Bruton's Tyrosine Kinase Inhibitor PCI-32765 Abrogates BCR- and Nurselike Cell-Derived Activation of CLL Cells In Vitro and In Vivo.
Authors: Ponader Sabine , Buggy Joseph , O'Brien Susan , Wierda William G. , Keating Michael , Burger Jan A. 
Published: October 2019

Abstract
Abstract 45
Bruton's tyrosine kinase (Btk) plays a crucial role in development and function of normal B cells. Mutations in the gene encoding Btk cause a B cell defect, which manifests in boys during early childhood as X-linked agammaglobulinemia, a primary immunodeficiency originally described by Bruton in 1952. In normal B cells, Btk is involved in B-cell antigen receptor (BCR) signaling, but its function in chronic lymphocytic leukemia (CLL) cells has not yet been explored. PCI-32765 is a potent (IC50=0.5 nM), selective, and irreversible Btk inhibitor, which binds to the Cys-481 residue of Btk, causing an irreversible inhibition of Btk's enzymatic activity (Honigberg et al, Proc Natl Acad Sci USA 107(29):13075-80). The clinical activity of PCI-32765 is currently under investigation in patients with B cell malignancies, and preliminary results indicated significant clinical activity in CLL patients (Advani R. et al., ASCO 2010 meeting,JCO 28: 8012, 2010).
We investigated the effect of PCI-32765 (conc. 1 mM) on CLL cell viability and activation after BCR triggering with anti-IgM and in co-cultures with nurselike cells (NLC). After 24h incubation with PCI-32765, the viability of anti-IgM-stimulated CLL cells decreased significantly to 79.4% ± 4.6% of respective controls (n=10). In NLC co-cultures, CLL cell viability also was significantly decreased in the presence of PCI-32765. For example, after 24h incubation with PCI-32765, the mean viability of CLL cells was 78.7 ± 5.1% compared to respective controls (see Fig. A).
The chemokines CCL3 and CCL4 are secreted by CLL cells after BCR triggering and in NLC co-cultures, and function as surrogate markers of BCR-derived activation of CLL cells (Burger JA et al., Blood 113:3050-8, 2009). Therefore, we measured the effect of Btk inhibition by PCI-32765 on secretion of these chemokines in vitro and in vivo. In CLL cells co-cultured with NLC, the secretion of these chemokines was significantly reduced after 24 hours incubation with 1 mM PCI-32765 from 393 ± 172 pg/mL to 54 ± 46 pg/mL (CCL3) or from 2550 ± 678 pg/mL to 394 ± 188 pg/mL (CCL4, mean ± SD, n=11). Plasma samples from CLL patients treated with PCI-32765 (ongoing Phase 1 study) revealed high pre-treatment CCL3/4 levels, and these levels were significantly decreased after treatment. For example, 24 hours following the first dose of PCI-32765, CCL3 levels decreased from 60 ± 29 pg/mL to 16 ± 13 pg/mL, and CCL4 pre-treatment levels decreased from 106 ± 55 pg/mL to 23 ± 12 pg/mL (mean ± SD, n=6, see Fig. B).
The clinical activity of PCI-32765 included a rapid (24 hour) reduction in lymphadenopathy accompanied by a transient lymphocytosis, suggesting that the drug might affect cell homing or migration to factors in tissue microenvironments. So we examined the impact of PCI-32765 on CLL cell chemotaxis towards the chemokines CXCL12 and CXCL13. Chemotaxis was significantly reduced by 1 mM PCI-32765 to levels that were 57 ± 9% (CXCL12) or 46 ± 5% (CXCL13) of respective controls (mean ± SD, n=10). This data is consistent with a compartmental shift of CLL cells from tissues to the blood, explaining the lymphocytosis early during treatment with PCI-32765.
Collectively, our data demonstrate that PCI-32765 is an effective inhibitor of BCR- and NLC-derived survival signals, and interferes with CLL cell chemotaxis. PCI-32765 rapidly downregulates BCR-derived CLL cell activation in vivo, using CCL3/4 plasma levels as surrogate markers. These studies provide insight into the activity of PCI-32765 in CLL and help explaining the fascinating clinical activity of this new agent.
Figure (A) CLL cell viability in suspension and nurselike (NLC) co-culture after 24 hours incubation with 1mM PCI-32765, n=7 and (B) CCL3 concentrations in blood plasma of CLL patients after treatment with PCI-32765 (n=6, Mean+/−SEM). Figure. (A) CLL cell viability in suspension and nurselike (NLC) co-culture after 24 hours incubation with 1mM PCI-32765, n=7 and (B) CCL3 concentrations in blood plasma of CLL patients after treatment with PCI-32765 (n=6, Mean+/−SEM).

Disclosures:
Buggy: Pharmacyclics, Inc.: Employment.