Figure 6.
Figure 6. Targeting IDH2 and proteasome activities triggers synergistic inhibition of human MM cells growth ex vivo and in vivo with low toxicity to normal human cells. (A) Buffy coats derived from bone marrow aspirates of MM patients were treated with CFZ (2.5 nM) in combination or not with AGI-6780 (5 μM). Cell viability was estimated by flow cytometry measuring annexin V− and CD138+ cells 96 hours posttreatment. Histograms represent the percentage of viable cells normalized vs DMSO samples. Data are the means ± SEM of 9 independent MM patients. (B) PBMC and KMS-27 were treated with AGI-6780 and increasing doses of CFZ. PBMC were derived from 4 healthy donors. Cell viability was measured by TMRM staining-flow cytometry 48 hours posttreatment. Data are the means ± SD (*P < .05; **P < .01; ***P < .001). (C) KMS-27-TK cells (expressing DsRed fluorescent protein) were co-cultured with HS-5 bone marrow/stroma cell line and treated with CFZ, AGI-6780, or a combination. Percentage of live DsRed+ cells was measured overtime. Data are the means ± SD of 3 independent experiments (CFZ vs CFZ+AGI-6780, **P < .01). (D) Growth patterns of KMS-27-TK-IDH2-A4 cells injected subcutaneously into the flanks of NSG mice. Tumor masses of 0.5 cm diameter mice were randomized for treatment with vehicle (n = 6), 4 mg/kg CFZ (n = 8), 0.1 mg/mL DOXY (n = 10), or a combination of both compounds (n = 10) over 3 weeks. Administration of either agent had a substantial effect on tumor growth compared with control mice (P < .0001). Combination of IDH2 silencing with CFZ further reduced tumor growth in relation to single treatments (CFZ vs CFZ/DOXY, P = .0244; DOXY vs CFZ/DOXY, P = .0238). Each data point represents the average tumor volume (mean ± SEM) for the indicated treatment condition. The timeline shows the schedule of treatment followed for in vivo treatments. (E) Kaplan-Meier survival plot showing survival for mice treated with vehicle (n = 6), 4 mg/kg CFZ (n = 6), 0.1 mg/mL DOXY (n = 8), or a combination (n = 6). CFZ plus DOXY-treated mice show significantly increased survival (49 days) in comparison with vehicle-treated mice (26 days; P < .0001), CFZ alone (35 days; P = .0007), and DOXY alone (38 days; P = .0472). PBMC, Peripheral blood mononuclear cell; SEM, standard error of the mean.

Targeting IDH2 and proteasome activities triggers synergistic inhibition of human MM cells growth ex vivo and in vivo with low toxicity to normal human cells. (A) Buffy coats derived from bone marrow aspirates of MM patients were treated with CFZ (2.5 nM) in combination or not with AGI-6780 (5 μM). Cell viability was estimated by flow cytometry measuring annexin V− and CD138+ cells 96 hours posttreatment. Histograms represent the percentage of viable cells normalized vs DMSO samples. Data are the means ± SEM of 9 independent MM patients. (B) PBMC and KMS-27 were treated with AGI-6780 and increasing doses of CFZ. PBMC were derived from 4 healthy donors. Cell viability was measured by TMRM staining-flow cytometry 48 hours posttreatment. Data are the means ± SD (*P < .05; **P < .01; ***P < .001). (C) KMS-27-TK cells (expressing DsRed fluorescent protein) were co-cultured with HS-5 bone marrow/stroma cell line and treated with CFZ, AGI-6780, or a combination. Percentage of live DsRed+ cells was measured overtime. Data are the means ± SD of 3 independent experiments (CFZ vs CFZ+AGI-6780, **P < .01). (D) Growth patterns of KMS-27-TK-IDH2-A4 cells injected subcutaneously into the flanks of NSG mice. Tumor masses of 0.5 cm diameter mice were randomized for treatment with vehicle (n = 6), 4 mg/kg CFZ (n = 8), 0.1 mg/mL DOXY (n = 10), or a combination of both compounds (n = 10) over 3 weeks. Administration of either agent had a substantial effect on tumor growth compared with control mice (P < .0001). Combination of IDH2 silencing with CFZ further reduced tumor growth in relation to single treatments (CFZ vs CFZ/DOXY, P = .0244; DOXY vs CFZ/DOXY, P = .0238). Each data point represents the average tumor volume (mean ± SEM) for the indicated treatment condition. The timeline shows the schedule of treatment followed for in vivo treatments. (E) Kaplan-Meier survival plot showing survival for mice treated with vehicle (n = 6), 4 mg/kg CFZ (n = 6), 0.1 mg/mL DOXY (n = 8), or a combination (n = 6). CFZ plus DOXY-treated mice show significantly increased survival (49 days) in comparison with vehicle-treated mice (26 days; P < .0001), CFZ alone (35 days; P = .0007), and DOXY alone (38 days; P = .0472). PBMC, Peripheral blood mononuclear cell; SEM, standard error of the mean.

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