Bortezomib Reverts Stroma-Mediated Resistant to APO2L/TRAIL in Multiple Myeloma.

Abstract 2806

Poster Board II-782

Hematopoietic stroma provides a sanctuary for multiple myeloma (MM) cells and promotes resistance to immune control mediated by APO2L/TRAIL in part by increasing synthesis of the anti-apoptotic protein c-FLIP (J Immunol, 180: 3; 2008). Bortezomib, a reversible 26S proteosome inhibitor, sensitizes cancer cells to APO2L/TRAIL induced apoptosis in a variety of malignancies including MM. In addition, it has been well established that bortezomib interferes with the protective interaction between cancer cells and the bone marrow stroma. In this study we tested whether bortezomib can reverse the APO2L/TRAIL environmental mediated-immune resistant (EM-IR). Using MM cell lines (RPMI 8226 and U266) or CD138+ positive selected cells from MM patient's bone marrow, we found that exposure to HS5 stroma cells by direct adhesion or in a transwell system induced resistance to bortezomib (10nM for 24 hours) and that pretreatment with bortezomib (10nM for 20 hours) effectively overcomes APO2L/TRAIL resistance (10ng/mL for 4 additional hours). Conditioned medium made from a 14-day culture of MM patient's bone marrow stroma induced resistance to bortezomib and pretreatment with bortezomib sensitized cells to APO2L/TRAIL induced cell death as shown with HS5 cells. Bortezomib showed not to be cytotoxic to HS5 stroma cells and only MCP-2-3 and IL-10 levels were altered in the stroma-MM milieu within cytokines measured by ELISA array. IL-6, a cytokine shown to induce APO2L/TRAIL mediated resistance, remained unchanged with bortezomib treatment. We found that bortezomib increased expression of TRAIL receptors (DR5, DCR1 and DCR2), but such expression did not predict for sensitivity to apoptosis as DR4 demonstrated to be the receptor responsible for activation of APO2L/TRAIL. Soluble factor(s) released by HS5 stroma increased expression of c-FLIP and induced STAT-3 and ERK phosphorylation in myeloma cells. However, only c-FLIP protein expression was effectively reduced by bortezomib. c-FLIP quantitative-PCR found that HS5 increased transcription only after 20 hours bortezomib treatment and significantly reduces soluble factor(s) induced c-FLIP transcription. In a transwell assay, HS5 stroma cells induced NF-κB activation and the addition of bortezomib diminished its activation at 20 hours. These findings provide the rationale to combine bortezomib and APO2L/TRAIL to disrupt the influence of the stroma microenvironment on myeloma cells.