The Kinin-Kallikre System in Chronic Lymphocytic Leukemia - A Potential Target for Therapy?

Abstract 3904

CLL remains an incurable disease with standard therapy regimens. The hyper reactivity of the B cell Receptor (BCR) to unknown antigen ligation plays a pivotal role in B-cell survival. Recent clinical trials of BCR-targeted therapies prompted the need for a better understanding of the biology of BCR signaling. We previously employed proteomics to study protein expression changes associated with BCR ligation. Using 2-dimensional gel electrophoresis (2DE) with MALDI-TOF mass spectrometry (MS) we identified that Kininogen (KNG) was upregulated in 3/3 “poor prognosis” clinical samples upon BCR stimulation. KNG (a critical protein of Kinin-Kallikrein System) is the precursor for Kinins which act via the B₁ and B₂ receptors (B₁R and B₂R, respectively) and is known to play a critical role in cell migration, proliferation, vascular permeability, inflammation and intracellular Ca²⁺ influx. Consequently we hypothesized that there is a functional role for B₁R and B₂R, Kinins and their precursor KNG in CLL B-cell survival which may offer a potential therapeutic target.

Following ethical approval, blood samples were collected from CLL patients. Time to first treatment, clinical stage, IgVh status, CD38 and ZAP-70 data were available. “Poor prognosis” samples were defined as unmutated IgVh status, high WBC and hyper-responsive BCR (p-ERK expression ≥ 2.0- fold increase upon stimulation). Immunoblotting was employed to confirm KNG upregulation and Tissue Kallikrein expression in CLL B-cells. A series of 59 CLL samples was screened for constituent KNG expression using immunoblotting. The presence of mRNA transcripts for KNG was assessed by PCR. An Automated One-stage Factor Assay on the Instrumentation Laboratory ACL-TOP analyser was used to determine the level of Plasma Kallikrein in CLL patients vs healthy controls. Fluorescence Activated Cell Sorting (FACS) was utilized for CLL B-cell enrichment according to CD20 status. Isolated CLL B-cells were subjected to immunodetection and flow cytometry for B₁R and B₂R cell surface expression identification.

Proteomic analysis by 2DE/MALDI-TOF MS previously revealed upregulation of KNG after 5.5 hours of in vitro BCR stimulation in 3/3 clinical samples. Upregulation of KNG after stimulation was confirmed by immunoblotting in 5 samples, including all 3 which were previously analyzed using proteomics. There are 2 forms of KNG: HMWK and LMWK. The analysis of constitutive KNG by immunoblotting revealed positive expression in 72.9% (43/59) and 80% (28/35) for LMWK and HMWK, respectively. No statistical significance was found with preliminary clinical correlations, however, there was a trend towards shorter median survival in LMWK positive cases (152 months vs. 264 months for LMWK negative cases) (p=0.161). PCR confirmed the presence of KNG transcript in CLL samples. An automated one-stage factor assay revealed that 47.2% (26/55) CLL patients expressed elevated Plasma Prekallikrein (the zymogen form of Plasma Kallikrein) level. Tissue Kallikrein overexpression was confirmed in 14 CLL samples, using an anti-KLK 6 antibody. We investigated the presence of inducible B₁R and constitutive B₂R on CD20⁺ CLL B-cells and demonstrated that B₁R and B₂R are expressed by CLL B-cells, however, B₁R expression was highly upregulated.

We demonstrate for the first time that CLL B-cells express differing levels of KNG protein and that the expression can be significantly increased after BCR ligation. Consequently, high concentration of KNG is achieved in near proximity to B₁R and B₂R, identified on the surface of CLL B-cells. Therefore, we show that CLL B-cells comprise the complete system for the synthesis and liberation of Kinins which in turn mediate the activation of B₁R and B₂R via an autocrine loop. We suggest, that simultaneous and sustained activation of B₁R and B₂R, which is known to be advantageous for immediate triggering of ERK_1/2 global phosphorylation and PLCγ2-dependent intracellular Ca²⁺ mobilization, play a functional role in the induction of pathologic signal transduction leading to B-cells survival and that Ca²⁺ influx-dependent transcriptional activation and MEK₁/ERK_1/2 pathway signaling may be dependent upon simultaneous complimentary stimuli input from BCR, B₁R and B₂R and possibly other disease-associated stimuli. The inhibition of Kinin receptor function may be a potential target for combined therapy in CLL.