Increased Levels of Soluble RAGE in CLL Patient Plasma Has a Protective Effect on CLL Disease Progression

Background: Receptor for advanced glycation end-products (RAGE) is the primary receptor for the inflammatory molecule HMGB1. We previously demonstrated that HMGB1 differentiates nurse like cells (NLC), and that increased levels HMGB1 in the plasma of CLL patients and overexpression of RAGE in CLL lymph nodes is associated with a poorer clinical outcome. Moreover, we have shown that HMGB1 induced activation of NF-κB in primary CLL cells in vitro . Soluble RAGE (sRAGE) is a reported antagonistic decoy receptor of plasma HMGB1, and limits chronic inflammation. The pool of plasma sRAGE originates from 2 main sources - alternative splicing to produce endogenous secreted RAGE (esRAGE), or from surface RAGE ectodomain shedding catalyzed by ADAM10. In a healthy setting, HMGB1 and sRAGE plasma levels are inversely correlated, with increased levels of sRAGE limiting inflammatory responses. RAGE shedding in response to saturating levels of ligand (i.e. HMGB1) establishes a negative feedback loop, dampening inflammatory signaling by sequestering RAGE ligands. In chronic disease, the HMGB1-sRAGE inverse relationship is frequently reversed. The functional impact of sRAGE is poorly documented in cancer, and its role in CLL is currently unknown.

We hypothesize that sRAGE could have a protective effect in CLL and could dampen HMGB1-mediated disease progression. The aim of this study is to characterise the clinical significance of sRAGE in CLL and investigate whether RAGE undergoes membrane shedding to confer a protective effect in CLL.

Methods: sRAGE plasma levels were measured by ELISA in 54 CLL patients and 15 healthy controls. sRAGE protein mass was assessed by Western blotting and compared to matched patient ADAM10 whole cell expression. ADAM10 inhibitor studies and HMGB1-induced RAGE shedding was performed in RAGE-transfected HEK293 cell line and primary CLL cells; sRAGE production was analyzed by ELISA and surface RAGE expression determined by flow cytometry. sRAGE containing media was produced by HEK293 cell line transfected with esRAGE isoform gene sequence; sRAGE containing media (2ng/ml) was used to assess the effect on primary CLL NLC differentiation.

Results: sRAGE plasma levels were significantly higher in CLL patients compared to healthy controls (median 60.0pg/ml ± 79.5 interquartile range and 31.0pg/ml ±46.9, respectively; P <0.01), but fell below levels capable of full HMGB1 inhibition for the same patient cohort (72.2ng/ml ±111.4). Absence of a negative correlation between HMGB1 and sRAGE suggested a breakdown in the negative feedback loop in CLL, compared to the classical inverse trend observed for the healthy controls. Patients with high levels of plasma sRAGE had an increased time to first treatment (P=0.0134; n=26) and sRAGE levels were increased in patients who had reached remission following treatment compared to pre-treatment samples, suggesting that sRAGE provides a protective effect on CLL disease progression.

CLL plasma sRAGE is of shorter length than cellular full-length RAGE and esRAGE from primary CLL cells, suggesting sRAGE originates from ectodomain shedding. ADAM10 expression, the main RAGE sheddase, is independent of sRAGE plasma levels assessed by Western blotting. ADAM10 stimulation with ionomycin induced RAGE ectodomain shedding into cell culture medium in RAGE-transfected HEK293 cell line (2270.0ng/ml) compared to untreated cells, without altering surface RAGE or ADAM10 expression. Ionomycin-induced RAGE shedding was abrogated by pre-treatment with ADAM10 inhibitor, GI254023X (45.5ng/ml). Ionomycin induced apoptosis in primary CLL cells, so could not be investigated for RAGE shedding, but our data demonstrates that saturating HMGB1 levels induces RAGE shedding in CLL. Finally, sRAGE-containing media did not reduce primary CLL NLC differentiation in vitro after 1-2 weeks culture.

Conclusions: Our data demonstrates for the first time that plasma sRAGE limits CLL disease progression by prolonging time to first treatment and sustaining stable disease following treatment. sRAGE originates from ectodomain shedding, is dependent on ADAM10 and inducible by HMGB1. Although sRAGE failed to block NLC differentiation in vitro, it contributes to the extracellular mileu 'cocktail' shaping the CLL microenvironment. We therefore propose that HMGB1 and sRAGE levels could be useful plasma markers for inflammatory microenvironment and therapy monitoring.