Bortezomib Targets Stroma-Mediated APO2L/TRAIL Apoptosis Resistance in Multiple Myeloma.

TRAIL mediates graft versus tumor responses while sparing normal tissues. Previous studies in our laboratory showed that myeloma:stroma microenvironment interactions confer resistance to TRAIL-induced apoptosis, a process referred to as environmental mediated-immune resistance (EM-IR) by increasing synthesis of the anti-apoptotic protein c-FLIP (J Immunol, 180: 3; 2008). Since bortezomib influences TRAIL signaling proteins and targets stroma induced survival signals, we studied whether it reverses TRAIL EM-IR. Myeloma cells (RPMI 8226 and U266) were treated either adhered to HS5 stroma cells or exposed to HS5-released soluble factors in a transwell system with myeloma cells in the upper well and HS-5 stroma cells in the lower well. Pretreatment with bortezomib (10nM for 20 hours) not only induced synergistic cytotoxicity in myeloma but did overcome TRAIL (10mg/mL for 4 hours) apoptosis resistance induced by HS5-released soluble factors. Mechanistic studies showed that bortezomib is not cytotoxic to HS5 stroma cells and does not alter HS5 release of IL-6, a cytokine known to mediate APO2L/TRAIL EM-IR (J Immunol, 180: 3; 2008). Cytokine array testing showed that stroma-myeloma co-culture lead to a significant increase in IL-10 levels but bortezomib had no effect on IL-10. Bortezomib only decreased MCP-2 and MCP-3 levels without affecting other inflammatory cytokines tested. Expression of DR5 receptor (agonistic) and DCR1-DCR2 receptors (antagonist) were increased at 20 hours bortezomib treatment. Pretreatment with DR4/DR5 blocking antibodies suggested that TRAIL signals trough DR4, not DR5, in these cells and increased of decoy receptors suggest that receptor expression could not predict TRAIL sensitivity. Among the proteins involved in TRAIL signaling that are bortezomib targets, HS5-released factors increased only c-FLIP expression, without affecting Heat Shock proteins, BcL-2 and/or IAP protein families. In the presence of HS5 stroma cells, bortezomib reduced c-FLIP thereby contributing to overcome apoptosis resistance. Since c-FLIP is a NF-κB target gene, we studied whether stroma cells lead to NF-κB activation. Gel shift assay showed increased NF-κB activation in myeloma cells treated with HS5 stroma. Bortezomib decreased NF-κB activation in myeloma cells in suspension, but was not effective in the presence of stroma. Since bortezomib abrogates cytokine-induce kinase activation and understanding that c-FLIP may be under control of these kinases, we next studied the effect of stroma and/or bortezomib on proteins involved in IL-6 signaling. HS5-released factor(s) induced STAT-3 and ERK phosphorylation but not AKT activation, although bortezomib effects on c-FLIP shown to be independent of these pathways. Our findings provide the rationale to combine bortezomib to APO2L/TRAIL treatment to abrogate the positive influence of the stroma microenvironment on myeloma cell survival.