Figure 1.
Activity of novel DOT1L inhibitors in leukemia cell lines in vitro. (A) Table of 14 leukemia cell lines used in this study indicating the type of leukemia, oncogenic driver, and 50% inhibitory concentration (IC50) values for EPZ5676, compound 10, and compound 11. IC50 was determined at day 10 after initiation of treatment by counting of viable cells using flow cytometry (dead cell exclusion by 4′,6-diamidino-2-phenylindole staining). Cell lines are sorted by sensitivity to EPZ5676. (B) Dose-response curves of cell lines toward compound 10 (top) and compound 11 (bottom) grouped by EPZ5676-sensitive (red), -intermediate (blue), and -insensitive (gray) cell lines. (C) Cell-surface expression of the myeloid differentiation markers CD11b (top) and Gr-1 (bottom) on murine MLL-AF9 leukemia cells at 3 days of DOT1L inhibitor treatment at the indicated doses. Statistical analysis was performed using unpaired t test. (D) Representative cytospin pictures of murine MLL-AF9 leukemia cells at 6 days of treatment with EPZ5676 (500 nM), compound 10 (100 nM), and compound 11 (100 nM). Fixation and staining was performed using the "Dip Quick Stain Kit" (Jorgensen Labs). (E) Western blot for H3K79me2 and histone H3 (control) in whole cellular lysates of MOLM13 cells treated with 5 different doses of compound 10 or 11 for 3 days. (F-G) Meta-plots of H3K79me2 chromatin immunoprecipitation sequencing (ChIPseq) in MOLM13 cells after treatment with EPZ5676 (1 μM), compound 10 (100 nM), or dimethyl sulfoxide (DMSO) over the gene body of all protein coding genes (F) and MLL-AF9 target genes (G). ChIPseq data were normalized using drosophila spike-in chromatin. (H) Volcano plots of differentially expressed genes (DEGs; red) in MOLM13 cells treated with 100 nM of compound 10 for 3, 6, and 9 days after initiation of drug treatment. Shown in the graph are protein coding genes only. DEGs were called using the DeSeq2 algorithm after alignment of reads using STAR. A gene was considered a DEG if the adjusted P value was <.05. (I) Gene set enrichment analysis (GSEA) of MLL-AF9 target genes after treatment of MOLM13 cells with compound 10 (100 nM; top) or EPZ5676 (1 μM; bottom) for 3, 6, and 9 days. (J) Correlation between the magnitude of changes (displayed as log2-fold change) in DEGs after compound 10 vs EPZ5676 treatment. Pearson correlation was used for statistical analysis. (K) Western blot of H3K79me2 and histone H3 (control) in whole cellular lysates of MOLM13 cells treated with VTP50469, compound 10, compound 11, or EPZ5676 at the indicated doses for 4 days. (L) Representative ChIPseq tracks of DOT1L (left) and Menin (right) at core MLL-AF9 target genes derived from the same experimental setting as shown in panel H. ChIPseq was performed as previously described.5 ***P < .001, ****P < .0001. AML, acute lymphoblastic leukemia; CML, chronic myeloid leukemia; FDR, false discovery rate; MFI, mean fluorescence intensity; NES, normalized enrichment score.

Activity of novel DOT1L inhibitors in leukemia cell lines in vitro. (A) Table of 14 leukemia cell lines used in this study indicating the type of leukemia, oncogenic driver, and 50% inhibitory concentration (IC50) values for EPZ5676, compound 10, and compound 11. IC50 was determined at day 10 after initiation of treatment by counting of viable cells using flow cytometry (dead cell exclusion by 4′,6-diamidino-2-phenylindole staining). Cell lines are sorted by sensitivity to EPZ5676. (B) Dose-response curves of cell lines toward compound 10 (top) and compound 11 (bottom) grouped by EPZ5676-sensitive (red), -intermediate (blue), and -insensitive (gray) cell lines. (C) Cell-surface expression of the myeloid differentiation markers CD11b (top) and Gr-1 (bottom) on murine MLL-AF9 leukemia cells at 3 days of DOT1L inhibitor treatment at the indicated doses. Statistical analysis was performed using unpaired t test. (D) Representative cytospin pictures of murine MLL-AF9 leukemia cells at 6 days of treatment with EPZ5676 (500 nM), compound 10 (100 nM), and compound 11 (100 nM). Fixation and staining was performed using the "Dip Quick Stain Kit" (Jorgensen Labs). (E) Western blot for H3K79me2 and histone H3 (control) in whole cellular lysates of MOLM13 cells treated with 5 different doses of compound 10 or 11 for 3 days. (F-G) Meta-plots of H3K79me2 chromatin immunoprecipitation sequencing (ChIPseq) in MOLM13 cells after treatment with EPZ5676 (1 μM), compound 10 (100 nM), or dimethyl sulfoxide (DMSO) over the gene body of all protein coding genes (F) and MLL-AF9 target genes (G). ChIPseq data were normalized using drosophila spike-in chromatin. (H) Volcano plots of differentially expressed genes (DEGs; red) in MOLM13 cells treated with 100 nM of compound 10 for 3, 6, and 9 days after initiation of drug treatment. Shown in the graph are protein coding genes only. DEGs were called using the DeSeq2 algorithm after alignment of reads using STAR. A gene was considered a DEG if the adjusted P value was <.05. (I) Gene set enrichment analysis (GSEA) of MLL-AF9 target genes after treatment of MOLM13 cells with compound 10 (100 nM; top) or EPZ5676 (1 μM; bottom) for 3, 6, and 9 days. (J) Correlation between the magnitude of changes (displayed as log2-fold change) in DEGs after compound 10 vs EPZ5676 treatment. Pearson correlation was used for statistical analysis. (K) Western blot of H3K79me2 and histone H3 (control) in whole cellular lysates of MOLM13 cells treated with VTP50469, compound 10, compound 11, or EPZ5676 at the indicated doses for 4 days. (L) Representative ChIPseq tracks of DOT1L (left) and Menin (right) at core MLL-AF9 target genes derived from the same experimental setting as shown in panel H. ChIPseq was performed as previously described.5  ***P < .001, ****P < .0001. AML, acute lymphoblastic leukemia; CML, chronic myeloid leukemia; FDR, false discovery rate; MFI, mean fluorescence intensity; NES, normalized enrichment score.

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