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

Abstract

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.

Keywords: CPT; TRAIL; bortezomib; intrinsic/extrinsic apoptotic; multiple myeloma; non-canonical NF-κB pathway.

FIGURE 1

The circularly permutated TRAIL (CPT)/bortezomib regimen synergistically induces apoptosis in multiple myeloma (MM) cells. (A‐D) U266, 8226, H929 and bortezomib‐resistant U266 cells (PS‐R) cells were exposed (48 h) to indicated doses of CPT and resistant to bortezomib (BTZ) treatment, followed by flow cytometric analysis of cell death after staining with 7‐AAD. The percentages of 7‐AAD (+) cells are presented. (E and F) U266 and PS‐R cells were exposed (24 h) to varying concentrations of CPT ± BTZ at a non‐fixed ratio, after which the percentage of 7‐AAD⁺ cells was determined. Combination Index (CI) values less than 1.0 denote a synergistic interaction; *p < 0.05; **p < 0.01; ***p < 0.001

FIGURE 2

The circularly permutated TRAIL (CPT)/resistant to bortezomib (BTZ) regimen activates of the extrinsic/intrinsic apoptotic pathway. (A) U266 were incubated with CPT ± BTZ for 48 h. Caspase‐8, Caspase‐3, poly‐ADP ribose polymerase (PARP), Fas‐associated protein with death domain (FADD), and cellular FLICE‐inhibitory protein (c‐FLIP) were monitored by immunoblotting analysis. CF = cleavage fragment. α‐tubulin was assayed to ensure equivalent loading and transfer. (B) U266/EV, U266/DN‐FADD and U266/DN‐Casp 8 were treated with the indicated concentrations of CPT ± BTZ for 48 h. FADD, Caspase 8 and PARP were monitored by immunoblotting analysis. CF = cleavage fragment. α‐tubulin was assayed to ensure equivalent loading and transfer. (C) Cells were treated as mentioned in B, followed by flow cytometric analysis of cell death after staining with 7‐AAD. The percentages of 7‐AAD (+) cells are presented. (D) U266 cells were incubated with indicated doses of CPT ± BTZ for 48 h. Cytochrome C and second mitochondrial activator of caspases (SMAC) were monitored by immunoblotting analysis. (E) U266 cells were incubated with of CPT ± BTZ for 48 h. Caspase 9 monitored by immunoblotting analysis. (F, left panel) U266 cells were pre‐treated for 30 min with caspase nine inhibitor (Z‐LEHD‐FMK, 20 μM) and then incubated with BTZ (2 nM) + CPT (30 ng/ml) for 48 h. After treatment, cells were subjected to flow cytometry to determine the percentage of death (7‐AAD⁺ cells). (F, right panel) Immunoblotting analysis was then performed to monitor levels of cleaved Caspase‐3. (G) U226/EV, U226/BCL‐X_L, and U226/BCL‐2 cells were incubated with CPT (10 ng/ml) ± BTZ (3 nM) for 48 h. BCL‐X_L, BCL‐2 and PARP were monitored by immunoblotting analysis. CF = cleavage fragment. (H) The percentages of 7‐AAD (+) cells are presented. Glyceraldehyde‐3‐Phosphate Dehydrogenase, β‐actin, or α‐tubulin was assayed to ensure equivalent loading and transfer. **p < 0.01; ***p < 0.001

FIGURE 3

The circularly permutated TRAIL (CPT)/resistant to bortezomib (BTZ) regimen inhibits non‐canonical NF‐κB signaling pathway (A,B) U266 or PS‐R cells were incubated with CPT ± BTZ for 48 h (A) p‐p65 (S536) was monitored by immunoblotting analysis. (B) DNA binding of NF‐kB (p65 subunit) was determined by using a TransAM assay for NF‐kB. (C) U266 or PS‐R cells were treated with CPT ± BTZ for 40 h. DNA binding of NF‐kB (p52 subunit). (D) Cells were treated as mentioned in A. TNF receptor associated factors 3 (TRAF3) and p‐p100/p52 (S864) were monitored by immunoblotting analysis. (E) U266 or PS‐R cells were treated with indicated doses of CPT ± BTZ for 48 h. NF‐κB inducing kinase (NIK) and p52 were monitored by immunoblotting analysis. (F) U266/shNC and U266/shRNA targeting TNF receptor associated factor 3 (shTRAF3) cells were exposed to the indicated concentrations of CPT ± BTZ for 48 h. Immunoblotting analysis was then performed to monitor levels of TRAF3, B‐cell lymphoma‐extra large (BCL‐X_L), Caspase‐8 and poly‐ADP ribose polymerase (PARP). CF = cleavage fragment. α‐tubulin or β‐actin was assayed to ensure equivalent loading and transfer. (G) Cells were treated as mentioned in F, followed by flow cytometric analysis of cell death after staining with 7‐AAD. The percentages of 7‐AAD (+) cells are presented. *p < 0.05

FIGURE 4

The circularly permutated TRAIL (CPT)/resistant to bortezomib (BTZ) regimen inhibits primary multiple myeloma (MM) cell growth ex vivo and MM cell growth in vivo. (A) Patient‐derived bone marrow mononuclear cells were isolated and treated with indicated doses of CPT ± BTZ for 24 h, after which the cells were stained with CD138‐PE. Flow cytometric analysis was performed to determine the CD138+ population. Combination index (CI) values less than 1.0 denote a synergistic interaction. (B) Representative primary bone marrow cells from a patient with MM (RR, relapse and refractory; prior BTZ) were exposed to 10 ng/ml CPT +/− 1.5 nM BTZ for 24 h, after which the cells were stained with CD138‐PE and annexin V‐fluorescein isothiocyanate (FITC). Images were obtained with an IX71‐Olympus inverted system microscope at × 40 magnification. (C) Experiments were carried out with 4 primary cord blood (CB) CD34⁺ samples. p > 0.05. (D–G) 20 SPF‐grade NOD‐SCID mice were inoculated via flank s.c. with 10 × 10⁶ patient‐derived MM cells. Mice were randomized to 4 groups (n = 5/group). Treatment was initiated after the tumor size was about 40–100 mm³. Mice were administered subcutaneously with BTZ 0.5 mg/kg (at day 1, 4, 8 and 11) ± CPT 10 mg/kg (daily). Tumor growth and body weight were monitored weekly (D, G). At day 36, tumors were harvested and dissected into small pieces. Immunoblotting analysis was then performed to monitor levels of c‐PARP, cellular FLICE‐inhibitory protein (c‐FLIP), and TNF receptor associated factors 3 (TRAF3). α‐tubulin was assayed to ensure equivalent loading and transfer (E, F)