ISG20L2 suppresses bortezomib antimyeloma activity by attenuating bortezomib binding to PSMB5
Yan Yang, Yuhan Gao, Jingcao Huang, Zhuang Yang, Hongmei Luo, Fangfang Wang, Juan Xu, Yushan Cui, Hong Ding, Zhimei Lin, Xinyu Zhai, Ying Qu, Li Zhang, Ting Liu, Lingqun Ye, Ting Niu, Yuhuan Zheng
Yan Yang, Yuhan Gao, Jingcao Huang, Zhuang Yang, Hongmei Luo, Fangfang Wang, Juan Xu, Yushan Cui, Hong Ding, Zhimei Lin, Xinyu Zhai, Ying Qu, Li Zhang, Ting Liu, Lingqun Ye, Ting Niu, Yuhuan Zheng
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Research Article Hematology

ISG20L2 suppresses bortezomib antimyeloma activity by attenuating bortezomib binding to PSMB5

Abstract

The proteasome inhibitors (PIs) bortezomib and carfilzomib, which target proteasome 20S subunit beta 5 (PSMB5) in cells, are widely used in multiple myeloma (MM) treatment. In this study, we demonstrated the role of interferon-stimulated 20 kDa exonuclease-like 2 (ISG20L2) in MM PI resistance. Gain- and loss-of-function studies showed that ISG20L2 suppressed MM cell sensitivity to PIs in vitro and in vivo. Patients with ISG20L2lo MM had a better response to PIs and a longer overall survival than patients with ISG20L2hi MM. Biotinylated bortezomib pull-down assays showed that ISG20L2 competed with PSMB5 in binding to bortezomib. The surface plasmon resonance assay confirmed the direct binding of bortezomib to ISG20L2. In ISG20L2hi MM cells, ISG20L2 attenuated the binding of bortezomib to PSMB5, resulting in lower inhibition of proteasome activity and therefore less bortezomib-induced cell death. Overall, we identified a potentially novel mechanism by which ISG20L2 conferred bortezomib resistance on MM. The expression of ISG20L2 correlated with MM PI responses and patient treatment outcomes.

Authors

Yan Yang, Yuhan Gao, Jingcao Huang, Zhuang Yang, Hongmei Luo, Fangfang Wang, Juan Xu, Yushan Cui, Hong Ding, Zhimei Lin, Xinyu Zhai, Ying Qu, Li Zhang, Ting Liu, Lingqun Ye, Ting Niu, Yuhuan Zheng

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Figure 5

ISG20L2 competes with PSMB5 in BTZ binding.

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ISG20L2 competes with PSMB5 in BTZ binding. (A) Molecular structure of b...
(A) Molecular structure of biotinylated BTZ (BTZ-b). (B) Flow cytometry–based apoptosis assay to examine the cytotoxicity of BTZ-b in CTR-KD versus ISG-KD (sh1) ARD cells. The cells were treated with BTZ-b (2 μM) for 24 hours. DMSO- or biotin-treated cells served as CTRs (n = 3). Student’s t test was performed. *P ≤ 0.05. (C) BTZ-b pull-down assay using ARD cell lysate in the presence of BTZ to examine BTZ competition with BTZ-b in PSMB5 binding. BTZ was added to the cell lysate to a final concentration of 200 μM and incubated with BTZ-b–coated beads for competition. (D) Biotinylated BTZ pull-down assay using CTR-KD and ISG-KD ARD cell lysates. Biotin served as a negative control for pull-down. The pull-down precipitate and the whole-cell lysates (input) were subjected to Western blotting. (E) BTZ-b pull-down assay using CTR and ISG-OE AMO1 cell lysates. (F) BTZ-b pull-down assay using recombinant ISG20L2 (rISG) and PSMB5 (rB5) proteins. The recombinant protein was dissolved in PBS. The final concentration of rB5 was 0.5 μg/mL (+), while the final concentration of rISG was 0.25 μg/mL (+) or 1.5 μg/mL (++). Using the gray intensity of PSMB5 pull-down by BTZ-b alone as a control, ImageJ software (NIH) was used to determine the levels of PSMB5 pull-down by BTZ-b with rISG competition. Numbers indicated on the lane. (G) BTZ binds to ISG20L2 as shown by SPR measurements. Graphs of equilibrium response unit (RU) responses versus compound concentrations were plotted. The estimated KD is 26.71 μM, as shown by the vertical dotted line. (H) Schematic mechanism of ISG20L2-induced MM BTZ resistance.