The bruton tyrosine kinase inhibitor ibrutinib (PCI-32765) blocks hairy cell leukaemia survival, proliferation and B cell receptor signalling: a new therapeutic approach

Abstract

B cell receptor (BCR) signalling plays a critical role in the progression of several B-cell malignancies, but its role in hairy cell leukaemia (HCL) is ambiguous. Bruton tyrosine kinase (BTK), a key player in BCR signalling, as well as B cell migration and adhesion, can be targeted with ibrutinib, a selective, irreversible BTK inhibitor. We analysed BTK expression and function in HCL and analysed the effects of ibrutinib on HCL cells. We demonstrated uniform BTK protein expression in HCL cells. Ibrutinib significantly inhibited HCL proliferation and cell cycle progression. Accordingly, ibrutinib also reduced HCL cell survival after BCR triggering with anti-immunoglobulins and abrogated the activation of kinases downstream of the BCR (PI3K and MAPK). Ibrutinib also inhibited BCR-dependent secretion of the chemokines CCL3 and CCL4 by HCL cells. Interestingly, ibrutinib inhibited also CXCL12-induced signalling, a key pathway for bone marrow homing. Collectively, our data support the clinical development of ibrutinib in patients with HCL.

Keywords: B cell receptor; bruton tyrosine kinase; hairy cell leukaemia; ibrutinib; microenvironment.

Figure 1. Ibrutinib reduces phosphorylated BTK expression upon BCR stimulation in HCL cells

(A) Immunoblots from 9 primary HCL cells, which were stimulated for 10 min with 10 μg/ml of anti Igs (anti IgA, anti IgG and anti IgM) in the presence or absence of 0.1 μM, 0.5 μM or 1 μM ibrutinib, as indicated. P indicates immunobloting for the active phosphorylated form of BTK (lower band). GAPDH was using as a loading control. (B) Occupancy test, assessed by the ability of BTK to bind to the fluorescence labelled probe (PCI-33380) in the presence of dose escalation of ibrutinib in the HCL cell lines, ESKOL and HC-1. Total BTK levels were determined by immunobloting. DOHH2 cell line lysates labelled with probe (100% and 10%) were used as a control.

Figure 2. Ibrutinib inhibits cell growth and proliferation and reduces the percentage of cells in S-phase in a dose-dependent manner

(A) Cell growth curves for untreated HCL cell lines ESKOL (left) and HC-1 (right), indicated as the control, and treated with ibrutinib (0.5 μM, 1 μM or 5 μM); at the indicated time points, cells were counted by flow cytometry, the graphs represent the mean and SEM, as indicated by the lines and error bars with ** as p<0.01 (n=3). (B) Left-hand graph depicts the effect of ibrutinib on the proliferation of HCL cell lines, ESKOL (black bars) and HC-1 (grey bars), using dose escalation, as assessed by XTT assay after 72 h of incubation., bar diagrams represent the mean ± SEM of 3 independent experiments with ** as p<0.01. The right-hand graph shows the percentage of cell cycle phase distribution, assessed by BrdU and 7-AAD incorporation, in the HC-1 cell line after 48 h of incubation with ibrutinib (0.5 μM, 1 μM or 5 μM), untreated cells are indicated as a control. The bars represent the mean of the percentage of cells in G0/G1 (lighter grey) S-phase (grey) and G2/M (darker grey) (n=3).

Figure 3. BCR-induced cell survival signalling in HCL cells is decreased after treatment with ibrutinib

(A) Contour plots of a representative case, showing HCL cell viabilities after 48 h of incubation with anti Igs (anti IgA, anti IgG and anti IgM), following incubation for 1 h with ibrutinib (0.5 μM, 1 μM or 5 μM), as indicated below the plots. The gates in each plot highlight the viable cell population, defined as DiOC₆ positive and PI negative. (B) The left-hand graph represents the mean relative HCL cell viabilities after 24 and 48 h of incubation with anti Igs in the presence or absence of ibrutinib (indicated as control); the right-hand graph shows the XTT assay performed in parallel; results were normalized relative to the untrea1ted controls samples (100%). Displayed are the mean ± SEM, with ** as p<0.01 (n=8). (C) The bar diagrams represent the mean relative viabilities of the HCL cell lines, ESKOL (on the left) and HC-1 (on the right), incubated for 24, 48 and 72 h with medium alone, indicated as control or ibrutinib (0.5 μM, 1 μM and 5 μM). Viabilities in treated samples were normalized to the viabilities in the controls (100%) at their respective time points. The bars represent the mean ± SEM; * p<0.05; ** p<0.01 (n=3).

Figure 4. Ibrutinib down-regulates BCR signalling and inhibits the secretion of the chemokines CCL3 and CCL4 in HCL cells

(A) Immunoblots from HCL primary cells (HCL13, HCL21 and HCL18) stimulated with anti Igs (anti IgA, anti IgG and anti IgM) for BCR triggering in the presence or absence of 0.1 μM, 0.5 μM or 1 μM ibrutinib, as indicated. P indicates immunoblots for the active phosphorylated form. GAPDH was used as a protein loading control. (B) The concentration of the chemokines CCL3 and CCL4 secreted in the supernatants of primary hairy cells was measured after BCR stimulation with anti Igs (anti IgA, anti IgG and anti IgM) in the presence or absence of ibrutinib as indicated, after 48 h of incubation. The bars represent the mean ± SEM (n=8) for CCL3 (left) and CCL4 (right).

Figure 5. CXCR4 signalling is down-regulated by ibrutinib

(A) Left: overlay histogram plots that depict the mean CXCR4 intensity florescence ratio (MIFR) in the HCL cell lines, ESKOL and HC-1, and CD19-gated primary cells, as indicated above the histograms. Light grey shaded area represents the corresponding isotype control and dark grey line indicates CXCR4 expression. Right: MIFR for CXCR4 for 13 primary HCL cases; the mean and SEM are indicated by the bars and error lines respectively. (B) Representive immunoblots from HCL primary cells (HCL14, HCL15 and HCL16) stimulated for 10 min with CXCL12 in the presence or absence of 0.1 μM, 0.5 μM or 1 μM ibrutinib as indicated. pERK refers to the phosphorylated form of the protein ERK. GAPDH was used as a protein loading control.

Figure 6. Schematic diagram depicting how ibrutinib may affect BCR and CXCR4 signalling in HCL

Upon antigen stimulation, BCR signalling induces LYN and SYK phosphorylation (P in orange circles refer to phosphorylation) that initiate a signalling cascade causing downstream BTK activation. BTK is recruited into the signalling complex at the plasma membrane via the docking of its pleckstrin homology domain to phosphatidylinositol 3,4,5 triphosphate (PIP3). BTK becomes phosphorylated and therefore induces calcium release, which initiates a signalling cascade with the consequent induction of cell proliferation and survival. BTK is also involved in signalling of chemokine receptor CXCR4, which is important in cell migration and could be a suitable target for ibrutinib.