Figure 3. EZH2 inhibition enhances apoptosis of cytotoxic agent–treated AML cells. A, THP-1, KG-1, and CD34⁺ AML primary cells (sample #40) were treated with doxorubicin alone (0.1, 0.3, or 0.5 μmol/L), or in combination with GSK126 (5 μmol/L), for 48 hours. Apoptosis (caspase-3/7 activity) was analyzed by flow cytometry. Graph bars represent fold change ± SEM in the percentage of caspase-3/7 activation over the untreated control. Unpaired t test: *, P < 0.05; **, P < 0.01; ***, P < 0.001; B, Combination Indexes (C.I.) of GSK126/doxorubicin-induced apoptosis in THP-1 and KG-1 cells. THP-1 or KG-1 cells were treated with three different concentrations of GSK126 and/or doxorubicin. Left, dose–response matrices showing the percentage of viable cells (±SEM) after treatment with increasing doses of doxorubicin, GSK126, and combinations of drug pair doxorubicin/GSK126. Colors in the dose–response matrices indicate different levels of responses (from white to red, decreasing viable cells) of THP-1 and KG-1 cells. Right, viability data elaborated using CompuSyn software to calculate C.I. plotted on the y-axis of a 2D graph. The x-axes show increasing concentrations of doxorubicin, whereas the different colors indicate increasing concentrations of GSK126 (see labels). C.I. = 0.90–1.10 indicates additive effects; C.I. = 0.1–0.9 indicates synergistic effects. C, H3K27me3 accumulation on nascent DNA (left) and caspase-3/7 activation in AML sample #40 CD34+CD38high or CD34+CD38low cells treated with GSK126 (5 μmol/L), doxorubicin (0.1 μmol/L), or the two drugs in combination. Figure 3. EZH2 inhibition enhances apoptosis of cytotoxic agent–treated AML cells. A, THP-1, KG-1, and CD34+ AML primary cells (sample #40) were treated with doxorubicin alone (0.1, 0.3, or 0.5 μmol/L), or in combination with GSK126 (5 μmol/L), for 48 hours. Apoptosis (caspase-3/7 activity) was analyzed by flow cytometry. Graph bars represent fold change ± SEM in the percentage of caspase-3/7 activation over the untreated control. Unpaired t test: *, P < 0.05; **, P < 0.01; ***, P < 0.001; B, Combination Indexes (C.I.) of GSK126/doxorubicin-induced apoptosis in THP-1 and KG-1 cells. THP-1 or KG-1 cells were treated with three different concentrations of GSK126 and/or doxorubicin. Left, dose–response matrices showing the percentage of viable cells (±SEM) after treatment with increasing doses of doxorubicin, GSK126, and combinations of drug pair doxorubicin/GSK126. Colors in the dose–response matrices indicate different levels of responses (from white to red, decreasing viable cells) of THP-1 and KG-1 cells. Right, viability data elaborated using CompuSyn software to calculate C.I. plotted on the y-axis of a 2D graph. The x-axes show increasing concentrations of doxorubicin, whereas the different colors indicate increasing concentrations of GSK126 (see labels). C.I. = 0.90–1.10 indicates additive effects; C.I. = 0.1–0.9 indicates synergistic effects. C, H3K27me3 accumulation on nascent DNA (left) and caspase-3/7 activation in AML sample #40 CD34+CD38high or CD34+CD38low cells treated with GSK126 (5 μmol/L), doxorubicin (0.1 μmol/L), or the two drugs in combination. More

Figure 5. PIM inhibition potentiates CDC and ADCP of rituximab. Increased CDC triggered by rituximab in MEN1703-treated cells. DHL4 and RAJI were pretreated with DMSO or 1.5 μmol/L MEN1703 for 48 hours and then treated with rituximab (1 hour) in the presence of human AB Rh⁺ serum. Cell death was determined with PI staining and flow cytometry. Error bars, ±SD of two independent replicates in a representative experiment. Combination indexes (CI) were calculated using CompuSyn software. Statistical significance was determined using two-way ANOVA. B, PIM inhibition in DLBCL cells potentiates rituximab-dependent phagocytosis. After 72 hours of treatment with DMSO or MEN1703 (1 μmol/L), CFSE-labeled DHL4 and DHL6 cells were incubated with human peripheral blood–derived macrophages in the presence of IgG isotype control or rituximab (1 μg/mL). Presence of fluorescently labeled DLBCL cells within the macrophages was assessed by immunofluorescence microscopy (arrows). Bar graphs present the mean phagocytic index, determined as the number of ingested cells per 100 macrophages. Error bars, ±SD of three independent replicates in a representative experiment. P values were determined using the two-sided t test: **, P < 0.01. Data in A–B are representative of three independent experiments. Figure 5. PIM inhibition potentiates CDC and ADCP of rituximab. Increased CDC triggered by rituximab in MEN1703-treated cells. DHL4 and RAJI were pretreated with DMSO or 1.5 μmol/L MEN1703 for 48 hours and then treated with rituximab (1 hour) in the presence of human AB Rh+ serum. Cell death was determined with PI staining and flow cytometry. Error bars, ±SD of two independent replicates in a representative experiment. Combination indexes (CI) were calculated using CompuSyn software. Statistical significance was determined using two-way ANOVA. B, PIM inhibition in DLBCL cells potentiates rituximab-dependent phagocytosis. After 72 hours of treatment with DMSO or MEN1703 (1 μmol/L), CFSE-labeled DHL4 and DHL6 cells were incubated with human peripheral blood–derived macrophages in the presence of IgG isotype control or rituximab (1 μg/mL). Presence of fluorescently labeled DLBCL cells within the macrophages was assessed by immunofluorescence microscopy (arrows). Bar graphs present the mean phagocytic index, determined as the number of ingested cells per 100 macrophages. Error bars, ±SD of three independent replicates in a representative experiment. P values were determined using the two-sided t test: **, P < 0.01. Data in A–B are representative of three independent experiments. More

Figure 6. Synergistic effect of YUM70 in combination with topotecan and vorinostat. MIA PaCa-2 cells were treated with YUM70 with or without topotecan (Topo) and vorinostat (SAHA), at stated concentrations, and kept in culture until colonies were observed in DMSO-treated control. A, A representative image is shown (one concentration). B and C, The number of colonies was quantified using Image Studio ver3.1 software from three independent experiments (more than one concentration). Graphical data is presented as mean ± SD. *, P < 0.05; **, P < 0.01. The P value of the combination was calculated and compared with YUM70 alone. D and E, The combined effect was calculated using CompuSyn software. CI <1 is defined as synergism. F, Combination regimen causes apoptosis in MIA PaCa-2 and PANC-1 cells. Top, cells in the bottom left quadrant of each panel (Annexin V–negative, PI-negative) are viable, whereas cells in the bottom right quadrant (Annexin V–positive, PI-negative) are in the early stage of apoptosis, and cells in the top right quadrant (Annexin V–positive, PI-positive) are in the late stage of apoptosis/necrosis. Bottom, the percentage of apoptotic cells is shown in a histogram. A representative image of three independent experiments is shown. Figure 6. Synergistic effect of YUM70 in combination with topotecan and vorinostat. MIA PaCa-2 cells were treated with YUM70 with or without topotecan (Topo) and vorinostat (SAHA), at stated concentrations, and kept in culture until colonies were observed in DMSO-treated control. A, A representative image is shown (one concentration). B and C, The number of colonies was quantified using Image Studio ver3.1 software from three independent experiments (more than one concentration). Graphical data is presented as mean ± SD. *, P < 0.05; **, P < 0.01. The P value of the combination was calculated and compared with YUM70 alone. D and E, The combined effect was calculated using CompuSyn software. CI <1 is defined as synergism. F, Combination regimen causes apoptosis in MIA PaCa-2 and PANC-1 cells. Top, cells in the bottom left quadrant of each panel (Annexin V–negative, PI-negative) are viable, whereas cells in the bottom right quadrant (Annexin V–positive, PI-negative) are in the early stage of apoptosis, and cells in the top right quadrant (Annexin V–positive, PI-positive) are in the late stage of apoptosis/necrosis. Bottom, the percentage of apoptotic cells is shown in a histogram. A representative image of three independent experiments is shown. More