Ibrutinib Inhibits VLA-4-Dependent Adhesion in CLL – Reply

We thank Zucchetto et al. for their valuable comments on our manuscript (1). They make two points: first, in their hands, in contrast to our report, inhibition of integrin beta-1 (CD29) and integrin alpha-4 (CD49d) were equally effective in reducing CLL cell adhesion. Repeating our original assay (Figure 5B) with three anti-CD29 antibodies (clones: 4B7R, P5D2 and P4G11; R&D systems), we found that our experiments may have underestimated the effect of CD29 blockade on cell adhesion, confirming Zucchetto’s findings. However, for each of the clones, there was considerable interpatient and interclonal variability in the effect size.

Secondly, Zucchetto et al. note that ibrutinib in vitro reduced but did not abolish adhesion. The results of in vitro assays presented in our Supplementary Figure 2A/B are in agreement with this conclusion (1). In contrast, in vivo, ibrutinib abrogated the ability of CLL cells to adhere to fibronectin as measured in ex vivo assays with cells obtained from patients on day 28 of ibrutinib treatment (Figure 2A/B) (1). This appears to be the result of cumulative drug exposure, given that adhesion of cells collected on day 2, after just one dose of ibrutinib, was only partially inhibited (1). Thus, treatment with ibrutinib in vivo has a much stronger inhibitory effect on cell adhesion then captured by experiments (both ours and Zucchetto’s) that drug cells in vitro.

We used samples from patients treated with single agent ibrutinib to investigate the basis for our previous observation that upon starting ibrutinib treatment most patients develop a transient rise in absolute lymphocyte count that is due to release of cells from the lymph node (2). VLA-4-dependent adhesion is regulated by chemokines and B-cell receptor (BCR) signaling, referred to as “inside-out signaling”, affecting both conformation (PKC/DAG dependent) and substrate affinity (IP3/Ca+ dependent) of the integrin (3). BCR signaling and chemokine binding occur primarily in the microenvironment of the secondary lymphoid tissues. In vivo, ibrutinib potently inhibits BCR and chemokine signaling (4, 5). We posit that sustained in vivo inhibition of inside out signaling leads to more profound changes in VLA-4 function than short term drugging of cells in vitro resulting in a more profound reduction in integrin mediated adhesion. Future experiments could address this hypothesis and we thank Zucchetto et al. for suggesting a tool that could be used to evaluate changes in VLA-4 confirmation and affinity.