Alemtuzumab induces caspase-independent cell death in human chronic lymphocytic leukemia cells through a lipid raft-dependent mechanism

Abstract

Alemtuzumab is a humanized IgG1 kappa antibody directed against CD52, a glycosyl-phosphatidylinositol linked cellmembrane protein of unknown function. Herein, we demonstrate that alemtuzumab promotes rapid death of chronic lymphocytic leukemia (CLL) cells in vitro, in a complement and accessory cell free system. Using minimal detergent solubilization of CLL membranes, we found that CD52 colocalizes with ganglioside GM-1, a marker of membrane rafts. Fluorescence microscopy revealed that upon crosslinking CD52 with alemtuzumab þ anti-Fc IgG, large patches, and in many cases caps, enriched in CD52 and GM-1 formed upon the CLL cell plasma membrane. Depletion of membrane cholesterol or inhibition of actin polymerization significantly diminished the formation of alemtuzumab-induced caps and reduced alemtuzumab-mediated CLL cell death. We compared alemtuzumab-induced direct cytotoxicity, effector cell-mediated toxicity and complement-mediated cytotoxicity of CLL cells to normal T cells. The direct cytotoxicity and observed capping was significantly greater for CLL cells as compared to normal T cells. Cell-mediated and complement-mediated cytotoxicity did not significantly differ between the two cell types. In summary, our data support the hypothesis that alemtuzumab can initiate CLL cell death by crosslinking CD52-enriched lipid rafts. Furthermore, the differential direct cytotoxic effect suggests that CD52 directed antibodies could possibly be engineered to more specifically target CLL cells.

Treatment of primary CLL cells with bezafibrate and medroxyprogesterone acetate induces apoptosis and represses the pro-proliferative signal of CD40-ligand, in part through increased 15dΔ12,14,PGJ2

“…In addition, in western blot analyses we did not always observe caspase cleavage products in CLL cells that were undergoing apoptosis (data not shown). Caspase-independent apoptosis has been reported in several cell models, including CLL 22 and it cannot be ruled out that BEZ þ MPA induces caspase-independent apoptosis in some samples. The drugs each inhibited the CD40 L -driven proliferation of CLL cells with the combination having more activity than either drug alone.…”

Divergence in CD19-Mediated Signaling Unfolds Intraclonal Diversity in Chronic Lymphocytic Leukemia, Which Correlates with Disease Progression

“…CLL cells (1 3 10 6 ) were separately mixed in the presence or in the absence of the following: 25 or 50 mg/ml anti-human CD21 (clone Bu32), 2.5 mg/ml anti-human CD62L (clone DREG-56), and 40 mg/ml anti-human b 2 integrin (clone P4H9-A11), all for 30 min; 10 mM MbCD for 5, 10, and 30 min; 20 mM cytochalasin B for 30 min; 10, 20, or 50 mM Syk inhibitor II, 70 mM genistein, 3 mM herbimycin A, or 1, 5, or 10 mM edelfosine or perifosine for 1 or 24 h; and 1, 5, 10, or 20 mM PP2 for 15 min or 2 h. These concentrations were established according to previous publications (17)(18)(19)(20)(21)(22)(23) and were titrated in this study to obtain a maximal effect without killing the cells. Controls used included Abs as isotype controls.…”

Management guidelines for the use of alemtuzumab in chronic lymphocytic leukemia

“…CD52 is a glycoprotein of unknown function that is highly expressed on cell surfaces of normal and malignant lymphocytes. 13 Binding of alemtuzumab to CD52 on lymphocytes induces complement-dependent cytotoxicity, 14,15 antibody-dependent cell-mediated cytotoxicity 14 and direct cytotoxicity (likely apoptotic cell death), [16][17][18] although a direct cytotoxic effect of alemtuzumab was not observed in other studies and therefore remains controversial. 19,20 Alemtuzumab received accelerated approval in the United States and Europe in 2001 for patients who had been treated with alkylating agents and in whom fludarabine therapy had failed.…”