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

Figure 2. DOT1L inhibition downregulates a subset of MLL-AF4 targets including the leukemic oncogene FLT3.

(A) MA plot showing genes differentially expressed in MV4;11 cells treated with 100 nM pinometostat or DMSO 7 days as log2-mean of expression (FPKM) of the DMSO and pinometostat-treated samples versus the log2-fold change of the mean normalized pinometostat versus DMSO-treated FPKM for three independent replicates. Red represents genes that meet the significance threshold, with an FDR-adjusted p ≤ 0.5. (B) Venn diagram depicting overlapping genes between those downregulated by 100 nM pinometostat and MV4;11 MLL-AF4 targets identified by Kerry et al., 2017, p-value computed by two-tailed Fisher Exact test. (C) Venn diagram displaying the overlap between genes downregulated 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). p-Value computed by two-tailed Fisher Exact test. (D) Bar plot depicting upregulated genes with the highest fold changes from RNA-seq analysis of three independent experiments of DMSO- (blue) or pinometostat-treated (red) MV4;11 cells with uncertainty presented as the standard deviation computed by CuffDiff (Trapnell et al., 2012) with immune response genes outlined in gray. (E) RT-qPCR analysis showing the fold-change for HLA-DRA, HLA-DRB1, and CIITA gene expression in MV4;11 cells ± 100 nM pinometostat treatment for 7 days. Results are shown as mean ± S.E.M. of three independent experiments. Student’s t-test (*****p ≤ 0.00001). (F) Bar plot depicting the top pinometostat-downregulated genes from the RNA-seq analysis that are previously described MLL-AF4 targets (Guenther et al., 2008) including the oncogenes MEF2C, FLT3, and PBX3. (G) RT-qPCR analysis of MEF2C, FLT3, and PBX3 expression in MV4;11 cells ± with 100 nM pinometostat for 7 days. Results are displayed as mean fold-change ± S.E.M. of three independent experiments; Student’s t-test (**** p ≤ 0.0001). (H) Western blot for FLT3 with RBBP5 as loading control in MV4;11 cells treated with 100 nM pinometostat for 5 or 7 days. (I) Venn diagram displaying the overlap between genes upregulated in MV4;11 cells by 100 nM pinometostat treatment (7 days) and genes downregulated in leukemic cells from patients with FLT3-ITD vs normal FLT3 karyotypically normal AML (Cauchy et al., 2015). p-Value computed by two-tailed Fisher Exact test.

Figure 2.

Figure 2—figure supplement 1. DOT1L inhibition upregulates components of the interferon gamma pathway and markers of differentiation.

Figure 2—figure supplement 1.

(A) Gene Ontology analysis (DAVID) (Huang et al., 2009a; Huang et al., 2009b) of pinometostat-upregulated genes showing top functional classification categories and the number of genes in each category that are significantly upregulated. (B) Bar graph of Cuffdiff (Trapnell et al., 2012) output for the expression of INFG (IFN-γ) from RNA-seq in MV4;11 cells ± 100 nM pinometostat. Values are represented as FPKM + 1 for three independent experiments with standard deviation. Student’s t-test (ns p > 0.05). (C) Bar graph of Cuffdiff (Trapnell et al., 2012) output for the expression of ITGAM (CD11b), ITGAX (CD11c), and CD86 macrophage cell surface marker expression in MV4;11 cells ± 100 nM pinometostat for 7 days. Values are represented as FPKM + 1 for three independent experiments with standard deviation. Student’s t-test (** p < 0.01, *** p ≤ 0.001). (D) GSEA (Subramanian et al., 2005; Mootha et al., 2003) of the set of differentially expressed genes in MV4;11 cells ± 100 nM pinometostat compared to KEGG_HEMATOPOIETIC_CELL_LINEAGE and IVANOVA_HEMATOPOIESIS_CELL_LINEAGE gene sets from the MSigDB data base. NES - normalized enrichment score. (E) RT-qPCR analysis of CSF3R and CSF1R expression in MV4;11 cells ± 100 nM pinometostat 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 (** p < 0.01, *** p ≤ 0.001). (F) Venn diagram displaying the overlap between genes downregulated in MV4;11 cells by 100 nM pinometostat treatment (7 days) and genes upregulated in leukemic cells from patients with FLT3-ITD vs normal FLT3 karyotypically normal AML (Cauchy et al., 2015). p-Value computed by two-tailed Fisher Exact test.