Anti-Proliferative and Apoptotic Activity of Lenalidomide in Combination with Rituximab in Follicular Lymphoma: An in Vitro and Ex Vivo Analysis in Follicular Lymphoma Cells.

Rituximab-containing chemoimmunotherapy regimens have become the standard of care for first line therapy of follicular lymphoma (FL). Despite such intensive treatments most FL patients will continue to relapse and prognosis for those not cured with initial therapy remains poor. Lenalidomide is an immunomodulatory agent; in several in vitro and ex vivo lymphoma models lenalidomide has shown an ability to induce both growth arrest and cell kill via both immune-mediated and direct cytotoxicity. A recent clinical study in relapsed/refractory FL heavily pretreated patients achieved a duration of response lasting >16.5 months in response to lenalidomide treatment (Witzig et al JCO in press). Preliminary results from smaller studies in FL also indicate that combining lenalidomide with rituximab (R2) can achieve remarkable responses in the newly diagnosed and in the relapsed/refractory settings. Here we aim to better characterize the non-immune mediated activity of lenalidomide in NHL cell lines and determine if lenalidomide-mediated cytotoxicity is affected by the presence of rituximab.

NHL cell lines used in the study included DoHH-2 (FL), Rec-1 (Mantle Cell Lymphoma) and Farage (Diffuse Large B cell Lymphoma). The range in concentrations of lenalidomide and rituximab was from 0.0001 to 100 μM, and from 0.0001 to 100 mg/ml, respectively, depending on the individual sensitivity of each cell line. In vitro tumor cell proliferation assays were done by incubating each cell line with each drug separately and in combination for 72 hrs, and measuring proliferative activity by ³H-thymidine incorporation. A proliferation inhibition index for each agent alone and combined was calculated via the Chou Talalay method (CompuSyn software) and expected additive effects were calculated using the fractional product method. Apoptosis was analyzed via Annexin V and propidium iodide (PI) staining and quantified using flow cytometry, which was also used to measure CD20 expression. Phospho-Bcl2 levels in drug-treated DoHH2 cells were assessed via western blot.

Lenalidomide induced approx. 40% and 20% inhibition in proliferative activity of both DoHH2 and Rec-1 cells, respectively. Rituximab inhibited the proliferation of DoHH2 and Rec-1 cells by approx. 50% and 20%, respectively, and additive antiproliferative activity (40%) resulted from treatment of Rec-1 cells with R2. The Farage DLBCL cell line showed approx. 25% inhibition in proliferation with lenalidomide and rituximab individually and additive anti-proliferative activity when treated with R2. However, synergistic inhibition in proliferation (85% inhibition) was achieved when DoHH2 cells were treated with R2. Further, lenalidomide did not affect surface CD20 expression of any cells examined. R2 inhibited proliferation of DoHH2 cells at combination index values ranging from 0.0045 to 0.023, and directly potentiated DoHH2 cells to undergo apoptosis. Synergy in apoptotic activity was also demonstrated ex vivo in cells from a FL patient treated with R2. In DoHH2 cells, addition of lenalidomide was found to potentiate the rituximab-mediated phosphorylation of Bcl-2, thus inactivating its activity and promoting a loss in mitochondrial membrane potential that results in release of cytochrome C to trigger caspase-mediated apoptosis.

These results indicate that R2 induces synergistic anti-proliferative and pro-apoptotic effects in both FL cell line and in primary cells. Evidence suggests that this direct cytotoxicity is mediated by signaling events involving Bcl-2 activation leading to cell death. These data support clinical findings showing a benefit in combining lenalidomide with rituximab and warrant further investigation of R2 in future lymphoma trials.

Gandhi:Celgene: Employment, Equity Ownership. Kang:Celgene: Employment. Capone:Celgene: Employment. Shafarenko:Celgene: Employment. Schafer:Celgene: Employment.