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X. Li et al.
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Figure 2. CUDC-907 and venetoclax synergize in primary acute myeloid leukemia cells and cooperatively prevent colony formation of acute myeloid leukemia pro- genitor cells while sparing normal hematopoietic progenitor cells ex vivo. (A) Primary acute myeloid leukemia (AML) patient samples were treated with vehicle con- trol, venetoclax (VEN), CUDC-907 (CUDC), or in combination for 24 hours. Flow cytometry analysis of Annexin-V-FITC/PI staining was performed. Results are shown as mean percent Annexin V+ cells ± standard error of the mean (SEM). Combination index (CI) values were calculated using CompuSyn software. **P<0.01 and ***P<0.001 compared to single drug treatments. (B) Primary AML patient samples were cultured with vehicle control, venetoclax, CUDC-907, or in combination for 24 hours and then plated in methylcellulose. After incubation for 10-14 days, the number of surviving AML cells capable of generating leukemia colonies (AML-CFU) were enumerated. Data are presented as mean ± SEM. #P<0.05, ##P<0.01, and ###P<001 compared to control. *P<0.05, *P<0.01, and ***P<0.001 compared to single drug treatments. Technical triplicates were performed. (C) Normal human bone marrow mononuclear cells from a single donor were cultured with vehicle con- trol, venetoclax, CUDC-907, or in combination for 24 hours, and then plated in methylcellulose. After incubation for 10-14 days, the number of surviving hematopoi- etic cells capable of generating colonies were enumerated. Total erythroid and myeloid colonies are presented as mean ± SEM (left panel). The number of BFU-E, CFU-E, CFU-G, CFU-M, CFU-GM, and CFU-GEMM colonies are presented as mean ± SEM (right panel).
activity of the combination (cell line characteristics are shown in the Online Supplementary Table S2). Co-treat- ment of AML cells with CUDC-907 and venetoclax increased the proportion of Annexin V positive cells (indicative of apoptosis) significantly compared to control and single treatment groups (Figure 1A). Combination index (CI) values demonstrate synergy between the two drugs in all AML cell lines tested. Western blots revealed increased cleavage of caspase 3 and PARP by combined
drug treatments compared to single treatments (Figure 1B), demonstrating that the cells underwent apoptosis and further confirming the flow cytometry data. In order to test efficacy and tolerance of CUDC-907 and venetoclax co-treatment in vivo, immunocompromised NSGS mice were inoculated with MV4-11 cells and treated with vehi- cle control, 85 mg/kg/inj venetoclax, and/or 100 mg/kg/inj CUDC-907. The mice were treated for 8 days, given a 4- day break, and then treated for 6 additional days. The 4-
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