Stimulation of T Cells by Lenalidomide Involves Putative Lenalidomide Binding Proteins CD3-Epsilon-Associated Protein and GDP-Mannose Pyrophosphorylase a

Introduction: Lenalidomide is approved in the US for the treatment of transfusion-dependent patients with anemia due to Low- or Intermediate-1-risk myelodysplastic syndromes associated with a del (5q) cytogenetic abnormality, with or without additional cytogenetic abnormalities. Lenalidomide is also approved for use in the US and Europe in combination with dexamethasone in previously treated multiple myeloma patients. In vitro and ex vivo studies have shown that lenalidomide has a direct antiproliferative effect against tumor cells and induces antiangiogenesis. It has also been shown to have immunomodulatory activity, including co-stimulatory effects on T and NK cells. Using the yeast 3-hybrid system, based on a leukemia cDNA library and biotinylated lenalidomide analog, we identified 16 putative lenalidomide-binding proteins from 700,000 clones screened. These included CD3-epsilon-associated protein (CAST) and GDP-mannose pyrophosphorylase A (GMPPA). CAST binds to the T cell receptor and is also part of the RNA polymerase 1 complex. GMPPA is a nucleotidyl transferase that converts mannose-1-phosphate and GTP to GDP-mannose, which is involved in the production of N-linked oligosaccharides. To determine whether these proteins are required for lenalidomide-induced immunomodulatory activity, we evaluated the lenalidomide-induced upregulation of interleukin-2 (IL-2) production in primary T cells transfected with siRNA against CAST and GMPPA.

Methods: Primary human peripheral blood T cells were transfected with CAST or GMPPA siRNA, or mock siRNA as a control. Reduction of CAST and GMPPA expression was confirmed by qRT-PCR. After 24 hours, transfected cells were stimulated with anti-CD3 mAb in the presence or absence of 0.1 and 1 μM lenalidomide 48 hours. Cells were lysed after 48 hours, and IL-2 mRNA and mature protein levels were quantified using qPCR and ELISA, respectively.

Results: CAST gene expression in T cells was reduced by 60% (p<0.05) in CAST siRNA-transfected cells. GMPPA gene expression was reduced by 66% (p<0.05) in GMPPA siRNA-transfected T cells. Lenalidomide-induced IL-2 mRNA production was reduced from 9-fold in controls to 6-fold in CAST siRNA transfectants (p<0.05) and from 15-fold in controls to 6-fold in GMPPA siRNA transfectants (p<0.05). Lenalidomide-induced IL-2 protein production was reduced from 22-fold in controls down to 8-fold in CAST siRNA transfectants (p<0.05) and from 5.6-fold in the controls down to 1.6-fold in GMPPA siRNA transfectants (p<0.05).

Conclusion: These findings support the hypothesis that CAST and GMPPA play an important role in the lenalidomide IL-2 induction mechanism in primary T cells.