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 ERK1/2 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/ERK1/2 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.
No relevant conflicts of interest to declare.
Asterisk with author names denotes non-ASH members.