The novel β2-selective proteasome inhibitor LU-102 decreases phosphorylation of I kappa B and induces highly synergistic cytotoxicity in combination with ibrutinib in multiple myeloma cells

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Abstract

Purpose: Proteasome-inhibiting drugs (PI) are gaining importance in hematologic oncology. The proteasome carries three proteolytically active subunits (β1, β2, β5). All established PI (bortezomib and carfilzomib), as well as experimental drugs in the field (dalanzomib, oprozomib, and ixazomib), by design target the rate-limiting β5 subunit. It is unknown whether β2-selective proteasome inhibition can also be exploited toward anticancer treatment. Combining PI with the pan B-cell-directed Bruton tyrosine kinase inhibitor ibrutinib appears a natural option for future improved treatment of multiple myeloma (MM) and B-cell lymphomas. However, bortezomib induces phosphorylation of IκB and activation of NF-κB in MM cells, while ibrutinib inhibits the IκB/NF-κB axis, suggesting antagonistic signaling. A β2-selective proteasome inhibitor may lack such antagonistic signaling effects. Methods: We recently introduced LU-102, the first β2-selective PI available for preclinical testing. We here compare bortezomib with carfilzomib and LU-102 in MM and MCL in vitro with regard to their effects on pIκB/NF-κB signaling and their cytotoxic activity in combination with ibrutinib. Results: LU-102 reduced phosphorylation of IκB, in contrast to bortezomib and carfilzomib, and was a superior inhibitor of NF-κB activation in MM cells. This translated into highly synergistic cytotoxicity between LU-102 and ibrutinib, which was able to overcome BTZ resistance and CFZ resistance. By contrast, BTZ lacked consistent synergistic cytotoxicity with ibrutinib. Conclusion: Ibrutinib is highly synergistic with β2-selective proteasome inhibition against MM and MCL in vitro. Novel β2-selective proteasome inhibitors may be exploited to overcome bortezomib/carfilzomib resistance and boost the activity of BTK inhibitors against B-cell-derived malignancies.

Figures

  • Fig. 1 BTK expression and ibrutinib-mediated cytotoxicity in MM cell lines. a Upper panel MM cell lines (AMO-1, AMO-BTZ, AMO-CFZ, INA-6, JK-6, L363, LP-1 MM.1R, MM.1S, RPMI 8226 and U-266), MCL cell lines (Granta-519 and Jeko-1), and AML cell line (THP-1) were analyzed with respect to protein expression of BTK. After cell lysis, equal amounts of protein were resolved by SDS-PAGE, and Western blots against BTK and activated BTK (p-BTK) were performed. Ponceau S staining of the same PVDF membrane that was
  • Fig. 2 Molecular effects of ibrutinib on the target proteins p-BTK/BTK, the downstream p-IκB/IκB activation and proteasome subunit activity. a Upper panel INA-6 cells were incubated with increasing ibrutinib concentrations (0–10 µM) for 4 h, and p-BTK and BTK proteins were assessed by Western blot. The bar graph illustrates the quantitative comparison of the fluorescence signals retrieved for p-BTK protein at the respective ibrutinib concentrations, relative to baseline (DMSO-treated). Lower panel INA-6 cells were incubated with increasing ibrutinib concentrations (0–10 µM) for 8 h, before IκB and activated IκB (p-IκB) proteins were determined by Western blot and quantified as described above. For a statistically significant quantitative difference from baseline, *p < 0.05; **p < 0.01. b After incubation with increasing ibrutinib concentrations (0–10 µM), active proteasome subunits in INA-6 cells were affinity-labeled using the fluorescent, pan-proteasome reactive, cell-permeable probe MV-151 for 1 h. After resolution by SDS-PAGE, the fluorescence signals representing active proteasome β1, β2, β5 polypeptides and the respective immuno-proteasome species β1i, β2i, β5i were visualized using a fluorescent reader and quantitated. Conventional Western blotting against GAPDH demonstrates equal protein load of the samples. The bar graph above illustrates the quantitative comparison of the specific fluorescence signals detected for the proteasomal β1(i)/β5(i) and β2(i) activities, relative to DMSO-treated baseline
  • Fig. 3 Molecular effects of bortezomib, carfilzomib, and the β2-specific proteasome inhibitor LU-102 on proteasome subunit activities, the p-IκB/IκB ratio and polyubiquitinated protein. Left panels After incubation with increasing proteasome inhibitor concentrations (bortezomib, carfilzomib: 0–33.3 nM; LU-102: 0–10 µM), active proteasome subunits in INA-6 cells were affinity-labeled using
  • Fig. 5 Cytotoxic effect of ibrutinib in combination with proteasome inhibitors. MCL cell lines (Jeko-1 and Granta-519) and MM cell lines (LP-1, MM.1R and RPMI 8226) were incubated with ibrutinib 10 µM (IBR), bortezomib 10 nM (BTZ), carfilzomib 10 nM (CFZ) and LU-102 3.3 µM (LU), or a combination of ibrutinib with one of the proteasome inhibitors for 48 h, and cell viability was assessed by MTT test. Results are expressed relative to cells treated with DMSO (0)
  • Table 1 Combination Index (CI) for cytotoxic activity, MTT test
  • Fig. 6 Visualization of ibrutinib-targeted proteins using a fluorescent synthetic ibrutinibanalog as activity-based covalent problem. a DLBCL type cell lines (HBL1, OciLy3, SU-DHL4), MM (INA-6, RPMI 8226) and the HaCat melanoma cell line were incubated with either DMSO, ibrutinib (IBR), or a mock version of ibrutinibABP lacking the active warhead (mock, 1 µM each), before cells were exposed to activitybased covalent affinity labeling (1 μM) by incubation with ibrutinib-ABP. After cell lysis, cellular protein was dissolved by SDS-PAGE and fluorescent signals visualized using a fluorescence reader. Western blotting for β-actin served as loading control from the same samples. b BTK-expressing (Granta-519, HL-60) cell lines and cell lines without BTK expression (Jeko-1, AMO-1, AMO-BTZ, as well as the breast cancer cell line BT-549) were incubated with DMSO (0), ibrutinib (IBR), or a mock treated as before, and ibrutinibreactive protein species were visualized as before. Western blotting for β-actin served as loading control

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Kraus, J., Kraus, M., Liu, N., Besse, L., Bader, J., Geurink, P. P., … Driessen, C. (2015). The novel β2-selective proteasome inhibitor LU-102 decreases phosphorylation of I kappa B and induces highly synergistic cytotoxicity in combination with ibrutinib in multiple myeloma cells. Cancer Chemotherapy and Pharmacology, 76(2), 383–396. https://doi.org/10.1007/s00280-015-2801-0

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