In Vivo Ibrutinib Abrogates BCR-Dependent Adhesion in Tumor Cells of Patients with Chronic Lymphocytic Leukemia

Ibrutinib, now FDA approved for use in both previously treated mantle cell lymphoma (MCL) and chronic lymphocytic leukemia (CLL), induces objective clinical responses and is well tolerated (Advani, JCO 2013; Byrd, NEJM 2013). As with other BCR-directed kinase inhibitors, (such as fostamatinib and idelalisib) ibrutinib leads to a treatment-induced transient lymphocytosis that resolves over the first 2-3 months on therapy in patients with CLL. The initial increase in absolute lymphocyte count (ALC) has been shown to be due to the release of tumor cells from the tissue compartments; specifically the lymph nodes, into the peripheral blood (Herman, Leukemia 2014). In vitro ibrutinib reduces the ability of CLL cells to migrate towards chemokines and prevents adhesion of BCR stimulated CLL cells (Ponader, Blood 2012; de Rooij, Blood 2012). We hypothesized that the initial rise in ALC is due to inhibition of CLL cell adhesion. We tested this ex vivo in patients enrolled on a single agent phase II trial of ibrutinib in CLL (NCT01500733) and investigated the mechanism. In order to evaluate the effect of ibrutinib on CLL cell adhesion in vivo we compared peripheral blood tumor cells collected from patients pre-treatment and on day 28 of treatment. Fluorescently labeled cells were plated on fibronectin-coated plates and allowed to adhere for 1 hour. Adhesion was then evaluated using florescence microscopy and quantified using the ImageJ software. After 28 days on ibrutinib, CLL cells displayed a severely reduced adhesion ability (99% reduction, P P =0.005, n=10). This suggests that even one dose of ibrutinib is sufficient to disrupt CLL cell adhesion. We next sought to determine if ibrutinib could directly interfere with adhesion by causing cells to detach from fibronectin. We found that after one hour of 1µM ibrutinib treatment in vitro there was a significant reduction in the number of CLL cells that remained attached (33% reduction, P =0.03, n=10). This suggests that ibrutinib can not only prevent CLL cell adhesion but can release adhered cells from the microenvironment, consistent with influx of cells from the lymph node into the peripheral blood within hours of starting treatment. To determine whether the observed defect in cell adhesion is due to changes in the expression of CD29/CD49d (VLA-4 or α4β1 integrin), or CD18/CD11a (LFA-1), we measured the expression of these known regulators of CLL cell adhesion in samples obtained pre-treatment and during treatment. While there was inter-patient variability, overall there was no significant change in expression of CD29 after 28 days on treatment. In contrast albeit minor, we found reductions in both CD49d and CD18 ( P In conclusion, ibrutinib abolished CLL cell adhesion at least in part due to direct inhibition of BTK- and BCR-mediated α4β1 integrin activity. Additionally, ibrutinib not only prevented adhesion but also caused the detachment of adhered cells from the extracellular matrix. Together, this suggests that disruption of cellular adhesion is a major contributor to the observed rise in ALC early on treatment. This work was supported by the Intramural Research Program of NHLBI, NIH. We thank our patients for donating blood samples to make this research possible. Disclosures Wiestner: Pharmacyclics Inc.: Research Funding.