Enhancement of death receptor-mediated apoptosis in multiple myeloma cells

Background: Multiple Myeloma (MM) is currently incurable despite many novel therapies. Tumour Necrosis Factor-Related Apoptosis-Inducing Ligand (TRAIL) is a potential anti-tumour agent although the effects as a single agent are limited. This investigation determined whether the Histone Deacetylase (HDAC) inhibitor SAHA, or inhibitors of the histone methyltransferases G9a and EZH2 (BIX 01294 and GSK343) and Nuclear export inhibitor (NEI) LMB, enhance TRAIL-induced apoptosis and overcome TRAIL resistance in both suspension culture, and 3D cell culture as a model of solid disseminated MM lesions that form in bone. Methods: The effects of TRAIL sensitizers and/or TRAIL treatment were investigated in both suspension cultures and in an alginate-based 3D culture model. Apoptosis was detected by assessment of nuclear morphology using Hoechst 33342/PI staining. TRAIL-resistant cells were generated by acute exposure of TRAIL sensitive cells to TRAIL followed by the selection of TRAIL-resistant cells (TRAILR). Apoptotic effects in quiescent cells (labelled as PKH26Hi) were also determined. Subsequently, an investigation was undertaken to identify potential mechanisms of action of these agents when used alone and in combination with TRAIL. Results: TRAIL significantly induced apoptosis in a dose-dependent manner in OPM2, RPMI 8226, NCI-H 929, U266, JJN3 human MM cell lines and ADC-1 plasma cell leukaemia cells. All epigenetic modifiers and NEI synergistically enhanced TRAIL responses in several lines and responses were potentiated in 3D culture. Interestingly, TRAILR cells were sensitive to BIX 01294 and LMB; however, TRAIL responses in cells that had been selected for TRAILR were not further enhanced by SAHA and GSK343. Quiescent PKH26Hi cells were resistant to dual therapy. Mechanistically, TRAIL and TRAIL sensitizers induced apoptosis via both extrinsic and intrinsic pathways in addition to decreasing the expression of oxidative enzyme catalase. Conclusions: Inhibitors of HDAC, EZH2 and G9a and NEI are potent sensitisers of TRAIL responses both in suspension, and crucially in 3D cell culture, which may mimic physiological aspects of bone metastases. These agents may be a therapeutic option in combination with TRAIL and may increase TRAIL sensitivity in insensitive cells, but not in cells that have specifically been selected for acquired TRAIL-resistance, and not in quiescent cells.