n ⫽ 3) were injected directly into the tumors twice a week
for 2 weeks. A relatively higher dose of lenalidomide was used in
this experiment to compensate for a short half-life of the drug and
to ensure a promptly evaluable pharmacologic effect in the
leukemia tumors. The mice were killed and tumors excised to
evaluate the effects of lenalidomide. The average size of the
lenalidomide-treated tumors (57.5 ⫾ 0.06 mm) was significantly
decreased compared with the vehicle-treated tumors (166.8 ⫾ 1.08
mm; P ⫽ .008; paired t test; Figure 7B-C). The tumor sizes after
lenalidomide administration were decreased by approximately
3-fold. Consistent with our previous results, lenalidomide-treated
tumors displayed increased miR-181a expression (Figure 7D). The
expression of miR-181a was found to be nearly 10-fold higher
(P ⬍ .05) in the tumors treated with lenalidomide compared with
the vehicle-treated tumors.
In summary, the tumor-suppressing effect of lenalidomide, was
accompanied by an increase of miR-181a expression, which is
analogous to tumor growth suppression by constitutive ectopic
expression of miR-181a, thereby suggesting antitumorigenic properties for this miRNA.
Discussion
We and others reported that CN-AML patients carrying mutations
in CEBPA have better clinical outcomes compared with patients
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BLOOD, 3 JANUARY 2013 䡠 VOLUME 121, NUMBER 1
Figure 7. Lenalidomide treatment in vivo induces miR-181a-mediated inhibition
of xenograft AML tumor growth. (A) THP-1 cells were transiently transfected with
empty expression vector (control), or construct expressing the ectopic miR-181a and
injected subcutaneously into NOD/SCID mice (5 mice per construct). Six weeks later
the tumors were excised (left) and their sizes were determined (right). (B-E) THP-1
cells were xenografted subcutaneously to NOD/SCID mice (1 tumor per mouse).
Four weeks later, tumors were directly injected with lenalidomide (lenalid.; 50 mg/kg;
n ⫽ 5) or vehicle control (n ⫽ 3), twice a week for 2 weeks. Six weeks after
transplantation mice were killed and tumors excised. (B) Xenograft tumors assessed
at the onset (white bars) and the end (black bars) of the treatment. Measurements
were plotted as the relative percentages of the tumors at the beginning of the
treatment. (C) Dissected tumors after the lenalidomide treatment (lenalid.; on the
right) or vehicle control (on the left). (D) Quantitative real-time RT-PCR assessment of
miR-181a expression for the xenografts after treatment with either lenalidomide
(lenalid.; black bar) or vehicle (white bar). (E) The relative expression of C/EBP␣-p42
and C/EBP␣-p30 in lenalidomide (lenalid.) or vehicle-treated xenograft tumors was
evaluated by Western blot of nuclear extracts prepared from the tumors. Blot was
stained with C/EBP␣ antibody and staining with Ku70 served as a loading control.
Both lanes were from the same blot and same exposure. Vertical line has been
inserted to indicate repositioned gel lanes.
with wild-type CEBPA.2-5 We also showed a strong association of
CEBPA mutations and high miR-181a levels5 and better outcomes
of CN-AML patients with higher miR-181a levels.22-24 The favorable impact of CEBPA mutations is restricted to only those patients
with double mutations (usually concurrent N and C-terminal
mutations) and not observed in patients with single mutations,
among which the majority are found only within the C-terminal
region.3,4 A hallmark of AML with CEBPA N-terminal mutations
present in ⬎ 90% of CEBPA mutated patients is that these mutations
prevent the expression of the full-length C/EBP␣-p42 isoform, whereas
the expression of the truncated C/EBP␣-p30 isoform is undisturbed, or
even enhanced.14 In general, C/EBP␣-p30 acts as a dominant-negative
isoform of C/EBP␣-p42 function,9,14,45 but proteomic approaches
identified exclusive targets of C/EBP␣-p30 (UBC9 and PIN1).33,46
Thus, we questioned whether C/EBP␣-p30 could exert any positive
activity on the regulation of miR-181a expression. Indeed, we
LENALIDOMIDE INDUCES C/EBP␣-p30 AND miR-181a IN AML
167
found that although both C/EBP␣ isoforms can directly bind to the
miR-181a-1 promoter, the N-terminally truncated C/EBP␣ is more
potent in its transactivation. To the best of our knowledge, in
contrast to UBC9 and PIN1, the miR-181a-1 gene located on
chromosome 1 is the first reported direct target of C/EBP␣-p30.
Because higher miR-181a expression is associated with a
favorable response to treatment and better outcomes,22-24 we
decided to pursue a strategy that increases the endogenous expression of this miRNA in myeloid blasts. Giving the similarities
between the gene expression profile predictive of response to
lenalidomide and the gene profile associated with CEBPA mutational status, lenalidomide was selected as a potential modulator of
miR-181a expression via C/EBP␣-dependent mechanisms. Indeed,
in both preclinical models and treated AML patients, we showed
that lenalidomide treatment of AML blasts harboring wild-type
CEBPA led to induction of both C/EBP␣-p30, C/EBP␣-p42, and
miR-181a. Lenalidomide has clinical activity in AML.39,40 Thus, in
addition to its immunomodulatory properties, it is also possible that
this activity occurs via 2 mechanisms that implicate C/EBP␣. One
mechanism involves the C/EBP␣-p30–mediated increased expression of miR-181a, which contributes to better chemotherapy
response29 (Figure 6), thereby improving the outcomes of AML
patients with wild-type CEBPA expressing either, nonfunctional
(phosphorylated on serine 21),31,37 or insufficient levels of C/EBP␣p42 to allow for granulocytic maturation.14 Because lenalidomide
also increased C/EBP␣-p42 levels, it is probable that a second
antileukemic mechanism may involve up-regulation of the
differentiation-promoting C/EBP␣-p42 polypeptide. Of course,
lenalidomide’s immunomodulatory activity may also mediate clinical response in AML patients, especially in those patients who
previously have undergone allogeneic stem cell transplantation.39
The mechanism of C/EBP␣ up-regulation by lenalidomide remains
to be elucidated, but preliminary results suggest that lenalidomide
modulates the activity of translation elongation factors responsible
for C/EBP␣-p30 expression (C. Hickey, unpublished data).
Different biologic functions of C/EBP␣ isoforms have been
reported relating to myeloid proliferation and differentiation.14,45,47
C/EBP␣-p30 lacks a differentiation-inducing and antimitotic activities15,48 Paradoxically, the lack of a cell-cycle inhibitory function of
C/EBP␣-p30 serves as an attractive feature when treating AML
with chemotherapeutic agents, such as Ara-C that are most
effective in dividing cells. Our data argue that the favorable impact
of C/EBP␣-p30 may also related to its ability to induce miR-181a,
which in turn can sensitize AML cells to chemotherapy.29 In
agreement, we showed here that lenalidomide can also sensitize
THP-1 cells to Ara-C. This lenalidomide activity is inhibited in the
presence of miR-181a antagomiR, thereby supporting that
lenalidomide-induced sensitivity to Ara-C is dependent on its
ability to increase miR-181a. To prove this principle, a phase 1 trial
of lenalidomide followed by the Ara-C induction chemotherapy is
currently ongoing at our institution (NCT01132586).
In summary, we presented a model for C/EBP␣-p30–dependent
miR-181a up-regulation. The absence of the C/EBP␣-p30 antiproliferative property15,48 and higher levels of miR-181a,22,24 that have
been shown to sensitize leukemia cells to chemotherapy (this
study),29 may partly explain the improved outcomes in CEBPA
mutated CN-AML patients, who usually present with N-mutations
predictive of C/EBP␣-p30 expression. One may raise the question
as to why patients with wild-type CEBPA have lower levels of
miR-181a than those with mutations affecting the N-terminal
region of the protein. Indeed, the C/EBP␣-p42 isoform, which is
linked to differentiation, was found to be expressed concurrently
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HICKEY et al
with the truncated C/EBP␣-p30 isoform among lenalidomidetreated AML patients (Figure 5, supplemental Figures 3 and 4). This
polypeptide also contributes to miR-181a regulation in vitro (Figure 4).
It is possible that in patients with wild-type CEBPA, lower miR-181a
levels are because of a silenced CEBPA49 or nonfunctioning C/EBP␣
protein.31,37 Thus, these patients may benefit from pretreatment
with agents such as lenalidomide alone, or in combination with
hypomethylating agents (for epigenetically silenced CEBPA),49 or
kinase inhibitors (for nonfunctioning serine 21 phosphorylated
C/EBP␣-p42).33 These therapies would increase C/EBP␣-p30
expression and in turn miR-181a, thereby leading to increase
sensitivity to chemotherapy and better clinical outcome.
Acknowledgments
The authors thank Donna Bucci, manager of the OSU and Alliance
(formerly Cancer and Leukemia Group B, CALGB) Leukemia
Tissue Banks, for sample processing and storage services, and Lisa
J. Sterling and Colin G. Edwards for data management. FLAGtagged C/EBP␣ expression vectors were kindly provided by Daniel
G. Tenen (Harvard Medical School, Boston, MA).
This work was supported in part by National Cancer Institute
grants CA101140, CA114725, CA31946, CA33601, CA16058,
CA77658, CA35279, CA03927, and CA41287; the Harry T.
Mangurian Jr Foundation; the Coleman Leukemia Research Foundation; the Leukemia & Lymphoma Society (SCOR grant); and
the National Science Foundation (EEC-0425626). H.B. was supported by the Deutsche Krebshilfe–Dr Mildred Scheel Cancer
Foundation.
Authorship
Contribution: C.J.H., H.S.R., S.S., and G.M. participated in
designing the research and drafting the paper; C.H., S.S., H.S.R.,
A.M.D., A.M.E., S.R., A.M., Y.-Z.W., X.Z., and H.B. performed
experiments; K.M. and D.N. performed the statistical analyses;
H.A., K.M., S.W.P., L.-C.W., D.P., R.B., M.A.C., J.C.B., J.L.,
C.M.C., C.D.B., and R.G. provided technical insight and critically
reviewed the paper; and W.B.,T.A.F., and R.V. participated in
clinical trials, provided clinical samples, and critically reviewed the
paper.
Conflict-of-interest disclosure: The authors declare no competing financial interests.
Correspondence: Guido Marcucci, The Ohio State University,
Comprehensive Cancer Center, Biomedical Research Tower 410, 460 W
12th Ave, Columbus, OH 43210; e-mail: [email protected].
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