Figure 3. Combination with proteasome inhibitors overcomes selinexor resistance in cell lines.

A. HT-1080-R cells were treated with 1 μM of selinexor and/or 100 nM bortezomib for 12 hours. The cells were fixed and the cellular localization of IκB-α was evaluated by immunofluorescence microscopy. Selinexor treatment showed minimal nuclear entrapment of IκB-α, but combined treatment with selinexor and bortezomib further increased nuclear retention of IκB-α. B. HT-1080-R cells were pre-treated with 3μM of selinexor (Sel) and/or 100nM bortezomib (Bortz) for 2 hours and then exposed to 20ng/mL TNFα for 4 hours in serum free media followed by evaluation for DNA binding activity. TNFα treatment induced NF-κB transcriptional activity by 8-fold, whereas treatment with the combination of selinexor and bortezomib reduced the activity by 60% compared to 20% and 40% by the single agent respectively. The error bars indicate standard deviation and the Student's t-test was used to calculate p values. *p<0.05. C. Sub-cellular localization of XPO1 cargos in HT-1080-R cells treated with 1 μM of selinexor and/or 100 nM bortezomib for 12 hours was evaluated by cellular fractionation and Western blotting. The combination treatment of selinexor and bortezomib increased nuclear levels of XPO1 cargos compared to either single agent treatment. Lamin B served as a nuclear protein marker; GAPDH as a cytosolic protein marker. D. HT-1080-R and ASPS-KY cells were treated with 1 μM of selinexor and/or 50 nM bortezomib for 24 hours. The combination of selinexor and bortezomib was more cytotoxic than either one of the single agents as indicated by pronounced cleavage of PARP and Caspase 3 with the combination.