Figure 3.

Selective inhibition of MLL-AF6–transformed cells by EPZ004777. (A) Inhibition of cellular H3K79me2 levels in MLL-AF6, MLL-AF9, or Hoxa9/Meis1a transformed hematopoietic progenitor cells following 7 days of treatment with the indicated concentrations of EPZ004777 as measured by immunoblot analysis of extracted histones with an anti-H3K79me2 antibody. (B) Representative results from the growth kinetics of MLL-AF6, MLL-AF9, and HoxA9/Meis1a transformed murine bone marrow cells exposed to 10 µM of EPZ004777. Viable cells were counted and replated at equal cell numbers in fresh media with fresh compound every 3-4 days. Results were plotted as percentage of split-adjusted viable cells in the presence of 10 µM EPZ004777 compared with dimethylsulfoxide (DMSO) vehicle control. Results are representative of 3 independent experiments. (C) Time course of HoxA9 and Meis1 mRNA expression in MLL-AF6–transformed cells over 10 days of incubation with 10 µM EPZ004777 as measured by quantitative real-time PCR. Expression levels were normalized to Gapdh and expressed relative to those at d 0 (set to 100%). Error bars represent the standard error of the mean (n = 3 independent experiments). (D) Representative graph of cell cycle changes (BrdU/7-AAD flow cytometry) in MLL-AF6–transformed bone marrow cells after being treated with 10 µM EPZ004777 for 0, 4, 8, or 10 days. Similar results were obtained in 2 independent experiments. (E) Annexin V staining in MLL-AF6–transformed bone marrow cells 10 days after treatment with 10 µM EPZ004777 or DMSO control (n = 2 independent experiments). Error bars represent standard error of the mean.