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. Ó2014 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 Ó2014 American Association for Cancer Research. clone (14–17). 1796 Mol Cancer Res; 12(12) December 2014 Downloaded from mcr.aacrjournals.org on October 2, 2021. © 2014 American Association for Cancer Research. Published OnlineFirst July 24, 2014; DOI: 10.1158/1541-7786.MCR-14-0056-T 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 soft- ware (https://www.roche-appliedscience.com/sis/rtpcr/upl). ÀDD 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