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

Figure 7. STAT5A-CA overexpression rescues the viability of MV4;11 cells treated with MLL1 inhibitors.

Proliferation assay of MLL-r cell lines treated with (A) 250 nM MI-503 (MLL1-Menin interaction inhibitor) or (B) 10 µM MM-401 (MLL1 histone methyltransferase inhibitor) for 7 days. Viability was measured by CellTiter Glo 2.0 assay and results are displayed as the fraction of luminescence of inhibitor-treated over DMSO-treated cells. Means ± SE are shown for three independent experiments. Student’s t-test (* p < 0.05, ** p < 0.01, *** p < 0.001). (C) H3K4me3 histone methylation density from −2000 to +2000 of the TSS from quantitative ICeChIP-seq from MV4;11 cells treated with 100 nM pinometostat for 7 days for genes up- or downregulated by 100 nM pinometostat, the most highly expressed genes, MLL-AF4 target genes (Kerry et al., 2017) as well as those MLL-AF4 targets downregulated by 100 nM pinometostat. (D and E) Proliferation assay of MV4;11 STAT5A-CA clonal isolates induced to express STAT5A-CA or eGFP with 1 µg/mL doxycycline and treated with D. 250 nM MI-503 or (E) 10 µM MM-401. Viability was measured and results displayed as in A and B. Means ± SE are shown for three independent experiments. Student’s t-test (* p < 0.05, ** p < 0.01, *** p < 0.001). (F) Gene expression analysis by RT-qPCR of MLL-fusion and STAT5a targets in MV4;11 cells treated with 250 nM MI-503 MLL1 inhibitor, 100 nM pinometostat DOT1L inhibitor or a combination for 7 days. Means ± S.E.M. are shown for three independent experiments (* p < 0.05). (G) Gene expression analysis by RT-qPCR of MLL-fusion and STAT5A targets in WT and STAT5A-CA MV4;11 cells treated with 250 nM MI-503 MLL1 inhibitor for 7 days. Means ± S.E.M. are shown for technical replicates of individual experiments.

Figure 7.

Figure 7—figure supplement 1. MLL1 inhibitors reduce STAT5A phosphorylation.

Figure 7—figure supplement 1.

(A) Western blots of whole cell extract from MV4;11 cells treated with 100 nM pinometostat for 7 days and blotted for H3K4me3 or LEDGF as a loading control. (B) Western blots of whole cell extract from MV4;11 cells treated with MLL1 inhibitors MI-503 (250 nM) or MM-401 (10 µM) or DMSO for 7 days and blotted for histone H3 lysine four trimethylation (H3K4me3) or GAPDH as a loading control. (C) Western blots of MV4;11 cell extract treated with MLL1 inhibitors MI-503 (250 nM) or MM-401 (10 µM) for 7 days and blotted for phosphorylated STAT5, histone H3 lysine 79 dimethylation (H3K79me2) or GAPDH as a loading control. (D) H3K79me2 meta promoter profiles ± 2000 bp of the TSS as in Figure 7C, representing 4 days, rather than seven days of 100 nM pinometostat treatment versus DMSO control. (E) ICe-ChIP-qPCR analysis of the percent H3K4me3 at the promoters of several genes both upregulated (BCL6, CSF3R, HLA-DRA) and downregulated (MEF2C, PIM1, ARID3B, and FLT3) by pinometostat as well as MLL-AF4 targets (HOXA9 and MEIS1) and an intergenic region (with little change) from two experiments as well as ICe-ChIP-seq (hashed lines) in MV4;11 cells ± 100 nM pinometostat for 7 days. Note that ICeChIP qPCR values are often slightly inflated as due to the capture of dinucleosomes that are over-represented due to avidity bias that are normally filtered out in ICeChIP. (F) MV4;11 cells were treated with DOT1L or MLL1 inhibitors alone or in combination for 7 days. Viability was analyzed using CellTiter Glo 2.0, showing the luminescence fraction of inhibited over DMSO-treated cells. Means ± SE are shown for three independent experiments. Student’s t-test of day 7 values: 100 nM pinometostat vs. 100 nM pinometostat + 250 nM MI-503 ** p < 0.01 100 nM pinometostat vs. 100 nM pinometostat + 10 µM MM-401 * p < 0.05. (G) Model of MLL-fusion-mediated activation of HOXA9/MEIS1 and STAT5A co-targets in MLL-r, FLT3-ITD+ leukemia. MLL-AF4 activates HOXA9, MEIS1, and FLT3-ITD gene expression through recruitment of DOT1L and H3K79me2 hypermethylation (fuchsia). FLT3-ITD phosphorylates STAT5A allowing it to translocate to the nucleus to cooperatively bind HOXA9/MEIS1 targets with PBX3 and facilitate gene activation. (H) Scatterplot from ICeChIP-seq of MV4;11 cells ± 100 nM pinometostat of the log2 (fold-change HMD of H3K79me2 vs. H3K4me3) of all expressed genes showing MLL-AF4 targets (red) and MLL-AF4 targets downregulated by pinometostat (purple). (I) The average fold-change in H3K4me3 HMD from ICeChIP-seq of MV4;11 cells ± 100 nM pinometostat at expressed genes in 20 bins grouped by decreasing expression (bin 1–20 sorted by highest to lowest FPKM of DMSO-treated cells).