Figure 3.
Overexpression of mutant IDH1, DOT1L, or dominant-negative DOT1L results in downregulation of DOT1L-dependent genes. (A) Analysis of KMT2A-target genes after introduction of mIDH1. Quantitative reverse-transcription polymerase chain reaction (qRT-PCR) of DOT1L targets genes (Hoxa9, Hoxa10, and Meis1) 5 days after introducing IDH1R132 into 2 individual murine KMT2A-MLLT3 leukemias. Expression levels are shown as fold change over empty vector control cells. Error bars represent SD. (B-C) Analysis of KMT2A-target genes after introduction of mIDH1 (RNA-seq). RNA was collected 5 days after introducing IDHR132 into 4 individual murine KMT2A-MLLT3 leukemias and submitted to sequencing. (B) GSEA plot showing genes that are downregulated upon DOT1L deletion1 are negatively enriched when IDH1R132 is overexpressed. (C) GSEA plot showing negative enrichment of HOXA9-bound genes44 when IDH1R132 is overexpressed. (D-E) Effect of directly increasing H3K79 methylation through overexpression of DOT1L on growth of murine KMT2A-MLLT3 AML cells. Western blot analysis for H3K79me2 (D) and growth of murine KMT2A-MLLT3 AML cells (E) when DOT1L is overexpressed for 4 days. DOT1L-DN served as positive control, decreasing H3K79 methylation and growth of murine KMT2A-MLLT3 AML cells. Murine HOXA9/MEIS1 AML cells serve as negative control for the growth assay, because the key KMT2A targets HOXA9 and MEIS1 in these cells are driven by the MSCV retroviral promoter and are insensitive to epigenetics regulation via the modulation of H3K79me2.1 Growth of DOT1L- or DOT1L-DN–transduced KMT2A-MLLT3 and HOXA9/MEIS1 leukemia cells as a percentage of empty vector control–transduced cells at day 12. Error bars represent SD. (F-G) Effect of indirectly increasing H3K79 methylation through overexpression of AF10, a cofactor of DOT1L, on the growth of the human KMT2A-MLLT3–rearranged AML cell line THP1, as well as the CALM-AF10–rearranged U937 and 31P cell lines. Western blot analysis of H3K79me2 (F) and growth of THP1, U937, and 31P cells treated with doxycycline for 3 days to induce AF10 expression (G). Error bars represent SD. *P < .01, **P < .001, ****P < .00001; Student t test. iAF10, induced AF10.

Overexpression of mutant IDH1, DOT1L, or dominant-negative DOT1L results in downregulation of DOT1L-dependent genes. (A) Analysis of KMT2A-target genes after introduction of mIDH1. Quantitative reverse-transcription polymerase chain reaction (qRT-PCR) of DOT1L targets genes (Hoxa9, Hoxa10, and Meis1) 5 days after introducing IDH1R132 into 2 individual murine KMT2A-MLLT3 leukemias. Expression levels are shown as fold change over empty vector control cells. Error bars represent SD. (B-C) Analysis of KMT2A-target genes after introduction of mIDH1 (RNA-seq). RNA was collected 5 days after introducing IDHR132 into 4 individual murine KMT2A-MLLT3 leukemias and submitted to sequencing. (B) GSEA plot showing genes that are downregulated upon DOT1L deletion1  are negatively enriched when IDH1R132 is overexpressed. (C) GSEA plot showing negative enrichment of HOXA9-bound genes44  when IDH1R132 is overexpressed. (D-E) Effect of directly increasing H3K79 methylation through overexpression of DOT1L on growth of murine KMT2A-MLLT3 AML cells. Western blot analysis for H3K79me2 (D) and growth of murine KMT2A-MLLT3 AML cells (E) when DOT1L is overexpressed for 4 days. DOT1L-DN served as positive control, decreasing H3K79 methylation and growth of murine KMT2A-MLLT3 AML cells. Murine HOXA9/MEIS1 AML cells serve as negative control for the growth assay, because the key KMT2A targets HOXA9 and MEIS1 in these cells are driven by the MSCV retroviral promoter and are insensitive to epigenetics regulation via the modulation of H3K79me2.1  Growth of DOT1L- or DOT1L-DN–transduced KMT2A-MLLT3 and HOXA9/MEIS1 leukemia cells as a percentage of empty vector control–transduced cells at day 12. Error bars represent SD. (F-G) Effect of indirectly increasing H3K79 methylation through overexpression of AF10, a cofactor of DOT1L, on the growth of the human KMT2A-MLLT3–rearranged AML cell line THP1, as well as the CALM-AF10–rearranged U937 and 31P cell lines. Western blot analysis of H3K79me2 (F) and growth of THP1, U937, and 31P cells treated with doxycycline for 3 days to induce AF10 expression (G). Error bars represent SD. *P < .01, **P < .001, ****P < .00001; Student t test. iAF10, induced AF10.

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