REDOX SIGNALING HYPERSENSITIVITY DISTINGUISHES CHRONIC LYMPHOCYTIC LEUKEMIA PATIENTS WITH FAVORABLE PROGNOSIS

Background. Chronic lymphocytic leukemia (CLL) patients exhibit a variable clinical course, with some patients having indolent disease and others experiencing a more accelerated course, treatment resistance and dismal outcome. The B-cell receptor (BCR) signaling is a key determinant of heterogeneous clinical behavior of CLL and is a target for therapeutic interventions. Endogenously produced H2O2 is thought to fine tune the level of BCR signaling by reversibly inhibiting phosphatases. However, relatively little is known about how CLL cells sense and respond to such redox cues. Aims. In this study, we used phospho-specific flow cytometry to compare BCR signaling responses to H2O2 in prognostic groups of CLL patients. Methods. The phosphorylation levels of five proteins downstream of the BCR signaling, namely SYK, NF-κB p65, ERK1/2, p38 and JNK, were analyzed at the single-cell level in 26 CLL cell samples using phospho-specific flow cytometry. Protein phosphorylation was measured in the basal condition and following stimulation with H2O2. Circulating B cells from healthy individuals were analyzed as controls. The two-sample Wilcoxon’s rank sum test was used to compare protein phosphorylation in groups of patients. Time to first treatment (TTFT) was calculated from the date of diagnosis to the date of initial therapy. TTFT curves estimated using the Kaplan- Meier method for the respective groups of patients were compared using the log-rank test. Results. In CLL cells, stimulation with H2O2 induced a statistically significant increase in phosphorylation of all analyzed signaling proteins with the exception of SYK. Moreover, the extents of responses to H2O2 were significantly higher in CLL than normal B cells for all signaling proteins but SYK. Comparison of H2O2 signaling response in prognostic groups of patients defined by IGHV mutational status, CD38 or ZAP-70 expression, showed that median phosphorylation response of ERK1/2 to H2O2 was significantly higher in the patient subset defined by the mutated IGHV status (M-CLL) (P=0.031). No significant correlations were observed between H2O2 responsiveness of BCR signaling proteins and ZAP-70 or CD38 expression. Kaplan-Meier curves showed statistically significant slower progression (longer time to first treatment, TTFT) in patients with higher p-ERK1/2 and p-NF-B p65 responses to H2O2, indicating that lower responsiveness of these signaling proteins to H2O2 correlated with more rapid progression [median TTFT was 41.6 and 115 months for patients with lower and higher NK-κB p65 responsiveness to H2O2, respectively (log-rank test P=0.0011); median TTFT was 38.7 and 117.0 months for patients with lower and higher ERK1/2 responsiveness to H2O2, respectively (log-rank test P=0.0008)] (Figure 1). Interestingly, the ability of H2O2 responsiveness signaling to define prognostic groups is comparable to that of IGHV mutational status (log-rank test P=0.0003). Conclusions. This study reveals that a novel H2O2 signaling response distinguishes a prognostic group of CLL patients with favorable prognosis. Specifically, higher H2O2 responsiveness of ERK1/2 or NF-κB is predictive of longer TTFT, thus highlighting ERK and NF-κB as biologically and clinically relevant signaling nodes in CLL.