Lenalidomide Inhibits Proliferation Of Chronic Lymphocytic Leukemia Cells Via a Cereblon/p21WAF1/Cip1-Dependent Mechanism
Authors: Fecteau Jessie-Farah , Corral Laura G , Futalan Diahnn , Gaidarova Svetlana , Bharati Ila , Cathers Brian , Ghia Emanuela M. , Schwaederlé Maria , Lopez-Girona Antonia , Messmer Davorka , Kipps Thomas J. 
Published: October 2019

Abstract
Lenalidomide has demonstrated clinical activity in patients with chronic lymphocytic leukemia (CLL). However, its mechanism of action is not fully elucidated. Lenalidomide is not directly cytotoxic to CLL cells in vitro, but can alter the capacity of CLL cells to interact with cells in its microenvironment. This has led to the speculation that its primary activity is indirect, although direct effects on CLL cells have been observed.
In this study, we examined the direct effects of lenalidomide on CLL cells. We performed transcriptome analysis on primary CLL cell samples exposed to lenalidomide in vitro for 6 and 24hrs and found a significant upregulation of the cyclin-dependent kinase inhibitor p21WAF1/Cip1 (p21), which was also confirmed at the protein level. Since p21 can inhibit the progression of cells from G1 to S phase of the cell cycle, this suggests that lenalidomide may render CLL cells less responsive to proliferation stimuli from the microenvironment. To test this hypothesis, we induced CLL cells to proliferate in vitro using accessory cells made to express CD154 and media containing human interleukin (IL)-4 and IL-10. We monitored for proliferation of CLL cells cultured with and without lenalidomide using carboxyfluorescein succinimidyl ester (CFSE). In repeated experiments, we observed that lenalidomide consistently and significantly inhibited the proliferation of CLL cells in a dose-dependent manner, at concentrations that are achieved in treated patients. Evaluation of the DNA content of CLL cells using propidium iodide and flow cytometry also revealed that lenalidomide significantly decreased the fraction of CLL cells in S and in G2/M phases of the cell cycle and concomitantly increased the percentage of CLL cells in G0/G1 phases. This block in cell proliferation also was accompanied by the upregulation of p21, and the extent of which correlated with the degree to which leukemia-cell proliferation was inhibited. In additional studies, we used small interfering RNA (siRNA) to silence p21 in CLL cells and measured the effect of silencing on cell proliferation in the presence of lenalidomide. We observed that the ability of lenalidomide to inhibit CLL cell proliferation was significantly reduced in CLL cells silenced for p21 compared to CLL cells transfected with control siRNA, supporting a role for p21 expression in mediating the proliferation block induced by lenalidomide. We did not however observe the induction of p53 expression following lenalidomide exposure, suggesting that lenalidomide may upregulate p21 via a p53-independent mechanism.
Cereblon (CRBN) is the only known molecular target of lenalidomide. To functionally interrogate the potential role of CRBN in lenalidomide activity on CLL cells, we used siRNA to silence CRBN in primary CLL cells and monitored the impact of silencing on the ability of lenalidomide to upregulate p21 and to inhibit proliferation. We observed lower levels of p21 expression in CRBN-silenced cells exposed to lenalidomide compared to cells transfected with non-specific siRNA, suggesting that CRBN may play a role in p21 upregulation. Furthermore, we observed that CRBN silencing significantly abrogated the anti-proliferative effect of lenalidomide. These results indicate the involvement of CRBN in the anti-proliferative activity of lenalidomide, which is mediated, at least in part, by p21.
This study demonstrates that lenalidomide inhibits the proliferation of CLL cells in a CRBN/p21-dependent manner. This direct effect of lenalidomide on CLL cells may account in part for the highly infrequent observation of disease progression in patients receiving long-term maintenance therapy with this agent (Blood, 2013, PMID:23801633).

Disclosures:
Fecteau: Celgene: Research Funding. Corral:Celgene: Employment. Gaidarova:Celgene: Employment. Cathers:Celgene: Employment. Lopez-Girona:Celgene: Employment. Messmer:Celgene: Research Funding. Kipps:Celgene: Membership on an entity’s Board of Directors or advisory committees, Research Funding.