Hematopoietic Stroma Confers Resistance to Immune Control by TRAIL in Multiple Myeloma through Induction of FLIP.

TNF-related apoptosis inducing ligand/Apo-2L (TRAIL), a member of the TNF superfamily of death ligands, is preferentially cytotoxic against neoplastic cells, while sparing normal tissue. TRAIL has been implicated in tumor immune-surveillance and mediates murine allogeneic graft-versus-tumor responses. The bone marrow microenvironment provides tumor protection from chemotherapy and Fas-death receptor mediated apoptosis through a process known as environmental mediated death resistance (EM-DR). We tested whether the tumor microenvironment inhibits TRAIL-mediated killing of myeloma cells, as this process may contribute to multiple myeloma escape from immune-surveillance. Three drug-sensitive myeloma cell lines (RPMI-8226, U266 and MM1s) exhibit apoptosis resistance to recombinant human (rh) TRAIL while adhered to HS5 stromal cells that expresses green-fluorescence-protein (HS5-GFP). Apoptosis resistance to TRAIL was time- and dose- dependent. Evaluation of TRAIL apoptosis in a transwell (TW) assay, with HS5-GFP cells on the bottom and RPMI-8226 cells in the upper well (TW+HS5) revealed that HS5-GFP stromal cells blocked TRAIL-induced apotosis through soluble factors. We then assessed the modifications in the TRAIL signaling pathway induced by TW+HS5 and responsible for the observed phenotype. RPMI-8226 treated in TW+HS5 exhibited attenuated pro-caspase-8, pro-caspase-3, PARP, and BID cleavage, with diminished mitochondrial membrane potential changes, but without alterations on TRAIL receptors and other Bcl-2 family members. We found increased levels of Fas-associated death domain like IL-1 converting enzyme-like inhibitory protein (FLIP), a regulatory factor that competes with caspase-8 inhibiting apoptosis. Subcellular fractionation of RPMI-8226 cells showed that FLIP_L is maintained in or associated with organelle membranes and is released to the cytosol when exposed to soluble factor (TW+HS5) suggesting that that soluble factor signaling may influence FLIP_L localization and availability. Furthermore, FLIP reduction by FLIP-RNA interference increases TRAIL sensitivity of RPMI-8226 treated in TW+HS5. Bortezomib is a proteosome inhibitor that inhibits NF-KB and reduces FLIP levels. To this end, pretreatment of RPMI-8226 cells with bortezomib for 20 hours followed by TRAIL for 4 hours overcame TRAIL EM-DR through inhibition of FLIP, thus providing rationale for testing this drug combination in a clinical trial. In addition, rh-IL-6 induced FLIP levels in RPMI-8226 cells conferring resistance to TRAIL mediated apoptosis. Our results suggest that IL-6 and other soluble factors produced by marrow stromal cells promote myeloma cell survival by upregulating FLIP, thereby mitigating the influence of the microenvironment on TRAIL-induced apoptosis. We conclude that the cytotoxic effect of TRAIL may be enhanced by FLIP inhibitors.