In Vivo Ibrutinib Abrogates BCR-Dependent Adhesion in Tumor Cells of Patients with Chronic Lymphocytic Leukemia
Authors: Herman Sarah E. M. , Mustafa Rashida Z , Farooqui Mohammed , Wiestner Adrian 
Published: October 2019

Abstract
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<0.001, n=13). Next, we tested the effect of ibrutinib on CLL cell adhesion to a confluent stromal cell layer. Although less dramatic, we found a similar reduction in the ability of cells to adhere to the stromal cell layer in all three patients evaluated. As the initial rise in ALC occurs very rapidly in some patients, we evaluated the ability of cells collected on day 2 of ibrutinib therapy and again found a significant inhibition of cell adhesion to fibronectin (81% reduction, 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<.05). When analyzing cells obtained on day 2, we found no significant changes suggesting that alteration of adhesion molecule expression does not account for the rapid increase in ALC. Integrin α4β1 is activated by PKC downstream of BTK. We thus sought to determine whether stimulation with PMA, an activator of PKC, could restore the ability of CLL cells to adhere to fibronectin. Indeed, PMA stimulation of CLL cells obtained from patients after 28 days on ibrutinib partially restored their ability to adhere to fibronectin, suggesting that reduced α4β1 integrin activity contributes to inhibition of adhesion by ibrutinib.
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.