American Society of Hematology

Figure 1

$Figure 1. MLN4924 is highly active in preclinical AML models and primary AML cells. (A) MLN4924 potently reduces the viability of AML cells. MOLM-13, PL-21, MV411, and HL-60 cell lines and PBMCs from healthy donors were treated with the indicated concentrations of MLN4924 for 72 hours. Viability was assessed by the ATPLite assay. Results shown represent the mean of 3 experiments. Error bars represent SD. (B) MLN4924 has activity in primary AML cells. Primary cells were obtained from 5 AML patients. Of these patients, 1 had therapy-related AML and another had primary refractory AML indicating aggressive clinical disease. Primary cells were treated with MLN4924 for 72 hours. Viability was assessed by the ATPLite assay. (C) Dose-dependent inhibition of clonogenic survival after treatment with MLN4924. MOLM-13 and MV411 human AML cells were treated with the indicated concentrations of MLN4924 for 24 hours. Drug was washed away, cells were seeded in Methocult, and colonies were scored on day 10. n = 3; bars represent the mean ± SD. (D) Quantification of drug-induced apoptosis. MV411 cells and PBMCs from healthy donors were treated with the indicated concentrations of MLN4924 for 48 hours. Percentages of cells with subdiploid DNA were determined by PI/FACS. n = 3; bars represent the mean ± SD. (E) Activation of apoptosis after treatment with MLN4924. Cells were treated with the indicated concentrations of MLN4924 for 24 hours. Protein lysates were subjected to SDS-PAGE, blotted, and probed with an active caspase-3 specific antibody. Tubulin documented equal loading. (F) Effect of stromal coculture on the proapoptotic activity of MLN4924. MV411 and HL-60 cells were treated with the indicated concentrations of MLN4924 for 48 hours in the presence and absence of HS-5 bone marrow stromal cells. Percentages of apoptotic cells were determined by PI/FACS. n = 3; bars represent the mean ± SD. (G) Impact of FLT3 expression on the sensitivity of AML cells to MLN4924. MV411 cells with and without stable shRNA-mediated knockdown of FLT3 expression were treated with the indicated concentrations of MLN4924 for 48 hours. Percentages of apoptotic cells were determined by PI/FACS. n = 3; bars represent the mean ± SD. Immunoblotting was used to confirm knockdown of FLT3 expression. (H) Effects of MLN4924 on NEDDylated substrates and downstream effectors. Cells were treated with MLN4924 for 24 hours, lysed, subjected to SDS-PAGE, and probed with NEDD8, p27, CDT-1, NRF-2, pCHK1, total CHK1, pIκBα, total IκBα, BCL-2, BCL-xL, FLIP, and SOD2-specific antibodies. Tubulin documented equal loading. Densitometry analysis was carried out by quantifying the band density for each protein relative to the band density of tubulin using an Alpha Innotech FluorChem HD2 gel documentation system (Alpha Innotech). The densitometry values for all controls were normalized to 1.0. Densitometry values are indicated above each band and reflect the ratio of the change in protein expression from control levels for each respective protein. (I) Effects of MLN4924 on NFκB (p65) DNA-binding activity. Cells were treated with MLN4924 as indicated for 24 hours. Relative p65 DNA-binding activity was quantified using a chemiluminescent detection method. (J) Quantification of MLN4924-induced ROS generation. MV411 cells were treated with MLN4924 for 12 hours, and ROS production was evaluated by staining with dichlorofluorescein (DCF) followed by FACS analysis. n = 3; bars represent the mean ± SD. (K) Impact of antioxidant treatment on MLN4924-induced apoptosis. Drug-induced apoptosis was quantified after 48 hours of exposure to MLN4924 in the presence and absence of the antioxidant NAC. n = 3; bars represent the mean ± SD.$

MLN4924 is highly active in preclinical AML models and primary AML cells. (A) MLN4924 potently reduces the viability of AML cells. MOLM-13, PL-21, MV411, and HL-60 cell lines and PBMCs from healthy donors were treated with the indicated concentrations of MLN4924 for 72 hours. Viability was assessed by the ATPLite assay. Results shown represent the mean of 3 experiments. Error bars represent SD. (B) MLN4924 has activity in primary AML cells. Primary cells were obtained from 5 AML patients. Of these patients, 1 had therapy-related AML and another had primary refractory AML indicating aggressive clinical disease. Primary cells were treated with MLN4924 for 72 hours. Viability was assessed by the ATPLite assay. (C) Dose-dependent inhibition of clonogenic survival after treatment with MLN4924. MOLM-13 and MV411 human AML cells were treated with the indicated concentrations of MLN4924 for 24 hours. Drug was washed away, cells were seeded in Methocult, and colonies were scored on day 10. n = 3; bars represent the mean ± SD. (D) Quantification of drug-induced apoptosis. MV411 cells and PBMCs from healthy donors were treated with the indicated concentrations of MLN4924 for 48 hours. Percentages of cells with subdiploid DNA were determined by PI/FACS. n = 3; bars represent the mean ± SD. (E) Activation of apoptosis after treatment with MLN4924. Cells were treated with the indicated concentrations of MLN4924 for 24 hours. Protein lysates were subjected to SDS-PAGE, blotted, and probed with an active caspase-3 specific antibody. Tubulin documented equal loading. (F) Effect of stromal coculture on the proapoptotic activity of MLN4924. MV411 and HL-60 cells were treated with the indicated concentrations of MLN4924 for 48 hours in the presence and absence of HS-5 bone marrow stromal cells. Percentages of apoptotic cells were determined by PI/FACS. n = 3; bars represent the mean ± SD. (G) Impact of FLT3 expression on the sensitivity of AML cells to MLN4924. MV411 cells with and without stable shRNA-mediated knockdown of FLT3 expression were treated with the indicated concentrations of MLN4924 for 48 hours. Percentages of apoptotic cells were determined by PI/FACS. n = 3; bars represent the mean ± SD. Immunoblotting was used to confirm knockdown of FLT3 expression. (H) Effects of MLN4924 on NEDDylated substrates and downstream effectors. Cells were treated with MLN4924 for 24 hours, lysed, subjected to SDS-PAGE, and probed with NEDD8, p27, CDT-1, NRF-2, pCHK1, total CHK1, pIκBα, total IκBα, BCL-2, BCL-xL, FLIP, and SOD2-specific antibodies. Tubulin documented equal loading. Densitometry analysis was carried out by quantifying the band density for each protein relative to the band density of tubulin using an Alpha Innotech FluorChem HD2 gel documentation system (Alpha Innotech). The densitometry values for all controls were normalized to 1.0. Densitometry values are indicated above each band and reflect the ratio of the change in protein expression from control levels for each respective protein. (I) Effects of MLN4924 on NFκB (p65) DNA-binding activity. Cells were treated with MLN4924 as indicated for 24 hours. Relative p65 DNA-binding activity was quantified using a chemiluminescent detection method. (J) Quantification of MLN4924-induced ROS generation. MV411 cells were treated with MLN4924 for 12 hours, and ROS production was evaluated by staining with dichlorofluorescein (DCF) followed by FACS analysis. n = 3; bars represent the mean ± SD. (K) Impact of antioxidant treatment on MLN4924-induced apoptosis. Drug-induced apoptosis was quantified after 48 hours of exposure to MLN4924 in the presence and absence of the antioxidant NAC. n = 3; bars represent the mean ± SD.

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