Treatment with Lenalidomide (Revlimid^®) Upregulates CD40 Expression on CLL Cells and Increases Their Sensitivity to CD40-Mediated Cytotoxicity in Vitro

Lenalidomide, an approved drug for the treatment of multiple myeloma and myelodysplastic syndromes with 5q deletion is currently undergoing clinical investigation in chronic lymphocytic leukemia (CLL). Although the exact mechanism of action of lenalidomide in CLL is not known, it was shown to augment anti-CD40 induced cytotoxicity in human multiple myeloma cells (Tai et al. Cancer Res. 2005) and is currently being evaluated in combination with an anti-CD40 agent in a multiple myeloma clinical trial. While signaling of the costimulatory receptor CD40 promotes survival, proliferation, and differentiation of normal B cells, it can cause activation-induced cell death in malignant B cells. In addition, ligation of CD40 by CD40L (CD154) was shown to sensitize B-CLL cells to apoptosis (Dicker et al. Blood 2006). In a phase II clinical study (Ferrajoli et al. Blood 2008), blood samples from 12 CLL patients were collected at baseline and after 1 week, 4 weeks, 3 months and 6 months of treatment with lenalidomide. Samples were shipped overnight to (San Diego) for the phenotypic monitoring of CD40 expression on CD19+CD5+ B-CLL cells by flow cytometry. Changes in CD40 expression were also measured in B-CLL cells treated in vitro with lenalidomide. In addition, samples were used for in vitro cytotoxicity assays that included treatment with fludarabine or valproic acid in combination with lenalidomide; and a CD40-induced cytotoxicity assay using CD40 Ligand (CD154)-transfected murine fibroblasts and lenalidomide. In 6 out of 12 patients, lenalidomide therapy significantly upregulated the expression of CD40 on B-CLL cells above pretreatment levels (4.5±4.5% vs. 49.5±32.7 %, p=0.020). Moreover, B-CLL cells obtained at baseline from 6 patients and treated in vitro with 1 μM of lenalidomide (a concentration corresponding to clinically achievable plasma levels) for 24h and/or 48h exhibited significantly higher expression of CD40 than B-CLL cells exposed to DMSO vehicle (1.95±0.23 fold increase; p=0.038). Exposure to lenalidomide in vitro neither caused apoptosis of B-CLL cells nor increased their sensitivity to fludarabine or valproic acid cytotoxicity (n=9). However, when B-CLL cells obtained at baseline from 6 patients were treated in vitro with lenalidomide, they became more sensitive to apoptosis induced by CD40 Ligand (CD154)-transfected murine fibroblasts than DMSO treated cells (62.6±16.8% lenalidomide vs. 43.2±16.1% DMSO; p=0.032). Lenalidomide had no significant effect on the viability of B-CLL cells co-cultured with control non-transfected murine fibroblasts. Our data suggest a potential therapeutic use of lenalidomide in combination with CD40 ligation in CLL and warrant further investigation.