Environmental Mediated-Immune Resistance (EM-IR) to APO2L/TRAIL Mediated Apoptosis.
Authors: Perez Lia E. , Parquet Nancy , Molisano Marina , Shain Ken , Alsina Melisa , Anasetti Claudio , Dalton William 
Published: October 2019

Abstract
Resistance to chemotherapy arises in a complex microenvironment and it is essential to study how it influences apoptosis in order to identify novel therapeutic targets. APO2L/TRAIL anti-tumor activity is attractive since it spares normal tissue. Previous studies in our laboratory have shown that cancer:stroma microenvironment interactions confer resistance to immune control via APO2L/TRAIL, a process referred to as environmental mediated-immune resistance (EM-IR). We studied EM-IR in multiple myeloma because it is a disease subject to bone marrow environmental influences and expresses the specific APO2L/TRAIL receptor(s). We have shown in a transwell assay, with myeloma (RPMI-8226, U266 and MM1s) in the upper well and HS5 stromal cells in the lower well, that APO2L/TRAIL (5 to 50ng/mL for 5 hours) apoptosis is reduced when compared to cells exposed to medium in the lower well. Additional studies using conditioned medium from HS5 and myeloma patient’s stroma (n=3) grown for 14 days, confirmed that soluble factor(s) produced by tumor bone marrow microenvironment protected cells from APO2/TRAIL apoptosis. APO2/TRAIL resistance was reversible as sensitivity was restored after HS5 stroma cells were removed (20 min). APO2/TRAIL signaling pathway studies showed that baseline levels of c-FLIP, an anti-apoptotic factor, increased in RPMI-8226 (2.86 fold), in U266 (9.5 fold increase) and in MM1s (1.34 fold increase) myeloma cell lines. Inhibition of c-FLIP by means of RNA interference using siRNA duplex that targeted both c-FLIP isoforms, c-FLIP long and short, increased APO2/TRAIL sensitivity of RPMI-8226 treated in the presence of HS5 stroma. Sub-cellular fractionation showed that c-FLIP is maintained in the membrane fraction and is not significantly increased in the cytosol when exposed to soluble factor(s). Pre-treatment (19 hours) with cyclohexamide, a protein synthesis inhibitor, restored APO2L/TRAIL sensitivity in association with down regulation of c-FLIP while maintained anti-apoptotic c-IAP, Bcl2 and Mcl-1 levels suggesting that c-FLIP synthesis, not intracellular traffic, is essential for soluble factors to regulate c-FLIP. To dissect which soluble factor(s) are involved in apoptosis resistance we have tested the effects of HS27a stroma cells. HS27a lack IL-6 and/or IL-1 production compared to HS5 stroma and do not confer APO2/TRAIL resistance suggesting that these cytokines may be involved in apoptosis resistance. Treatment with neutralizing IL-6 specific antibody of HS5 transwell decreased IL-6 levels and partially restored sensitivity to APO2L/TRAIL in myeloma. Treatment with rh-IL-6 (0.001ng/mL to 10ng/mL) with APO2L/TRAIL conferred a dose-dependent resistance to APO2L/TRAIL in myeloma cells associated with increased c-FLIP levels. IL-1 receptor antagonist (IL-1ra) either by pre-treatment (1hour–18 hours) or co-administration with APO2L/TRAIL did not reverse APO2L/TRAIL resistance. These findings suggest that IL-6 in part mediates APO2L/TRAIL EM-IR by increasing c-FLIP levels among other unknown mechanisms. We conclude that c-FLIP up-regulation inhibitors and/or targeting hematopoietic soluble factor(s) may contribute to enhanced APO2/TRAIL function.