High Level Phosphatase Activity Revealed in Chronic Lymphocytic Leukemia Cells That Use Mutated Immunoglobulin Heavy Chain Variable Region Genes and Lack High-Level Expression of the Zeta-Associated Protein 70 (ZAP-70).

Signals propagated through the B cell receptor (BCR) guide the maturation and survival of B cells and might factor in the pathogenesis and progression of chronic lymphocytic leukemia (CLL). BCR signaling in CLL cells was investigated at the single-cell level using multiparametric flow cytometry. Concurrent analysis was performed using fluorochrome-conjugated antibodies specific for B-cell surface antigens and a panel of antibodies recognizing specific phospho-peptide epitopes within a selected group of intracellular signaling proteins. CLL samples from patients (N=6) showed weak or minimal signaling activity at p72^SYK/p70^ZAP, Erk1/2, B-Cell linker protein (BLNK) and phospholipase-Cγ-2, (PLCγ2) when stimulated only at the BCR with anti-μ crosslinking, whereas a robust signal was observed in a control Ramos B cell line. The low-level signaling in CLL cells could be accounted for by either a defect in activation of a key protein required for signaling, or by enhanced inhibition mediated by phosphatases such as SHIP-1, SHIP-2, SHP-1, or SHP-2. To determine whether phosphatases were preventing or dampening BCR activation in CLL samples, CLL cells were treated with hydrogen peroxide (H₂O₂), a physiologic phosphatase inhibitor generated during BCR signaling that has been used previously to reveal dysregulated BCR signaling in follicular lymphoma (Sing et al., Cell 2005, Reth., Nat. Immunol. 2002, Irish et al., Blood 2006). H₂O₂ treatment of CLL cells that had molecular features associated with indolent disease (e.g. use of mutated immunoglobulin heavy chain variable region genes (IgV_H) and a low level expression of ZAP70) induced high-levels of phosphorylated p72^SYK/p70^ZAP, ERK1/2, BLNK, and PLCγ2, independent of surface F(ab)₂anti-μ ligation. In contrast, CLL-B cells that had molecular features associated with more aggressive disease (expression of unmutated IgV_H and high-level expression of ZAP-70) were significantly less responsive to this treatment, even in the presence of F(ab)₂anti-μ. Exposure of blood B cells from healthy donors to H₂O₂ failed to elicit a substantial increase in phosphorylation of these same intracellular signaling proteins. These studies reveal a previously unrecognized, constitutive high-level phosphatase activity in CLL cells that express mutated IgV_H and low levels of ZAP-70, possibly contributing to the attenuated signaling observed in these cells following surface IgM ligation. Because this high level of constitutive phosphatase activity was not observed in CLL cells that express unmutated IgV_H with high-level expression of ZAP70, flow cytometric measurement of the increased phosphorylation triggered by H₂O₂ could provide for a useful means with which to distinguish leukemia cells from patients with indolent versus aggressive CLL. Evaluation of additional samples is ongoing to test this hypothesis.