The role of α4β1 in the pathogenesis of chronic lymphocytic leukaemia; insights from the adhesome

Abstract

Despite recent advances in treating chronic lymphocytic leukaemia (CLL), the disease remains incurable. Signalling through the B cell receptor (BCR), and from the microenvironment are both critical in the pathophysiology of the disease. One of the molecules involved in the interaction between CLL cells and the microenvironment is α4β1 integrin. Integrins are heterodimeric receptors that interact with proteins in the extracellular matrix (ECM), forming a mechanical link between the ECM and the actin cytoskeleton. Cells can sense mechanical cues in the microenvironment and transduce these cues downstream. α4β1 is normally expressed in B cells but is absent from cells in a subset of CLL patients. Expression of the integrin is an independent marker of poor prognosis and correlates with lymphadenopathy, a feature of patients with high-grade disease. Moreover, the mutation status of the immunoglobulin heavy chain gene (IgHV) of the BCR plays a significant role in the prognosis of CLL; patients whose cells express unmutated IgHV (UM-CLL) have poorer outcomes than those with mutated IgHV (M-CLL). Evidence suggests that although both α4β1 and BCR are independent markers of patients with poor prognosis, the two molecules co-operate in vivo. This study aimed to discover if α4β1 and BCR are functionally integrated and to investigate wider mechanisms of integrin and BCR crosstalk in mutated and unmutated CLL.

In order to examine this crosstalk, we used integrin adhesion complex (IAC) enrichment approaches. These enable the isolation of complexes at the sites of cell-substrate adhesion. These complexes contain a mixture of structural and signalling components. Following removal of the cell body, the IACs are harvested and then processed for mass-spectrometry. We used this approach to examine the complexes formed following the adhesion of mantle cell lymphoma MCL and CLL cells to VCAM-1 (a ligand for α4β1), anti-IgM (to stimulate the BCR), and VCAM-1 + anti-IgM. We used the two MCL cell lines (MAVER-1 and JeKo-1) as a model of CLL, as well as M- and UM-CLL cases. Interestingly, our results clearly demonstrate that co-signalling from VCAM-1 clearly alters the IgM signal (and vice versa) both in a positive and negative manner. Some of these changes were similar between all samples, whereas others differed between the two MCL cell lines and between M and UM-CLL.

Using pathway analysis of the proteins identified under the different conditions outlined above, we identified several pathways which were altered when BCR and α4β1 were co-engaged in both CLL and MCL cells. When examining the effect of α4β1 on the BCR-regulated IAC components of MCL cells, we noticed that among the pathways positively affected by the engagement of α4β1 was BCR receptor trafficking. By contrast, interrogation of the BCR pathways affected by α4β1 in primary CLL cells indicated that pathways involved in cell migration were amongst those affected. Interestingly, Vav1, a guanine nucleotide exchange factor (GEF) for the Rho/Rac family of GTPases, was identified as a key protein in the BCR, endocytosis, and cell migration pathways. Thus, Vav1 may act as a key mediator of α4β1 and BCR crosstalk. Subsequently, we confirmed the increase of recruitment of Vav1 to the α4β1 and BCR and that this Vav1 was activated. We are currently investigating the functional consequences of this increased recruitment and activation of Vav1 as a result of α4β1 and BCR crosstalk.

In conclusion, our data has shown that α4β1 and BCR, two molecules important for the pathogenesis of CLL, cooperate in affecting pathways known to be essential for the disease process. Furthermore, our data identified proteins affected by α4β1-BCR receptor crosstalk, and therefore has potential to identify new therapeutic approaches for this difficult to treat group of patients.