Uncovering Thalidomide Mechanism of Action: Neuropilin 1 Represents the Target of Antiangiogenic and Immunomodulatory Effect in Chronic Lymphocytic Leukemia

Abstract 4608

In chronic lymphocytic leukemia (CLL) thalidomide was found to significantly decrease the percentage of regulatory T cells (Tregs) as well the number of CLL cells in vivo. In combination with fludarabine, thalidomide was effective both in refractory/relapse and naïve CLL patients. In our recent clinical trial, we also observed a reduction of vascular endothelial growth factor (VEGF) levels during therapy that were correlated with the reduction of Tregs (r²=0.47, p<0.05). Furthermore, gene expression profiles associated with thalidomide response in CLL revealed several genes involved in angiogenesis (Giannopoulos et al. Leukemia 2009).

To further characterize the thalidomide mechanism of action in CLL we assessed expression of neuropilin 1 (NRP1), which is a membrane-bound coreceptor to the tyrosine kinase receptor for both vascular endothelial growth factor (VEGF) and semaphorin (SEMA3A) family members. NRP1 plays versatile roles in angiogenesis, cell survival, migration, and invasion. Recently, NRP1 was also found expressed on plasmacytoid dendritic cells (PDC) as well as on Tregs, two immune cell subpopulations involved in tolerance mechanisms commonly deregulated in tumorigenesis. Our analysis showed NRP1 expression on CLL cells, Tregs and PDC of 38 CLL patients. Using five parameter flow cytometry we found increased expression of NRP1 in CLL when compared to cells derived from healthy volunteers. NRP1 expression was 22.7% on CD5⁺CD19⁺ CLL cells vs. 6.2% on CD19⁺ B cells from controls, p=0.03. Furthermore, we found expression of NRP1 on Tregs as well as PDC with a median expression of NRP1 on Tregs of 42.6 % (range: 10 – 100%). NRP1 was expressed on almost all PDC with a median expression of 100% (range: 98.2 – 100%). In functional studies, we found that NRP1 expression might be regulated by VEGF expression levels. Magnetically separated CLL cells increased expression of NRP1 after cell culture with VEGF. Here, VEGF levels of 0.1 – 0.5ng/ml, which are also observed in primary CLL patient samples, effectively induced expression of NRP1. In accordance, we also observed that VEGF upregulates NRP1 expression on magnetically separated Tregs. However, higher VEGF concentrations inhibited NRP1 expression in CLL cells probably due to a negative feedback loop.

In conclusion, we found expression of NRP1 on CLL cells, Tregs as well as PDC in patients with CLL, and we could demonstrate that the expression of NRP1 is regulated by VEGF expression levels. Thus, our previously observed thalidomide-induced reduction of VEGF levels along with a reduced percentage of Tregs in CLL might in part be explained by down-regulation of the NRP1 expression.