Synergistic effects of the fenretinide (4-HPR) and anti-CD20 monoclonal antibodies on apoptosis induction of malignant human B cells

Retinoids have been shown to be clinically useful in the biological therapy of certain myeloid and T-cell malignancies, whereas CD20 has proven to be an effective target in B-cell lymphoma immunotherapy. Both retinoic acid de rivatives and anti-CD20 monoclonal antibodies have also been shown to induc e apoptosis of malignant cells in vitro. Retinoid-induced apoptosis is thou ght to be mediated by nuclear retinoid receptor binding and transcriptional activation, whereas CD20 ligation appears to initiate transmembrane Ca2+ i nflux with resultant programmed cell death. In this report, we evaluate the in vitro effects of N-(4-hydroxyphenyl) retinamide (4-HPR) with and withou t anti-CD20 antibodies in B-cell lymphoma lines. We demonstrate that 4-HPR inhibits the growth of malignant B-cells beyond that of all-trans-retinoic acid and 13-cis-retinoic acid. We also show that this 4-HPR-mediated growth inhibition is attributable to apoptosis, is consistent across a variety of malignant B-cell lines (Ramos, Ramos AW, SU-DHL4, and Raji), peaks at 96 t o 144 h, and is attainable with concentrations as low as 2 muM. As with CD2 0-mediated apoptosis, we show that the final common pathway includes caspas e activation that can be blocked by 2-val-Ala-Asp-fluoromethyl ketone (z-VA D), a specific inhibitor of caspase function. Coincubation of a 2 muM conce ntration of 4-HPR and the anti-CD20 antibodies rituximab and tositumomab ex hibited a supraadditive increase in levels of apoptosis induction of 24% (P = 0.009) and 42% (P = 0.0019) relative to expected additive levels of thes e same agents. These in vitro findings suggest that the potential in vivo s ynergy of these well-tolerated drugs may augment the previously demonstrate d clinical activity of anti-CD20 monoclonal antibodies in the treatment of B-cell malignancies.