Letter to the Editor Ibrutinib Inhibits VLA-4–Dependent Adhesion in CLL—Letter Antonella Zucchetto 1 , Erika Tissino 1 , Tanja Nicole Hartmann 2 , Alexandre Chigaev 3 , Giovanni Del Poeta 4 , Alfonso Colombatti 5 , and Valter Gattei 1 We read with interest the article by Herman and colleagues (1), where the Authors demonstrate that in vitro and in vivo inhibition of the B-cell receptor (BCR) pathway by ibrutinib in chronic lymphocytic leukemia (CLL) impairs the adhesive properties of CLL cells via inhibition of the very late antigen-4 (VLA-4) integrin function. VLA-4, a heterodimer composed of the a4/CD49d and b1/CD29 integrin subunits, is a key molecule mediating both cell–matrix and cell–cell adhesion through interaction with fibronectin and the vascular cell adhesion molecule-1 (VCAM-1), respectively (2). On resting lymphocytes, VLA-4 is present in an inactive conformation, while upon lymphocyte activation by different stimuli, including those mediated by BCR triggering (2), VLA-4 conformation rapidly changes and increases its ligand-binding capacity. It is highly conceivable that inhibition of the BCR pathway by ibrutinib, by negatively affecting this inside-out VLA-4 activation, also impairs the VLA-4–mediated adhesive properties of cells. To demonstrate this line of reasoning, the Authors show adhesion assays, in which CLL cells, exposed or not to ibrutinib, are left to adhere onto VLA-4 substrates in the presence or not of bona fide blocking Ab against the a4 and b1 chains of VLA-4. Ibrutinib or anti-a4 Abs similarly hampered CLL cell adhesion, whereas anti-b1 Abs seemed ineffective in this setting (see Fig. 5B in ref. 1). By performing similar adhesion assays, we found, compared with Herman and colleagues (1), the following differences (Fig. 1): (i) pretreatment of VLA-4–expressing primary CLL cells with anti-b1 blocking Abs completely inhibited adhesion onto VLA-4 substrates; (ii) ibrutinib exposure reduced, but did not abolish, the adhesive capability of VLA-4–expressing CLL cells. It is well known that integrins, including VLA-4, functionally operate as heterodimers and that the b1 chain plays a key role in substrate recognition (see example in ref. 3). In this regard, the direct involvement of the VLA-4 b1 chain in adhesion may have practical implications, as the modulation of VLA-4 activity can be easily monitored by flow cytometry by using a confor- mation sensitive anti-b1 Ab (HUTS21) employed in conjunc- tion with small peptides (e.g., an LDV-containing peptide) mimicking the binding site of the VLA-4–specific ligand VCAM-1 (4). Moreover, our observation that ibrutinib did not completely block the adhesion of VLA-4–expressing CLL cells, in keeping with a recent study (5), may provide the rationale for a combined use of ibrutinib with other BCR pathway inhibitors, such as idelalisib, to totally inhibit the inside-out integrin activation in CLL. Disclosure of Potential Conflicts of Interest No potential conflicts of interest were disclosed. Grant Support This study was supported in part by Progetto Giovani Ricercatori no. GR-2011-02346826, Ministero della Salute (Rome, Italy) and the Associa- zione Italiana Ricerca Cancro (AIRC), Investigator Grant IG-17622 (Milan, Italy). Received January 8, 2016; accepted February 9, 2016; published online July 1, 2016. 1 Clinical and Experimental Onco-Hematology Unit, Centro di Riferi- mento Oncologico, I.R.C.C.S., Aviano, Italy. 2 Laboratory for Immuno- logical and Molecular Cancer Research, 3rd Medical Department with Hematology, Medical Oncology, Hemostaseology, Infectious Diseases and Rheumatology,Oncologic Center, Paracelsus Medical University, Salzburg, Austria. 3 Department of Pathology and Cancer Center, University of New Mexico, Albuquerque, New Mexico. 4 Division of Hematology, S. Eugenio Hospital and University of Tor Vergata, Rome, Italy. 5 Experimental Oncology 2, Centro di Riferimento Oncologico, I.R.C.C.S., Aviano, Italy. Corresponding Author: Valter Gattei, Clinical and Experimental Onco-Hema- tology Unit, Centro di Riferimento Oncologico of Aviano, Via F. Gallini 2, Aviano 33081, Italy. Phone: 39-0434-659-410; Fax: 39-0434-659-409; E-mail: vgattei@cro.it doi: 10.1158/1078-0432.CCR-16-0050 Ó2016 American Association for Cancer Research. P = 0.002 P = 0.004 P = 0.06 7 6 5 4 3 2 1 0 Irrelevant Adhesion ratio (VCAM-1/BSA) + Anti-α4 + Anti-β1 + IB Figure 1. Modulation of adhesion of VLA-4–expressing CLL cells by anti-a4 and anti-b1 chain blocking Abs and ibrutinib. Cells from VLA-4–expressing CLL cases (n ¼ 10), treated or not treated with 1 mmol/L ibrutinib (IB) for 1 hour, were labeled with the vital fluorochrome calcein AM, seeded onto 96-well VCAM-1–coated plates, and incubated for 30 minutes at 37  C in the presence of either an irrelevant Ab, the anti-a4 (clone P1H4), or the anti-b1 (clone 4B4) blocking Abs (10 mg/mL). Ibrutinib concentration, Abs concentration, and incubation times were as in ref. 1. The relative number of adherent cells was evaluated by fluorescence detection, and results were expressed as relative fold change in fluorescence intensity compared with controls carried out by seeding cells onto BSA. All statistics were determined by a Wilcoxon matched pairs test. Clinical Cancer Research Clin Cancer Res; 22(13) July 1, 2016 3410 on June 5, 2020. © 2016 American Association for Cancer Research. clincancerres.aacrjournals.org Downloaded from