Figure 5
A novel inhibitor of mutant IDH1 inhibits human AML cell growth. (A) Structural model of the IDH1R132H dimer showing binding of HMS-101 and NAD phosphate (NADP) to the active center of IDH1. (B) Cell viability of HoxA9+IDH1wt and HoxA9+IDHmut cells incubated with increasing concentrations of HMS-101 (mean ± SEM of 4 independent experiments). (C) Ratio of R-2HG and S-2HG in HoxA9+IDHmut-transduced bone marrow cells incubated with 10 µM HMS-101 for 72 hours (mean ± SEM, n = 3). (D) Induction of apoptosis by HMS-101 (10 µM) indicated by the proportion of annexin V positive cells in murine bone marrow cells transduced with HoxA9 CTL, HoxA9 IDH1wt, and HoxA9 IDH1mut after 72 hours of treatment (mean ± SEM, n = 3). (E) Representative FACS blots of cell-cycle phases in HoxA9 CTL, HoxA9 IDH1wt, and HoxA9 IDH1mut cells treated with HMS-101 (10 µM) for 72 hours. Cell-cycle phases were determined by analysis of BrdU/7AAD-stained cells by FACS. The upper quadrant represents cells in S phase, the lower left quadrant represents apoptotic cells, the lower middle quadrant represent cells in G0/G1 phase, and the lower right quadrant represents cells in G2/M phase of the cell cycle. (F) Quantitative analysis of cell-cycle distribution after treatment with HMS-101 (10 µM) in the HoxA9 CTL, HoxA9 IDH1wt, and HoxA9 IDH1mut cell lines for 72 hours as described in Figure 5E (mean ± SEM, n = 3). (G) Representative western blot of in vitro–cultured cells treated with HMS-101 for the indicated murine cell lines using antibodies against phospho-ERK, total-ERK, and β-actin as loading control. (H) Induction of apoptosis by HMS-101 (10 µM) in the indicated cell types relative to dimethylsulfoxide (DMSO)-treated cells after suspension culture for 72 hours. (I) Inhibition of colony formation in CFC assays using human cells from healthy donors or AML patients with wild-type or mutant IDH1 by HMS-101 relative to DMSO-treated cells (mean ± SEM). *P < .05; **P < .01; ns, not significant.

A novel inhibitor of mutant IDH1 inhibits human AML cell growth. (A) Structural model of the IDH1R132H dimer showing binding of HMS-101 and NAD phosphate (NADP) to the active center of IDH1. (B) Cell viability of HoxA9+IDH1wt and HoxA9+IDHmut cells incubated with increasing concentrations of HMS-101 (mean ± SEM of 4 independent experiments). (C) Ratio of R-2HG and S-2HG in HoxA9+IDHmut-transduced bone marrow cells incubated with 10 µM HMS-101 for 72 hours (mean ± SEM, n = 3). (D) Induction of apoptosis by HMS-101 (10 µM) indicated by the proportion of annexin V positive cells in murine bone marrow cells transduced with HoxA9 CTL, HoxA9 IDH1wt, and HoxA9 IDH1mut after 72 hours of treatment (mean ± SEM, n = 3). (E) Representative FACS blots of cell-cycle phases in HoxA9 CTL, HoxA9 IDH1wt, and HoxA9 IDH1mut cells treated with HMS-101 (10 µM) for 72 hours. Cell-cycle phases were determined by analysis of BrdU/7AAD-stained cells by FACS. The upper quadrant represents cells in S phase, the lower left quadrant represents apoptotic cells, the lower middle quadrant represent cells in G0/G1 phase, and the lower right quadrant represents cells in G2/M phase of the cell cycle. (F) Quantitative analysis of cell-cycle distribution after treatment with HMS-101 (10 µM) in the HoxA9 CTL, HoxA9 IDH1wt, and HoxA9 IDH1mut cell lines for 72 hours as described in Figure 5E (mean ± SEM, n = 3). (G) Representative western blot of in vitro–cultured cells treated with HMS-101 for the indicated murine cell lines using antibodies against phospho-ERK, total-ERK, and β-actin as loading control. (H) Induction of apoptosis by HMS-101 (10 µM) in the indicated cell types relative to dimethylsulfoxide (DMSO)-treated cells after suspension culture for 72 hours. (I) Inhibition of colony formation in CFC assays using human cells from healthy donors or AML patients with wild-type or mutant IDH1 by HMS-101 relative to DMSO-treated cells (mean ± SEM). *P < .05; **P < .01; ns, not significant.

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