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. 2021 Jul 15;10:e64960. doi: 10.7554/eLife.64960

Figure 1. Low doses of DOT1L inhibitor ablate bulk H3K79me2 and curtail MV4;11 proliferation without impacting expression of canonical target genes.

(A) Conventional model depicting how DOT1L methyltransferase activity activates transcription of key proliferative oncogenic transcription factors (Okada et al., 2005; Bernt et al., 2011; Guenther et al., 2008; Armstrong et al., 2002; Zeisig et al., 2004; Kroon et al., 1998). (B) Proliferation assay of MV4;11 cells treated with the indicated concentrations of the DOT1L inhibitor pinometostat (EPZ5676). Cell viability was assayed every 2 days, starting 1 day after treatment commenced using the CellTiter Glo 2.0 reagent. Relative cell viability is presented as the mean fraction of pinometostat versus cells treated with the equivalent volume of DMSO from three independent experiments ± S.E.M. (C) Western blots for H3K79me2 with H4 or MBD3 loading controls in MV4;11 cells treated with 10–200 nM pinometostat for 5 or 7 days. (D) RT-qPCR analysis of HOXA9 and MEIS1 expression fold-change in MV4;11 cells treated with 100 nM or 1 µM pinometostat for 7 days. Results are shown as mean ± S.E.M. of three independent experiments. Student’s t-test (ns p > 0.05, ** p ≤ 0.01, *** p ≤ 0.001). (E) Western blot of HOXA9 and MEIS1 with H4 as a loading control from MV4;11 cells treated with 100 nM pinometostat for 7 days.

Figure 1.

Figure 1—figure supplement 1. Low-dose DOT1L inhibition has little effect on Hox gene expression.

Figure 1—figure supplement 1.

(A) Proliferation assay of MV4;11 cells treated with the DOT1L inhibitor SGC0946 using CellTiter-Glo 2.0 to measure viability represented as the luminescence fraction of treated over mock-treated cells. Data are represented as mean ± SE of three independent experiments. (B) RT-qPCR analysis of HOXA9 and MEIS1 expression in MV4;11 cells ± 50 nM SGC0946 DOT1L inhibitor for 7 days. Results are displayed as mean fold-change vs. DMSO-treated cells ± S.E.M. of three independent experiments. Student’s t-test (ns p > 0.05). (C) Bar graph of Cuffdiff (Trapnell et al., 2012) output for non-native RNA ‘spike-ins’ for ± 100 nM pinometostat cDNA libraries used for RNA-seq from MV4;11 cells. Bar graphs represent the average of three independent experiments ± S.E.M. Student’s t-test (ns p > 0.05). (D) Venn diagram displaying the overlap between genes upregulated in MV4;11 cells by 100 nM pinometostat treatment (7 days) and treatment with 3 µM of the pinometostat-related compound EPZ004777 for 6 days (Daigle et al., 2011). (E) Bar graph of Cuffdiff (Trapnell et al., 2012) output for the expression of HOXA cluster genes and HOX domain-containing oncogenes MEIS1, RUNX1, and PBX3 from RNA-seq in MV4;11 cells ± 100 nM pinometostat. Values are represented as log10 (FPKM + 1) for three independent experiments with standard deviation. Student’s t-test (ns p > 0.05, * p < 0.05, ** p < 0.01). (F) RT-qPCR analysis of SPI1 and CEBPA expression from three independent experiments of MV4;11 cells treated with 100 nM pinometostat for 7 days. Fold change over DMSO-treated cells is depicted ± S.E.M. Student’s t-test (ns p > 0.05, ** p < 0.01).