UNC1999, but not GSK126, efficiently suppresses H3K27me3 in MLL-rearranged leukemia cells and inhibits their growth. (A) Immunoblots of the global H3K27me3 level after treatment of DB lymphoma cells (top) or MLL-AF9–transformed murine leukemia progenitors (bottom) with 2 μM of the indicated compounds for 5 days. General H3 serves as control. (B) Relative proliferation of DB cells treated with a range of concentrations of GSK126 (top) or UNC1999 (bottom) for the indicated duration. Y-axis represents the relative percentage of accumulative cell numbers normalized to DMSO treatment, and is presented as the mean of triplicates ± SD. (C) Flow cytometry analysis of H3K27me3 in MLL-AF9–transformed murine leukemia progenitors following treatment with various concentrations of GSK126 (top) or UNC1999 (bottom) for 4 days. DMSO serves as control. (D) Relative proliferation of MLL-AF9–transformed leukemia progenitors treated with a range of concentrations of GSK126 (top) or UNC1999 (bottom) for the indicated duration. Y-axis represents the relative percentage of cell numbers after normalization to DMSO treatment, and is presented as the mean of triplicates ± SD. (E) Relative proliferation of a panel of leukemia or lymphoma cell lines treated with various concentrations of UNC1999 for 16 days. Y-axis, presented as the mean of triplicates ± SD, represents the relative percentage of accumulative cell numbers after normalization to DMSO treatment. Shown as a dashed line is DB, an EZH2-mutated (Y641N) lymphoma line known to be sensitive to EZH2 inhibition.²²  (F) Summary of EC₅₀ of a panel of cell lines in response to UNC1999. m-MLL-AF9 and m-MLL-ENL represent murine leukemia lines established by MLL-AF9 and MLL-ENL, respectively. (G-I) Relative proliferation of murine MLL-ENL–bearing leukemia cells (G) and EOL-1 (H) and LOUCY (I) human leukemia cells treated with a range of UNC1999 concentrations for the indicated duration. Y-axis represents relative percentage of accumulative cell numbers after normalization to DMSO treatment, and is presented as the mean of triplicates ± SD. (J) Immunoblot of H3K27me3 and general H3 in EOL-1, LOUCY, and DB cells. (K) Relative proliferation of murine leukemia cells bearing MLL-AF9 or MLL-ENL after treatment with UNC1999 or UNC2400 and normalization to DMSO treatment. *P < .05; **P < .01; ***P < .001; ns, not significant.