Abstract 2578: Synergistic activity of lenalidomide and interleukin-21 in chronic lymphocytic leukemia
Authors: Browning Rebekah L. , Jones Jeffrey A. , Johnson Amy J. , Muthusamy Natarajan , Byrd John C. 
Published: May 2015

Abstract
Interleukin-21 (IL-21) is an immunomodulatory cytokine with cytotoxic activity against chronic lymphocytic leukemia (CLL) B cells. Its cytotoxic activity has been shown in part to directly correlate with level of surface expression of the IL-21 receptor (IL-21R) on CLL cells. Although CpG and CD40 ligand have been show to induce upregulation of IL-21R, their limited availability for clinical use prompted us to investigate alternative immunomodulatory agents such as lenalidomide. We observed that treatment of CLL B cells with 0.5 μM lenalidomide upregulated IL-21R expression in 7/10 patients (samples with &lt;20% induction were considered not responsive), with maximum induction of protein at 48 hours (mean 67% increase in responsive samples, median 50% increase). Peripheral blood mononuclear cells from two patients receiving lenalidomide showed similar, if less pronounced, induction of IL-21R transcript and protein, with an average 42.5% increase of mRNA by Day 3 of treatment and 34% increase of protein by Day 8. In primary CLL B cells, induction of IL-21R by lenalidomide with subsequent treatment with IL-21 generally leads to an increase in phosphorylation of STAT1 (signal transducer and activator of transcription 1) as compared to treatment with IL-21 alone, and rarely an increase in phosphorylation of STAT3.
We next assessed if pre-treatment with lenalidomide resulted in enhanced killing of CLL B cells by IL-21. Whereas lenalidomide produced minimal cytotoxicity and IL-21 modest effect, combination of the two exhibited synergistic cytotoxic effects against CLL B cells (p=0.028, n=8). The synergistic interaction between lenalidomide and IL-21 treatment was further revealed by a distinct expression pattern of miR and mRNA signatures in CLL cells treated in vitro with combined lenalidomide and IL-21, as compared to vehicle, lenalidomide, or IL-21 alone. Analysis of the comprehensive mRNA expression data revealed pro-apoptotic protein BID (BH3-interacting domain death agonist) as a potential mediator of the enhanced killing observed with combined lenalidomide and IL-21. Western blot analysis revealed that treatment of CLL B cells with either lenalidomide or soluble CD154 can increase BID protein levels by 48 hours; however, while CD40 blocking antibody diminished BID induction by CD154 in all patients tested, it diminished BID induction by lenalidomide in only 2/6 patient samples. This indicates an alternative pathway of BID induction in a subset of patients and presents a possible mechanism for the enhanced cytotoxicity seen with the combination of lenalidomide and IL-21. Further exploration of this synergistic combination is ongoing. These data demonstrate that lenalidomide can up-regulate IL-21R and leads to increased sensitization to IL21 treatment. These studies justify further clinical investigation of the combination of IL-21 and lenalidomide in CLL and related lymphoid malignancies.
Citation Format: Rebekah L. Browning, Jeffrey A. Jones, Amy J. Johnson, Natarajan Muthusamy, John C. Byrd. Synergistic activity of lenalidomide and interleukin-21 in chronic lymphocytic leukemia. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2578. doi:10.1158/1538-7445.AM2014-2578