Combined SUMO and proteasome inhibition acts synergistically in MM cell lines. (A) Combination of subasumstat with the proteasome inhibitors bortezomib (BTZ) and CFZ has a synergistic effect on the viability of THE indicated MM cells. Synergy score has been determined by SynergyFinder using the Zero Interaction Potency method (ZIP). The presented ZIP synergy scores are the average of n = 3 independent biological experiments with n = 3 technical replicates. Cells were treated with single and combination treatments using a 4 × 6 matrix. (B) Landscape plots depicting the synergistic area of concentrations for subasumstat and CFZ combination treatment in JJN3, OPM2, and AMO1 cells. Cells were treated for 72 hours with the indicated concentrations (4 × 6 matrix) of subasumstat and CFZ, and cell viability was measured by CellTiterGlo. Subsequently, cell viability data were used to generate landscape plots using SynergyFinder. (C) Bar diagrams showing the effect on cell viability after 72 hours of treatment with CFZ, subasumstat, and the combination thereof in JJN3 (2 nM CFZ, 200 nM subasumstat), OPM2 (2 nM CFZ, 200 nM subasumstat), and AMO1 (4 nM CFZ, 200 nM subasumstat) cells. Statistical testing was determined by one-way analysis of variance. (D) Bar diagram showing the effect on cell viability after 72 hours of treatment with 6 nM (JJN3) or 12 nM (AMO1) CFZ, 200 nM (JJN3) or 1 μM (AMO1) subasumstat, and the combination thereof in parental (P) and CFZ-resistant (R) cells. Statistical testing was determined by one-way analysis of variance. ∗P ≤ .05, ∗∗P ≤ .01, ∗∗∗∗P ≤ .0001. Suba, subasumstat.