C/EBPb Is a Critical Mediator of Resistance to IMiD® Immunomodulatory Compounds and Affected by IMiD Compounds Via Control of Protein Translation

Lenalidomide and pomalidomide are IMiD® immunomodulatory compounds that have been shown to be highly active in the treatment of multiple myeloma (MM). IMiD compounds exert their anti-tumor effects via acting on costimulatory proteins of T cells and NK cells, augmenting both the adaptive and innate immune system. But the mechanisms by which IMiD compounds directly inhibit MM cell proliferation are still unclear. Here we focused on the direct effects of IMiD compounds alone on MM cells.

We found that IMiDs, at concentrations as low as 0.01 μ M, induce significant inhibition of DNA synthesis in MM cells as shown by thymidine uptake. Since our previous work demonstrated that C/EBPβ is an important transcription factor which controls the growth and proliferation of myeloma cells, we analyzed the effects of IMiD compounds on C/EBPβ. We found that both pomalidomide and lenalidomide significantly decreased the protein level of C/EBPβ LAP-isoform in MM cell lines and primary MM cells. IMiD compound-induced suppression of C/EBPβ protein expression led to impaired transcription of the downstream IRF4, and subsequently to downregulation of BLIMP1 and XBP1, which are all critical for MM survival. To confirm our findings in vivo, we analyzed IRF4 expression by double labeling (IRF4⁺/CD138⁺) immunohistochemical staining of bone marrow biopsy samples of 23 myeloma patients prior to therapy and during therapy with lenalidomide. During lenalidomide therapy, the bone marrow MM cells showed a significantly weaker staining intensity for IRF4 in comparison to prior therapy. This was quantified by a significant (p<0.001) decrease of the staining score from 176 to 152, respectively. To confirm the critical role of C/EBPβ in MM we stably overexpressed C/EBPβ in MM cells. Overexpression of C/EBPβ prevented IMiD compound-induced inhibition of MM cell proliferation, indicating that C/EBPβ is critical in mediating resistance to IMiD compounds. This was supported by the fact that C/EBPβ was not down regulated in IMiD-resistant cell lines by IMiD treatment. Dissection of the C/EBPβ protein regulation revealed that IMiD compounds shut down C/EBPβ protein translation by decreasing eIF-4E. Knockdown experiments of eIF-4e resulted in downregulation of C/EBPβ, suggesting that C/EBPβ is under translational control in MM.

Our studies, for the first time, provide evidence that IMiD compounds inhibit MM cell proliferation and survival by affecting the translation of C/EBPβ and subsequently multiple downstream transcription factors including IRF4, BLIMP1 and XBP1. Due to the critical role of C/EBPβ in mediating effects of IMiD compounds in MM, it might be a target to overcome drug resistance to IMiD compounds. The fact that pomalidomide can overcome resistance to lenalidomide in MM requires still further evaluation.

Schafer:Celgene Corporation: Employment, Equity Ownership. Mapara:Gentium: Equity Ownership. Lentzsch:Celgene Corp: Research Funding.