MM cells are sensitive to E. chrysanthemi ASNase treatment. (A) HMCLs were treated with increasing doses of l-asparaginase (ASNase) from E. coli or E. chrysanthemi (from 0.0001 to 1 U/mL). After 48 hours, cell viability was assessed, and data were expressed as % of the value obtained with untreated cells. For each HMCL, IC₅₀ for E. coli ASNase and for the E. chrysanthemi enzyme are shown. (B) HMCLs were treated with increasing doses of bortezomib (from 1.77 to 10 nM) or vehicle in the presence or in the absence of E. chrysanthemi ASNase (0.1 U/mL). After 48 hours, cell viability was assessed, and data were expressed as % of the value obtained with the cells treated with vehicle. For panels A and B, data are means ± SD of 3 experiments with 3 determinations each. (C) RPMI 8226 were treated with increasing doses of bortezomib (from 1 to 16 nM), increasing doses of E. chrysanthemi ASNase (from 0.0625 to 1 U/mL), or the combination of the 2 drugs (16:1) or vehicle. After 48 h, cell viability was assessed, and the data were analyzed as % of the value obtained with the cells treated with vehicle. Combination index analysis was then performed using CompuSyn software. Isobologram for ED₇₅ represents means ± SEM of 3 experiments with 5 determinations each. (D-E) RPMI 8226 and JJN3 cells were treated for 24 hours with BPTES (40 µM), CB-839 (1 µM), or ASNase from E. coli (1 U/mL), or ASNase from E. chrysanthemi (0.1 U/mL), or bortezomib (10 nM), or ASNase from E. chrysanthemi (0.1 U/mL) and bortezomib (10 nM), or vehicle. For panel D, cell expression of Apo 2.7 was then evaluated by flow cytometry. The graph shows the mean % plus SD (n = 3) of Apo 2.7 positive cells for each condition after the subtraction of the value obtained in control. For panel E, cells expression of cleaved forms of caspase 3 in HMCLs, evaluated by Western blot. β-Actin was used for loading control. For panels A, B, and D, *P < .05, ***P < .001 vs control.