The role of phosphatidylinositol 3-kinase-δ in the immunomodulatory effects of lenalidomide in chronic lymphocytic leukemia

Abstract

In patients with chronic lymphocytic leukemia (CLL), lenalidomide can promote humoral immune responses but also induces a distinct disease-specific toxicity of tumor flare and cytokine release. These CLL-specific events result from increased expression of costimulatory molecules on B cells. Here we demonstrate that lenalidomide activation of CLL cells depends on the phosphatidylinositol 3-kinase p110δ (PI3K-δ) pathway. Inhibition of PI3K-δ signaling by the PI3K-δ-inhibiting drug, CAL-101, or by siRNA knockdown of p110δ, abrogates CLL cell activation, costimulatory molecule expression, and vascular endothelial growth factor and basic fibroblast growth factor gene expression that is induced by lenalidomide. In addition, CAL-101 attenuates lenalidomide-mediated increases in immunoglobulin M production by normal B cells. Collectively, these data demonstrate the importance of PI3K-δ signaling for lenalidomide immune modulation. These findings may guide development of strategies for the treatment of CLL that combine lenalidomide with CAL-101, with other inhibitors of the PI3K-δ pathway, or with other agents that target downstream kinases of this signaling pathway.

Figure 1

Lenalidomide leads to activation of the PI3K pathway via a PI3K-δ-dependent mechanism. (A) CD19⁺ cells from CLL patients (N = 9) treated with or without 0.5μM lenalidomide were examined for PI3K activity with and without the addition of 1 or 10μM CAL-101 to the lysate. Results were calculated relative to micrograms of protein. (B) CD19⁺ cells from CLL patients (N = 6) were incubated with or without 0.5μM lenalidomide and/or CAL-101 for 48 hours. AKT phosphorylation at Ser473 was assessed by immunoblot. Results are shown from one of 6 experiments. (C) CD19⁺ cells from CLL patients (N = 4) were incubated with or without 0.5μM lenalidomide and/or CAL-101 for 48 hours. GSK3β phosphorylation at Ser9 was assessed by immunoblot. Results are shown from one of 4 experiments. (D) CD19⁺ cells from CLL patients (N = 3) were transfected with siRNA targeted to PI3K-δ, PI3K-γ, or a nonsense target. p110δ protein expression was assessed by immunoblot. Results are shown from one of 3 experiments. (E) CD19⁺ cells from CLL patients (N = 3) were transfected with siRNA targeted to PI3K-δ, PI3K-γ, or a nonsense target and then incubated with or without 0.5μM lenalidomide for 48 hours. AKT phosphorylation at Ser473 was assessed by immunoblot. Results are shown from one of 3 experiments. (B-E) Quantification was done using the Alpha Innotech FluorChemQ MultiImage III System.

Figure 2

Inhibition of PI3K-δ prevents CLL cell immune activation induced by lenalidomide. (A) CD19⁺ cells from CLL patients (N = 25) were treated with or without 0.5μM lenalidomide and/or 10μM CAL-101 for 48 hours. Surface expression of CD20, CD40, or CD86 was evaluated by flow cytometry using CD20-phycoerythrin, CD40-phycoerythrin, or CD86-phycoerythrin antibodies and IgG1-phycoerythrin isotype control. (B) CD19⁺ cells from CLL patients (N = 15) were treated with or without 0.5μM lenalidomide and/or 10μM CAL-101 for 48 hours. RNA was extracted and converted to cDNA, and RT-PCR analysis was done to determine quantities of CD40, CD86, and CD154 mRNA. (C) CD19⁺ cells from CLL patients (N = 6) were treated with or without 0.5μM lenalidomide and/or CAL-101 for 48 hours. CLL cells were irradiated (20 Gy) and placed in culture with purified B cells, in the absence or presence of 5 μg/mL pokeweed mitogen. Quantification of IgM was determined by enzyme-linked immunosorbent assay. (D) CD19⁺ cells from CLL patients (N = 15) were treated with or without 0.5μM lenalidomide and/or 10μM CAL-101 for 48 hours. RNA was extracted and converted to cDNA, and RT-PCR analysis was done to determine quantities of bFGF. (E) CD19⁺ cells from CLL patients (N = 13) were treated with or without 0.5μM lenalidomide and/or 10μM CAL-101 for 48 hours. RNA was extracted and converted to cDNA, and RT-PCR analysis was done to determine quantities of VEGF.