In Vivo Evidence That Ibrutininb Deregulates Chemokine Receptor CXCR4 Surface Membrane Expression and Signaling, Along with Inhibiting B Cell Antigen Receptor Signaling, As Causes for Defective Homing and Impaired Retention of CLL Cells in Tissues
Authors: Chen Shih-Shih , Chang Betty Y. , Chang Stella , Tong Timothy , Zou Yong-Rui , Sherry Barbara , Buggy Joseph J. , Burger Jan A. , Rai Kanti R. , Chiorazzi Nicholas 
Published: October 2019

Abstract
The Bruton’s tyrosine kinase (BTK) inhibitor ibrutinib has been approved by the FDA for the treatment of chronic lymphocytic leukemia (CLL) and mantle cell lymphoma. Patients receiving this treatment often develop lymphocytosis and concomitant reduced organomegaly. These actions of ibrutinib are believed due to egress of CLL cells from lymphoid compartments, although the mechanism(s) responsible for this are not clear.
Overexpressed surface membrane chemokine receptor CXCR4 (smCXCR4) is a hallmark of CLL cells and is involved in CLL cell migration and interaction with protective niches. Surface expression of CXCR4 is regulated by phosphorylation at serine/theronine residues in the receptor’s cytoplasmic tail. Kinases including GRK and PIM are known to regulate phosphorylation and surface expression of CXCR4. The transcript and protein levels of BTK, GRK and PIM also correlate with smCXCR4 expression.
Here we studied the effect of ibrutinib on CLL cell distribution using TCL1-192 cells, a clonal murine cell line that mimics aggressive CLL and involves active B-cell antigen receptor (BCR) signaling. Similar to patients, TCL1-192-bearing mice receiving ibrutinib developed almost immediately (≤ 1 hour) lymphocytosis that consisted of both non-divided and recently-divided cells. While TCL1-192 cells overexpress smCXCR4, ibrutinib promoted lymphocytosis was associated with a fall in smCXCR4 to a level similar to that on normal B cells from wild type C3H/B6 mice. Reduced levels of smCXCR4 were also observed at the later timepoints when mice still had the continued release of cells into circulation even though lymphocytosis was no longer evident. Despite this change in smCXCR4 expression, total CXCR4 was not changed after ibrutinib treatment.
Importantly, cells obtained from ibrutinib treated animals that had reduced levels of smCXCR4 also failed to respond to their ligand CXCL12 in vitro, as measured by absence of smCXCR4 internalization or calcium mobilization. Furthermore, re-expression of smCXCR4 after withdrawal of CXCL12 was not observed in treated cells. We therefore next studied in vitro the mechanism whereby ibrutinib lowered smCXCR4 expression. Western blot analysis using cells treated with ibrutinib at the dose ≥0.1μM at 37⁰C for 2 hours in the presence of CXCL12 showed significantly reduced levels of phosphorylated CXCR4 at Ser339. Phosphorylation of Ser339 in the CXCR4 intracellular domain is known to be essential for normal receptor recycling, hyper-phosphorylated CXCR4 on Ser339 is associated with overexpressed smCXCR4 in CLL patient cells. Correlating with this, TCL1-192 cells treated with ibrutinib at 0.1μM not only de-phosphorylated CXCR4 on Ser339, but also significantly enhanced internalization of smCXCR4 after CXCL12 stimulation.
We then examined the kinases responsible for the de-phosphorylation of CXCR4. Western blot analysis of CLL cells from animals treated with ibrutinib for 4 weeks showed minimal levels of total BTK protein. There was a direct positive correlation in between BTK protein expression and smCXCR4 expression. In addition to BTK, reduced levels of kinases known to regulate CXCR4 phosphorylation on Ser339 were also observed in cells collected from ibrutinib treated mice. Together, these results suggest blocking BTK, directly or indirectly causes de-phosphorylation of CXCR4, resulting in impaired smCXCR4 recycling and blocked CXCL12 signaling.
The function of CXCR4 is dependent on its location on the cell membrane. Impaired CXCR4 recycling after ibrutinib treatment would lead to defective cell trafficking. Indeed, cell transfer studies involving TCL1-192 cells previously treated with ibrutinib indicated that persistent decrease in smCXCR4, which was ibrutinib-dependent, blocked the return of CLL cells to solid tissue niches. Finally, ibrutinib-fed mice eventually developed lower lymphocyte counts, reduced organomegaly, and prolonged survival. Ibrutinib-fed mice also had defective response to BCR stimulation, including abolished calcium mobilization, blocked cell proliferation and survival after anti-IgM antibody stimulation. Together, the data provide direct in vivo evidence of ibrutinib impairing CLL cell homing and retention and suggest a mechanism by which BTK targeted therapy affects not only BCR signaling but also CXCR4 phosphorylation and signaling, with the latter contributing to defective smCXCR4 expression.

Disclosures
Chang: Pharmacyclics, Inc: Employment, Equity Ownership. Chang:Pharmacyclics: Employment. Buggy:Pharmacyclics: Employment. Burger:Pharmacyclics: Consultancy, Honoraria, Research Funding.