Lenalidomide Enhancement of NK Cell-Mediated Antibody-Dependent Cellular Cytotoxicity (ADCC) Is Mediated by Granzyme B and FasL and Is Associated with Modification of SHIP-1, PLC-g2 and pERK and Enhanced Chemokine Production.

Abstract

Lenalidomide is an oral anti-angiogenic, anti-proliferative and immunomodulatory drug that is approved for the treatment of transfusion-dependent patients with anemia due to low- or intermediate-1-risk MDS associated with a del 5q cytogenetic abnormality with or without additional cytogenetic abnormalities, and in combination with dexamethasone for the treatment of previously treated multiple myeloma patients. Encouraging early results suggest a potential for clinical efficacy in B cell non-Hodgkin's lymphoma (NHL) and chronic lymphocytic leukemia (CLL). Lenalidomide has been shown to enhance Th1-type cytokines and T cell and NK cell activation markers in patients with advanced cancers. Lenalidomide has been shown to enhance rituximab-mediated protection in a SCID mouse lymphoma model in vivo and we have previously shown that in an in vitro ADCC system lenalidomide is able to directly enhance human NK cell and monocyte function in response to rituximab (chimeric anti-CD20 mAb) and other IgG1 antibodies. We have now addressed the effect of lenalidomide on NK cell signaling pathways and have shown that compared to the levels seen with FC gR stimulation (with IgG) plus IL-12, there is an inhibitory effect of lenalidomide on SHIP-1 with concomitant enhancement of PLC g2 and pERK. This is associated with increased secretion of a variety of chemokines as well as GM-CSF and decreased IL-6 production. In functional ADCC assays, NHL cell lines were pre-coated with rituximab and exposed to either NK cells or monocytes pre-treated with lenalidomide. After 4 (NK cells) or 15 (monocytes) hours in culture the viability of the tumor cells was assessed. The consistent enhancement of ADCC in the presence of lenalidomide is associated with greatly increased expression of granzyme B and FasL, although perforin is maximally elevated without lenalidomide. We found that ADCC is totally blocked by an inhibitor of granzyme B and is partially blocked by anti-FasL. In conclusion, we have shown that the ability of lenalidomide to enhance ADCC-mediated killing of rituximab treated CD20+ve NHL cell lines is mediated by granzyme B and FasL and is associated with clear effects on NK cell signaling and chemokine production.