Anti-CD44 antibodies induce cell death in CLL cells by reduction of MCL1 levels and by effector caspase activation. (A) Treatment with IM7 leads to reduction of MCL1 protein levels in murine CLL cells. Shown is a representative immunoblot of 4 independent experiments with splenocytes (isolated from TCL1 transgenic leukemic mice of age ≥ 12 months) that were treated for 24 hours in culture with 10 µg/mL IM7 or isotype control. MCL1 protein expression was quantified by densitometry and normalized to actin control. MCL1 protein in cells treated with IM7 was 0.50 ± 0.05 as compared with isotype (mean ± SEM, P = .0211). (B) Reduction of MCL1 protein levels upon A3D8 ligation in human CLL. Shown is 1 out of 7 representative cases. Numbers indicate densitometric values relative to β-actin. (C) A cocktail of IL-4/CD40L blunted increases of caspase-3/7 activity associated with spontaneous apoptosis in human CLL cells (7 cases; mean ± SEM, 0.40 ± 0.04). Caspase activity is significantly increased by the CD44-targeting clone A3D8 (mean ± SEM, 1.79 ± 0.29; P < .0001) compared with stimulation with the natural CD44 ligands HA and CS, showing mean reductions of 93% (HA) and 92% (CS) (both P = .0006) compared with A3D8-specific isotype. (D) Caspase inhibitors prevent anti-CD44–induced apoptosis (A3D8: mean 1.57 ± 0.20, dose 5 µg/mL; Z-VAD-FMK: mean 1.23 ± 0.20, dose 25 µM; Z-VAD-FMK: mean 0.97 ± 0.07, dose 50 µM; Z-DEVD-FMK: mean 1.05 ± 0.08). Incubation time was 36 hours. Plotted are mean values with SEM. (E) Cleavage of caspase 3 and PARP upon anti-CD44 ligation by A3D8 indicate induction of apoptosis. Shown are 2 exemplary cases out of 7 CLL patients.