2 Progenitors are Affected by the ETV6-RUNX1...

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Cytoskeletal Regulatory Gene Expression and Migratory Properties of B Cell 1

Progenitors are Affected by the ETV6-RUNX1 Rearrangement 2

Chiara Palmi1, Grazia Fazio1, Angela M. Savino1, Julia Procter2, Louise Howell2, 3

Valeria Cazzaniga1, Margherita Vieri1, Giulia Longinotti1, Ilaria Brunati1, Valentina 4

Andrè1, Pamela Della Mina3, Antonello Villa3, Mel Greaves2, Andrea Biondi1, 5

Giovanna D’Amico1, Anthony Ford2 and Giovanni Cazzaniga1. 6

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1Centro Ricerca Tettamanti, Clinica Pediatrica, Università di Milano-Bicocca, Monza, 8

Italy. 9

2Haemato-Oncology Research Unit, Division of Molecular Pathology, The Institute of 10

Cancer Research, Sutton, Surrey SM2 5NG, UK. 11

3Microscopy and Image Analysis Consortium, Università di Milano-Bicocca, Monza, 12

Italy. 13

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RUNNING TITLE 15

ETV6-RUNX1 inhibits CXCL12 driven cell migration. 16

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KEYWORDS 18

ETV6-RUNX1, pediatric BCP-ALL, pre-leukemia, CXCL12, CXCR4. 19

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CORRESPONDING AUTHOR 21

Andrea Biondi, Clinica Pediatrica, Università di Milano Bicocca, Ospedale San 22

Gerardo, Via Pergolesi 33, 20900 Monza (MB), Italy. 23

E-mail: [email protected] 24

Tel: +39 (0)39 233.3661 25

on May 22, 2018. © 2014 American Association for Cancer Research. mcr.aacrjournals.org Downloaded from

Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on July 24, 2014; DOI: 10.1158/1541-7786.MCR-14-0056-T

http://mcr.aacrjournals.org/

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Fax: +39 (0)39 233.2167 26

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AUTHORS’ DISCLOSURES OF POTENTIAL CONFLICTS OF INTEREST 29

Nothing to disclose 30

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WORD COUNT: 4655 32

TOTAL NUMBER OF FIGURES AND TABLES: 7 (5 figures and 2 tables) 33

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ABSTRACT 51

Although the ETV6-RUNX1 fusion is a frequent initiating event in childhood 52

leukemia, its role in leukemogenesis is only partly understood. The main impact of 53

the fusion itself is to generate and sustain a clone of clinically silent pre-leukemic B 54

cell progenitors (BCP). Additional oncogenic hits, occurring even several years later, 55

are required for overt disease. The understanding of the features and interactions of 56

ETV6-RUNX1 positive cells during this “latency” period may explain how these silent 57

cells can persist, and whether they could be prone to additional genetic changes. In 58

this study, two in vitro murine models were employed to investigate whether ETV6-59

RUNX1 alters the cellular adhesion and migration properties of BCP. ETV6-RUNX1 60

expressing cells showed a significant defect in the chemotactic response to CXCL12, 61

caused by a block in CXCR4 signaling, as demonstrated by inhibition of CXCL12-62

associated calcium flux and lack of ERK kinase phosphorylation. Moreover, the 63

induction of ETV6-RUNX1 caused changes in the expression of cell-surface 64

adhesion molecules. The expression of genes regulating the cytoskeleton was also 65

affected, resulting in a block of CDC42 signaling. The abnormalities described here 66

could alter the interaction of ETV6-RUNX1 pre-leukemic BCP with the 67

microenvironment and contribute to the pathogenesis of the disease. 68

IMPLICATIONS: Alterations in the expression of cytoskeletal regulatory genes and 69

migration properties of BCP represent early events in the evolution of the disease, 70

from the pre-leukemic phase to the clinical onset, and suggest new strategies for 71

effective eradication of leukemia. 72

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INTRODUCTION 76

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ETV6-RUNX1, generated by the t(12;21) chromosome translocation, is the most 78

common fusion gene in childhood cancer, selectively associated with B cell 79

precursor acute lymphoblastic leukemia (BCP-ALL) (1-3). The t(12;21) translocation 80

fuses the protein dimerization domain of ETV6 with essentially all of the DNA binding 81

and activating regions of RUNX1, generating an aberrant transcription factor (2, 4). 82

Observations on clinical samples, normal cord blood (5), monozygotic twins (6) and 83

animal modeling (7-13) indicate that this oncogene induces a pre-leukemic 84

phenotype which is insufficient for overt leukemogenesis. Indeed, ETV6-RUNX1 85

fusion generated during fetal hemopoiesis produces a clinically covert pre-leukemic 86

clone that can persist post-natally for at least 15 years (4). Additional genetic 87

abnormalities observed at diagnosis of ETV6-RUNX1 positive ALL are generally 88

considered to be secondary events associated with the transition of silent pre-89

leukemic cells to overt ALL (4). 90

The understanding of which cellular signaling pathways are corrupted by ETV6-91

RUNX1 to sustain this persistent pre-leukemic state might help to explain the 92

vulnerability of its constituent stem cells to secondary genetic changes. 93

We have previously shown evidence that ETV6-RUNX1 compromised the TGFβ 94

signaling pathway, providing a plausible basis for both the persistence and 95

maintenance of covert pre-leukemic clones in patients and their competitive positive 96

selection in an inflammatory context (12). We and other investigators have also 97

described an increased level of heat-shock proteins, survivin, has-mir-125b-2 and 98

erythropoietin receptor in ETV6-RUNX1 positive cells, factors that could provide the 99

survival advantage to the pre-leukemic clone (14-17). 100




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However, in addition to proliferative advantage and resistance to apoptotic signals, 101

the site of localization and interaction with the microenvironment is crucial to sustain 102

the hematopoietic stem cells in quiescence and the survival of both normal and pre-103

leukemic cells (18). Moreover, alterations in adhesive and chemotactic responses to 104

normal stimuli have been described in BCR-ABL1 positive chronic myeloid 105

leukaemia (19-21). 106

Interestingly, some genes involved in cellular adhesion and cytoskeleton 107

organization are listed among the RUNX1 target genes (22-23), and changes in the 108

expression of genes belonging to this functional pathway are described in ETV6-109

RUNX1 positive ALL (24-26). 110

The aim of this work was to investigate whether the ETV6-RUNX1 pre-leukemic 111

clone showed alterations in its adhesive and migratory properties that could provide 112

a rationale for its persistence and proliferation. 113

The pre-leukemic phase is usually clinically silent, while the ETV6-RUNX1 leukemic 114

clone at ALL diagnosis carries additional genetic abnormalities (4); for these 115

reasons, we used two alternative murine experimental systems: the Ba/F3 pro-B cell 116

line transduced with a hormone inducible ETV6-RUNX1 (12) and pre-BI primary 117

cells from fetal liver (27-28) stably transduced with the pMIGR1-ETV6-RUNX1-IRES-118

GFP construct. 119

In both model systems, we found evidence that ETV6-RUNX1 alters the expression 120

of cytoskeletal regulatory genes, resulting in a block of CDC42 signaling pathway, 121

and compromises the chemotactic response to CXCL12. 122

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MATERIALS AND METHODS 124

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Cell culture and ETV6-RUNX1 expression 126

The GeneSwitch system (Life Technologies, Carlsbad, CA, USA), a Mifepristone-127

regulated expression system for mammalian cells, was used to produce inducible 128

expression of the ETV6-RUNX1 gene in the IL-3-dependent murine pro-B cell line 129

Ba/F3, as previously described (12). Briefly, cells were transfected with the pSwitch 130

plasmid (Invitrogen, Carlsbad, CA, USA) expressing a GAL4 regulatory fusion 131

protein (Control cells). Positive clones were then transfected with pGene plasmid 132

(Life Technologies) containing the ETV6-RUNX1 cDNA fused to V5 epitope tag 133

controlled by a promoter regulated by the GAL4 regulatory fusion protein (inducible 134

ETV6-RUNX1 cells). All Ba/F3 cells were cultured in RPMI medium supplemented 135

with 10% fetal calf serum (FCS), 2% MoIL-3 conditioned medium as a source of IL-3 136

(29), 10µM 2-mercaptoethanol and 0.2mg/ml Hygromycin B. In addition, cells 137

inducible for ETV6-RUNX1 were cultured in the presence of 0.05mg/ml Zeocin. 138

The expression of ETV6-RUNX1 was induced by adding to the culture medium 139

0.0125nM Mifepristone (Invitrogen) for 3 days. Efficiency of ETV6-RUNX1 induction 140

was verified by flow cytometry using an anti-V5 antibody (Life Technologies), as 141

previously described (12). 142

Pre-BI cells are murine primary cKIT+/B220+/CD19+ cells isolated from fetal liver 143

(gift of Prof. A. Rolink, University of Basel, Switzerland) and were transduced by the 144

bicistronic retroviral vectors pMSCV-IRES-GFP (MIGR-GFP) or pMSCV-ETV6-145

RUNX1-IRES-GFP, which allows the expression of ETV6-RUNX1 cDNA fused to c-146

Myc epitope tag and GFP (MIGR- ETV6-RUNX1), as previously described (27). At 147

day +3 from transduction, cell sorting for GFP fluorescence was performed by the 148

FACS Aria instrument (BD Biosciences, Franklin Lakes, NJ, USA). 149




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The pre-BI cells were cultured on OP9 bone marrow stroma cells in Iscove’s 150

modified Dulbecco’s medium (IMDM) supplemented with 2% FCS, 0.03% w/v 151

primatone and 100 units/ml IL-7 (27). 152

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Antibodies and flow cytometry 154

Phycoerythrin-conjugated antibodies anti-CD18 (M18/2), anti-CD11a (M17/4), anti-155

CD54 (YN1/1.7.4), anti-CD135 (A2F10), anti-CD29 (HMb 1-1), anti-CD49d (R1-2), 156

anti-CD49e (HMa 5-1) (e-Bioscience Inc, San Diego, CA, USA) and anti-CXCR7 157

(8F11-M16) (Biolegend, San Diego, CA, USA) were used. Allophycocyanin-158

conjugated antibodies anti-CD44 (IM7) and anti-CXCR4 (2B11) (e-Bioscience Inc) 159

were used. Data were analyzed using CellQuest Software (BD Biosciences). 160

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Quantitative PCR Array and real-time PCR (RQ-PCR) 162

The RT² Profiler™ Assay Cytoskeleton Regulators PCR Array (SuperArray 163

Bioscience, Frederick, MD, USA) was performed following the manufacturer’s 164

recommendation. 165

Real-time analysis was done on a Light Cycler 480II with Universal Probe Master 166

System (Roche Diagnostics; F. Hoffmann-La Roche Ltd., Basilea, Swizerland). 167

Optimal primers and probe for amplification were selected by the Roche ProbeFinder 168

software (https://www.roche-appliedscience.com/sis/rtpcr/upl). 169

Data were expressed using the comparative 2−DDCt method (30), with Hprt gene as 170

reference; for each gene studied the transcript level was always referred to that of 171

control cells. A fold change <0.75 or >1.5 was considered as threshold for down- or 172

up-regulation, respectively. 173

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Adhesion assays 175

96 well plates were coated with fibronectin, murine stroma cell line OP9 (kindly 176

provided by Prof. A. Rolink, University of Basel, Switzerland), murine fresh stroma or 177

murine endothelial cell lines 1G11 and MELC2 (a gift of Prof. A. Vecchi, Istituto 178

Clinico Humanitas, Rozzano, Italy). Details of the coating procedures: 50 µl 179

fibronectin (Sigma-Aldrich), at a concentration of 25ng/µl in each 96 well, was allow 180

to air dry for 2h and the cell lines and murine fresh stroma were grown in each well 181

until confluent. OP9 were grown in IMDM with 20% FCS, the murine fresh stroma 182

was isolated from BM of a C57BL/6 wt mouse and grown in IMDM with 20% FCS at 183

33°C and 5% CO2, the cell lines 1G11 and MELC2 were grown on a gelatin coating 184

(Sigma-Aldrich) in DMEM supplemented with 4.5 g/L glucose, FCS (20% for 1G11, 185

10% for MELC2), 1% non-essential aminoacids, 1mM sodium pyruvate, 100 µg/ml 186

Endothelial cell growth supplement (ECGS, Sigma-Aldrich), 100 µg/ml heparin 187

(PharmaTex, Milano, Italy) and, for MELC2 only, 10% murine Sarcoma 180 188

conditioned medium. The endothelial cell lines were stimulated or not for 24h with 189

inflammatory cytokines IL1β (25ng/ml, PeproTech), IL6 (20ng/ml, ImmunoTools, 190

Friesoythe, Germany) and TNFα (50ng/ml, ImmunoTools) before being used for the 191

adhesion assay. 192

After 3 days-induction, control and ETV6-RUNX1 expressing Ba/F3 cells were 193

stained with 12.5μM Calcein AM (Sigma-Aldrich) and resuspended in adhesion 194

medium (RPMI with 5% FCS and 10nM HEPES). Cells were then added to 96 well 195

plates coated with the substrates indicated above. After 30 minutes of incubation at 196

37°C and 5% CO2, non-adherent cells were removed by washing three times with the 197

adhesion medium. The adhesion index was measured as ratio of fluorescence 198




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detected before and after washing by the fluorescence reader TECAN GENios 199

(Tecan, Mannedorf, Switzerland). 200

Cell adhesion to VCAM1 and ICAM1 recombinant proteins was performed as 201

previously described (28). Briefly, 15mm round coverslips were coated with 202

recombinant mouse VCAM1-Fc protein (25 µg/ml; R&D Systems) or recombinant 203

mouse ICAM1-Fc protein (25 µg/ml; R&D Systems) and placed in 12-well dishes 204

containing 1.5x105 3 days-induced control or ETV6-RUNX1 expressing Ba/F3 cells. 205

After o/n incubation at 37°C and 5% CO2, the coverslips were washed to eliminate 206

the non-adherent cells and were mounted on slides in the presence of DAPI. Each 207

coverslip was analyzed by a fluorescence microscope, acquiring 30 representative 208

fields, and the adherent cells were counted. 209

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Immunoblotting 211

Western blot analysis of CDC42 protein was performed by lysing cells in the Lysis 212

Buffer (Thermo Scientific, Waltham, MA, USA) with Protease inhibitor cocktail 213

(Sigma-Aldrich, St. Louis, MO, USA). Mouse anti-CDC42 antibody was used at 214

working dilution 1:167 (Thermo Scientific), mouse anti-beta-actin antibody at 1:1000 215

(AC-15, Sigma-Aldrich, St. Louis, MO, USA) and the secondary goat anti-mouse IgG 216

(Fc-specific) Peroxidase antibody at working dilution 1:20000 (Sigma-Aldrich). A 217

StripAblot Stripping Buffer (Euroclone S.p.A., Pero, Italy) was used to recover 218

membranes. Densitometry analyses were performed using Alliance instrument and 219

Uviband software (Uvitec Cambridge, UK). 220

For p-ERK, total ERK, p-PAK2 and total PAK2 protein analysis, cells were starved 221

for 1 h in RPMI without serum, then 2x106 cells were stimulated with 100ng/ml 222

hCXCL12 (Peprotech, London, UK) in RPMI at 37°C. At different time points, cells 223




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were washed with ice-cold PBS and pellets were lysed in 20mM Tris HCl/NaCl pH 224

7.4 containing 2mM EDTA, 0.2 mM Na3VO4, 1% Triton x-100, 25 mM β-225

glycerophosphate, 25mM NaF, 1mM phenylmethylsulfonyl fluoride and Protease 226

inhibitor cocktail at 4°C for 30 min. Rabbit anti-phospho-p44/42 MAPK (ERK1/2) 227

(Thr202/Tyr204) antibody, rabbit anti phospho-PAK1 (Thr423) / PAK2 (Thr402) 228

antibody, rabbit anti-p44/42 MAPK (ERK1/2) antibody and rabbit PAK2 (C17A10) 229

antibody were used at working dilution 1:1000 (Cell Signaling, Danvers, MA, USA), 230

the mouse anti-GAPDH antibody at 1:200 (6C5, Santa Cruz Biotechnology, Dallas, 231

Texas, USA) and the secondary goat anti-rabbit IgG (H+L) HRP at 1:10000 dilution 232

(Thermo Scientific). 233

Western blot analysis of c-Myc epitope tag, fused to ETV6-RUNX1 cDNA in MIGR- 234

ETV6-RUNX1 PreBI cells, was performed using the rabbit anti-c-Myc (A-14) antibody 235

at working dilution 1:200 (Santa Cruz Biotechnology). 236

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Migration assay 238

Transwell plates (8.0 μm for Ba/F3 cells and 5.0 μm for Pre-BI cells) were used; 5 × 239

105 cells were loaded in the upper chamber in 100 μL of RPMI with 1% FCS 240

(migration medium) with or without 10μg/ml of the anti-CXCR7 antibody (8F11-M16) 241

(Biolegend). 600 μl of migration medium with or without hCXCL12 (100 ng/ml), 242

FLT3L (10ng/ml) (ImmunoTools) or 10% FCS was added in the lower chamber. After 243

3 h at 37°C and 5% CO2, cells in the lower chamber were collected and counted by 244

fluorescence-activated cell sorting (FACS). The migration index (M.I.) was defined as 245

the ratio between the number of cells migrated to the lower chamber of the transwell 246

in response to the chemokine stimulus and in its absence. 247




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Migration assay in presence of EGF: migration assay of PreBI cells towards 100 248

ng/ml hCXCL12 was performed in presence or absence of 20ng/ml of EGF 249

(Peprotech) homogeneously present in the upper and lower chambers of the 250

transwell. After 4h the GFP positive cells migrated to the lower well were counted by 251

FACS. The migration index (M.I.) was defined as the ratio between the number of 252

cells migrated to the lower chamber of the transwell in response to CXCL12 and in 253

its absence. 254

Migration assay in presence of CCL2: migration assay of Ba/F3 cells towards 100 255

ng/ml CXCL12 was performed in absence or presence of 100ng/ml of CCL2 256

(Peprotech) in the upper chamber of the transwell. The migration index (M.I.) was 257

defined as the ratio between the number of cells migrated to the lower chamber of 258

the transwell in response to CXCL12 and in its absence. 259

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Protein array analysis. Protein array was performed on cell supernatant, obtained 261

from Ba/F3 control and ETV6-RUNX1 positive cells, using RayBio® Cytokine 262

Antibody Arrays - Mouse Array III-IV (Raybiotech Inc), following the manufacturer’s 263

protocol. Densitometry analyses were done using using Kodak image station (Kodak 264

SpA). 265

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Calcium mobilization analysis 267

After o/n IL3 starvation, 0.6 × 106 3-days induced cells were loaded with FluoForte 268

dye-loading solution (Enzo Life Sciences, Farmingdale, NY, USA) in RPMI with 10% 269

FCS for 45 min at 37°C and then 15 min at RT. Baseline calcium levels were 270

established for about 2 min prior to the addition of 300 ng/ml CXCL12. Data were 271




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collected for a total of 512 sec and analyzed on a FACSCalibur using CellQuest 272

Software (BD Biosciences). 273

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Generation of Ba/F3 over-expressing CDC42 proteins 275

The cDNA of wild-type, constitutively active (Q61L) and dominant negative (T17N) 276

CDC42 were cloned into the retroviral vector pMSCV-IRES-GFP using In-Fusion HD 277

Cloning kit (Clontech Laboratories, Mountain View, CA, USA) and following the 278

manufacturer’s recommendation. Ba/F3 cells were transfected by nucleofection 279

following the Amaxa protocol, as previously described (12). 280

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RESULTS 282

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ETV6-RUNX1 alters the expression of cell-surface adhesion molecules and 284

adhesion properties of Ba/F3 cell 285

After ETV6-RUNX1 expression in Ba/F3 cells, flow cytometric analysis indicated 286

alterations of the cell-surface expression levels of several molecules involved in cell 287

adhesion and migration of BCP (31). In detail, ETV6-RUNX1 positive cells expressed 288

higher levels (MFI ratio) of the following adhesion molecules: CD44 (average of 289

increase in independent experiments: 58%, range 19-118%, p<0.05), CD18 290

(average: 121%, range: 19-206%, p<0.05), CD11a (average: 182%, range: 54-291

334%, p<0.01), and CD54 (average: 145%, range: 80-207%, p<0.05). On the other 292

hand, they expressed lower levels of the integrin CD29 (average of decrease: 22%, 293

range: 8-34%, p<0.01) (Figure 1A), but did not show a significant difference in the 294

expression of CD49d and CD49e (data not shown). 295




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The transcription level of genes coding some of these antigens tested by RQ-PCR 296

confirmed the immunophenotype results (Supplemental Figure 1). 297

We observed an increase in the adhesion of ETV6-RUNX1 positive cells to the 298

murine endothelial cell lines 1G11 and MELC2 (Adhesion index of ETV6-RUNX1 299

positive cells vs. control cells: 1.34± 0.26 (p=0.0431) on 1G11 and 1.39± 0.26 300

(p=0.0286) on MELC2) (Figure 1B). The stimulation of the endothelial cell lines with 301

inflammatory cytokines did not modify the adhesion index (data not shown). 302

However, we did not observe any difference in the adhesion abilities to several other 303

substrates: fibronectin, CD54/ICAM1 and VCAM1 molecule, murine stroma cell line 304

OP9 and murine fresh stroma. 305

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ETV6-RUNX1 causes altered expression of genes regulating the cytoskeleton 307

We explored whether the ETV6-RUNX1 fusion gene affected key genes in the 308

organization of the cytoskeleton. A panel of 84 genes involved in the biogenesis and 309

organization of the cytoskeleton was examined using the RT² Profiler™ Assay 310

Cytoskeleton Regulators PCR Array, plus single candidate gene transcripts tested by 311

RQ-PCR. 312

We identified 9 genes over-expressed and 7 genes repressed in Ba/F3 ETV6-313

RUNX1 positive cells compared to control cells (Table 1A and 1B). These were 314

genes involved in cell shape, formation of pseudopodia, cell migration, actin and 315

microtubule organization. Interestingly, several of these differentially expressed 316

genes belonged to the CDC42 pathway, a key element for the regulation of the 317

cytoskeleton and for the directional migration of the cells: Cdc42ep2, Cdc42ep3, 318

Map3k11, Was, Nck2, Nck1 and Mmp9 (32-37). In particular, Cdc42ep2 and 319

Cdc42ep3, negative regulators of Cdc42 (32), were among the most up-regulated 320




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genes in ETV6-RUNX1 positive cells (Table 1A). Moreover, after induction of the 321

fusion gene (Figure 2A), we observed a reduction of CDC42 at the transcription and 322

at the protein level by RQ-PCR and western-blot analyses (Figure 2B and 2C). 323

Consistently, we verified that CDC42 signaling was perturbed by ETV6-RUNX1. As 324

shown in Figure 2D, the CDC42 downstream effector kinase PAK2 (38) was less 325

expressed in ETV6-RUNX1 positive cells and cell stimulation with CXCL12 induced 326

a marked increase in the phosphorylation of PAK2 only in Ba/F3 control cells 327

(densitometry analyses of pPAK2 after normalization on the amount of PAK2 total 328

and beta-actin: +2.36 fold increase in Ctr cells vs -0.15 fold decrease in ETV6-329

RUNX1 positive cells). 330

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ETV6-RUNX1 impairs migration towards CXCL12 332

By applying a transwell migration assay, we observed a significant defect in the 333

chemotactic response of ETV6-RUNX1 expressing Ba/F3 cells to CXCL12, a potent 334

chemoattractant for B cells precursors (Figure 3A). In detail, the induction of ETV6-335

RUNX1 expression caused a decrease in the migration index (M.I.) of 86.2% 336

(average of 5 independent experiments, range: 75.1%-92.6%, p<0.001), although 337

the expression of the CXCR4 receptor on the cell-surface was unaffected or even 338

increased (MFI increase average: 31%, range: 3-100%, p<0.05) (Supplemental 339

Figure 2A). 340

We then explored the possible role of CXCR7 and CCR2 on the migration defect of 341

ETV6-RUNX1 positive cells. These two receptors, that negatively regulate the 342

CXCL12 signaling cascade in BCP cells (39-41), are both up-regulated in Ba/F3 cells 343

after ETV6-RUNX1 induction (MFI increase average of CXCR7: 16%, range: 6-27%, 344




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p<0.05 (Supplemental Figure 2B); fold change of Ccr2 gene: 12.55, p<0.01 345

(Supplemental Figure 3A)). 346

In Ba/F3 control cells CXCL12-mediated chemotaxis was modulated by both an anti-347

CXCR7 antibody and CCL2, the ligand of CCR2 (Supplemental Figure 4A and 348

Supplemental Figure 3B). However, in ETV6-RUNX1 positive cells, the migration 349

towards CXCL12 remained defective after CXCR7 blocking (Supplemental Figure 350

4A), thus excluding a role of CXCR7 in the inhibition of their migration ability. On the 351

other hand, in presence of CCL2, we observed a more marked decrease in the 352

ability to migrate to CXCL12 in Ba/F3 ETV6-RUNX1 -induced cells (Supplemental 353

Figure 3B). Interestingly, CCL2 is secreted at higher amount by ETV6-RUNX1 354

positive Ba/F3 cells (Supplemental Figure 3C). However, no difference in migration 355

towards CXCL12 was observed in control cells upon pretreatment with the ETV6-356

RUNX1 positive Ba/F3 supernatant, thus also excluded a pivotal role of the secreted 357

CCL2 in the inhibition of the migration ability of ETV6-RUNX1 positive cells. 358

We previously observed high expression levels of the FLT3L receptor (CD135) in the 359

immature hematopoietic cells from ETV6-RUNX1 transgenic mice (12). FLT3L plays 360

an important role in cellular proliferation and survival, but it also enhances migration 361

towards CXCL12 (42). However, although we confirmed higher level of CD135 362

protein expression in Ba/F3 cells after ETV6-RUNX1 induction (Supplemental Figure 363

2C), we did not observe an increase in their migration ability, unlike the control cells 364

(Supplemental Figure 4B), thus excluding that FLT3L could recover the ability of 365

ETV6-RUNX1 positive cells to migrate to CXCL12. 366

Finally, since we showed above that ETV6-RUNX1 deregulated CDC42 signaling, 367

we tested if the overexpression of Cdc42 could counteract the impaired migration of 368

ETV6-RUNX1 positive cells. In Supplemental Figure 4C we showed that while the 369




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over-expression of wild-type (WT) or constitutively active (CA) Cdc42 caused a 370

marked increase in the migration towards CXCL12 in control cells, only a slight 371

increase was measured in ETV6-RUNX1 positive cells (MFI increase average in 372

CDC42 CA cells: 3.49 ± 0.13 in Ctr cells vs. 1.29 ± 0.08 in ER cells). This result 373

demonstrated that neither the enhancement of the CDC42 activity was able to 374

recuperate the migration property towards CXCL12 of ETV6-RUNX1 positive cells. 375

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ETV6-RUNX1 positive cells do not have a general defect of movement 377

We wondered whether ETV6-RUNX1 positive Ba/F3 cells were unable to specifically 378

migrate towards CXCL12, or whether they presented any general defect in 379

movement. By transwell migration assay using 10% FCS as a general stimulus, we 380

found that not only ETV6-RUNX1 positive cells were not inhibited in movement, but 381

they migrated more than control cells (Figure 3B). 382

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ETV6-RUNX1 positive cells present a defect in CXCR4 signaling 384

Although the expression of CXCR4 receptor on the cell surface was unaffected or 385

even increased (Supplemental Figure 2A), ETV6-RUNX1 induction inhibited the 386

mobilization of intracellular calcium flux after CXCL12 stimulation in Ba/F3 cells 387

(Figure 4A). Moreover, the phosphorylation of extracellular signal regulated kinase 388

(ERK) in response to CXCL12 was absent in ETV6-RUNX1 positive cells (Figure 389

4B). 390

Thus, the expression of ETV6-RUNX1 in Ba/F3 cells resulted in the block of ERK 391

phosphorylation, early downstream to CXCR4 signaling. 392

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ETV6-RUNX1 alters the expression of genes regulating the cytoskeleton and 394

migration properties of primary Pre-BI cells 395

The effect of ETV6-RUNX1 on the expression of genes regulating the cytoskeleton 396

and migration properties was confirmed in primary Pre-BI cells, purified from fetal 397

liver of wild-type mice, a more physiological murine model. These cells were stably 398

transduced with a retroviral vector (pMIGR1) containing the ETV6-RUNX1 cDNA 399

upstream of the IRES-GFP, and isolated by GFP sorting (Material and Methods) 400

(Figure 5A). 401

Pre-BI MIGR-ETV6-RUNX1 cells presented a higher cell-surface expression levels 402

(MFI ratio) of adhesion molecules such as CD18 (average of increase: 64%, range 403

15-146%, p<0.01), CD11a (average: 90%, range 19-203%, p<0.01) and CD54 404

(average: 27%, range 14-85%, p<0.05) (Supplemental Figure 5A, 5B and 5C) and 405

lower levels of CD62L (average of decrease: 68%, range 7-83%, p<0.05) 406

(Supplemental Figure 5D) compared to MIGR-GFP control cells. 407

As in Ba/F3 cell line, the expression of ETV6-RUNX1 in Pre-BI cells caused 408

alteration in the expression of genes involved in the modulation of the cytoskeleton 409

(Table 2A and 2B), including the over-expression of Cdc42ep2 and Cdc42ep3, the 410

negative regulators of Cdc42, and a reduction of Cdc42 transcription (Figure 5B). 411

Moreover, the CDC42 signaling was perturbed in the Pre-BI ETV6-RUNX1 positive 412

cells since the stimulation with CXCL12 induced an increase in PAK2 413

phosphorylation only in the control MIGR-GFP cells (Figure 5C). 414

Interestingly, also Pre-BI MIGR-ETV6-RUNX1 cells showed a significant defect in 415

their ability to migrate towards CXCL12, with 73.5% decrease of M.I. (average of 5 416

independent experiments, range: 57.0%-88.9%, p<0.001) (Figure 5D). However, the 417




18

expression of CXCR4 receptor on the cell-surface was unaffected (Supplemental 418

Figure 5E). 419

Unlike Ba/F3 cells, Pre-BI cells express the EGF receptor (EGFR), a strong activator 420

of CDC42 pathway (43). As shown in Supplemental Figure 6, although we observed 421

an increased M.I. towards CXCL12 in the control cells in presence of EGF stimulus, 422

however, the migration towards CXCL12 remained defective in ETV6-RUNX1 423

positive cells. 424

425

DISCUSSION 426

Although the t(12;21) translocation is a frequent prenatal initiating mutation in BCP-427

ALL (1-3, 6), the cellular signaling pathways corrupted by ETV6-RUNX1 in the pre-428

leukemic clone remain unknown. In the present paper we consistently showed in two 429

in vitro models that ETV6-RUNX1 de-regulates the cytoskeleton and compromises 430

the chemotactic response to CXCL12. 431

It has been increasingly recognized that cancer initiation and progression is not 432

solely a cancer cell autonomous process. Primary tissue cells live in complex 433

microenvironments, characterized by heterotypic signaling between ancillary cells 434

and hematopoietic cells (44). This signaling is considered to play a role in the 435

regulation of the behavior of stem and precursor hematopoietic cells, including their 436

survival, proliferation and differentiation. For this reason, alterations in the 437

environment or in the abilities of the stem and progenitors cells to interact with the 438

innate niches play a crucial role in tumor initiation and progression. 439

We have employed two different model systems to study the ETV6-RUNX1 pre-440

leukemic phase, and in particular to examine whether ETV6-RUNX1 altered the 441

cellular adhesion and migration properties of BCP. Indeed, this type of study is not 442




19

feasible in clinical samples at diagnosis of ALL, where the analysis of the fusion 443

function is confounded by the additional genetic abnormalities. We have therefore 444

used a murine progenitor cell line (Ba/F3), with hormone inducible ETV6-RUNX1 445

expression, a model developed in the past to demonstrate the impact of the fusion 446

gene on the inhibitory response to TGF-beta (12). Next, we confirmed our results in 447

pre-BI cells, primary cells derived from a wild-type mouse fetal liver, already 448

successfully used for functional analysis of BCP-ALL associated fusion transcripts 449

(27-28). 450

We observed that the expression of ETV6-RUNX1 in Ba/F3 cell line resulted in 451

changes in the cellular phenotype: several molecules involved in cell adhesion were 452

deregulated in expression. We observed an increase in the adhesion of ETV6-453

RUNX1 positive cells to murine endothelial cell lines. 454

To understand the reason for the reported alterations, we explored the effect of the 455

ETV6-RUNX1 fusion, an aberrant transcription factor, on the transcription of a panel 456

of genes involved in the biogenesis and organization of the cytoskeleton. Indeed, we 457

showed that the expression of ETV6-RUNX1 in Ba/F3 cell line caused alteration in 458

the expression of genes regulating cell shape, formation of pseudopodia, cell 459

migration, actin and microtubule organization. In particular, among the most over-460

expressed genes in ETV6-RUNX1 positive cells, we identified two negative 461

regulators of CDC42. Moreover, we observed a reduction of CDC42 at the 462

transcription and protein level and a block of CDC42 signaling pathway. CDC42 not 463

only has a pivotal role in cell cycle progression (in agreement with this, we previously 464

described an increase in the proportion of ETV6-RUNX1 expressing cells in G0/G1) 465

(12), but also in cytoskeleton rearrangement during directional migration. 466




20

In parallel, we investigated the migration abilities of ETV6-RUNX1 inducible Ba/F3 467

cells. Interestingly, we found that the fusion gene significantly impaired the 468

chemotactic response to CXCL12, although the cell-surface expression of the 469

receptor CXCR4 was unaffected. Indeed, the CXCL12 chemotaxis defect was not 470

due to a general impairment of movement of ETV6-RUNX1 positive cells, as their 471

migration towards a general stimulus was instead increased. 472

We then excluded a possible role in this migration defect of several players of 473

CXCL12/CXCR4 pathway, including CXCR7, a receptor with CXCL12-scavenging 474

activity (39), FLT3L receptor, a positive regulator of CXCL12 migration (42), the 475

GTPase CDC42, as well as the CCL2/CCR2 and EGF/EGFR axes. These two axes 476

are both involved in the modulation of CXCL12-mediated chemotaxis. Indeed, it has 477

been reported that the expression of CCR2 negatively regulates the cytoskeletal 478

rearrangement and migration of immature B cells and that the control of B cell 479

homing by CCR2 is mediated by its ligand, CCL2, which is secreted by B cells and 480

down-regulates the CXCL12 signaling cascade (40-41). On the contrary, a 481

synergistic effect of CXCR4 and the EGF receptor EGFR on promoting cancer 482

metastasis has been reported (45). In particular, EGF was described to promote 483

breast cancer cell chemotaxis in CXCL12 gradients, while CXCL12 alone failed to 484

stimulate the migration of these cells (46). 485

We were able to demonstrate that ETV6-RUNX1 impairs the calcium flux, a very 486

proximal CXCR4 signaling event, and the phosphorylation of ERK kinase, as a 487

downstream event. 488

Further analyses are needed to fully define the mechanism of migration defect and to 489

establish how ETV6-RUNX1 inhibits the CXCL12-CXCR4 signaling pathway. In this 490

regard, it was reported in the literature that ETV6-RUNX1 positive ALL patients 491




21

presented at diagnosis lower level of CD9 than the negative ALL patients (47). 492

Importantly, the tetraspanin CD9 has been described to regulate migration, 493

adhesion, homing of human cord blood CD34+ cells (48). In light of these 494

observations, we believe that it will be important to explore whether the low 495

expression of CD9 is a property not only of the overt leukemic blasts, but also of the 496

pre-leukemic cells and whether this feature may play a key role in the CXCL12-497

migration defect of ETV6-RUNX1 positive B precursor cells described here. 498

Noteworthy, the results observed in the Ba/F3 ETV6-RUNX1 inducible expression 499

system were reproducible in primary pre-BI cells. Indeed, after transduction of the 500

chimeric gene, we confirmed an altered expression of genes involved in cytoskeleton 501

modulation, including Cdc42 with its regulators, and several adhesion molecules. 502

Moreover, we consistently observed the same block of CDC42 signaling and the 503

same significant defect of pre-BI ETV6-RUNX1 positive cells to migrate towards 504

CXCL12. 505

Interestingly, similar alterations in the expression of adhesion molecules and defects 506

in CXCL12 migration have been reported in BCR-ABL1 positive leukemia (19-21). In 507

this context it has been hypothesized that these aberrations could contribute to the 508

homing and retention defects in the bone marrow typical of immature myeloid cells in 509

chronic myelogenous leukemia (49). 510

In light of the findings described here, we can sustain the hypothesis that the ETV6-511

RUNX1 pre-leukemic clone, compared to its normal counterpart, might have an 512

altered interaction with the bone marrow microenvironment which results in a greater 513

tendency to migrate to the periphery. A direct demonstration of their ability to leave 514

the bone marrow, in spite of their immature status, is represented by the detection of 515

pre-leukemic clones in peripheral blood at birth (in Guthrie cards, cord blood and 516




22

peripheral blood) (5, 50). Appropriate in vivo studies in murine models must be 517

afforded to further exploit the characteristics of the pre-leukemia phase and to define 518

the role of microenvironmental factors in the preleukemic state induced by ETV6-519

RUNX1. 520

In conclusion, the abnormalities we observed in the expression of genes regulating 521

the cytoskeleton and in migration towards CXCL12 may represent early events in the 522

pathogenesis of the disease, not necessary associated to the progress towards overt 523

leukaemia unless additional hits occur (4). Indeed, our observation raises the 524

question as to how ETV6-RUNX1 pre-leukemic cells interact with the 525

microenvironment. We believe that identification of the precise localization of these 526

cells, their cell-cell contacts and gene regulation are crucial to providing a better 527

understanding of the mechanisms that allow the pre-leukemic clone to persist 528

covertly in an individual for several years, maybe prone to additional genomic 529

events. This will be decisive in helping to develop strategies for their effective 530

eradication and leukaemia prevention. 531

532

ACKNOWLEDGEMENTS 533

We thank Cristina Bugarin for cell sorting by Flow Cytometry and Marta Galbiati for 534

her help in VCAM and ICAM adhesion assay analyses. 535

This study was supported by grants from: Fondazione Tettamanti (Monza), Comitato 536

Maria Letizia Verga, Comitato Fiori di Lavanda, Associazione Italiana Ricerca sul 537

Cancro (AIRC) (to AB and GC), MIUR (to AB), Fondazione Cariplo (to AB and GC), 538

Leukaemia & Lymphoma Research (UK) (to MG and AF). 539

540

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697

698

699

700

701

702

703

704

705

706

707

708

709

710

711

712




30

TABLES 713

714

FC T testCdc42ep2 5.41 0.00005Arhgef11 2.72 0.00006Cdc42ep3 2.61 0.00019Map3k11 1.49 0.00169

Fscn2 1.86 0.00303Dstn2 1.61 0.00188Was 1.47 0.00033Nck2 2.68 0.00003Nck1 1.56 0.00785

FC T testRock1 0.18 0.00002Ppp3cb 0.47 0.00000Stmn1 0.64 0.00170Clip1 0.68 0.00015

Cyfip2 0.76 0.00011Mylk 0.65 0.00031

Mmp9 0.34 0.00006

Table 1A. Up-regulated genes inETV6-RUNX1+ Ba/F3 cells.

Gene name

Table 1B. Down-regulated genes inETV6-RUNX1+ Ba/F3 cells.

Gene nameRQ-PCR

RQ-PCR

715

716

717

718

719

720

721

722

723

724




31

FC T testCdc42ep2 1.46 0.02215Arhgef11 1.86 0.00036Cdc42ep3 3.1 0.00114

FC T testRock1 0.66 0.00028Stmn1 0.75 0.01874Clip1 0.75 0.00008

Cyfip2 0.78 0.02114Mylk 0.47 0.00005

Mmp9 0.16 0.00022Mmp2 0.16 0.00256Cdh2 0.3 0.00357

Table 2A. Up-regulated genes inETV6-RUNX1+ PreBI cells.

Table 2B. Down-regulated genes inETV6-RUNX1+ PreBI cells.

RQ-PCR

RQ-PCRGene name

Gene name

725

726

727

728

729

730

731

732

733

734

735

736

737

738

739




32

FIGURE LEGENDS 740

741

Figure 1: Phenotypic and adhesion analyses of control and ETV6-RUNX1 742

positive Ba/F3 cells. 743

(A) Overlay analyses of the expression on cell-surface of the indicated antigen 744

measured as MFI levels by FACS. The figure shows a representative experiment. 745

(B) Adhesion analyses of control and ETV6-RUNX1 positive Ba/F3 cells to murine 746

endothelial cell lines 1G11 and MELC2. After 3 days-induction, control and ETV6-747

RUNX1 expressing Ba/F3 cells were stained with Calcein AM and added to 96 well 748

plates coated with the cell lines. After 30 minutes of incubation, non-adherent cells 749

were removed by washing three times with the adhesion medium. The adhesion 750

index was measured as ratio of fluorescence detected before and after washing. Ctr: 751

control cells; ER: ETV6-RUNX1 positive cells. T test: *, p<0.05. 752

753

Figure 2: Cdc42 pathway analysis in control and ETV6-RUNX1 positive Ba/F3 754

cells. 755

Ba/F3 cells were induced to express ETV6-RUNX1 for 3 days. (A) Flow cytometry 756

analysis of the intra-cellular expression of V5 epitope tag, fused to ETV6-RUNX 757

cDNA. (B) cDNA was subjected to TaqMan RQ-PCR and normalized to Hprt 758

expression. Transcript level of Cdc42 gene in ETV6-RUNX1 positive cells relative to 759

control cells is shown as an average of triplicates. Ctr: control cells; ER: ETV6-760

RUNX1 positive cells. T test: **, p<0.01. (C) Cell lysates were analysed by Western 761

blot with anti-CDC42 antibody. The blot was later stripped and re-probed with an 762

anti-β actin antibody. CDC42 protein expression level in ETV6-RUNX1 positive cells 763

was quantified by densitometry, normalized to β-actin, and indicated in the figure as 764




33

the percentage with respect to control cells. M: Marker; - and +: negative and 765

positive control for CDC42 protein, respectively. (D) Western-blot analysis of PAK2 766

phosphorylation, total PAK2 and beta-actin after 2’ of CXCL12 stimulation. 767

768

Figure 3: Migration analyses of control and ETV6-RUNX1 positive Ba/F3 cells. 769

Ba/F3 control and ETV6-RUNX1 positive cells were incubated for 3 days in the 770

presence of the inducer before performing the migration assays towards (A) 100 771

ng/ml CXCL12, (B) 10% FCS. After 3h the cells migrated to the lower well were 772

collected and counted by FACS. The migration index (M.I.) was defined as the ratio 773

between the number of cells migrated to the lower chamber of the transwell in 774

response to the chemokine stimulus and in its absence. Error bars correspond to 775

standard deviation from triplicates of a representative experiment. T test: *, p<0.05; 776

**, p<0.01. Ctr: control cells; ER: ETV6-RUNX1 positive cells. 777

778

Figure 4: Mobilization of intracellular calcium and activation of ERK in 779

response to CXCL12 in control and ETV6-RUNX1 positive Ba/F3 cells. 780

(A) Calcium flux in response to stimulation with CXCL12 as indicated by the arrow. 781

(B) Western-blot analysis of ERK phosphorylation, total ERK and GAPDH after 782

CXCL12 stimulation (the numbers indicate the minutes of stimulation). Ctr: control 783

cells; ER: ETV6-RUNX1 positive cells. 784

785

Figure 5: Cdc42 pathway and migration analysis of Pre-BI MIGR-GFP and 786

MIGR-ETV6-RUNX1 cells 787

(A) Western-blot analysis of c-Myc epitope tag, fused to ETV6-RUNX cDNA. (B) 788

cDNA of pre-BI cells was subjected to TaqMan RQ-PCR and normalized to Hprt 789




34

expression. Transcript levels of Cdc42 gene in MIGR-ETV6-RUNX1 (MIGR-ER) 790

relative to MIGR-GFP cells are shown as an average of triplicates. T test: *, p<0.05. 791

(C) Western-blot analysis of PAK2 phosphorylation, total PAK2 and beta-actin after 792

2’ of CXCL12 stimulation. GFP: Pre-BI MIGR-GFP cells; ER: Pre-BI MIGR-ETV6-793

RUNX1 cells. (D) Migration assays towards 100 ng/ml CXCL12. After 3h the GFP 794

positive cells migrated to the lower well were counted by FACS. The migration index 795

(M.I.) was defined as the ratio between the number of cells migrated to the lower 796

chamber of the transwell in response to CXCL12 and in its absence. Error bars 797

correspond to standard deviation from triplicates of a representative experiment. T 798

test: ***, p<0.001. 799

800 801 802




Figure 1A

B

1 392

3

dex

**

= Control cells = ETV6-RUNX1 positive cells

1.0 1.0

1.34 1.39

0

1

2

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Figure 5

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Published OnlineFirst July 24, 2014.Mol Cancer Res Chiara Palmi, Grazia Fazio, Angela M. Savino, et al. ETV6-RUNX1 RearrangementProperties of B Cell Progenitors are Affected by the Cytoskeletal Regulatory Gene Expression and Migratory

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