Retention of inside-out VLA-4 Integrin Activation upon B-Cell Receptor Triggering in in-Vitro and in-Vivo Ibrutinib Treated Chronic Lymphocytic Leukemia Cells: Clinical Implication

Background. Ibrutinib (IB), an orally administered Bruton tyrosine kinase (BTK) inhibitor antagonizing B-cell receptor (BCR) signaling, has demonstrated high response rates in chronic lymphocytic leukemia (CLL). Transient lymphocytosis is a common, although variable, clinical feature during IB treatment, which has been related to the efflux of CLL cells from lymphoid organs, due to impairment of microenvironmental interactions. CD49d, a strong negative prognosticator in CLL, is the alpha-chain of the integrin heterodimer CD49d/CD29 (VLA-4), a key player of CLL microenvironmental interactions. The adhesive properties of VLA-4 can be rapidly inside-out activated by BCR signals, although the role of BCR as VLA-4 activator in CLL is largely unknown.

Aim. To understand whether the inhibition of the BCR signaling by IB exposure of CLL cells affects VLA-4 activation.

Methods. The study included 28 refractory/relapsed CLL patients (15 CD49d+, 23 IGHV unmutated, 22 del11q/del17p) treated with IB on a compassionate basis (IB-CLL), and 11 additional CD49d+ CLL cases (7 IGHV unmutated, 1 del17p). Levels of the active VLA-4 were assessed by flow cytometry using an LDV-containing small molecule as VLA-4 specific ligand, along with the conformation sensitive anti-CD29 HUTS-21 mAb, and measuring the VLA-4 receptor occupancy (VLA4-RO) in values ranging from 0.0 (no RO) to 1.0 (100% RO) as in Chigaev et al. (J Biol Chem, 284,14337, 2009). According to this measurement higher RO indicates larger fraction of high affinity VLA-4 receptors. BCR engagement was performed using goat F(ab′)2 anti–human IgM. CD49d, CD29, IgM expression, the phosphorylation state of ERK (pERK) and BTK (pBTK), and Ca⁺⁺ release were cytometrically analysed. Adhesion assays were performed on VCAM-1-coated slides.

Results. CD49d+ IB-naïve cells from 18 CLL cases, including 7 pre-treatment IB-CLL, were stimulated with anti-IgM. Although with variability, in all cases cells responded to BCR triggering in terms of increased pERK (mean anti-IgM/control MFI ratio: 1.7), pBTK (mean anti-IgM/control MFI ratio: 1.3), and Ca⁺⁺ release (mean Ca⁺⁺ release increase: 11.9%). IgM stimulation resulted in an increased VLA-4 RO (mean 0.52, range 0.40-0.73, vs. mean 0.38, range 0.22-0.63; p=0.0002) in CLL, but not in residual T cells (mean 0.34 vs. 0.35). Consistently, VLA-4-dependent CLL cell adhesion to VCAM-1 increased upon IgM stimulation (mean values of adherent cells/control= 4.6 vs 3.7, p=0.008). IB treatment in vitro (1µM, 1 hour) overall impaired BCR signaling, as shown by lower increases of pERK (mean anti-IgM/control MFI ratio: 1.1), pBTK (mean anti-IgM/control MFI ratio: 1.2) and Ca⁺⁺ release (mean increase: 7.4%). However, VLA-4 RO increased from a mean of 0.37 (range 0.18-0.65) to 0.47 (range 0.15-0.65) after IgM stimulation (p=0.0007), and a parallel increased adhesion was observed (mean values of adherent cells/control= 2.4 vs 3.7). We then moved to cells collected from IB-CLL on t30 of IB treatment (n=7), which showed slightly decreased (79-86%) CD49d, CD29, and IgM MFI levels compared to pre-treatment cells. Despite an overall impaired BCR signaling (mean Ca⁺⁺ release increase: 12.4% at pre-treatment, 3.6% at t30), IgM stimulation still increased both VLA-4 RO (mean 0.27, range 0.01-0.45 compared to 0.53, range 0.41-0.84, p=0.010) and CLL cell adhesion. Similarly, cells from IB-CLL on day 60-90 of treatment (n=3) kept showing an increased VLA-4 RO upon IgM stimulation (from 0.14, range 0.10-0.17 to 0.34, range 0.20-0.56), although Ca⁺⁺ release was relevantly decreased (from 14.8% at pre-treatment to 4.3% at t60-90). Finally, the impact of CD49d expression on the IB-induced lymphocyte redistribution in the 28 IB-CLL was evaluated. Absolute lymphocyte count (ALC) at baseline and after 30-120 days of IB therapy were lower in CD49d+ than CD49d- CLL (see Figure), with a lower median percentage ALC rise from baseline (0% vs +132%, p=0.0044). Consistently, at day t30/t60,  >50% reduction of lymph nodes/spleen from pre-treatment stage only occurred in CD49d- CLL (8/13 cases), while CD49d+ CLL usually experienced reductions <50% (11/12 cases).

Conclusions. BCR triggering in CLL cells activates VLA-4 via an inside-out pathway at least in part independent to IB blockage of BTK. This observation has clinical implication in the kinetics of ALC, shrinkage of CLL masses, and should be considered in the design of IB therapies.