doi:10.1182/blood-2007-03-078303Prepublished online May 2, 2007;   

 Briercheck, Mattia Ronchetti, Denis C. Roy, Bruno Calabretta, Michael A. Caligiuri and Danilo PerrottiJi Suk Chang, Ramasamy Santhanam, Rossana Trotta, Paolo Neviani, Anna M Eiring, Edward 

-driven Myeloid DifferentiationαE2-dependent suppression of C/EBPHigh levels of the BCR/ABL oncoprotein are required for the MAPK-hnRNP

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High levels of the BCR/ABL oncoprotein are required for the MAPK-hnRNP E2-

dependent suppression of C/EBPα-driven Myeloid Differentiation.

Running Title: BCR/ABL levels determine the CML cell phenotype.

Ji Suk Chang1, Ramasamy Santhanam1, Rossana Trotta1, Paolo Neviani1, Anna M. Eiring1, Edward Briercheck1, Mattia Ronchetti2, Denis C. Roy3, Bruno Calabretta2, Michael A. Caligiuri1 and Danilo Perrotti1# 1Human Cancer Genetics Program, Dept. of Microbiology Virology and Medical Genetics and The Comprehensive Cancer Center, The Ohio State University, Columbus OH 43210; 3Department of Microbiology and Immunology, Kimmel Cancer Institute, Thomas Jefferson University, Philadelphia PA 19107; 2Division of Hematology-Immunology, Maisonneuve-Rosemont Hospital Research Center, Montreal, QC, Canada. (#) To whom correspondence should be addressed: Danilo Perrotti, The Ohio State University Comprehensive Cancer Center, 2001 Polaris Parkway, Room 205. Columbus, OH 43240. Phone: 614-293-5739; Fax: 614-293-5952; E-Mail: danilo.perrotti@osumc.edu

Blood First Edition Paper, prepublished online May 2, 2007; DOI 10.1182/blood-2007-03-078303

Copyright © 2007 American Society of Hematology

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ABSTRACT

The inability of myeloid chronic myelogenous leukemia blast crisis (CML-BC)

progenitors to undergo neutrophil differentiation depends on suppression of C/EBPα

expression through the translation inhibitory activity of the RNA binding protein hnRNP-

E2. Here we show that “oncogene dosage” is a determinant factor for suppression of

differentiation in CML-BC. In fact, high levels of p210-BCR/ABL are required for

enhanced hnRNP-E2 expression, which depends on phosphorylation of hnRNP-E2

serines 173, 189, 272 and threonine 213 by the BCR/ABL-activated MAPKERK1/2.

Serine/threonine to alanine substitution abolishes hnRNP-E2 phosphorylation and

markedly decreases its stability in BCR/ABL-expressing myeloid precursors. Similarly,

pharmacologic inhibition of MAPKERK1/2 activity decreases hnRNP-E2 binding to the

5′UTR of C/EBPα mRNA by impairing hnRNP-E2 phosphorylation and stability. This,

in turn, restores in vitro and/or in vivo C/EBPα expression and G-CSF-driven

neutrophilic maturation of differentiation-arrested BCR/ABL+ cell lines, primary CML-

BCCD34+ patient-cells and lineage-negative mouse bone marrow cells expressing high

levels of p210-BCR/ABL. Thus, increased BCR/ABL oncogenic tyrosine kinase activity

is essential for suppression of myeloid differentiation of CML-BC progenitors as it is

required for sustained activation of the MAPKERK1/2-hnRNP-E2-C/EBPα differentiation-

inhibitory pathway. Furthermore, these findings suggest the inclusion of clinically-

relevant MAPK inhibitors in the therapy of CML-BC.

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INTRODUCTION

Impaired differentiation is a common feature of many hematological malignancies

including blast crisis chronic myelogenous leukemia (CML). CML is a

myeloproliferative disorder caused by the constitutive tyrosine kinase activity of p210-

BCR/ABL oncoprotein, the product of the t(9;22)(q34;q11) translocation1. In the initial

chronic phase (CML-CP), BCR/ABL provides a survival advantage but does not affect

the differentiation of myeloid progenitors1. However, progression from chronic phase to

the acute and fatal blast crisis (CML-BC) stage impairs the ability of myeloid progenitors

to terminally differentiate into mature neutrophils1. Myeloid differentiation is controlled

by a coordinate network of several transcription factors which regulate the expression of

important differentiation-related genes, including those encoding growth factors and their

receptors2,3. The transcriptional factor CCAAT/enhancer binding protein α (C/EBPα) is

crucial for the differentiation of multipotent myeloid progenitors into granulocytic

precursors4-6; a process which depends, in part, on the C/EBPα-mediated transcriptional

regulation of genes essential for granulocytic differentiation (e.g. G-CSF receptor,

myeloperoxidase and neutrophil elastase)7-10.

We recently reported that downregulation of C/EBPα expression occurs in Ph1(+)

myeloid progenitors from CML-BC patients11. The importance of loss of C/EBPα activity

as a central mechanism leading to differentiation arrest of CML-BC myeloid blasts is

supported by two lines of evidence: a) ectopic C/EBPα expression induces maturation of

differentiation-arrested BCR/ABL+ myeloid precursors11-14; and b) a CML-BC-like

process emerges in mice transplanted with BCR/ABL-transduced Cebpa-null, but not

heterozygous or wild type fetal liver cells15.

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Genetic or functional inactivation of C/EBPα is also a common event occurring in

differentiation-arrested acute myeloid leukemia blasts16,17. Dominant-negative mutations

that abrogate transcriptional activation of C/EBPα have been found in 10% of acute

myeloid leukemia (AML) with normal cytogenetics18,19. By contrast, no mutation of the

CEBPA gene was detected in a screening of 95 CML-BC patients20. In fact, loss of

C/EBPα in CML-BC depends on the BCR/ABL-regulated activity of the RNA binding

protein hnRNP-E2 (PCBP2; poly(rC)-binding protein 2) that, upon interaction with the 5'

untranslated region of CEBPA mRNA, inhibits CEBPA translation11. C/EBPα protein but

not mRNA expression is downmodulated in primary bone marrow cells from CML-BC

patients and inversely correlates with BCR/ABL levels11. Accordingly, hnRNP-E2

expression inversely correlates with that of C/EBPα11, as hnRNP-E2 levels are abundant

in CML-BC but undetectable in CML-CP mononuclear marrow cells. Furthermore, in

myeloid precursors expressing high levels of p210-BCR/ABL, hnRNP-E2 levels are

downmodulated by imatinib treatment11, suggesting that BCR/ABL-generated signals

suppress differentiation by affecting hnRNP-E2 expression/function.

Herein, we show that hnRNP-E2 expression is induced by BCR/ABL in a dose- and

kinase-dependent manner through constitutive activation of MAPKERK1/2. This, in turn,

post-translationally increases hnRNP-E2 protein stability. Furthermore, in vitro and in

vivo suppression of hnRNP-E2 phosphorylation/expression by inhibition of MAPKERK1/2

activity restores C/EBPα expression and rescues G-CSF-driven differentiation of 32D-

BCR/ABL cells, patient-derived CML-BCCD34+ progenitors and primary lineage-negative

(lin-) bone marrow cells ectopically expressing high levels of p210-BCR/ABL

oncoprotein.

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MATERIALS AND METHODS

Cell cultures and primary cells. Philadelphia1-positive K562 and EM-3 cell lines were

maintained in culture in IMDM supplemented with 10% FBS and 2mM L-glutamine. IL-

3-dependent 32Dcl3 myeloid precursor and its derivative cell lines were maintained in

culture in IMDM supplemented with 10% FBS, 2mM L-glutamine, and 10% WEHI-

conditioned medium as a source of mIL-3. For assays requiring cell starvation, cells were

washed four times in PBS and incubated for 8 h in IMDM supplemented with 10% FBS

or 0.1% FBS and 2mM L-glutamine. 32Dcl3- and 32D-BCR/ABL-derived cell lines were

generated by retroviral infections followed by antibiotics-mediated selection or FACS-

mediated sorting of GFP+ cells as described11. Newly established 32D-BCR/ABLhigh cells

were grown in the presence of IL_3 during selection and the cultures of 17-25 days after

retroviral transduction were used in differentiation assays. Normal murine hematopoietic

marrow cells were obtained from the femurs of untreated or 5-FU-treated C57BL/6 mice

after hypotonic lysis. Mononuclear cells (from 5-FU-treated mice) or lineage-negative

cells (Lin-) (Miltenyi Biotech isolation protocol) were kept for 2 days in IMDM

supplemented with 10 % FBS, 2 mM L-glutamine, and murine recombinant cytokines (2

ng/ml IL-3, 2 ng/ml IL-6, 10 ng/ml SCF, 5 ng/ml GM-CSF, and 5 ng/ml Flt3) (R&D

systems) prior to infection with MigRI-GFP or MigRI-GFP-BCR/ABL (W. Pear,

University of Pennsylvania, Philadelphia, PA). Frozen samples of CD34+ bone marrow

cells (NBM) from different healthy donors were purchased from Cincinnati Children’s

Hospital Division of Experimental Hematology (Cincinnati, OH). All studies performed

with human specimens obtained from the Ohio State University (OSU) Leukemia Tissue

Bank (Columbus, OH), and the Division of Hematology, Maisonneuve-Rosemont

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Hospital (Montreal, Canada) were done with approval from the OSU Institutional Review

Board. Mononuclear hematopoietic cells of CML patients were ficoll-separated and

cultured in IMDM supplemented with 30% FBS, 2 mM L-glutamine, and a recombinant

human cytokine cocktail (IL-3 (20 ng/ml), IL-6 (20 ng/ml), Flt3-ligand (100 ng/ml), SCF

(100 ng/ml)) (Stem Cell Technologies, Vancouver, Canada). The CD34+ fraction was

enriched by using a MACS CD34 kit (Miltenyi Biotec., Auburn, CA). Where indicated,

cells were treated for the indicated time with the following enzyme inhibitors: 1-2 µM

imatinib mesylate (STI571, kindly provided by Novartis Oncology), 25 µM U0126

(Promega), 3-10 µM CI-1040 (kindly provided by Pfizer), 2 µM U73122 (Biomol), 50

µM PD98059, 20 nM Rapamycin, 50 µM LY294002, 25 µM ALLN, and 25 µM ALLM

(Calbiochem).

Plasmids. pMAL-PBCP2/hnRNP-E2 was a kind gift of R. Andino (University of

California, San Francisco). pMAL-hnRNP-E2S173A, pMAL-hnRNP-E2S189A, pMAL-

hnRNP-E2T213A, pMAL-hnRNP-E2S272A, and pMAL-hnRNP-E2S173A,S189A,T213A,S272A,

which carry single or quadruple serine/threonine to alanine mutations in the hnRNP-E2

consensus ERK phosphorylation sites [(S/T)P] were generated by site-directed

mutagenesis of pMAL-hnRNP-E2 with Quikchange Mutagenesis system (Stratagene). To

construct plasmids MigRI-HA-hnRNP-E2 and MigRI-HA-hnRNP-

E2S173A,S189A,T213A,S272A, wild-type hnRNP-E2 and hnRNP-E2S173A,S189A,T213A,S272A cDNAs

were PCR-amplified using an upstream primer containing a BamHI site followed by the

hemagglutinin (HA) epitope sequence at the 5’ end of hnRNP-E2, and a downstream

primer containing a XhoI site. The PCR products were digested with BamHI and XhoI

and subcloned into the MigRI vector. MigRI-HA-hnRNP-E2S173D,S189D,T213E,S272D was

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generated by site-directed mutagenesis of MigRI-HA-hnRNP-E2. All regions

mutagenized or amplified by PCR were verified by DNA sequencing. MigRI-ERK1,

MigRI-ERK1K71R, MigRI-ERK2, and MigRI-ERK2K52R were previously described21.

Differentiation assays. For induction of neutrophilic differentiation, cells were washed

three times in PBS and seeded in IMDM supplemented with 10% FBS, 2 mM L-

glutamine, and 10% U87MG-conditioned medium as a source of crude G-CSF or 25

ng/ml of human recombinant G-CSF (Abazyme, Needham, MA). For differentiation

assay of Lin- BMC, cells were seeded in IMDM supplemented with 10% FBS, 2 mM L-

glutamine, 0.2 ng/ml IL-3, 5 ng/ml GM-CSF, 5 ng/ml Flt3-ligand and 25 ng/ml G-CSF.

Morphologic differentiation was assayed by May-Grumwald/Giemsa staining or flow

cytometric detection of the differentiation marker Gr-1 with a specific phycoerythrin

(PE)-conjugated mouse monoclonal antibody (Pharmingen, San Diego, CA). ). The

microscopy was performed using a Zeiss Axioskop 2 Plus microscope (Thornwood, NY)

equipped with a Plan-Neo 40x /0.75 NA objective and a Canon Powershot A70 digital

camera (Lake Success, NY). Images were acquired using Canon Remote Capture

software (Lake Success, NY) and prepared for figures in Adobe Photoshop CS (San Jose,

CA).

Western blot and immunoprecipitation. Cells (1x107) were harvested, washed with

ice-cold PBS, and lysed in 100 µl of 50 mM Tris-HCl, pH 7.5, 1% Triton X-100, 1%

sodium deoxycholate, 0.1 % SDS, 150 mM NaCl, 1mM phenylmethylsulfonyl fluoride

supplemented with a protease inhibitor cocktail (Roche). For C/EBPα detection, cells

(1x106) were washed with ice-cold PBS, lysed directly in 50 µl of Laemmli buffer, and

denatured (5 min, 100°C) prior to SDS-PAGE (4-15%). Antibodies used were: polyclonal

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anti-hnRNP-E2 (R. Andino, University of California, San Francisco, CA), rabbit

polyclonal anti-C/EBPα, anti-G-CSF, monoclonal anti-Abl(24-11) (Santa Cruz

Biotechnology, Santa Cruz, CA), anti-phosphotyrosine, clone 4G10 (Upstate, Lake

Placid, NY), anti-HSP90, anti-GRB2 (BD transduction laboratories, Lexington, KY),

monoclonal anti-MBP (New England Biolabs, Beverly, MA), anti-HA (Convance,

Princeton, NJ), anti-ERK1/2, and anti-pERKThr202/Tyr204 (Cell Signaling, Danvers, MA).

Immunoprecipitations were performed in 20 mM HEPES, pH 7.0, 150 mM NaCl, 0.2 %

NP-40 supplemented with protease and phosphatase inhibitors. Lysates were precleared

with protein G-agarose beads and immunoprecipitated with antibody-coated beads for 3h

at 4°C. After washings, immunoprecipitates were subjected to Western blot analysis.

Recombinant MBP-fusion protein purification and in vitro kinase assay. Overnight

BL21 cultures were diluted and grown in LB+AMP at 30 °C to an OD600 of 0.5. Protein

expression was IPTG induced for 3 h at 30 °C. Cells were harvested, washed,

resuspended in MBP buffer (20 mM Tris-Cl, 200 mM NaCl, 1 mM EDTA, 1 mM DTT)

supplemented with 0.1 M PMSF, 5 mM DTT, and a protease inhibitor cocktail and lysed

by sonication. The lysates were incubated with amylose beads (Sigma, St Louis, MO) for

4 h at 4 °C. After washings, MBP-fusion proteins were eluted with 100 mM maltose in

MBP buffer. To detect phosphorylation of MBP-hnRNP-E2 by ERK1/2 in vitro,

immunoprecipitated ERK1/2 or recombinant ERK1/2 (Upstate, Lake Placid, NY) were

used in kinase assay. Kinase reactions were initiated by addition of 50 µM ATP, 20 µCi

of [γ-32P]ATP (Perkin-Elmer), and 1 µg of affinity-purified MBP fusion proteins in a

final volume of 30 µl. After incubation (30 min; 30 °C), reactions were terminated by

adding 4x Laemmli buffer..

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Pulse-chase experiments. 32D-BCR/ABL cells (2 x 107 cells) expressing HA-hnRNP-

E2, HA-hnRNP-E2S173A,S189A,T213A,S272A or HA-hnRNP-E2S173D,S189D,T213E,S272D were

washed (4X) and incubated for 60 min in methionine-free RPMI 1640 containing 10%

dialyzed FBS. Cells (5x106 cells/ml) were labeled in medium containing of [35S]-

methionine (250 µCi/ml; 1.5 h). After labeling, cells were washed (2X) with methionine-

containing RPMI and cultured (105 cells/ml) for 24 h in RPMI containing L-methionine

(15 µg/ml). At different times, cells (5x106 cells) were harvested and lysed in isotonic

buffer and immunoprecipitations were performed as described above. The

immunoprecipitated proteins were visualized by autoradiography or western blot analysis

and analyzed by densitometry. The values of radioactivity were normalized.

In vivo 32P labeling. 32D-BCR/ABL cells (1.5x107 cells) expressing HA-hnRNP-E2 or

HA-hnRNP-E2S173A,S189A,T213A,S272A were washed (3X) with 0.9% NaCl pH7.4,

resuspended in phosphate-free DMEM containing 0.1% bovine serum albumin and 10

mM HEPES pH7.4, and labeled with [32P]orthophosphate (0.5 mCi/ml; 3 h). Following

labeling, cells were washed (3X) with 0.9% NaCl pH7.4 and lysed in isotonic buffer.

HA-hnRNP-E2 immunoprecipitations were carried out as described. Immunoprecipitated

proteins were visualized by autoradiography or western blot analysis.

RNA electrophoretic mobility shift assay (REMSA). Cytoplasmic extracts from

parental and BCR/ABL-expressing 32Dcl3 cells were prepared and used in REMSA as

described previously11,22. 107 cells were harvested and lysed in 100 µl of binding buffer

(10 mM HEPES-KOH, pH 7.5, 14 mM KCl, 3 mM MgCl2, 5 % glycerol, 0.2 % NP-40, 1

mM DTT, 1 mM PMSF, 5 mM benzamidine, 1 mM Na3VO4, 50 mM NaF, 10 mM β-

glycerophosphate) supplemented with protease inhibitors, and the cytoplasmic fraction

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was purified by centrifugation (2 min, 8500xg, 4°C). For REMSA, 20 µg of cytoplasmic

proteins were incubated with 50,000 cpm of 32P-labeled WT-uORF of CEBPA ribo-

oligonucleotide for 30 min at RT. After additional 20 min incubation with heparin, the

reactions were resolved on 5 % PAGE.

RT-PCR. Nuclear RNA (1 µg) obtained from sucrose gradient-purified nuclei by acid-

phenol/guanidinium-mediated extraction was treated with DNAse I and subjected to

reverse transcription using 200U M-MLV reverse transcriptase (Gibco-BRL), 200 µM

dNTPs, 0.25 U/ml random hexamers (Pharmacia) and sets of primers specific for

hnRNP-E2 or hnRNP-E1 or that recognize both hnRNP-E2 and E1 mRNA. As an

internal control, all cDNA samples were adjusted to yield relatively equal amplification

of β-actin.

In vivo effect of CI-1040 on neutrophilic maturation of differentiation-arrested

BCR/ABL+ cells. 6.15-MigR1 and 6.15-WT-uORF cells were injected (5x106

cells/mouse) intravenously into 10 week-old SCID mice (n=6 per group). Mice were

monitored for engraftment a week after cell injection by nested RT-PCR-mediated

detection of BCR/ABL transcripts23. CI-1040 was dissolved in 10% Cremophor EL

(Sigma), 10% ethanol, and 80% water as previously reported24,25. Mice were divided into

2 groups; one received for two weeks intraperitoneal injections of G-CSF (Neupogen,

Amgen) (100µg/kg/day)26,27 and CI-1040 (100mg/kg/mouse/twice a day)24 using dosage

and schedules previously described by other groups, whereas the other received G-CSF

and vehicle–only for the same time period. After the end of treatment, mice were

sacrificed and tissue sections from spleens were fixed in formalin and embedded in

paraffin blocks. Slides were stained with hematoxylin/eosin (H&E) or with chloroacetate

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esterase (Leder staining) to assess presence of differentiated myeloid cells. The

microscopy was performed using a Zeiss Axioskop 2 Plus microscope (Thornwood, NY)

equipped with a Plan-Neo 40x /0.75 NA objective and a Canon Powershot A70 digital

camera (Lake Success, NY). Images were acquired using Canon Remote Capture

software (Lake Success, NY) and prepared for figures in Adobe Photoshop CS (San Jose,

CA).

RESULTS

BCR/ABL post-translationally enhances hnRNP-E2 expression. HnRNP-E2

expression is induced upon retroviral transduction of the 32Dcl3 myeloid cells with p210-

BCR/ABL and directly correlates with BCR/ABL levels and kinase activity in primary

CML progenitors11. Similarly, doxycycline treatment (1µg/ml, 24h) enhances hnRNP-E2

expression in the TonB210.1 lymphoid precursor cells expressing a tetracycline-inducible

p210-BCR/ABL(not shown).

To determine the molecular mechanism(s) whereby BCR/ABL increases hnRNP-E2

levels, hnRNP-E2 mRNA and protein expression were assessed in parental and

BCR/ABL-expressing 32Dcl3 cells cultured in the presence of IL-3 or IL-3-deprived for

8 hours. Regardless of the presence or absence of IL-3, hnRNP-E2 protein but not

mRNA levels were enhanced in 32D-BCR/ABL cells when compared to parental cells

(Fig. 1A). Notably, 32Dcl3 exhibited high levels of hnRNP-E2 mRNA but low levels of

protein that further decreased upon IL-3 deprivation (Fig. 1A). These data suggest that

the primary mechanism by which myeloid cells regulate hnRNP-E2 expression is post-

transcriptional. Interestingly, treatment of IL3-deprived 32Dcl3 cells with the

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proteosome/calpain inhibitor ALLN restored, albeit not totally, hnRNP-E2 expression

(Fig. 1B, lanes 1-3). Because the calpain inhibitor ALLM was unable to rescue hnRNP-

E2 expression in parental cells (Fig 1B, lane 4), and neither ALLN nor ALLM affected

hnRNP-E2 expression in 32D-BCR/ABL (Fig. 1B, lanes 5-8), increased protein stability

may account for hnRNP-E2 overexpression in BCR/ABL cells. Indeed, a more rapid

decline of hnRNP-E2 levels was observed in parental 32Dcl3 than 32D-BCR/ABL cells

treated with the protein synthesis inhibitor cycloheximide (CHX; 25 µg/ml) (Fig. 1C). As

expected11, mRNA and protein expression of the closely related hnRNP-E1 protein28 was

not affected by BCR/ABL expression (Fig. 1A and 1C). Thus, p210-BCR/ABL enhances

hnRNP-E2 expression by inducing post-translational modifications that increase hnRNP-

E2 stability most likely by delaying its proteasome-dependent turnover.

Increased hnRNP-E2 expression requires phosphorylation by the BCR/ABL-

activated MAPKERK1/2. To determine the mechanism(s) whereby BCR/ABL induces

hnRNP-E2 upregulation, the CML-BC-derived EM-3 myeloid cell line was exposed to

various inhibitors of BCR/ABL-activated pathways1,29-33. HnRNP-E2 levels were

markedly reduced by treatment with the MEK inhibitor PD098059 (50µM) but not with

inhibitors of PI-3K- (LY204002, 50µM), PLCγ- (U73122, 2µM) and mTOR/S6K-

(rapamycin, 20nM) dependent signals (Fig. 2A), suggesting that enhanced hnRNP-E2

expression may depend on BCR/ABL-induced activation of MAPK (i.e. ERK1/2).

Indeed, hnRNP-E2 levels were also inhibited by treatment with the MEK1/2 inhibitor

U0126 (25µM ) in K562 and 32D-BCR/ABL cells and by ectopic expression of

dominant-negative ERK1K71R and ERK2K52R mutants34 (Fig. 2A). Furthermore, decreased

hnRNP-E2 levels (~ 80% reduction) were observed upon treatment of primary CML-

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BCCD34+ progenitors (n=3) with U0126 (Fig. 2A). As expected, inhibition of BCR/ABL

activity by imatinib also led to suppression of hnRNP-E2 expression (Fig. 2A).

Consistent with the existence of a BCR/ABL-MAPK pathway that post-translationally

regulates hnRNP-E2 expression, sequence analysis of hnRNP-E2 revealed the presence

of four consensus ERK phosphorylation sites (S/T-P)35,36 at amino acid residues 173, 189,

213, and 272 (Fig. 2B). Thus, wild type and serine/threonine to alanine mutant HA-

tagged and MBP-fusion hnRNP-E2 proteins were generated and used to assess in vivo

and in vitro MAPK-dependent phosphorylation. In [32P]-labeled 32D-BCR/ABLcells,

wild type HA-tagged hnRNP-E2 was abundantly phosphorylated (Fig. 2C, lane 1). By

contrast, alanine substitution of residues 173, 189, 213 and 272 completely abolished

hnRNP-E2 in vivo phosphorylation (Fig. 2C, lane 3), which was also markedly reduced

by inhibition of MAPK activity with U0126 (Fig. 2C, lane 2). Furthermore, recombinant

MBP-hnRNP-E2 fusion protein was efficiently phosphorylated in vitro by the BCR/ABL-

activated ERK1/2 immunoprecipitated from lysates of 32D-BCR/ABL cells (Fig. 2D left,

upper panel, lane 2). Likewise, in vitro kinase assays showed that ERK1/2-dependent

phosphorylation of rMBP-hnRNP-E2 was significantly reduced by single

serine/threonine to alanine substitutions and almost totally abolished in the quadruple

hnRNP-E2 mutant (Fig.2D, right panels). Note that the effect of ERK2 on hnRNP-E2

phosphorylation was more pronounced than that of ERK1 (Fig. 2D, right panel). Because

ERK2 activity is higher than that of ERK1 in CML-BCCD34+ progenitors21, these data

demonstrate that serines/threonine at residues 173, 189, 213, and 272 are crucial for

ERK-dependent phosphorylation of hnRNP-E2 and suggest that MAPKERK2 is the kinase

responsible for hnRNP-E2 phosphorylation in BCR/ABL+ cells.

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To determine whether ERK phosphorylation accounts for increased hnRNP-E2 protein

stability, expression of HA-tagged wild type hnRNP-E2, non-phosphatable hnRNP-

E2S173A,S189A,T213A,S272A (Ser/Thr�Ala) and phospho-mimetic hnRNP-

E2S173D,S189D,T213E,S272D (Ser/Thr�Asp/Glu) was assessed in parental and BCR/ABL-

transduced 32Dcl3 cells IL-3-deprived for 8 hours. Expression of wild type and non-

phosphatable hnRNP-E2 was low or undetectable in 32Dcl3 cells (Fig. 3A, top panel).

Likewise, hnRNP-E2S173A,S189A,T213A,S272A expression was also less abundant than wild

type in BCR/ABL-expressing cells (Fig. 3A, bottom panel). Conversely, levels of the

phosphomimetic hnRNP-E2S173D,S189D,T213E,S272D were abundant in 32Dcl3 cells and

comparable to those of wild type in 32D-BCR/ABL cells (Fig 3A), further suggesting

that increased hnRNP-E2 stability depends on ERK-mediated phosphorylation at these

indicated amino acids. Indeed, pulse chase experiments revealed that the half-life of

newly synthesized 35S-hnRNP-E2S173A,S189A,T213A,S272A (t1/2 ≅ 5.5 h) was shorter than that

of wild type (t1/2 ≅ 7 h) and phosphomimetic (t1/2 ≅ 14 h) hnRNP-E2 proteins in

metabolically-labeled 32D-BCR/ABL cells (Fig 3B). Accordingly, expression of the

hnRNP-E2S173A,S189A,T213A,S272A mutant was more rapidly downmodulated than that of

wild type and phosphomimetic hnRNP-E2 in CHX-treated 32D-BCR/ABL-expressing

cells (Fig. 3C). Altogether, these data indicate that ERK-dependent phosphorylation of

hnRNP-E2 at serines 173, 189, 272 and threonine 213 is responsible for increased

hnRNP-E2 protein stability in BCR/ABL-transformed cells.

In vitro and/or in vivo suppression of ERK-dependent hnRNP-E2 phosphorylation

restores C/EBPα expression and rescues G-CSF-driven neutrophilic maturation of

BCR/ABL+ cell lines, primary Lin- bone marrow cells and CML-BCCD34+

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progenitors. To further assess the importance of BCR/ABL expression in the hnRNP-

E2-mediated suppression of myeloid differentiation, 32D-BCR/ABL single cell clones

expressing low or high levels of p210-BCR/ABL were isolated upon transduction of

32Dcl3 cells with the MigR1-p210-BCR/ABL expression plasmid. 32D-BCR/ABL cells

expressing high BCR/ABL levels showed a marked increase of hnRNP-E2 levels and

suppression of C/EBPα expression (Fig. 4A, left panel lanes 1-4). This correlated with

the development of growth factor independence and differentiation arrest (not shown). By

contrast, low BCR/ABL-expressing cells exhibited high C/EBPα expression and barely

detectable hnRNP-E2 levels (Fig. 4A left panel, lanes 5-8), were IL-3-dependent and

underwent differentiation upon treatment with G-CSF (not shown).

Since increased hnRNP-E2 expression depends on its phosphorylation by the BCR/ABL-

activated MAPKERK1/2 kinases, we investigated the in vitro and in vivo effects of

MEK1/2 inhibition by U0126 or by the clinically-relevant CI-104037-40 (PD184352;

Pfizer) on the BCR/ABL-MAPKERK1/2-hnRNP-E2-C/EBPα axis and granulocytic

differentiation of myeloid progenitors (primary and cell lines) expressing high levels of

p210-BCR/ABL.

In newly established 32D-BCR/ABLhigh cells cultured in the presence of G-CSF,

inhibition of MAPKERK1/2 activity by U0126 (32 h) (Fig. 4A, right panel) and by CI-1040

(48 h) (not shown) abolished the expression of hnRNP-E2 and rescued that of C/EBPα.

Accordingly, REMSA assays with the 32P-labeled C-rich uORF/spacer region of CEBPA

mRNA and cytoplasmic lysates of U0126-treated 32D-BCR/ABLhigh cells revealed that

inhibition of MAPK activity decreases binding of hnRNP-E2 to the translation inhibitory

element contained in the 5’UTR of CEBPA mRNA (Fig. 4B, left panel). Furthermore,

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32D-BCR/ABLhigh cells treated with U0126 (25µM) or CI-1040 (10µM) in the presence

of G-CSF underwent terminal granulocytic differentiation as indicated by increased

expression of the myeloid differentiation marker Gr-1 after 4-day culture in G-CSF and

by the appearance of polymorphonuclear cells after 7-day culture in G-CSF (Fig. 4B right

panel and 5C). Likewise, suppression of BCR/ABL activity by imatinib caused molecular

and morphological effects similar to those induced by MEK1/2 inhibition (Fig. 4B and

4C). As expected, parental 32Dcl3 cells expressed C/EBPα but not hnRNP-E2 and

underwent G-CSF-driven granulocytic differentiation (Fig. 4).

Rescue of C/EBPα expression and restoration of neutrophilic maturation by

pharmacologic inhibition of ERK activity were not only observed in differentiation-

arrested 32D-BCR/ABLhigh cells but also in lineage-negative (Lin-) mouse bone marrow

cells (BMC) transduced with the MigR1-GFP-BCR/ABL retrovirus11 and sorted for low

and high GFP expression. As expected, GFP levels correlated with BCR/ABL expression

and activity (Fig. 5A, left panel). Consistent with the knowledge that differentiation arrest

is a characteristic of CML-BC myeloid progenitors expressing high BCR/ABL and

hnRNP-E2 levels11, graded p210-BCR/ABL expression resulted in a dose-dependent

induction of MAPKERK1/2 activity and hnRNP-E2 expression that inversely correlated

with levels of C/EBPα and of the C/EBPα-regulated G-CSF receptor (G-CSFR) (Fig. 5A,

left panel). Note that total MAPKERK1/2 levels were not affected by BCR/ABL (Fig. 5A,

left panel). Furthermore, Lin- BMC-BCR/ABLlow but not Lin- BMC-BCR/ABLhigh cells

underwent granulocytic differentiation within 6 days of culture in the presence of G-CSF

(25 ng/ml) (Fig. 5A, right panel). Notably, downregulation of hnRNP-E2 levels and

restoration of C/EBPα expression were also observed in G-CSF-cultured mouse Lin-

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BMC-BCR/ABLhigh progenitors (Fig. 5A, middle panel) and patient-derived CML-

BCCD34+ cells (n=3) (Fig. 5B, left panel) exposed for 48h to the MEK1/2 inhibitor CI-

1040 and/or U0126. Moreover, U0126- or CI-1040-treated differentiation-arrested Lin-

BMC-BCR/ABLhigh and CML-BCCD34+ developed into mature neutrophils upon exposure

to G-CSF (25 ng/ml) (Fig. 5A and 5B, right panels).

To assess whether inhibition of MAPKERK1/2 activity rescues in vivo neutrophilic

maturation of differentiation-arrested BCR/ABL+ myeloid blasts, we used a 32D-

BCR/ABL long-term cultured cell clone (6.15 cells) that exhibits abnormally high levels

of BCR/ABL and hnRNP-E2 expression but is unable to undergo G-CSF-driven

differentiation because of transcriptional suppression of CEBPα expression11. Thus, 6.15

cells were retrovirally transduced with either a MigRI-GFP-wt-uORF/spacer-C/EBPα

(6.15-WT-uORF) plasmid containing the hnRNP-E2 negative translational regulatory

element11 or with an empty MigRI-GFP vector (6.15-MigRI). SCID mice (n=6 per group)

were thereafter intravenously (i.v.) injected with 6.15-wt-uORF and 6.15-MigRI cells

(5x105 GFP-sorted cells/mouse), and engraftment was assessed one week after cell-

injection by nested RT-PCR-mediated detection of p210-BCR/ABL transcripts in

peripheral blood cells (not shown). Thereafter, mice were co-treated with either G-CSF

(100 µg/kg/day)26,27 and CI-1040 (100 mg/kg/twice a day)24 or G-CSF and vehicle for 14

days. At the end of treatment, spleen tissue sections from untreated, CI-1040-treated, and

control (age-matched) mice were subjected to histopathologic examination. Consistent

with the almost complete lack of translational control of C/EBPα expression in 6.15

cells11, Hematoxylin/eosin-stained sections of spleens from untreated 6.15-MigRI-

injected mice showed massive infiltration of myeloid blasts with a low degree of myeloid

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differentiation (≤1% neutrophils), which was further confirmed by the scarce

chloroacetate esterase (Leder staining) positivity (Fig. 6, bottom panels). Terminally

differentiated myeloid cells were occasionally observed in spleens from CI-1040-treated

6.15-MigRI-injected mice (2-5% neutrophils) (Fig. 6, bottom panels), suggesting the

involvement of MAPK in additional mechanisms leading to suppression of myeloid

differentiation. By contrast, spleens from CI-1040-treated 6.15-wt-uORF-injected mice

showed marked infiltration of mature neutrophils (40-70% neutrophils) and myeloid

precursors at the post-mitotic stages of differentiation (Fig. 6, top panels). Accordingly, a

high degree of chloroacetate esterase-positivity was observed in spleen section of CI-

1040-treated 6.15-wt-uORF-injected mice (Fig 6, bottom panels). However, 5-10% of

terminally differentiated myeloid cells were also observed in spleens of vehicle (DMSO)-

treated 6.15-wt-uORF-injected mice (Fig. 6, top panels) as a consequence of the leaky

nature of the MigR1-wt-uORF-C/EBPα plasmid that allows low levels of

C/EBPα expression. Note that histopathologic analysis of H&E and Leder-stained spleen

sections from untreated and CI-1040-treated age-matched mice did not show signs of

myeloid cell infiltration (Fig. 6). Altogether, these results suggest that in vivo rescue of

granulocytic maturation of differentiation-arrested BCR/ABL+ cells by chemical ERK1/2

inactivation is likely due to decreased hnRNP-E2 expression that, in turn, reverses

translation inhibition of C/EBPα mRNA.

The p53 status does not affect BCR/ABL-dependent regulation of C/EBPα levels.

Genetic (mutations) or functional (MDM2-dependent degradation) inactivation of p53 is

a common feature of CML-BC41,42. Because it has been reported that C/EBPα is

positively regulated by wild type p5343, and loss of p53 activity is associated with and

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may promote blastic transformation44,45, we asked whether BCR/ABL–dependent

downregulation of C/EBPα levels may be affected by the p53 status. Thus, bone marrow

mononuclear cells were isolated from 5-FU-treated (4 days) wild type or p53-/- C57BL/6

mice and left untreated or transduced with the MigRI-p210-BCR/ABL retrovirus. After

sorting, GFP positive cells were expanded for 2 days in the presence of IL-3, KL, IL-6

and FLT3 ligands, washed and cultured in the presence of G-CSF for 3, 5 and 7 days.

Levels of C/EBPα were readily detectable after 3 days of culture in the presence of G-

CSF but rapidly declined in both cell cultures with similar kinetics (Fig 7). By contrast,

in both p53 -/- and +/+ mononuclear cells maintained in culture for the same length of

time, expression of C/EBPα was barely detectable at time 0 but increased with similar

kinetics upon G-CSF treatment for 2-8 days (Fig. 7). Altogether, these findings suggest

that the p53 status does not directly affect C/EBPα expression and does not cooperate

with BCR/ABL in promoting its downregulation.

DISCUSSION

The mechanisms responsible for transition of CML chronic phase into blast crisis remain

poorly understood, although it appears that the unrestrained activity of BCR/ABL in

hematopoietic stem/progenitor cells is the primary determinant of disease progression1,46.

Indeed, BCR/ABL expression specifically increases during blastic transformation in

hematopoietic stem cells and committed myeloid progenitors47-53clones with increasingly

malignant characteristics. In fact, augmented BCR/ABL activity alters the expression

and/or function of several factors important for proliferation, survival and maturation of

myeloid progenitors1,46.

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Despite the molecular heterogeneity of CML-BC, a distinctive feature of blast crisis

CML is the inability of myeloid progenitors to differentiate into mature granulocytes1.

We recently reported that impaired differentiation of myeloid CML-BC progenitors is

caused by the BCR/ABL-dependent suppression of C/EBPα expression through the

translation inhibitory activity of the RNA binding protein hnRNP-E211, and that hnRNP-

E2 protein levels specifically increase in CML-BC progenitors in a BCR/ABL kinase-

dependent manner11. By progressively increasing BCR/ABL expression in Lin- mouse

marrow progenitors and hematopoietic cell lines, we provided evidence that induction of

hnRNP-E2 expression requires high levels of p210-BCR/ABL. These findings are

consistent with the lack of C/EBPα expression in CML-BC and the low hnRNP-E2 and

BCR/ABL expression in CML-CP progenitors11. In BCR/ABL-expressing myeloid

precursors and primary CML-BC progenitors, the molecular mechanism whereby

BCR/ABL upregulates hnRNP-E2 expression is post-translational and depends on

increased hnRNP-E2 protein stability. This is consistent with the observation that

enhanced expression of various RNA binding proteins is among the many changes in

gene expression found in bone marrow-derived myeloid progenitors from CML-BC

patients and BCR/ABL-transformed murine myeloid progenitors11,54. This enhanced

expression of RNA binding proteins correlates with the levels of BCR/ABL and is

sensitive to imatinib treatment11,46,54. Molecular mechanisms responsible for such an

increase may involve the activation of a cascade of phosphorylation events leading to

either enhanced gene transcription (e.g. hnRNP K) or increased protein stability (e.g.

TLS/FUS, hnRNP A1, and La/SSB)11,21,42,55-58. Increased expression of these RNA

binding proteins correlates with enhanced post-transcriptional- and/or translational-

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regulatory activity, which is tightly controlled by different BCR/ABL-activated

pathways21,54,55,58. Indeed, we showed that hnRNP-E2 undergoes phosphorylation in

BCR/ABL-expressing cells. Furthermore, by treating BCR/ABL+ cell lines, primary

BCR/ABL-expressing Lin- mouse bone marrow progenitors and patient-derived CML-

BCCD34+ cells with chemical inhibitors of MEK1/2, including the clinically relevant CI-

104059,60, and by expressing ERK 1 and 2 constructs with dominant negative activity34,

we demonstrated that increased hnRNP-E2 protein stability requires the constitutive

activation of MAPKERK1/2. Furthermore, we also identified four hnRNP-E2 MAPKERK1/2-

phosphorylation sites and demonstrated that hnRNP-E2 is a bona fide MAPKERK1/2

substrate and that MAPKERK1/2-dependent phosphorylation of hnRNP-E2 at these amino

acid residues is essential for increased hnRNP-E2 expression in BCR/ABL-expressing

cells. The involvement of MAPKERK1/2 in the regulation of hnRNP-E2 expression in

CML-BC is not surprising, as constitutive MAPK activation is readily detectable in

CML-BCCD34+ progenitors21 while CML-CP progenitors are only capable of transient

MAPK activation in response to mitogenic and survival signals induced by extracellular

growth factors61. In fact, MAPKERK1/2 activation appears to depend on increased levels of

BCR/ABL activity, as graded BCR/ABL expression correlates with a progressive

increase in MAPKERK1/2 activity21. Interestingly, in BCR/ABL+ myeloid progenitor cells,

MAPKERK1/2 is also responsible for the increased expression and translational-regulatory

function of the shuttling RNA binding protein hnRNP K21. Like hnRNP-E2, hnRNP K

belongs to the subfamily of the K homology (KH)-domain containing poly(rC)-binding

proteins62. However, the molecular mechanism whereby MAPKERK1/2 enhances hnRNP K

translational-regulatory function appears different from that of hnRNP-E2. In fact,

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BCR/ABL promotes the cytoplasmic accumulation of hnRNP K by MAPKERK1/2-

dependent phosphorylation at serines 284 and 35321. Conversely, the subcellular

localization of hnRNP-E2 does not appear to be regulated by BCR/ABL or MAPKERK1/2

activity, as neither imatinib nor U0126 treatment induced hnRNP-E2 nuclear relocation

in BCR/ABL+ myeloid cells (not shown). Thus, enhancement of hnRNP-E2 translational-

regulatory activity largely results from the effect of BCR/ABL-activated MAPKERK1/2 on

hnRNP-E2 expression. Nonetheless, hnRNP-E2 and hnRNP K cooperate in the positive

regulation of MYC translation via binding to the IRES element located in the 5’UTR of

MYC mRNA21,63. Because hnRNP K translationally enhances MYC expression in a

BCR/ABL- and MAPKERK1/2-dependent manner21, and differentiation of myeloid

progenitors requires the C/EBPα-induced MYC downregulation64,65, it is conceivable that

BCR/ABL uses MAPKERK1/2 to activate two similar RNA binding proteins (i.e. hnRNP-

E2 and hnRNP K) that, in turn, simultaneously inhibit C/EBPα and enhance MYC

expression, two steps essential for the phenotype of leukemic myeloid progenitors14,64,66-

68. Indeed, C/EBPα expression decreases proportionally with a progressive increase of

BCR/ABL levels. Accordingly, suppression of MAPKERK1/2 activity impairs hnRNP-E2

binding to CEBPA mRNA, rescues C/EBPα and G-CSFR expression, and allows in vitro

and in vivo G-CSF-driven neutrophil maturation of differentiation-arrested marrow

progenitors expressing high levels of p210-BCR/ABL. Thus, constitutive MAPKERK1/2

activation is not only essential for transduction of mitogenic and survival signals but it

also promotes the activation of anti-differentiation signals (i.e hnRNP-E2

phosphorylation) leading to loss of C/EBPα expression in CML-BC progenitors. In

support of the relevance of MAPKERK1/2 activation in CML-BC rather than CML-CP, it

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was reported that levels of activated MAPKERK1/2 in the absence of exogenous cytokines

were similar in normal and CML-CP cells, and imatinib neither reduced nor altered

MAPKERK1/2 activity in both normal and CML-CPCD34+ progenitors61. Moreover,

MAPKERK1/2 activity appears to be required for post-translational inactivation of

C/EBPα69,70. MAPKERK1/2-mediated phosphorylation of C/EBPα on serine 21 inhibits its

transcriptional-activating function and leads to a block of granulocytic differentiation in

acute myeloid leukemia cells69,70. This observation is consistent with the differentiation-

promoting effects of MEK1 inhibitors in myeloid progenitors expressing high levels of

p210-BCR/ABL oncoprotein; in fact, rescued C/EBPα is not phosphorylated on serine 21

in BCR/ABL+ cells treated with U0126 and G-CSF (not shown). Hence, constitutive

activation of MAPKERK1/2 is a feature of CML-BC but not CML-CP in which myeloid

progenitors express C/EBPα11 and retain the ability to terminally differentiate into

apparently normal neutrophils.

In conclusion, our data formally demonstrate that impaired differentiation of CML-BC

myeloid progenitors requires increased expression/activity of p210-BCR/ABL

oncoprotein and depends on the constitutive activation of the inhibitory BCR/ABL-

MAPKERK1/2-hnRNP-E2-C/EBPα pathway. Moreover, the demonstration that

pharmacologic inhibition of MAPKERK1/2 activity rescues neutrophilic maturation of

differentiation-arrested BCR/ABL+ myeloid blasts portents the addition of clinically-

relevant MEK inhibitors to the current imatinib or dasatinib monotherapy for patients

with myeloid CML-BC.

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ACKNOWLEDGEMENTS We thank Novartis Oncology and Pfizer Inc. for providing imatinib mesylate and CI-1040, respectively. This work was supported by the National Cancer Institute CA095512 (D.P.), CA16058 and GRT8230100 (OSU-CCC); and the US Army, CML Research Program DAMD17-03-1-0184 (D.P.); JSC was in part supported by the Up-on-the-Roof Human Cancer Genetics Fellowship, The Ohio State University, Columbus OH. PN is a fellow of the American Italian Cancer Foundation (New York, NY). D.P. is a Scholar of the Leukemia and Lymphoma Society. DCR is supported by the Canadian Institutes of Health Research and Fonds de la Recherche en Sante du Quebec. Contributions: Ji Suk Chang: performed research and wrote paper Ramasamy Santhanam: performed research Rossana Trotta: Performed research Paolo Neviani: performed research Anna Eiring: performed research Edward Briercheck: performed research Mattia Ronchetti: performed research Denis C. Roy: contributed vital material Bruno Calabretta: supervised M.R. work and analyzed data Michael A. Caligiuri: supervised R.T. work and contributed vital material Danilo Perrotti: designed research, analyzed data, wrote paper and supervised work.

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Figure 1

Figure 1. BCR/ABL post-translationally enhances hnRNP-E2 expression by

increasing its protein stability. (A) Western blot (left panel) and RT-PCR (right panel)

analyses of hnRNP-E2 expression in parental and BCR/ABL-transduced 32Dcl3 cells

cultured in the presence or IL-3 deprived for 8 h. (B) Effect of ALLN (25 µM) and

ALLM (25 µM) on hnRNP-E2 levels. Note that 100 µg and 25 µg of lysates from 32Dcl3

and 32D-BCR/ABL cells were loaded for each lane, respectively. (C) Effect of the

protein synthesis inhibitor cycloheximide (CHX) on hnRNP-E2 and hnRNP-E1 levels in

parental and BCR/ABL-transduced 32Dcl3 cells.

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Figure 2

Figure 2. The BCR/ABL-activated MAPKERK1/2 is responsible for enhanced hnRNP-

E2 expression . (A) Effect of different chemical kinase inhibitors on hnRNP-E2 protein

levels in Ph1(+) EM-3, K562, 32D-BCR/ABL and primary CML-BCCD34+ progenitors.

Graph shows mean ± SD of hnRNP-E2 levels after normalization with HSP90 in

untreated and U0126-treated primary CML-BCCD34+ progenitors (n=3). Effect of wild

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type and dominant negative K71R ERK1 and K52R ERK2 expression on hnRNP-E2

levels in 32D-BCR/ABL cells. (B) Schematic diagram shows ERK phosphorylation sites

(S/T-P) in hnRNP-E2. (C) In vivo MAPK-dependent phosphorylation of hnRNP-E2.

Autoradiography (top) shows phosphorylated HA-hnRNP-E2 in 32P-labeled 32D-

BCR/ABL cells ectopically expressing HA-hnRNP-E2 (untreated and U0126-treated) and

HA-hnRNP-E2 S173A, S189A, T213A, S272A, respectively. Anti-HA Western blot (bottom)

shows expression of HA-hnRNP-E2 and HA-hnRNP-E2 S173A, S189A, T213A, S272A. Asterisks

indicate IgG chains. (D) In vitro MAPK-dependent phosphorylation of hnRNP-E2. (left

panel). Bacterially expressed and purified wild type MBP-hnRNP-E2 was subjected to an

in vitro kinase assay and western blot analysis. Autoradiography (top) shows

phosphorylated MBP-hnRNP-E2 by the anti-ERK1/2 immunoprecipitates. Western blot

(bottom) shows expression of MBP-hnRNP-E2 and MBP. Effect of serines/threonine to

alanine mutations on hnRNP-E2 phosphorylation (right panel). Wild type MBP-hnRNP-

E2 and its serine/threonine to alanine mutants were subjected to a kinase assay with

recombinant ERK1 (rERK1) and ERK2 (rERK2), respectively. Autoradiography show

phosphorylated MBP-hnRNP-E2 by ERK1 and ERK2, respectively. Coomassie blue

stained gels were used as controls for equal loading.

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Figure 3.

Figure 3. ERK-dependent phosphorylation controls hnRNP-E2 protein stability. (A)

hnRNP-E2 expression in parental and BCR/ABL-transduced 32Dcl3 cells expressing an

empty vector, HA-hnRNP-E2, HA-hnRNP-E2S173A,S189A,T213A,S272A (Ser/Thr�Ala) and

HA-hnRNP-E2S173D,S189D,T213E,S272D (Ser/Thr�Asp/Glu). Cells were IL-3 deprived for 8

h. (B) Stability of newly synthesized HA-tagged hnRNP-E2, hnRNP-

E2S173A,S189A,T213A,S272A and hnRNP-E2S173D, S189D,T213E,S272D in 35S-labeled 32D-BCR/ABL

cells. The half-life of hnRNP-E2 was assessed by pulse-chase assay and quantified by

densitometry. Each point on the graph represents the relative normalized amounts of

hnRNP-E2 during the chase period. (C) Stability of HA-tagged hnRNP-E2, hnRNP-

E2S173A,S189A,T213A,S272A and hnRNP-E2 S173D,S189D, T213E, S272D in 32D-BCR/ABL cells

treated with cycloheximide (CHX). HSP90 levels were used as control for equal loading.

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Figure 4.

Figure 4. Effect of ERK-dependent hnRNP-E2 downregulation on C/EBPα

expression and neutrophilic differentiation of 32D-BCR/ABLhigh cells. (A) Protein

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levels of C/EBPα and hnRNP-E2 in newly established 32Dcl3 cell clones expressing

different levels of p210-BCR/ABL (left panel). Levels of C/EBPα, hnRNP-E2, phospho-

ERK1/2, total ERK1/2, and BCR/ABL in 32Dcl3 cells and untreated, Imatinib-, and

U0126-treated 32D-BCR/ABL cells expressing high levels of the p210-BCR/ABL

oncoprotein (32D-BCR/ABLhigh) (right panel). Cells were cultured in the presence of IL-

3 or G-CSF. (B) REMSA with 32P-labeled WT-uORF/spacer CEBPΑ oligoribonucleotide

probe and cytosolic lysates from 32Dcl3 cells and untreated, Imatinib-, and U0126-

treated 32D-BCR/ABLhigh cells (left panel). Expression of Gr-1 on 32Dcl3 and untreated,

U0126, and CI-1040-treated 32D-BCR/ABLhigh cells upon G-CSF stimulation for 4 days

(right panel). (C) May-Grumwald/Giemsa staining of 32Dcl3 and 32D-BCR/ABLhigh

cells cultured with IL-3 or G-CSF for 7 days in the presence or absence of Imatinib,

U0126, or CI-1040.

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Figure 5

Figure 5. Effect of ERK-dependent suppression of hnRNP-E2 on C/EBPα

expression and neutrophilic differentiation of Lin- BMC-BCR/ABLhigh cells and

patient-derived CML-BCCD34+ progenitors. (A) Levels of hnRNP-E2, C/EBPα, G-

CSFR, phospho-ERK1/2 and total ERK1/2 in lineage-negative (Lin-) mouse bone

marrow cells (BMC) transduced with the empty vector (Lin-BMC-MigRI) or with a

p210-BCR/ABL retrovirus and sorted for low and high GFP expression (Lin-BMC-

BCR/ABLlow and -BCR/ABLhigh) (left panel). Effect of MAPK inhibition by U0126 on

hnRNP-E2 and C/EBPα expression and/or G-CSF-driven differentiation in Lin- BMC-

BCR/ABLhigh cells (middle and right panels). May-Grumwald/Giemsa stained

representative microphotographs of Lin- BMC-MigRI, BMC-BCR/ABLlow, untreated-

and U0126-treated BMC-BCR/ABLhigh cells (right panel). (B) Effect of MAPK inhibition

by U0126 and CI-1040 on hnRNP-E2 and C/EBPα expression in primary CD34+ human

normal bone marrow cells (NMBCD34+) and CML-BCCD34+ cells (left panel). May-

Grumwald/Giemsa staining of NBMCD34+ and untreated, U0126-, and CI-1040-treated

CML-BCCD34+ cells (right panel).

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Figure 6.

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Figure 6. In vivo effect of pharmacologic inhibition of ERK-dependent hnRNP-E2

expression on neutrophilic maturation of differentiation-arrested myeloid 32D-

BCR/ABL cells. H&E (top panel) and Leder (bottom panel) staining of the spleen

sections of untreated and CI-1040-treated control and 6.15-MigRI and 6.15-WT-uORF

cell-injected mice. In addition, all mice were co-treated with G-CSF. Microphotographs

are representative of data obtained by analyzing multiples sections of spleens from 3

mice per group.

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Figure 7.

Figure 7. Effect of the p53 genetic background on BCR/ABL-dependent regulation

of C/EBPα expression. C/EBPα levels at a different time course in non-transduced

(none) and BCR/ABL-transduced wild type and p53 -/- mouse bone marrow cells

(BMC) cultured in the presence of G-CSF for the indicated time.

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