Bruton tyrosine kinase represents a promising therapeutic target for treatment of chronic lymphocytic leukemia and is effectively targeted by PCI-32765

Abstract

B-cell receptor (BCR) signaling is aberrantly activated in chronic lymphocytic leukemia (CLL). Bruton tyrosine kinase (BTK) is essential to BCR signaling and in knockout mouse models its mutation has a relatively B cell-specific phenotype. Herein, we demonstrate that BTK protein and mRNA are significantly over expressed in CLL compared with normal B cells. Although BTK is not always constitutively active in CLL cells, BCR or CD40 signaling is accompanied by effective activation of this pathway. Using the irreversible BTK inhibitor PCI-32765, we demonstrate modest apoptosis in CLL cells that is greater than that observed in normal B cells. No influence of PCI-32765 on T-cell survival is observed. Treatment of CD40 or BCR activated CLL cells with PCI-32765 results in inhibition of BTK tyrosine phosphorylation and also effectively abrogates downstream survival pathways activated by this kinase including ERK1/2, PI3K, and NF-κB. In addition, PCI-32765 inhibits activation-induced proliferation of CLL cells in vitro, and effectively blocks survival signals provided externally to CLL cells from the microenvironment including soluble factors (CD40L, BAFF, IL-6, IL-4, and TNF-α), fibronectin engagement, and stromal cell contact. Based on these collective data, future efforts targeting BTK with the irreversible inhibitor PCI-32765 in clinical trials of CLL patients is warranted.

Figures

Figure 1

BTK protein but not mRNA expression is highly variable among CLL patients. (A) CD19+ cells from CLL patients (N = 30) were examined for BTK expression by immunoblot. Results are shown from 3 of 6 experiments. (B) RNA was extracted and converted to cDNA from CD19+ cells from CLL patients (N = 18) and CD19+ normal B cells (N = 5). RT-PCR analysis was done to determine quantities of BTK mRNA. Ct values are relative to 18S. Higher relative Ct values represent lower gene expression.

Figure 2

PCI-32765 induces selective cytotoxicity in CLL cells independent of IVGH mutational status or interphase cytogenetics. (A) CD19+ cells from CLL patients (N = 10) were incubated with or without increasing concentrations of PCI-32765 (0.01μM-100μM) for 48 hours. Viability was determined by MTT assay and was calculated relative to time-matched untreated controls. (B) CD19+ cells from CLL patients (N > 60) were incubated with or without PCI-32765 (0.1μM-10μM) for 48 hours. Viability was determined by annexin-V/PI flow cytometry. Dark lines represent averages. (C) CD19+ cells from CLL patients (N > 60) were incubated with or without 10μM PCI-32765 for 12-72 hours. Dark lines represent averages. (D) CD19+ cells from CLL patients (N > 60; minimum 10 per group) were incubated with or without 10μM PCI-32765 for 48 hours. Cytogenetics was determined independently of our laboratory. (E) CD19+ cells from CLL patients (N > 60; minimum 12 per group) were incubated with or without 10μM PCI-32765 for 48 hours. Mutational status was determined independently of our laboratory. In panels B through E viability was determined by annexin-V/PI flow cytometry, and was calculated relative to time-matched untreated controls.

Figure 3

PCI-32765 cytotoxicity against CLL cells is dependent on caspase pathway activation. (A) CD19+ cells from CLL patients (N = 4) were incubated with or without various concentrations of PCI-32765 (1μM-50μM) for 8 hours and PARP cleavage was assessed by immunoblot. Results are shown from 1 of 4 experiments. (B) CD19+ cells from CLL patients (N = 7) were incubated with or without various concentrations of PCI-32765 (1μM-50μM) for 8 hours. Cells were lysed and caspase activity was determined by the amino trifluoromethyl coumarin assay. Results were calculated relative to micrograms of protein. Each symbol represents an individual patient and dark lines represent averages. (C) CD19+ cells from CLL patients (N = 10) were incubated with or without various concentrations of PCI-32765 (1μM-50μM) and 100μM z-VAD-fmk for 48 hours. Viability was determined by annexin-V/PI flow cytometry, and is shown relative to time-matched untreated controls. Each symbol represents an individual patient and dark lines represent averages. (D) CD19+ cells from CLL patients (N = 3) were incubated with or without various concentrations of PCI-32765 (1μM-50μM) and 100μM z-VAD-fmk for 8 hours. PARP cleavage was assessed by immunoblot. Results are shown from 1 of 4 experiments.

Figure 4

PCI-32765 induces selective cytotoxicity in B cells compared with T cells, but alters activation induced T-cell cytokine production. (A) CD19+ cells from CLL patient cells (N > 60) and CD19+ cells from normal B cells (N = 10) were incubated with 10μM PCI-32765 for 48 hours. Viability was determined by annexin-V/PI flow cytometry, and was calculated relative to time-matched untreated controls. Dark lines represent averages. (B) CD3+ T cells (N = 3) and CD19+ B cells (N = 3) from normal volunteers were examined for BTK expression by immunoblot. (C) CD3+ T cells (N = 10) from normal volunteers were incubated with or without increasing concentrations of PCI-32765 (0.1μM-10μM) for 48 hours. Viability was determined by annexin-V/PI flow cytometry, and was calculated relative to time-matched untreated controls. (D) CD3+ T cells (N = 7) from normal volunteers were incubated with or without increasing concentrations of PCI-32765 (0.1μM-10μM) for 48 hours. Cells were stimulated using an anti-CD3 T-cell activation plate for 24 hours, and IL-6, IL-10, and TNF-α production were measured by ELISA. (E) RNA was extracted and converted to cDNA from CD19+ normal B cells and CD3+ normal T cells (N = 5; each). RT-PCR analysis was done to determine quantities of BTK mRNA.

Figure 5

PCI-32765 antagonizes BCR dependent signaling pathways and cell proliferation. (A) CD19+ cells from CLL patients (N = 12) were immunoprecipitated for 4G10. BTK phosphorylation at tyrosine sites was then evaluated by immunoblot analysis. Quantification was done using the Alpha Innotech FluorChemQ MultiImage III system. Dark line represents average. (B) CD19+ cells from CLL patients (N = 4) were treated with and without 10μM PCI-32765 for 2 hours. Cells were immunoprecipitated with 4G10 phospho-tyrosine antibody. BTK phosphorylation at tyrosine sites was then evaluated by immunoblot analysis. Results are shown from 1 of 4 experiments. (C) CD19+ cells from CLL patients (N = 7) were incubated with various concentrations of PCI-32765 for 1 hour. ERK phosphorylation at Thr202/Tyr204 was assessed by immunoblot. Results are shown from 1 of 3 experiments. (D) CD19+ cells from CLL patients (N = 4) were incubated with 1 or 10μM PCI-32765 and/or 1 μg/mL CD40L for 1 hour. AKT phosphorylation at ser473 was assessed by immunoblot. Results are shown from 1 of 3 experiments. (E) CD19+ cells from CLL patients (N = 3) were incubated with 10μM PCI-32765 and/or 1 μg/mL CD40L for 1 hour. EMSA analysis was done with nuclear extract using a radio-labeled oligonucleotide containing a consensus NF-κB binding site. Antibody shifts were performed from CD40L treated sample incubated with antibodies specific to the p65 or p50 subunits of NF-κB. The p65/p50 complex is indicated. Results are shown from 1 of 3 experiments. (F) CD19+ cells from CLL patients (N = 7) were incubated with or without 10μM PCI-32765 and 3.2μM CpG685. Proliferation was assessed by tritiated thymidine incorporation.

Figure 6

PCI-32765 antagonizes microenvironment stimuli. (A) CD19+ cells from CLL patients (N = 10) were incubated with or without 10μM PCI-32765 and 1 μg/mL CD40L or 50 ng/mL BAFF for 48 hours. (B) CD19+ cells from CLL patients (N = 5-10) were incubated with or without 10μM PCI-32765 and 20 ng/mL TNF-α, 40 ng/mL IL-6 or 800 U/mL IL-4 for 48 hours. (C) CD19+ cells from CLL patients (N = 7) were incubated with or without 10μM PCI-32765 on and off fibronectin coated plates for 48 hours. (D) CD19+ cells from CLL patients (N = 8) were isolated from peripheral blood and incubated with or without various concentration of PCI-32765 (0.1μM-10μM) in suspension or on an Hs5 cell layer for 48 hours. (A-D) Viability was determined by annexin-V/PI flow cytometry, and is shown relative to time-matched untreated controls for each group. Dark lines represent averages.