Mechanisms underlying synergism between circularized tumor necrosis factor‐related apoptosis inducing ligand and bortezomib in bortezomib‐sensitive or ‐resistant myeloma cells

Mechanisms underlying interactions between a novel, clinically relevant circularized tumor necrosis factor‐related apoptosis inducing ligand (TRAIL) agonist, circularly permuted TRAIL (CPT) have been examined in multiple myeloma (MM) cells sensitive or resistant to bortezomib (BTZ). Various MM cell lines for example, U266, including those resistant to bortezomib‐resistant U266 cells were exposed to low nanomolar concentrations of bortezomib ± CPT and apoptosis monitored. Circularly permuted TRAIL and bortezomib synergistically induced apoptosis in both BTZ‐naïve and ‐resistant cells. The regimen up‐regulated DR4 receptor internalization in MM cells, known to modulate both NF‐κB and extrinsic apoptotic pathways. CPT/BTZ disrupted the non‐canonical NF‐κB pathway, reflected by tumor necrosis factor (TNF) receptor associated factors 3 (TRAF3) up‐regulation, NF‐κB inducing kinase down‐regulation, diminished p52 and p50 processing, and B‐cell lymphoma‐extra large (BCL‐XL) down‐regulation, but failed to inactivate the canonical NF‐κB pathway, reflected by unchanged or increased expression of phospho‐p65. The regimen also sharply increased extrinsic apoptotic pathway activation. Cells exhibiting TRAF3 knock‐down, dominant‐negative Fas‐associated protein with death domain, knock‐down of caspase‐8, BCL‐2/BCL‐XL, or exposure to a caspase‐9 inhibitor displayed markedly reduced CPT/BTZ sensitivity. Concordant results were observed in bortezomib‐resistant cells. The regimen was also active in the presence of stromal cells and was relatively sparing toward normal CD34(+) hematopoietic cells. Finally, ex vivo results revealed synergism in primary MM primary cells, including those BTZ, and the CPT/BTZ regimen significantly decreased tumor growth in a patient‐derived MM xenograft model. These results indicate that the CPT/BTZ regimen acts via the non‐canonical NF‐κB as well as intrinsic/extrinsic apoptotic pathways to induce cell death in MM cells, and may represent an effective strategy in the setting of bortezomib resistance.