The Elastin Microfibril Interfacer-1 (EMILIN-1) Is a Ligand for CD49d in Chronic Lymphocytic Leukemia Cells
Authors: Tissino Erika , Benedetti Dania , Bomben Riccardo , Bo Michele Dal , Vaisitti Tiziana , Deaglio Silvia , Rossi Davide , Zaja Francesco , Pozzato Gabriele , Raimondo Francesco Di , Gaidano Gianluca , Del Poeta Giovanni , Zamò Alberto , Spessotto Paola , Doliana Roberto , Zucchetto Antonella , Colombatti Alfonso , Gattei Valter 
Published: October 2019

Abstract
Abstract 1772
CD49d (α4 integrin chain) is a strong negative prognosticator in chronic lymphocytic leukemia (CLL) with a key role in CLL cell microenvironmental interactions. CD49d triggering by its main ligands Vascular Cell Adhesion Molecule-1 (VCAM-1) and non-RGD sites (CS-1 fragments) of fibronectin (FN) activates signalling pathways delivering pro-survival signals, and promoting resistance to drug-induced apoptosis in CLL. Recently, the globular (g) C1q-like domain of Elastin MIcrofibriL INterfacer1 (EMILIN1), an adhesive extracellular matrix constituent, was described as a new ligand for CD49d, where it operates as a negative modulator of proliferation signals in substrate-adherent non-hematopoietic CD49d+ cells (Danussi et al, J Cell Biol, 2011). Here, we investigated the distribution of EMILIN1 in normal and CLL-involved tissues, and the effects of EMILIN1/CD49d interaction in CLL in terms of adhesion and survival.
By taking advantage of a specific anti-human EMILIN1 monoclonal antibody (Spessotto et al, J Biol Chem, 2003), exploratory staining in reactive lymphoid tissues (tonsil) indicated a clear extracellular EMILIN1 specific reactivity in the outer zone of the mantle/marginal areas. When investigated in lymph node tissues from CLL cases (n=3) by both immunohistochemical and immunofluorescence analyses, a clear EMILIN1 positive staining was detected intermingled with the neoplastic component. To verify whether EMILIN1 could promote CLL cell adhesion, we took advantage of the CLL-derived CD49d+ Mec-1 CLL-like cell model, previously demonstrated by us to adhere to both VCAM-1 and FN (Zucchetto et al, Leukemia, 2012). Results demonstrated similar adhesion levels of Mec-1 cells on VCAM-1, CS-1 fragment of FN and the gC1q-like EMILIN1 domain (mean number of adherent cells per field 267±24, 272±7 and 317±21, respectively).Consistently, adhesion experiments with primary CLL cells characterized by high and homogeneous CD49d expression confirmed similar levels of adhesion on both VCAM-1 and EMILIN1 (mean number of adherent cells per field 141±55 and 135±76, respectively). In all cases adhesion was specifically blocked by pre-treatment with the anti-CD49d HP1/2 blocking antibody. Immunofluorescence analysis with Mec-1 and primary CLL cells showed the recruitment of phospho-Vav-1 at the CD49d/EMILIN1 adhesion sites, with concomitant F-actin reorganization, confirming the activation of the integrin signalling pathway.
The effects of EMILIN1/CD49d interactions in CLL were next investigated performing short-term adhesion experiments (2 and 5 minutes) on gC1q-like EMILIN1 domain and VCAM-1 using CLL cells from three CD49d+ cases, and verifying the phosphorylation of Akt and ERK1/2 kinases, key mediators of survival signals by western blotting. In all cases, an increased intensity of Akt and ERK1/2 phosphorylation was documented after two-minutes of adhesion on both EMILIN1 (mean fold increase =1.2±0.1, and =5.0±1.0 as compared to controls, respectively) and VCAM-1 (mean fold increase =1.6±0.2, and =4.0±1.9, respectively). The phosphorylation was even greater after five minutes (mean fold increase =2.4±0.5, and =10.0±4.0, for EMILIN1 and =2.4±0.6 and =5.0±0.7 for VCAM-1, respectively). The concomitant increase of phospho-Vav-1 confirmed the activation of the integrin signalling pathway.
Finally, we verified whether CD49d/EMILIN1 interaction was able to protect CLL cells from spontaneous apoptosis, by culturing purified cells from 6 CLL cases on gC1q-like EMILIN1 domain, VCAM-1, or control substrate (1% BSA), and checking cell viability after 5 days. Both VCAM-1 and EMILIN1 were able to protect CLL cells from spontaneous apoptosis (p=0.03 and p=0.001, respectively), the viability obtained on EMILIN1 being also significantly higher than that observed on VCAM-1 (p=0.007).
In conclusion, we showed here for the first time that EMILIN1 is present in normal and CLL-involved lymphoid tissues, and it is able to efficiently bind to CD49d, as expressed by CLL cells. At variance of what demonstrated in non-hematopoietic models, EMILIN1 was shown to be able to deliver anti-apoptotic/pro-survival signals to circulating CLL cells. EMILIN1/CD49d interactions may have a role in the maintenance of the neoplastic clone in CD49d-expressing CLL.

Disclosures:
No relevant conflicts of interest to declare.