The Immunomodulatory Drugs Lenalidomide and Pomalidomide Inhibit Multiple Myeloma-Induced Osteoclast Formation and RANKL/OPG Ratio In Myeloma Microenvironment Targeting the Expression of Adhesion Molecules

Abstract 448

The increase of osteoclast formation and activation occurring into the bone marrow (BM) area of myeloma cells infiltration is the hallmark of multiple myeloma (MM). The increase of the RANKL/OPG ratio in BM stromal cells (BMSCs) and osteoprogenitor cells, induced by MM cells through the cell-to-cell contact, is critically involved in MM-induced osteoclast formation. In addition, MM cells also up-regulate RANKL expression and secretion by activated T in the MM BM microenvironment. Soluble factors such as CCL3/MIP-1α, IL-3 and IL-7 produced by MM cells contribute to the increase of osteoclast formation either directly or indirectly through RANKL stimulation. Recent data have suggested that thalidomide and the immunomodulatory drugs (IMiDs^®) lenalidomide directly inhibit osteoclast formation and maturation.

In the present study we have investigated the potential effects of lenalidomide and the more potent IMiD® pomalidomide on MM-induced osteoclast formation. First in a cell-to-cell contact co-culture system we found that both IMiDs^® at concentration ranging from 2 to 100 μM significantly blunted RANKL secretion by human BMSC/osteoprogenitor cells induced by MM cells decreasing the RANKL/OPG ratio level with a more potent effect of pomalidomide as compared to lenalidomide. Consistently the pro-osteoclastogenic property of the conditioned medium of MM cells co-cultured with BMSC/osteoprogenitor cells was reduced in the presence of IMiDs^®. On the other hand, we did not find any significant inhibitory effect of both drugs neither on the production of soluble pro-osteoclastogenic factors by MM cells as CCL3/MIP-1α, IL-3 and IL-7 nor on RANKL expression and secretion by T lymphocytes. To go further inside to the capacity of IMiDs^® to blunt MM-induced RANKL/OPG ratio, we performed a microarray analysis (using Affymetrix, GeneChip^®, HG-U133Plus2.0 platform) to investigate the effect of lenelidomide and pomalidomide on the transcriptional profile of both MM cells and BMSCs. We found that 40 and 83 genes were significantly modulated by lenalidomide and pomalidomide, respectively, in MM cells Interestingly, among the genes significantly modulated by IMiDs® we identified the downregulation of the adhesion molecules ITGA4 (CD49d), ITGA8 and ICAM2 (CD102). In human BMSCs, 71 genes and 214 genes were significantly modulated by lenalidomide and pomalidomide, respectively, including those belonging to focal adhesion, cell cycle, BMP2, TGF-beta and IL-6 signaling pathways.

Finally, using flow cytometry we confirmed the capacity of lenalidomide and pomalidomide to inhibit the expression of adhesion molecules as CD49d by MM cells co-cultured either in presence or absence of BMSCs showing that this effect is critically involved in the inhibition of RANKL/OPG ratio by IMiDs^®.

In conclusion our data strongly suggest that lenalidomide and pomalidomide inhibits MM-induced osteoclast formation through the inhibition of RANKL/OPG ratio targeting the expression of adhesion molecules by MM cells.