Lenalidomide Inhibits Proliferation of Chronic Lymphocytic Leukemia Cells in Vitro

Abstract 1775

Promising clinical responses have been observed in chronic lymphocytic leukemia (CLL) patients treated with lenalidomide as a single agent or in combination with other agents, The mechanisms of action of lenalidomide are under study; unlike most other anti-leukemia drugs, lenalidomide has no direct cytoxic effects in vitro on primary CLL cells, which typically are in G0/1 phase of the cell cycle. We examined the activity of lenalidomide on CLL cells that were induced to proliferate in vitro. To induce proliferation, CLL cells were cultured in media containing human interleukin (IL)-4 and IL-10 and with stromal cells (HeLa) made to express CD154. Labeling CLL cells with carboxyfluorescein succinimidyl ester (CFSE), allowed us to monitor for several rounds of induced CLL-cell division via flow cytometry. We found that addition of 0.33–10 micro M lenalidomide to such cultures resulted in a dose-dependent reduction in the number of leukemia cells induced to undergo cell-division. Moreover, we found that lenalidomide could significantly reduce the number of dividing CLL cells in each patient sample tested (n=4) by an average of 1.7 fold, reducing the fraction of dividing cells from 77% ± 27% to 44% ± 22% after 6 days of culture (mean +/− SD, P < 0.05). Evaluation of the DNA content of CLL cells using propidium iodide (PI) and flow cytometry revealed that lenalidomide significantly decreased the percentage of CLL cells in the G2/M phase of the cell cycle from 9% ± 2.7% to 5.1% ± 2.1% (mean +/− SD, n=4; P< 0.05) in control versus lenalidomide-treated cultures, respectively. Furthermore, lenalidomide appeared also to reduce the percentages of CLL cells in S phase from 12% ± 8% to 6.2% ± 3.1%. We found that the capacity of lenalidomide to inhibit CLL cell-division was associated with lenalidomide-induced leukemia-cell expression of p21/WAF/Cip, which can directly inhibit the activity of cyclin-dependent kinases required for progression from G1 into the S phase of the cell cycle. Gene expression analysis of CLL cells (n=10) revealed that lenalidomide induced increased leukemia-cell expression of p21/WAF/Cip at 6h and at 24h, an effect that also was noted for leukemia cells in the blood of patients treated with single-agent lenalidomide. Lenalidomide-induced expression of p21/WAF/Cip was associated with induced expression of the pro-apoptotic protein Bim, a downstream target of of p21/WAF/Cip. However, lenalidomide did not appear to induce leukemia-cell expression of TP53, which can induce p21/WAF/Cip, suggesting that lenalidomide induces p21/WAF/Cip via a TP53-independent mechanism. These results suggest that lenalidomide has effects on CLL cells that are distinct from those induced by CD40-ligation. Moreover, these studies reveal a potential mechanism for the anti-leukemia activity of lenalidomide, which might inhibit factors that potentially drive leukemia-cell proliferation in vivo.