The IAP antagonist birinapant potentiates bortezomib anti-myeloma activity in vitro and in vivo

Abstract

Background: Mechanisms by which Smac mimetics (SMs) interact with proteasome inhibitors (e.g., bortezomib) are largely unknown, particularly in multiple myeloma (MM), a disease in which bortezomib represents a mainstay of therapy.

Methods: Interactions between the clinically relevant IAP (inhibitor of apoptosis protein) antagonist birinapant (TL32711) and the proteasome inhibitor bortezomib were investigated in multiple myeloma (MM) cell lines and primary cells, as well as in vivo models. Induction of apoptosis and changes in gene and protein expression were monitored using MM cell lines and confirmed in primary MM cell populations. Genetically modified cells (e.g., exhibiting shRNA knockdown or ectopic expression) were employed to evaluate the functional significance of birinapant/bortezomib-induced changes in protein levels. A MM xenograft model was used to evaluate the in vivo activity of the birinapant/bortezomib regimen.

Results: Birinapant and bortezomib synergistically induced apoptosis in diverse cell lines, including bortezomib-resistant cells (PS-R). The regimen robustly downregulated cIAP1/2 but not the canonical NF-κB pathway, reflected by p65 phosphorylation and nuclear accumulation. In contrast, the bortezomib/birinapant regimen upregulated TRAF3, downregulated TRAF2, and diminished p52 processing and BCL-X_L expression, consistent with disruption of the non-canonical NF-κB pathway. TRAF3 knockdown, ectopic TRAF2, or BCL-X_L expression significantly diminished birinapant/bortezomib toxicity. The regimen sharply increased extrinsic apoptotic pathway activation, and cells expressing dominant-negative FADD or caspase-8 displayed markedly reduced birinapant/bortezomib sensitivity. Primary CD138⁺ (n = 43) and primitive MM populations (CD138^-/19⁺/20⁺/27⁺; n = 31) but not normal CD34⁺ cells exhibited significantly enhanced toxicity with combined treatment (P < 0.0001). The regimen was also fully active in the presence of HS-5 stromal cells or growth factors (e.g., IL-6 and VEGF). Finally, the regimen was well tolerated and significantly increased survival (P < 0.05 and P < 0.001) compared to single agents in a MM xenograft model. Combined treatment also downregulated cIAP1/2 and p52 while increasing PARP cleavage in MM cells in vivo.

Conclusions: Our data suggest that birinapant and bortezomib interact synergistically in MM cells, including those resistant to bortezomib, through inactivation of the non-canonical NF-κB and activation of the extrinsic apoptotic pathway both in vitro and in vivo. They also argue that a strategy combining cIAP antagonists and proteasome inhibitors warrants attention in MM.

Keywords: Bortezomib; IAP antagonist; Multiple myeloma; NF-κB.

Fig. 1

TL32711 interacts synergistically with Btz to induce apoptosis in both Btz-naïve MM cells and Btz-resistant MM cells. a–b Btz-naïve U266 cells or Btz-resistant PS-R cells were exposed to the indicated concentrations of Btz +/− TL for 24 h (U266) or 48 h (PS-R), followed by flow cytometric analysis of cell death after staining with 7-AAD. c U266 and PS-R cells were exposed (24 h or 48 h) to varying concentrations of Btz +/− TL at a fixed ratio (1:100 or 1:30), after which the percentage of annexin V+ cells was determined. Median dose-effect analysis was then employed to characterize the nature of the interaction between these agents with constant ratio. The fraction affected (FA) less than 1.0 reflect synergistic interactions. The results are representative of three separate experiments. d U266 and PS-R cells were incubated with Btz +/− TL for 24 h or 48 h, after which γH2A.X and cleavage of Caspase-3 and PARP were monitored by immunoblotting analysis. CF, cleavage fragment. β-actin was assayed to ensure equivalent loading and transfer. **P < 0.01; ***P < 0.001

Fig. 2

TL +/− Btz downregulates cIAP1/2 but does not inactivate the canonical NF-κB pathway. a U266 and PS-R cells were treated with Btz +/− TL for 24 h, after which cIAP1 and cIAP2 were monitored by immunoblotting analysis. α-Tubulin was assayed to ensure equivalent loading and transfer. b U266 cells were exposed to the indicated concentrations of Btz +/− TL for 16 h, after which nuclear protein was extracted from the cells. Immunoblotting analysis was then performed to monitor levels of p65. p84 was assayed to ensure equivalent loading and transfer. c U266 cells were incubated with 500 nM TL32711 +/− 3 nM Btz for 4 h, 8 h, and 16 h, after which p-p65 (S536) was monitored by immunoblotting analysis. β-actin was assayed to ensure equivalent loading and transfer. d Following treatment as in b, nuclear proteins were isolated using a Nuclear Extract Kit. DNA binding of NF-κB (p65 subunit) was determined using TransAM for NF-κB activity. ***P < 0.001; NS not significant

Fig. 3

Upregulation of TRAF3 plays a functional role in TL/Btz-induced apoptosis. a U266 cells were treated with Btz +/− TL for 24 h, after which TRAF3, TRAF2, p52, and BCL-X_L were monitored by immunoblotting analysis. β-actin was assayed to ensure equivalent loading and transfer. b U266 cells were exposed to the indicated concentrations of Btz +/− TL for 16 h, after which nuclear protein was extracted from the cells. Immunoblotting analysis was then performed to monitor levels of p52. p84 was assayed to ensure equivalent loading and transfer. c DNA binding of NF-κB (p52 subunit) was determined using a TransAM assay for NF-κB activity. d–e U266 cells were stably transfected with constructs encoding shRNA targeting TRAF3 (shTRAF3) or scrambled sequence as a negative control (shNC). Cells were treated with Btz +/− TL for 24 h, after which cell death was analyzed by flow cytometry following staining with 7-AAD (e). The results shown are representative of three separate experiments. Immunoblotting analysis was carried out to monitor TRAF3, p52, caspase-3, and PARP (d). A black line separates images acquired from different regions of the same gel with identical exposures. Densitometry analysis was performed using ImageJ. Values indicate fold-change of p52 versus untreated control (arbitrarily set as 1.0), after normalization to β-actin. CF, cleavage fragment. β-actin and GAPDH were assayed to ensure equivalent loading and transfer. *P < 0.05; **P < 0.01

Fig. 4

Overexpression of TRAF2 or BCL-X_L significantly diminishes TL/Btz-induced apoptosis. a–c U266/GFP-TRAF2 and U266/GFP cells were established by stably transfecting cells with full-length human TRAF2 cDNA or empty vector. Cells were treated with Btz +/− TL for 24 h. a Immunoblotting analysis was performed to monitor TRAF2 and p52. GAPDH was assayed to ensure equivalent loading and transfer. Endo, endogenous. b Cytospin slides were prepared, stained with 7-AAD, and counterstained with DAPI. Images were obtained with an IX71-Olympus inverted system microscope at × 200 magnification. c After drug treatment, cells were subjected to flow cytometry to determine the percentage of dead (7-AAD⁺) cells in GFP⁺ cells (*P < 0.05; **P < 0.01). Values represent the means ± SD for at least three independent experiments performed in triplicate. d–e U266/BCL-X_L and U266/EV cells were established by stably transfecting full-length human BCL-X_L cDNA or empty vector. Cells were treated with Btz +/− TL for 24 h. After drug treatment, cells were subjected to flow cytometry to determine the percentage of dead (7-AAD⁺) cells (**P < 0.01). Values represent the means ± SD for at least three independent experiments performed in triplicate. e Immunoblotting analysis was performed to monitor BCL-X_L and PARP. A black line separates images acquired from different regions of the same gel with identical exposures. CF, cleavage fragment. β-actin was assayed to ensure equivalent loading and transfer

Fig. 5

Blockade of FADD significantly reduces TL/Btz-induced apoptosis. a U266 and PS-R cells were treated with Btz +/− TL for 24 h. Immunoblotting analysis was carried out to monitor caspase-8 expression. CF cleavage fragment. β-actin was assayed to ensure equivalent loading and transfer. b–c U266/DN-FADD and U266/EV cells were established by stably transfecting human dominant-negative FADD cDNA or empty vector. Cells were treated with Btz +/− TL for 24 h. c After drug treatment, cells were subjected to flow cytometry to determine the percentage of dead (7-AAD⁺) cells (**P < 0.01). Values represent the means ± SD for at least three independent experiments performed in triplicate. Immunoblotting analysis was performed to monitor FADD, caspase-8, and PARP expression. Endo, endogenous; CF. cleavage fragment. GAPDH was assayed to ensure equivalent loading and transfer

Fig. 6

TL/Btz circumvents microenvironment-driven intrinsic resistance. a GFP-labeled PS-R cells co-cultured with or without BM stromal HS-5 cells and were incubated with Btz +/− TL for 48 h. Apoptosis of GFP⁺ cells was analyzed by multi-color flow cytometry of 7-AAD staining. b Cells were stained with 7-AAD to monitor death of GFP⁺ cells. Bright field images were captured with an IX71-Olympus inverted system microscope at × 200 magnification. NS, not significant

Fig. 7

The TL/Btz regimen is active against primary CD138⁺ MM cells and diminishes primitive progenitor cell-enriched CD138⁻/CD19⁺/CD20⁺/CD27⁺ populations while sparing normal CD34⁺ cells. a Representative primary bone marrow cells from a patient with MM (RR, relapse and refractory; prior Btz) were exposed to 500 nM TL +/− 3 nM Btz for 24 h, after which the cells were stained with CD138-PE and annexin V-FITC. Images were obtained with an IX71-Olympus inverted system microscope at × 200 magnification. Flow cytometric analysis was performed to determine the CD138⁺ population. b After exposure to 500 nM TL +/− 3 nM Btz for 24 h, the percentage of primitive CD138⁻/CD19⁺/CD20⁺/CD27⁺ cells in bone marrow mononuclear cells from a primary MM sample was determined by multi-color FCM. c Primary cells from a patient with newly diagnosed MM were exposed to 500 nM TL +/−  3 nM Btz for 24 h in the presence of HS-5, after which they were stained with CD138-PE and annexin V-FITC. Images were captured with an IX71-Olympus inverted system microscope at × 200 magnification. d Parallel experiments were carried out with 43 primary samples. Viability of CD138⁺ cells was analyzed by multi-color flow cytometry determination of 7-AAD. Lines indicate means and SD (****P < 0.001). e After exposure to 500 nM TL +/− 3 nM Btz for 24 h, apoptosis of CD138⁻/CD19⁺/CD20⁺/CD27⁺ cells (n = 31) was analyzed by 7-AAD staining and quantitated by multi-color flow cytometry (****P < 0.001). f Parallel experiments were carried out with eight primary cord blood (CB) CD34⁺ samples. ns, not significant

Fig. 8

Co-administration of TL and Btz suppresses tumor growth in a MM xenograft model. a–d NOD/SCID-γ (NSG) mice were subcutaneously (s.c.) inoculated in the right rear flank with 5 × 10⁶ luciferase-expressing U266 cells. TL and Btz were administered via intra-peritoneal (i.p.) injection at a dose of 15 mg/kg (TL) and 0.5 mg/kg (Btz). a Tumors were monitored every other day after i.p. injection with 150 mg/kg luciferin using an IVIS 200 imaging system. Mice were euthanized when tumor length reached 17 mm or humane endpoints were reached; Veh, vehicle. b Tumor size was measured every other day. *P < 0.05 vs Btz; ****P < 0.0001 vs TL. c Kaplan-Meier analysis was carried out to analyze survival. Inset, median survival days. Arrows indicate the time when treatment began (day 18) and was discontinued (day 48). **P < 0.01 vs Btz. d Western blot analysis was performed to monitor the indicated candidate proteins, identified from in vitro studies, in tumors excised from representative mice. Densitometry analysis was performed using ImageJ. Values indicate fold-change of p52 versus untreated control (arbitrarily set as 1.0), after normalization to β-actin. e Mice did not display significant body weight loss (≥ 20%) compared to initial weight (P > 0.05 vs each single agent) or other signs of toxicity over the course of treatment