Cytoskeletal Regulatory Gene Expression and Migratory Properties of B-Cell Progenitors Are Affected by the ETV6–RUNX1 Rearrangement

Published OnlineFirst July 24, 2014; DOI: 10.1158/1541-7786.MCR-14-0056-T

Molecular Cancer Genomics Research

Cytoskeletal Regulatory Gene Expression and Migratory Properties of B-cell Progenitors Are Affected by the ETV6–RUNX1 Rearrangement

Chiara Palmi1, Grazia Fazio1, Angela M. Savino1, Julia Procter2, Louise Howell3, Valeria Cazzaniga1, Margherita Vieri1, Giulia Longinotti1, Ilaria Brunati1, Valentina Andre1, Pamela Della Mina4, Antonello Villa4, Mel Greaves2, Andrea Biondi1, Giovanna D'Amico1, Anthony Ford2, and Giovanni Cazzaniga1

Abstract Although the ETV6–RUNX1 fusion is a frequent initiating event in childhood leukemia, its role in leukemogenesis is only partly understood. The main impact of the fusion itself is to generate and sustain a clone of clinically silent preleukemic B-cell progenitors (BCP). Additional oncogenic hits, occurring even several years later, are required for overt disease. The understanding of the features and interactions of ETV6–RUNX1–positive cells during this "latency" period may explain how these silent cells can persist and whether they could be prone to additional genetic changes. In this study, two in vitro murine models were used to investigate whether ETV6– RUNX1 alters the cellular adhesion and migration properties of BCP. ETV6–RUNX1–expressing cells showed a signiﬁcant defect in the chemotactic response to CXCL12, caused by a block in CXCR4 signaling, as demonstrated by inhibition of CXCL12-associated calcium ﬂux and lack of ERK phosphorylation. Moreover, the induction of ETV6–RUNX1 caused changes in the expression of cell-surface adhesion molecules. The expression of genes regulating the cytoskeleton was also affected, resulting in a block of CDC42 signaling. The abnormalities described here could alter the interaction of ETV6–RUNX1 preleukemic BCP with the microenvironment and contribute to the pathogenesis of the disease. Implications: Alterations in the expression of cytoskeletal regulatory genes and migration properties of BCP represent early events in the evolution of the disease, from the preleukemic phase to the clinical onset, and suggest new strategies for effective eradication of leukemia. Mol Cancer Res; 12(12); 1796–806. 2014 AACR.

Introduction normal cord blood (5), monozygotic twins (6), and animal – ETV6–RUNX1, generated by the t(12;21) chromosome modeling (7 13) indicate that this oncogene induces a preleukemic phenotype which is insufﬁcient for overt leu- translocation, is the most common fusion gene in childhood – cancer, selectively associated with B-cell precursor acute kemogenesis. Indeed, ETV6 RUNX1 fusion generated lymphoblastic leukemia (BCP-ALL; refs. 1–3). The t during fetal hemopoiesis produces a clinically covert pre- (12;21) translocation fuses the protein dimerization domain leukemic clone that can persist postnatally for at least 15 years (4). Additional genetic abnormalities observed at of ETV6 with essentially all of the DNA binding and – – activating regions of RUNX1, generating an aberrant tran- diagnosis of ETV6 RUNX1 positive ALL are generally scription factor (2, 4). Observations on clinical samples, considered to be secondary events associated with the tran- sition of silent preleukemic cells to overt ALL (4). The understanding of which cellular signaling pathways 1Centro Ricerca Tettamanti, Clinica Pediatrica, Universita di Milano- are corrupted by ETV6–RUNX1 to sustain this persistent Bicocca, Monza, Italy. 2Centre for Evolution and Cancer, Division of Molecular Pathology, The Institute of Cancer Research, Sutton, Surrey, preleukemic state might help to explain the vulnerability United Kingdom. 3Haemato-Oncology Research Unit, Division of Molec- of its constituent stem cells to secondary genetic changes. ular Pathology, The Institute of Cancer Research, Sutton, Surrey, United We have previously shown evidence that ETV6–RUNX1 Kingdom. 4Microscopy and Image Analysis Consortium, Universitadi b Milano-Bicocca, Monza, Italy. compromised the TGF signaling pathway, providing a plausible basis for both the persistence and maintenance of Note: Supplementary data for this article are available at Molecular Cancer Research Online (http://mcr.aacrjournals.org/). covert preleukemic clones in patients and their competitive positive selection in an inﬂammatory context (12). We and Corresponding Author: Andrea Biondi, Clinica Pediatrica, Universitadi Milano Bicocca, Ospedale San Gerardo, Via Pergolesi 33, Monza 20900, other investigators have also described an increased level of Italy. Phone: 39-0-392333661; Fax: 39-0-392332167; E-mail: heat shock proteins, survivin, has-mir-125b-2, and eryth- [email protected] ropoietin receptor in ETV6–RUNX1–positive cells, factors doi: 10.1158/1541-7786.MCR-14-0056-T that could provide the survival advantage to the preleukemic 2014 American Association for Cancer Research. clone (14–17).

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ETV6–RUNX1 Inhibits CXCL12-Driven Cell Migration

However, in addition to proliferative advantage and resis- the bicistronic retroviral vectors pMSCV-IRES-GFP (MIGR– tance to apoptotic signals, the site of localization and GFP) or pMSCV-ETV6-RUNX1-IRES-GFP, which allows interaction with the microenvironment is crucial to sustain the expression of ETV6–RUNX1 cDNA fused to c-Myc the hematopoietic stem cells in quiescence and the survival of epitope tag and GFP (MIGR–ETV6–RUNX1), as previously both normal and preleukemic cells (18). Moreover, altera- described (27). At day þ3 from transduction, cell sorting for tions in adhesive and chemotactic responses to normal GFP fluorescence was performed by the FACS Aria instrument stimuli have been described in BCR-ABL1–positive chronic (BD Biosciences). myeloid leukemia (19–21). The pre-BI cells were cultured on OP9 bone marrow Interestingly, some genes involved in cellular adhesion and stroma cells in Iscove modified Dulbecco medium (IMDM) cytoskeleton organization are listed among the RUNX1 supplemented with 2% FCS, 0.03% w/v primatone, and target genes (22–23), and changes in the expression of genes 100 units/mL IL7 (27). belonging to this functional pathway are described in ETV6–RUNX1–positive ALL (24–26). Antibodies and flow cytometry The aim of this work was to investigate whether the Phycoerythrin-conjugated antibodies anti-CD18 (M18/ ETV6–RUNX1 preleukemic clone showed alterations in 2), anti-CD11a (M17/4), anti-CD54 (YN1/1.7.4), anti- its adhesive and migratory properties that could provide a CD135 (A2F10), anti-CD29 (HMb 1-1), anti-CD49d rationale for its persistence and proliferation. (R1-2), anti-CD49e (HMa 5-1; e-Bioscience Inc.), and The preleukemic phase is usually clinically silent, whereas anti-CXCR7 (8F11-M16; Biolegend) were used. Allophy- the ETV6–RUNX1 leukemic clone at ALL diagnosis carries cocyanin-conjugated antibodies anti-CD44 (IM7) and anti- additional genetic abnormalities (4); for these reasons, we CXCR4 (2B11; e-Bioscience Inc.) were used. Data were used 2 alternative murine experimental systems: the Ba/F3 analyzed using CellQuest Software (BD Biosciences). pro-B cell line transduced with a hormone-inducible ETV6– RUNX1 (12) and pre-BI primary cells from fetal liver (27, Quantitative PCR array and real-time PCR 28) stably transduced with the pMIGR1–ETV6–RUNX1– The RT2 Profiler Assay Cytoskeleton Regulators PCR IRES–GFP construct. Array (SuperArray Bioscience) was performed following the In both model systems, we found evidence that ETV6– manufacturer's recommendation. RUNX1 alters the expression of cytoskeletal regulatory Real-time analysis was done on a Light Cycler 480II with genes, resulting in a block of the CDC42 signaling Universal Probe Master System (Roche Diagnostics; F. pathway, and compromises the chemotactic response to Hoffmann-La Roche Ltd.). Optimal primers and probe for CXCL12. amplification were selected by the Roche ProbeFinder software (https://www.roche-appliedscience.com/sis/rtpcr/upl). DD Data were expressed using the comparative 2 Ct meth- Materials and Methods od (30), with the Hprt gene as a reference; for each gene Cell culture and ETV6–RUNX1 expression studied, the transcript level was always referred to that of The GeneSwitch System (Life Technologies), a mifepris- control cells. A fold change of <0.75 or >1.5 was considered tone-regulated expression system for mammalian cells, was as threshold for down- or upregulation, respectively. used to produce inducible expression of the ETV6–RUNX1 gene in the IL3-dependent murine pro-B cell line Ba/F3, as Adhesion assays previously described (12). Briefly, cells were transfected with Plates (96-well) were coated with fibronectin, murine the pSwitch plasmid (Invitrogen) expressing a GAL4 regu- stroma cell line OP9 (kindly provided by Prof. A. Rolink, latory fusion protein (control cells). Positive clones were then University of Basel), murine fresh stroma, or murine endo- transfected with pGene plasmid (Life Technologies) con- thelial cell lines 1G11 and MELC2 (a gift of Prof. A. Vecchi, taining the ETV6–RUNX1 cDNA fused to V5 epitope tag Instituto Clinico Humanitas, Rozzano, Italy). Details of the controlled by a promoter regulated by the GAL4 regulatory coating procedures: 50 mL fibronectin (Sigma-Aldrich), at a fusion protein (inducible ETV6-RUNX1 cells). All Ba/F3 concentration of 25 ng/mL in each of the 96 wells, was allow cells were cultured in RPMI supplemented with 10% FCS, to air dry for 2 hours and the cell lines, and murine fresh 2% MoIL3 conditioned medium as a source of IL3 (29), 10 stroma were grown in each well until confluent. OP9 were mmol/L 2-mercaptoethanol, and 0.2 mg/mL hygromycin B. grown in IMDM with 20% FCS, the murine fresh stroma In addition, cells inducible for ETV6–RUNX1 were cul- was isolated from bone marrow of a C57BL/6 wt mouse and tured in the presence of 0.05 mg/mL zeocin. grown in IMDM with 20% FCS at 33 C and 5% CO2.The The expression of ETV6–RUNX1 was induced by adding cell lines 1G11 and MELC2 were grown on a gelatin coating to the culture medium 0.0125 nmol/L mifepristone (Invi- (Sigma-Aldrich) in DMEM supplemented with 4.5 g/L trogen) for 3 days. Efficiency of ETV6–RUNX1 induction glucose, FCS (20% for 1G11, 10% for MELC2), 1% was verified by flow cytometry using an anti-V5 antibody nonessential amino acids, 1 mmol/L sodium pyruvate, 100 (Life Technologies), as previously described (12). mg/mL endothelial cell growth supplement (ECGS; Sigma- þ þ þ Pre-BI cells are murine primary cKIT /B220 /CD19 Aldrich), 100 mg/mL heparin (PharmaTex) and, for MELC2 cells isolated from fetal liver (gift of Prof. A. Rolink, Uni- only, 10% murine sarcoma 180 conditioned medium. The versity of Basel, Basel, Switzerland) and were transduced by endothelial cell lines were stimulated or not for 24 hours with

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inflammatory cytokines IL1b (25 ng/mL; PeproTech), IL6 Western blot analysis of c-Myc epitope tag, fused to ETV6- (20 ng/mL; ImmunoTools), and TNFa (50 ng/mL; Immu- RUNX1 cDNA in MIGR–ETV6–RUNX1 pre-BI cells, was noTools) before being used for the adhesion assay. performed using the rabbit anti-c-Myc (A-14) antibody at After 3 days of induction, control and ETV6–RUNX1– working dilution 1:200 (Santa Cruz Biotechnology). expressing Ba/F3 cells were stained with 12.5 mmol/L Calcein AM (Sigma-Aldrich) and resuspended in adhesion Migration assay medium (RPMI with 5% FCS and 10nmol/L HEPES). Transwell plates (8.0 mm for Ba/F3 cells and 5.0 mm for pre- Cells were then added to 96-well plates coated with the BI cells) were used; 5 105 cells were loaded in the upper substrates indicated above. After 30 minutes of incubation at chamber in 100 mL of RPMI with 1% FCS (migration m 37 C and 5% CO2, nonadherent cells were removed by medium) with or without 10 g/mL of the anti-CXCR7 washing 3 times with the adhesion medium. The adhesion antibody (8F11-M16; Biolegend). About 600 mL of migration index was measured as ratio of fluorescence detected before medium with or without hCXCL12 (100 ng/mL), FLT3L (10 and after washing by the fluorescence reader TECAN ng/mL; ImmunoTools), or 10% FCS was added in the lower GENios (Tecan). chamber. After 3 hours at 37 C and 5% CO2, cells in the Cell adhesion to VCAM1 and ICAM1 recombinant lower chamber were collected and counted by FACS. The proteins was performed as previously described (28). Briefly, migration index was defined as the ratio between the number 15-mm round coverslips were coated with recombinant of cells migrated to the lower chamber of the Transwell in mouse VCAM1-Fc protein (25 mg/mL; R&D Systems) or response to the chemokine stimulus and in its absence. recombinant mouse ICAM1-Fc protein (25 mg/mL; R&D Migration assay in the presence of EGF: migration assay of Systems) and placed in 12-well dishes containing 1.5 105 pre-BI cells toward 100 ng/mL hCXCL12 was performed in 3-days induced control or ETV6–RUNX1–expressing Ba/ presence or absence of 20 ng/mL of EGF (PeproTech) F3 cells. After overnight incubation at 37 C and 5% CO2, homogeneously present in the upper and lower chambers the coverslips were washed to eliminate the nonadherent of the Transwell. After 4 hours, the GFP-positive cells cells and were mounted on slides in the presence of 4',6- migrated to the lower well were counted by FACS. The diamidino-2-phenylindole (DAPI). Each coverslip was ana- migration index was defined as the ratio between the number lyzed by a fluorescence microscope, acquiring 30 represen- of cells migrated to the lower chamber of the Transwell in tative fields, and the adherent cells were counted. response to CXCL12 and in its absence. Migration assay in the presence of CCL2: migration assay Immunoblotting of Ba/F3 cells toward 100 ng/mL CXCL12 was performed in Western blot analysis of CDC42 protein was performed absence or presence of 100 ng/mL of CCL2 (PeproTech) in by lysing cells in the lysis buffer (Thermo Scientific) with the upper chamber of the Transwell. The migration index protease inhibitor cocktail (Sigma-Aldrich). Mouse anti- was defined as the ratio between the number of cells migrated CDC42 antibody was used at working dilution 1:167 to the lower chamber of the Transwell in response to (Thermo Scientific), mouse anti-b-actin antibody at CXCL12 and in its absence. 1:1,000 (AC-15, Sigma-Aldrich), and the secondary goat anti-mouse IgG (Fc-specific) peroxidase antibody at working Protein array analysis dilution 1:20,000 (Sigma-Aldrich). A StripAblot Stripping Protein array was performed on cell supernatant, obtained Buffer (Euroclone S.p.A.) was used to recover membranes. from Ba/F3 control and ETV6–RUNX1–positive cells, Densitometry analyses were performed using Alliance using RayBio Cytokine Antibody Arrays—Mouse Array instrument and Uviband software (Uvitec). III-IV (Raybiotech Inc), following the manufacturer's pro- For p-ERK, total ERK, p-PAK2, and total PAK2 protein tocol. Densitometry analyses were done using Kodak image analysis, cells were starved for 1 hour in RPMI without station (Kodak SpA). serum, then 2 106 cells were stimulated with 100 ng/mL hCXCL12 (PeproTech) in RPMI at 37C. At different time Calcium mobilization analysis points, cells were washed with ice-cold PBS and pellets were After overnight IL3 starvation, 0.6 106 3-day induced lysed in 20 mmol/L Tris-HCl/NaCl, pH 7.4, containing 2 cells were loaded with FluoForte dye loading solution (Enzo mmol/L EDTA, 0.2 mmol/L Na3VO4, 1% Triton X-100, Life Sciences) in RPMI with 10% FCS for 45 minutes at 25 mmol/L b-glycerophosphate, 25 mmol/L NaF, 1 mmol/L 37C and then 15 minutes at room temperature. Baseline phenylmethylsulfonyl fluoride, and protease inhibitor cock- calcium levels were established for about 2 minutes before tail at 4C for 30 minutes. Rabbit anti-phospho-p44/42 the addition of 300 ng/mL CXCL12. Data were collected for MAPK (ERK1/2; Thr202/Tyr204) antibody, rabbit anti a total of 512 seconds and analyzed on a FACSCalibur using phospho-PAK1 (Thr423)/PAK2 (Thr402) antibody, rabbit CellQuest software (BD Biosciences). anti-p44/42 MAPK (ERK1/2) antibody, and rabbit PAK2 (C17A10) antibody were used at working dilution 1:1,000 Generation of Ba/F3-overexpressing CDC42 proteins (Cell Signaling), the mouse anti-GAPDH antibody at 1:200 The cDNA of wild-type, constitutively active (Q61L), (6C5, Santa Cruz Biotechnology), and the secondary goat and dominant negative (T17N) CDC42 were cloned into anti-rabbit IgG (HþL) HRP at 1:10,000 dilution (Thermo the retroviral vector pMSCV-IRES-GFP using In-Fusion Scientific). HD Cloning kit (Clontech Laboratories) and following the

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ETV6–RUNX1 Inhibits CXCL12-Driven Cell Migration

manufacturer's recommendation. Ba/F3 cells were trans- We observed an increase in the adhesion of ETV6– fected by nucleofection following the Amaxa protocol, as RUNX1–positive cells to the murine endothelial cell lines previously described (12). 1G11 and MELC2 [adhesion index of ETV6–RUNX1– positive cells vs. control cells: 1.34 0.26 (P ¼ 0.0431) on 1G11 and 1.39 0.26 (P ¼ 0.0286) on MELC2; Fig. 1B]. Results The stimulation of the endothelial cell lines with inflam- ETV6–RUNX1 alters the expression of cell-surface matory cytokines did not modify the adhesion index (data adhesion molecules and adhesion properties of Ba/F3 cell not shown). However, we did not observe any difference in After ETV6–RUNX1 expression in Ba/F3 cells, flow the adhesion abilities to several other substrates: fibronectin, cytometric analysis indicated alterations of the cell-surface CD54/ICAM1 and VCAM1 molecule, murine stroma cell expression levels of several molecules involved in cell adhe- line OP9, and murine fresh stroma. sion and migration of BCP (31). In detail, ETV6–RUNX1– positive cells expressed higher levels (MFI ratio) of the ETV6–RUNX1 causes altered expression of genes following adhesion molecules: CD44 (average of increase regulating the cytoskeleton in independent experiments: 58%; range: 19%–118%; P < We explored whether the ETV6–RUNX1 fusion gene 0.05), CD18 (average: 121%; range: 19%–206%; P < 0.05), affected key genes in the organization of the cytoskeleton. CD11a (average: 182%; range: 54%–334%; P < 0.01), and A panel of 84 genes involved in the biogenesis and CD54 (average: 145%; range: 80%–207%; P < 0.05). On organization of the cytoskeleton was examined using the the other hand, they expressed lower levels of the integrin RT2 Profiler Assay Cytoskeleton Regulators PCR Array, CD29 (average of decrease: 22%; range: 8%–34%; P < plus single-candidate gene transcripts tested by RQ-PCR. 0.01; Fig. 1A) but did not show a significant difference in the We identified 9 genes overexpressed and 7 genes repressed expression of CD49d and CD49e (data not shown). in Ba/F3 ETV6–RUNX1–positive cells compared with The transcription level of genes coding some of these control cells (Table 1). These were genes involved in cell antigens tested by RT-PCR confirmed the immunopheno- shape, formation of pseudopodia, cell migration, actin, and type results (Supplementary Fig. S1). microtubule organization. Interestingly, several of these

A Counts Counts 0 200 Figure 1. Phenotypic and adhesion 0 150 100 101 102 103 104 100 101 102 103 104 analyses of control and ETV6– CD44 CD18 RUNX1–positive Ba/F3 cells. A, overlay analyses of the expression on cell surface of the indicated antigen measured as MFI levels by Counts FACS. The ﬁgure shows a Counts 0 200 representative experiment. B, 0 100 100 101 102 103 104 100 101 102 103 104 adhesion analyses of control and CD11a CD54 ETV6–RUNX1–positive Ba/F3 cells to murine endothelial cell lines 150 1G11 and MELC2. After 3-day induction, control and ETV6–

RUNX1–expressing Ba/F3 cells Counts

were stained with Calcein AM and 0 added to 96-well plates coated 100 101 102 103 104 with the cell lines. After 30 minutes CD29 of incubation, nonadherent cells = Control cells = ETV6-RUNX1–positive cells were removed by washing 3 times with the adhesion medium. The adhesion index was measured as B 3 ratio of ﬂuorescence detected before and after washing. Ctr, * control cells; ER, ETV6–RUNX1– 2 1.34* 1.39 Ctr positive cells. t test: , P < 0.05. ER 1.0 1.0 1 Adhesion index Adhesion index 0 1G11 MELC2

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Cdc42ep3, Map3k11, Was, Nck2 Nck1 Mmp9 Table 1. Upregulated and downregulated , ,and (32–37). In particular, Cdc42ep2 and Cdc42ep3,negative genes in ETV6–RUNX1–positive Ba/F3 cells regulators of Cdc42 (32),wereamongthemostupregu- lated genes in ETV6–RUNX1–positive cells (Table 1, RT-PCR upregulated genes). Moreover, after induction of the Gene name Fold change t test fusion gene (Fig. 2A), we observed a reduction of CDC42 Upregulated genes at the transcription and at the protein level by RT-PCR Cdc42ep2 5.41 0.00005 and Western blot analyses (Fig. 2B and C). Consistently, we verified that CDC42 signaling was perturbed by Arhgef11 2.72 0.00006 – Cdc42ep3 2.61 0.00019 ETV6 RUNX1.AsshowninFig.2D,theCDC42 downstream effector kinase PAK2 (38) was less expressed Map3k11 1.49 0.00169 – – Fscn2 1.86 0.00303 in ETV6 RUNX1 positive cells, and cell stimulation Dstn2 1.61 0.00188 with CXCL12 induced a marked increase in the phos- Was 1.47 0.00033 phorylationofPAK2onlyinBa/F3controlcells(densi- Nck2 2.68 0.00003 tometry analyses of pPAK2 after normalization on the amount of PAK2 total and b-actin: þ2.36-fold increase in Nck1 1.56 0.00785 – – Downregulated genes Ctr cells vs. 0.15-fold decrease in ETV6 RUNX1 Rock1 0.18 0.00002 positive cells). Ppp3cb 0.47 0.00000 – Stmn1 0.64 0.00170 ETV6 RUNX1 impairs migration toward CXCL12 Clip1 0.68 0.00015 By applying a Transwell migration assay, we observed fi asignificant defect in the chemotactic response of ETV6– Cy p2 0.76 0.00011 – Mylk 0.65 0.00031 RUNX1 expressing Ba/F3 cells to CXCL12, a potent Mmp9 0.34 0.00006 chemoattractant for BCP (Fig. 3A). In detail, the induction of ETV6–RUNX1 expression caused a decrease in the migration index of 86.2% (average of 5 independent differentially expressed genes belonged to the CDC42 path- experiments, range: 75.1%–92.6%; P < 0.001), although way, a key element for the regulation of the cytoskeleton and the expression of the CXCR4 receptor on the cell surface for the directional migration of the cells: Cdc42ep2, was unaffected or even increased (MFI increase average:

A B Cdc42 2.00

1.00

-DD Ct 1.00

2 0.43** Counts

0 300 0.00 0 1 2 3 4 10 10 10 10 10 Ctr ER V5 = Control cells = ETV6-RUNX1–positive cells C D – CXCL12 kDa M – + Ctr ER Ctr ER Ctr ER 25 pPAK2 CDC42 15 –59% PAK2 55 b-Actin 35 b-Actin

Figure 2. Cdc42 pathway analysis in control and ETV6–RUNX1–positive Ba/F3 cells. Ba/F3 cells were induced to express ETV6–RUNX1 for 3 days. A, flow cytometric analysis of the intracellular expression of V5 epitope tag, fused to ETV6–RUNX cDNA. B, cDNA was subjected to TaqMan RT-PCR and normalized to Hprt expression. Transcript level of the Cdc42 gene in ETV6–RUNX1–positive cells relative to control cells is shown as an average of triplicates. Ctr, control cells; ER, ETV6–RUNX1–positive cells. t test: , P < 0.01. C, cell lysates were analyzed by Western blotting with anti-CDC42 antibody. The blot was later stripped and reprobed with an anti-b-actin antibody. CDC42 protein expression level in ETV6–RUNX1–positive cells was quantified by densitometry, normalized to b-actin, and indicated in the figure as the percentage with respect to control cells. M, Marker; and þ, negative and positive controls for CDC42 protein, respectively. D, Western blot analysis of PAK2 phosphorylation, total PAK2, and b-actin after 2 minutes of CXCL12 stimulation.

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in Ba/F3 ETV6–RUNX1–induced cells (Supplementary A CXCL12 Fig. S3B). Interestingly, CCL2 is secreted at higher amount 15 by ETV6–RUNX1–positive Ba/F3 cells (Supplementary 10.6 Fig. S3C). However, no difference in migration toward CXCL12 was observed in control cells upon pretreatment 10 with the ETV6–RUNX1–positive Ba/F3 supernatant, thus also excluded a pivotal role of the secreted CCL2 in the inhibition of the migration ability of ETV6–RUNX1–pos- 5 itive cells.

Migration index ** 1.4 We previously observed high expression levels of the FLT3L receptor (CD135) in the immature hematopoietic cells from ETV6–RUNX1 transgenic mice (12). FLT3L 0 Ctr ER plays an important role in cellular proliferation and survival, but it also enhances migration toward CXCL12 (42). However, although we conﬁrmed higher level of CD135 B FCS 10% – 15 protein expression in Ba/F3 cells after ETV6 RUNX1 * induction (Supplementary Fig. S2C), we did not observe 10.4 an increase in their migration ability, unlike the control cells (Supplementary Fig. S4B), thus excluding that FLT3L 10 could recover the ability of ETV6–RUNX1–positive cells to migrate to CXCL12. 4.5 Finally, as we showed above that ETV6–RUNX1 5 deregulated CDC42 signaling, we tested whether the

Migration index overexpression of Cdc42 could counteract the impaired migrationofETV6–RUNX1–positive cells. In Supple- mentaryFig.S4C,weshowedthatwhiletheoverexpres- 0 sion of wild-type (WT) or constitutively active (CA) Cdc42 Ctr ER caused a marked increase in the migration toward CXCL12 in control cells, only a slight increase was – – Figure 3. Migration analyses of control and ETV6–RUNX1–positive Ba/F3 measured in ETV6 RUNX1 positive cells (MFI increase cells. Ba/F3 control and ETV6–RUNX1–positive cells were incubated for 3 average in CDC42 CA cells: 3.49 0.13 in Ctr cells vs. days in the presence of the inducer before performing the migration 1.29 0.08 in ER cells). This result demonstrated that assays toward (A) 100 ng/mL CXCL12, (B) 10% FCS. After 3 hours, the neither the enhancement of the CDC42 activity was able cells migrated to the lower well were collected and counted by FACS. The migration index was defined as the ratio between the number of cells to recuperate the migration property toward CXCL12 of migrated to the lower chamber of the Transwell in response to the ETV6–RUNX1–positive cells. chemokine stimulus and in its absence. Error bars, SD from triplicates of a representative experiment. t test: , P < 0.05; , P < 0.01. Ctr, control cells; ETV6–RUNX1–positive cells do not have a general – – ER, ETV6 RUNX1 positive cells. defect of movement We wondered whether ETV6–RUNX1–positive Ba/F3 31%, range: 3%–100%, P < 0.05; Supplementary Fig. cells were unable to specifically migrate toward CXCL12 or S2A). whether they presented any general defect in movement. By We then explored the possible role of CXCR7 and CCR2 Transwell migration assay using 10% FCS as a general on the migration defect of ETV6–RUNX1–positive cells. stimulus, we found that not only ETV6–RUNX1–positive These 2 receptors that negatively regulate the CXCL12 cells were inhibited in movement, but also they migrated signaling cascade in BCP cells (39–41) are both upregulated more than control cells (Fig. 3B). in Ba/F3 cells after ETV6–RUNX1 induction [MFI increase average of CXCR7: 16%, range: 6%–27%, P < 0.05 ETV6–RUNX1–positive cells present a defect in CXCR4 (Supplementary Fig. S2B); fold change of the Ccr2 gene: signaling 12.55, P < 0.01 (Supplementary Fig. S3A)]. Although the expression of CXCR4 receptor on the cell In Ba/F3 control cells, CXCL12-mediated chemotaxis surface was unaffected or even increased (Supplementary was modulated by both an anti-CXCR7 antibody and Fig. S2A), ETV6–RUNX1 induction inhibited the mobi- CCL2, the ligand of CCR2 (Supplementary Figs. S4A and lization of intracellular calcium flux after CXCL12 stimu- S3B). However, in ETV6–RUNX1–positive cells, the lation in Ba/F3 cells (Fig. 4A). Moreover, the phosphory- migration toward CXCL12 remained defective after lation of ERK in response to CXCL12 was absent in ETV6– CXCR7 blocking (Supplementary Fig. S4A), thus excluding RUNX1–positive cells (Fig. 4B). a role of CXCR7 in the inhibition of their migration ability. Thus, the expression of ETV6–RUNX1 in Ba/F3 cells On the other hand, in presence of CCL2, we observed a resulted in the block of ERK phosphorylation, early down- more marked decrease in the ability to migrate to CXCL12 stream to CXCR4 signaling.

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Ctr CALCIUM 0 200 400 600 800 1,000 0 200 400 600 800 1,000 Time (512.00 sec.)

ER Figure 4. Mobilization of

CALCIUM intracellular calcium and activation of ERK in response to CXCL12 in control and ETV6–RUNX1–positive 0 200 400 600 800 1,000 Ba/F3 cells. A, calcium ﬂux in 0 200 400 600 800 1,000 Time (512.00 sec.) response to stimulation with CXCL12 as indicated by the arrow. B, Western blot analysis of ERK CXCL12 phosphorylation, total ERK, and B Ctr ER GAPDH after CXCL12 stimulation (the numbers indicate the minutes kDa M 0 1' 2' 3' 5' 7' 10' 0 1' 2' 3' 5' 7' 10' of stimulation). Ctr, control cells; ER, ETV6–RUNX1–positive cells. 55 p-ERK 35

55 ERK

35 55 GAPDH 35 25

ETV6–RUNX1 alters the expression of genes regulating As in Ba/F3 cell line, the expression of ETV6–RUNX1 in the cytoskeleton and migration properties of primary pre-BI cells caused alteration in the expression of genes pre-BI cells involved in the modulation of the cytoskeleton (Table 2), The effect of ETV6–RUNX1 on the expression of genes including the overexpression of Cdc42ep2 and Cdc42ep3, the regulating the cytoskeleton and migration properties was negative regulators of Cdc42, and a reduction of Cdc42 confirmed in primary pre-BI cells, purified from fetal liver of transcription (Fig. 5B). Moreover, the CDC42 signaling was wild-type mice, a more physiological murine model. These perturbed in the pre-BI ETV6–RUNX1–positive cells, as cells were stably transduced with a retroviral vector the stimulation with CXCL12 induced an increase in (pMIGR1) containing the ETV6–RUNX1 cDNA upstream PAK2 phosphorylation only in the control MIGR–GFP of the IRES-GFP and isolated by GFP sorting (Material and cells (Fig. 5C). Methods; Fig. 5A). Interestingly, also pre-BI MIGR–ETV6–RUNX1 cells Pre-BI MIGR–ETV6–RUNX1 cells presented a higher showed a significant defect in their ability to migrate toward cell-surface expression levels (MFI ratio) of adhesion molecules CXCL12, with 73.5% decrease of M.I. (average of 5 inde- such as CD18 (average of increase: 64%, range: 15%–146%, pendent experiments, range: 57.0%–88.9%, P < 0.001; Fig. P < 0.01), CD11a (average: 90%, range: 19%–203%, P < 5D). However, the expression of CXCR4 receptor on the cell 0.01), and CD54 (average: 27%, range: 14%–85%, P < 0.05; surface was unaffected (Supplementary Fig. S5E). Supplementary Fig. S5A–S5C) and lower levels of CD62L Unlike Ba/F3 cells, pre-BI cells express the EGF receptor (average of decrease: 68%, range: 7%–83%, P < 0.05; (EGFR), a strong activator of the CDC42 pathway (43). As Supplementary Fig. S5D) than MIGR–GFP control cells. shown in Supplementary Fig. S6, although we observed an

1802 Mol Cancer Res; 12(12) December 2014 Molecular Cancer Research

ETV6–RUNX1 Inhibits CXCL12-Driven Cell Migration

A MIGR–ER MIGR–GFP B Cdc42 2.00

1.00 ETV6–RUNX1 1.00 -DD Ct 0.44* 2

0.00 MIGR–GFP MIGR–ER

C – CXCL12 D GFP ER GFP ER CXCL12 50 pPAK2 40 31.8 30 PAK2 20 10 ***8.1 Migration index b-Actin 0 MIGR–GFP MIGR–ER

Figure 5. Cdc42 pathway and migration analysis of pre-BI MIGR–GFP and MIGR–ETV6–RUNX1 cells. A, Western blot analysis of c-Myc epitope tag, fused to ETV6–RUNX cDNA. B, cDNA of pre-BI cells was subjected to TaqMan RT-PCR and normalized to Hprt expression. Transcript levels of the Cdc42 gene in MIGR–ETV6–RUNX1 (MIGR–ER) relative to MIGR–GFP cells are shown as an average of triplicates. t test: , P < 0.05. C, Western blot analysis of PAK2 phosphorylation, total PAK2, and b-actin after 2 minutes of CXCL12 stimulation. GFP, pre-BI MIGR–GFP cells; ER, pre-BI MIGR–ETV6–RUNX1 cells. D, migration assays toward 100 ng/mL CXCL12. After 3 hours, the GFP-positive cells that migrated to the lower well were counted by FACS. The migration index was defined as the ratio between the number of cells migrated to the lower chamber of the Transwell in response to CXCL12 and in its absence. Error bars, SD from triplicates of a representative experiment. t test: , P < 0.001. increased migration index toward CXCL12 in the control preleukemic clone remain unknown. In the present article, cells in presence of EGF stimulus; however, the migration we consistently showed in 2 in vitro models that ETV6– toward CXCL12 remained defective in ETV6–RUNX1– RUNX1 deregulates the cytoskeleton and compromises the positive cells. chemotactic response to CXCL12. It has been increasingly recognized that cancer initiation Discussion and progression are not solely a cancer cell autonomous Although the t(12;21) translocation is a frequent prenatal process. Primary tissue cells live in complex microenviron- initiating mutation in BCP-ALL (1–3, 6), the cellular ments, characterized by heterotypic signaling between ancil- signaling pathways corrupted by ETV6–RUNX1 in the lary cells and hematopoietic cells (44). This signaling is considered to play a role in the regulation of the behavior of stem and precursor hematopoietic cells, including their Table 2. Upregulated and downregulated survival, proliferation, and differentiation. For this reason, genes in ETV6–RUNX1–positive pre-BI cells alterations in the environment or in the abilities of the stem and progenitors cells to interact with the innate niches play a crucial role in tumor initiation and progression. RT-PCR We have used 2 different model systems to study the Gene name Fold change t test ETV6–RUNX1 preleukemic phase and in particular to – Upregulated genes examine whether ETV6 RUNX1 altered the cellular adhe- Cdc42ep2 1.46 0.02215 sion and migration properties of BCP. Indeed, this type of Arhgef11 1.86 0.00036 study is not feasible in clinical samples at diagnosis of ALL, Cdc42ep3 3.1 0.00114 where the analysis of the fusion function is confounded by Downregulated genes the additional genetic abnormalities. We have therefore used Rock1 0.66 0.00028 a murine progenitor cell line (Ba/F3), with hormone-induc- – Stmn1 0.75 0.01874 ible ETV6 RUNX1 expression, a model developed in the Clip1 0.75 0.00008 past to demonstrate the impact of the fusion gene on the b fi Cyfip2 0.78 0.02114 inhibitory response to TGF (12). Next, we con rmed our Mylk 0.47 0.00005 results in pre-BI cells, primary cells derived from a wild-type Mmp9 0.16 0.00022 mouse fetal liver, already successfully used for functional – – Mmp2 0.16 0.00256 analysis of BCP-ALL associated fusion transcripts (27 28). – Cdh2 0.3 0.00357 We observed that the expression of ETV6 RUNX1 in Ba/ F3 cell line resulted in changes in the cellular phenotype:

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Palmi et al.

þ several molecules involved in cell adhesion were deregulated in CD34 cells (48). In light of these observations, we believe expression. We observed an increase in the adhesion of ETV6– that it will be important to explore whether the low expres- RUNX1–positive cells to murine endothelial cell lines. sion of CD9 is a property not only of the overt leukemic To understand the reason for the reported alterations, we blasts but also of the preleukemic cells and whether this explored the effect of the ETV6–RUNX1 fusion, an aberrant feature may play a key role in the CXCL12 migration defect transcription factor, on the transcription of a panel of genes of ETV6–RUNX1–positive BCP described here. involved in the biogenesis and organization of the cytoskel- Noteworthy, the results observed in the Ba/F3 ETV6– eton. Indeed, we showed that the expression of ETV6– RUNX1–inducible expression system were reproducible in RUNX1 in Ba/F3 cell line caused alteration in the expression primary pre-BI cells. Indeed, after transduction of the of genes regulating cell shape, formation of pseudopodia, cell chimeric gene, we confirmed an altered expression of genes migration, actin, and microtubule organization. In partic- involved in cytoskeleton modulation, including Cdc42 with ular, among the most overexpressed genes in ETV6– its regulators, and several adhesion molecules. Moreover, we RUNX1–positive cells, we identified 2 negative regulators consistently observed the same block of CDC42 signaling of CDC42. Moreover, we observed a reduction of CDC42 at and the same significant defect of pre-BI ETV6–RUNX1– the transcription and protein levels and a block of the positive cells to migrate toward CXCL12. CDC42 signaling pathway. CDC42 not only has a pivotal Interestingly, similar alterations in the expression of adhe- role in cell-cycle progression (in agreement with this, we sion molecules and defects in CXCL12 migration have been previously described an increase in the proportion of ETV6– reported in BCR-ABL1–positive leukemia (19–21). In this – – RUNX1 expressing cells in G0 G1; ref. 12) but also in context, it has been hypothesized that these aberrations cytoskeleton rearrangement during directional migration. could contribute to the homing and retention defects in In parallel, we investigated the migration abilities of ETV6– the bone marrow typical of immature myeloid cells in RUNX1–inducible Ba/F3 cells. Interestingly, we found that chronic myelogenous leukemia (49). the fusion gene significantly impaired the chemotactic In light of the findings described here, we can sustain the response to CXCL12, although the cell-surface expression of hypothesis that the ETV6–RUNX1 preleukemic clone, com- the receptor CXCR4 was unaffected. Indeed, the CXCL12 pared with its normal counterpart, might have an altered chemotaxis defect was not due to a general impairment of interaction with the bone marrow microenvironment, which movement of ETV6–RUNX1–positive cells, as their migra- results in a greater tendency to migrate to the periphery. A tion toward a general stimulus was instead increased. direct demonstration of their ability to leave the bone marrow, We then excluded a possible role in this migration defect despite of their immature status, is represented by the detec- of several players of the CXCL12/CXCR4 pathway, includ- tion of preleukemic clones in peripheral blood at birth (in ing CXCR7, a receptor with CXCL12-scavenging activity Guthrie cards, cord blood, and peripheral blood; refs. 5, 50). (39), FLT3L receptor, a positive regulator of CXCL12 Appropriate in vivo studies in murine models must be afforded migration (42), the GTPase CDC42, as well as the to further exploit the characteristics of the preleukemia phase CCL2/CCR2 and EGF/EGFR axes. These 2 axes are both and to define the role of microenvironmental factors in the involved in the modulation of CXCL12-mediated chemo- preleukemic state induced by ETV6–RUNX1. taxis. Indeed, it has been reported that the expression of In conclusion, the abnormalities we observed in the CCR2 negatively regulates the cytoskeletal rearrangement expression of genes regulating the cytoskeleton and in and migration of immature B cells and that the control of B- migration toward CXCL12 may represent early events in cell homing by CCR2 is mediated by its ligand, CCL2, the pathogenesis of the disease, not necessary associated to which is secreted by B cells and downregulates the CXCL12 the progress toward overt leukemia unless additional hits signaling cascade (40–41). On the contrary, a synergistic occur (4). Indeed, our observation raises the question as to effect of CXCR4 and the EGFR on promoting cancer how ETV6–RUNX1 preleukemic cells interact with the metastasis has been reported (45). In particular, EGF was microenvironment. We believe that identification of the described to promote breast cancer cell chemotaxis in precise localization of these cells, their cell–cell contacts, and CXCL12 gradients, whereas CXCL12 alone failed to stim- gene regulation are crucial to providing a better understand- ulate the migration of these cells (46). ing of the mechanisms that allow the preleukemic clone to We were able to demonstrate that ETV6–RUNX1 persist covertly in an individual for several years, maybe impairs the calcium flux, a very proximal CXCR4 signaling prone to additional genomic events. This will be decisive in event, and the phosphorylation of ERK kinase, as a down- helping develop strategies for their effective eradication and stream event. leukemia prevention. Further analyses are needed to fully define the mechanism of migration defect and to establish how ETV6–RUNX1 Disclosure of Potential Conflicts of Interest inhibits the CXCL12–CXCR4 signaling pathway. In this No potential conflicts of interest were disclosed. regard, it was reported in the literature that ETV6– RUNX1–positive patients with ALL presented at diagnosis Authors' Contributions lower level of CD9 than the negative patients with ALL (47). Conception and design: C. Palmi, A. Biondi, A. Ford, G. Cazzaniga Development of methodology: V. Andre, A. Villa, A. Ford Importantly, the tetraspanin CD9 has been described to Acquisition of data (provided animals, acquired and managed patients, provided regulate migration, adhesion, homing of human cord blood facilities, etc.): L. Howell, G. Longinotti, A. Villa, A. Ford

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ETV6–RUNX1 Inhibits CXCL12-Driven Cell Migration

Analysis and interpretation of data (e.g., statistical analysis, biostatistics, compu- Grant Support tational analysis): C. Palmi, G. Fazio, A.M. Savino, J. Procter, V. Cazzaniga, M. Vieri, C. Palmi is supported by a grant from Beat Leukemia Foundation (www.beat- P.D. Mina, A. Villa, M. Greaves, G. D'Amico, A. Ford leukemia.org). This study was supported by grants from: Fondazione Tettamanti Writing, review, and/or revision of the manuscript: C. Palmi, M. Greaves, (Monza), Comitato Maria Letizia Verga, Comitato Fiori di Lavanda, Associazione G. D'Amico, A. Ford, G. Cazzaniga Italiana Ricerca sul Cancro (AIRC; to A. Biondi, G. D'Amico, and G. Cazzaniga), Administrative, technical, or material support (i.e., reporting or organizing data, MIUR (to A. Biondi), Fondazione Cariplo (to A. Biondi and G. Cazzaniga), constructing databases): I. Brunati, A. Villa Leukaemia & Lymphoma Research (UK) (to M. Greaves and A. Ford). Study supervision: A. Biondi, A. Ford, G. Cazzaniga The costs of publication of this article were defrayed in part by the payment of Other (provided confocal microscopy expertise and acquired images of immu- advertisement ﬂ page charges. This article must therefore be hereby marked in no uorescent stained cells): L. Howell accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Acknowledgments The authors thank Cristina Bugarin for cell sorting by ﬂow cytometry and Marta Received February 7, 2014; revised June 10, 2014; accepted June 30, 2014; Galbiati for her help in VCAM and ICAM adhesion assay analyses. published OnlineFirst July 24, 2014.

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1806 Mol Cancer Res; 12(12) December 2014 Molecular Cancer Research

Cytoskeletal Regulatory Gene Expression and Migratory Properties of B-cell Progenitors Are Affected by the ETV6 −RUNX1 Rearrangement

Chiara Palmi, Grazia Fazio, Angela M. Savino, et al.

Mol Cancer Res 2014;12:1796-1806. Published OnlineFirst July 24, 2014.

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