Bortezomib Partially Overcomes TNF-Related Apoptosis Inducing ligand/Apo-2L (TRAIL/Apo-2L) Environment Mediated-Drug Resistance (EM-DR).
Authors: Parquet Nancy , Nimmanapalli Ramadevi , Anasetti Claudio , Alsina Melissa , Dalton William , Perez Lia E. 
Published: April 2020

Multiple Myeloma (MM) is an incurable disease that warrants novel treatments. MM survival is dependent on resistance conferred by interactions with the bone marrow (BM) microenvironment (stroma and/or extracellular matrix proteins): Environment Mediated-Drug Resistance (EM-DR). We have shown that adhesion to fibronectin inhibits CD95-induced caspase-8 activation resulting in apoptosis by regulating the cellular localization and availability of c- FLIPL (Shain K. et al. J Immunol. 2002;168(5):2544–53). TNF-related apoptosis inducing ligand/Apo-2L (TRAIL/Apo-2L) is a member of the TNF superfamily of death ligands that induce apoptosis of resistant MM cell lines and patient’s cells while cultured in suspension. TRAIL/Apo-2L also inhibits the growth of human plasmocytomas in xenografted NOD/SCID mice (Mitsiades C. et al. Blood2001; 98(3):795–804). To explore if the tumor microenvironment confers resistance to TRAIL/Apo-2L mediated apoptosis, we treated RPMI 8226 cells with recombinant human killer TRAIL (Alexis Biochemicals). In a series of 3 experiments, we observed a 50.8 ± 10 % specific apoptosis, measured by annexin/PI staining, when cells were treated with TRAIL (10ng/mL) in suspension (Su) media. In contrast, a dramatic resistance to TRAIL mediated apoptosis (6.9 ± 1.2 %) was observed when RPMI 8226 cells were adhered (Ad) to stroma cells (HS-5 and HS-5 transfected with green fluorescent protein-GFP) suggesting the EM-DR phenotype. We hypothesized that EM-DR can potentially be reversed by Bortezomib, a proteosome inhibitor, via NF-κB inhibition resulting in reduced transcription of cellular adhesion proteins genes (Mitsiades et al. Blood2002; 99(11): 4079–4086). Resistance to drug-induced apoptosis was also observed when RPMI 8226 cells were adhered to HS-5 and HS5-GFP and treated with Bortezomib alone at 5, 10 and 15nM (55.6 ± 11.6 % Su and 12.1 ± 1.6 % Ad). Microarray analysis of RPMI 8226 cells treated with Bortezomib showed that death receptor 4 (DR4) and DR5 expression was up-regulated suggesting a potential interaction between Bortezomib and TRAIL pathway. When RPMI 8226 cells were treated with Bortezomib (10nM) for 20 Hrs and subsequently treated with TRAIL for additional 4 Hrs, we observed an enhancement to 77.7 ± 3.64 % (Su) and 47.22 ± 13.6 % (Ad) specific apoptosis. In addition, in a series of 3 experiments using transwell inserts, RPMI 8226 cells (upper well) separated from HS-5 (lower well) were treated with Bortezomib (10nM), TRAIL (10ng/ML) or Bortezomib and TRAIL combined as described above. Results were comparable with direct contact experiments described, suggesting that cytokines and/or chemokines released by stroma may be responsible for EM-DR phenotype (Figure 1). Further mechanistic studies elucidating TRAIL and Bortezomib combination are currently under investigation. These preclinical studies provide the basis for clinical trial testing the combination of these agents.

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