Journal: eLife

Article Title: Non-canonical H3K79me2-dependent pathways promote the survival of MLL-rearranged leukemia

doi: 10.7554/eLife.64960

Figure Lengend Snippet: ( A ) MA plot showing genes differentially expressed in MV4;11 cells treated with 100 nM pinometostat or DMSO 7 days as log 2 -mean of expression (FPKM) of the DMSO and pinometostat-treated samples versus the log 2 -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 , 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 . 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 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 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 . p-Value computed by two-tailed Fisher Exact test.

Article Snippet: DOT1L inhibitor pinometostat (EPZ5676, Cayman Chemical cat # 16175), EZH2 inhibitor EI1 (Cayman Chemical cat # 19146–1), FLT3 inhibitor tandutinib (MLN518) (Selleckchem cat # S1043), MI-503 (Selleckchem cat # S7817), and PIM1 inhibitor (MedChemExpress cat # HY-15604) were resuspended in DMSO.

Techniques: Expressing, Two Tailed Test, RNA Sequencing, Standard Deviation, Quantitative RT-PCR, Gene Expression, Western Blot, Control

Journal: eLife

Article Title: Non-canonical H3K79me2-dependent pathways promote the survival of MLL-rearranged leukemia

doi: 10.7554/eLife.64960

Figure Lengend Snippet: ( A ) Gene Ontology analysis (DAVID) ( ; ) 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 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 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 ( ; ) 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 . p-Value computed by two-tailed Fisher Exact test.

Article Snippet: DOT1L inhibitor pinometostat (EPZ5676, Cayman Chemical cat # 16175), EZH2 inhibitor EI1 (Cayman Chemical cat # 19146–1), FLT3 inhibitor tandutinib (MLN518) (Selleckchem cat # S1043), MI-503 (Selleckchem cat # S7817), and PIM1 inhibitor (MedChemExpress cat # HY-15604) were resuspended in DMSO.

Techniques: Functional Assay, Expressing, RNA Sequencing, Standard Deviation, Marker, Quantitative RT-PCR, Two Tailed Test

Journal: eLife

Article Title: Non-canonical H3K79me2-dependent pathways promote the survival of MLL-rearranged leukemia

doi: 10.7554/eLife.64960

Figure Lengend Snippet: ( A ) Scatterplot of the mean normalized log 2- FPKM (three independent replicates) of genes expressed in DMSO-treated MV4;11 cells plotted versus the log 2- HMD (H3K79me2) for +1000 bp from the TSS. Colors signify: red, MLL-AF4 targets ; purple, MLL-AF4 targets downregulated by 100 nM pinometostat. ( B ) (top) Quantitative measurement of H3K79me2 modification density from ICeChIP-seq of MV4;11 cells treated with 100 nM pinometostat for 7 days contoured over the promoters (−2 to +two kp from the TSS) of indicated gene sets, including genes up- or down-regulated by 100 nM pinometostat, the most highly-expressed genes, MLL-AF4 target genes as well as those MLL-AF4 targets downregulated by 100 nM pinometostat. (bottom) Heatmaps depicting H3K79me2 density (HMD) for the gene promoter regions shown above ranked by HMD. ( C ) Scatterplot of genes in MV4;11 cells downregulated by 100 nM pinometostat depicting log 2 -fold change H3K79me2 HMD (+1000 bp from TSS) versus the log 2 -fold change of the mean normalized FPKM (three independent replicates) for 100 nM pinometostat or DMSO treated cells. Colors signify: red, MLL-AF4 targets ; orange, FLT3-ITD upregulated genes ; blue, FLT3-ITD downregulated genes ; yellow, labeled genes in gray font. ( D ) H3K79me2 meta promoter profiles as in B , but including curves for 100 nM pinometostat treatment at 4 days, and the promoter set where this complex spreads . ( E ) The FLT3 locus as representative of an MLL-AF4 target ( ; ) downregulated by 100 nM pinometostat, displaying MV4;11 ICeChIP-seq tracks for H3K79me2 100 nM pinometostat 4- and 7-day treatment and H3K27me3 and H3K4me3 tracks from 100 nM pinometostat 7-day treatment as well as DMSO control-treated cells and an RNA-seq track (FPKM) from a single replicate of 100 nM pinometostat 7-day treatment and DMSO-treated cells.

Article Snippet: DOT1L inhibitor pinometostat (EPZ5676, Cayman Chemical cat # 16175), EZH2 inhibitor EI1 (Cayman Chemical cat # 19146–1), FLT3 inhibitor tandutinib (MLN518) (Selleckchem cat # S1043), MI-503 (Selleckchem cat # S7817), and PIM1 inhibitor (MedChemExpress cat # HY-15604) were resuspended in DMSO.

Techniques: Modification, Labeling, Control, RNA Sequencing

Journal: eLife

Article Title: Non-canonical H3K79me2-dependent pathways promote the survival of MLL-rearranged leukemia

doi: 10.7554/eLife.64960

Figure Lengend Snippet: ( A ) Plots of H3K79me2 density from ICeChIP-seq in MV4;11 cells treated with 100 nM pinometostat for 4 or 7 days. H3K79me2 modification density (HMD) is displayed from ± 2000 bp of the TSS of all genes expressed, non-MLL-AF4 targets downregulated by 100 nM pinometostat and MLL-AF4 targets not downregulated by 100 nM pinometostat. ( B ) Scatterplot of genes downregulated by 100 nM pinometostat plotted as the log 2 fold-change in gene expression vs the log 2 fold-change in HMD with MLL-AF4 targets in red, FLT3-ITD upregulated genes in orange, FLT3-ITD downregulated genes in cyan and labeled genes highlighted in yellow. Regression analysis of all genes (R 2 = 0.13) or the MLL-AF4 target subset (R 2 = 0.20) reveals poor correlation between the fold changes of H3K79me2 and RNA expression. ( C ) Scatterplot showing the reproducibility of H3K79me2 HMD from 0 to +2000 bp from the TSS from ICeChIP of (left) immunoprecipitation replicates performed simultaneously in MV4;11 cells treated with DMSO for 4 days; (Right) immunoprecipitation performed by different individuals on separate occasions from MV4;11 cells treated with DMSO for 4 or 7 days. ( D ) H3K79me2 HMD of ICeChIP replicates from MV4;11 cells ± 100 nM pinometostat from ± 2000 bp of the TSS of the gene groups identified, for comparison to replicate one presented in . ( E ) Scatterplots demonstrating the linearity of H3K79me2 calibrant immunoprecipitation from denaturative ICeChIP-seq from MV4;11 cells treated with 0 or 100 nM pinometostat for 7 days for replicate 1. Similar scatter plots for treated and untreated 7 day points for native ICeChIP of H3K4me3 and H3K27me3 ICeChIP are displayed.

Article Snippet: DOT1L inhibitor pinometostat (EPZ5676, Cayman Chemical cat # 16175), EZH2 inhibitor EI1 (Cayman Chemical cat # 19146–1), FLT3 inhibitor tandutinib (MLN518) (Selleckchem cat # S1043), MI-503 (Selleckchem cat # S7817), and PIM1 inhibitor (MedChemExpress cat # HY-15604) were resuspended in DMSO.

Techniques: Modification, Gene Expression, Labeling, RNA Expression, Immunoprecipitation, Comparison

Journal: eLife

Article Title: Non-canonical H3K79me2-dependent pathways promote the survival of MLL-rearranged leukemia

doi: 10.7554/eLife.64960

Figure Lengend Snippet: ( A ) MLL-rearranged leukemia lines with genotypes indicated were treated with 100 nM pinometostat (left panel, DOT1L inhibitor) or 30 nM tandutinib (right panel, FLT3 inhibitor MLN518), and relative growth monitored by CellTiter Glo 2.0 assay on the indicated days. Relative viability presented is the mean fraction of luminescence of treated versus side-by-side mock treated cultures (same volume of DMSO) for three independent replicates ± S.E.M. Student’s t-test (** p ≤ 0.01, *** p ≤ 0.001). ( B ) Western blots of phosphorylated STAT5 (active) or total STAT5A with H3 or HNRNPK as loading controls across the cell lines from panel A treated as indicated; H3K79me2 is monitored in pinometostat-treated lines to confirm inhibition. ( C ) Time course of gene expression by RT-qPCR, presented as mean fold-change of FLT3, PBX3, PIM1 , and MEF2C in MV4;11 cells ± 100 nM pinometostat at each time point indicated ± S.E.M.; n = 3; Student’s t-test (ns p > 0.05, * p ≤ 0.05, **** p ≤ 0.0001, ***** p < 0.00001). ( D-E ) DOT1L and FLT3 inhibition downregulate STAT5A targets in FLT3-ITD . RT-qPCR expression analysis presented as mean fold-change ± S.E.M. for the indicated transcript in MV4;11 cells treated with indicated inhibitor versus mock-treatment for 7 days. Student’s t-test (** p < 0.01, **** p < 0.0001, ***** p < 0.00001). ( F ) Proliferation assay as in panel A, with three clonal populations of MV4;11 cells virally transduced, selected, then induced to express shRNA to FLT3 or a scrambled shRNA control by 1 µg/mL doxycycline. Means of fractional viability relative to uninduced cells ± S.E.M. are shown for three independent experiments; Student’s t-test (** p < 0.01). ( G ) RT-qPCR analysis of PIM1, PIM2 , and ARID3B expression in MV4;11 cells expressing an inducible shRNA targeting FLT3 for 7 days. Results are depicted as fold-change expression of control cells expressing shRNA to GFP . ( H ) Proliferation assay of K562, PL-21, and EOL-1 cells treated with 10 µM or 100 nM pinometostat using CellTiter Glo 2.0 to measure viability, 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 (EOL-1 cells), day 9 (PL-21), or days 9 and 11 (K562 and PL-21) values: ns p > 0.05, * p < 0.05, **** p < 0.0001. ( I ) Gene expression analysis by RT-qPCR in PL-21 cells treated for 9 days with 10 µM pinometostat. Results are displayed as fold-change over DMSO-treated cells with means ± SE for three independent experiments (ND = not detected). Student’s t-test (**** p < 0.0001). ( J ) Western blots of (left) cell extract from PL-21 cells treated with 10 µM pinometostat for 9 days and (right) EOL-1 cells treated with 100 nM pinometostat for 7 days and then blotted for H3K79me2 and p-STAT5 with H2B or HNRNPK as a loading controls. ( K ) Gene expression analysis by RT-qPCR in EOL-1 cells treated for 7 days with 100 nM or 10 µM pinometostat. Results are displayed as fold-change over DMSO-treated cells with means ± SE for three independent experiments. Student’s t-test (ns p > 0.05, **** p < 0.0001).

Article Snippet: DOT1L inhibitor pinometostat (EPZ5676, Cayman Chemical cat # 16175), EZH2 inhibitor EI1 (Cayman Chemical cat # 19146–1), FLT3 inhibitor tandutinib (MLN518) (Selleckchem cat # S1043), MI-503 (Selleckchem cat # S7817), and PIM1 inhibitor (MedChemExpress cat # HY-15604) were resuspended in DMSO.

Techniques: Western Blot, Inhibition, Gene Expression, Quantitative RT-PCR, Expressing, Proliferation Assay, shRNA, Control

Journal: eLife

Article Title: Non-canonical H3K79me2-dependent pathways promote the survival of MLL-rearranged leukemia

doi: 10.7554/eLife.64960

Figure Lengend Snippet: ( A ) RT-qPCR analysis of HOXA9 and MEIS1 expression from three independent experiments of MOLM13 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). ( B ) (left) RT-qPCR analysis of FLT3 and PIM1 expression in MV4;11 cells ± 50 nM SGC0946 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 < 0.05, *** p ≤ 0.001). (right) Western blots from MV4;11 cells (left) treated with 50 nM SGC0946 for 7 days blotted for phosphorylated STAT5, H3K79me2, or histone H2B as a loading control. (right) MV4;11 cells treated with increasing concentrations of SGC0946 for 7 days blotted for H3K79me2 or H2B as a loading control. ( C ) Quantitative western blot of whole cell extracts from 3 experiments of MV4;11 cells ± 100 nM pinometostat for 7 days, stained with antibodies for phosphorylated STAT5 or histone H3 as a loading control. The blot includes a dilution series of the extract from the DMSO-treated sample one that encompasses the range of signal observed in each of the replicates and is used to calibrate a relative quantification of phosphorylated STAT5A signal integrated in ImageJ and shown in the bar graph at right as a 65 ± 8% reduction of the DMSO treated samples ± SD. Student’s t-test (** p < 0.01). ( D ) Gene Set Enrichment Analysis (GSEA) ( ; ) of the set of downregulated genes in MV4;11 cells ± 100 nM pinometostat compared to genes upregulated by exogenous expression of constitutively active STAT5A in the WIERENGA_STAT5A_TARGETS GROUP1 gene set from the MSigDB database. ( E ) Bar graph of Cuffdiff output for the expression of STAT5A targets ARID3B , PIM1 , and PIM2 from RNA-seq in MV4;11 cells ± 100 nM pinometostat. Values are represented as log(10) FPKM + 1 for three independent experiments with standard deviation. Student’s t-test (** p < 0.01, *** p < 0.001). ( F ) RT-qPCR analysis of PBX3 , PIM1, FLT3 , and MEF2C expression from three independent experiments of MOLM13 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 < 0.001, **** p < 0.0001). ( G ) Proliferation assay of MV4;11 cells treated with DOT1L or FLT3 inhibitors alone or in combination using CellTiter Glo 2.0 to measure viability, showing the luminescence fraction of inhibited over uninhibited cells. Data are represented as mean ± SE of three independent experiments. Student’s t-test for significance of day 7 values: 100 nM pinometostat vs. combined ** p < 0.01, 30 nM tandutinib vs combined *** p < 0.001. ( H ) Same as G but cells were treated with DOT1L and PIM1 inhibitors alone or in combination. Student’s t-test of day 7 values: 100 nM pinometostat vs. combined * p < 0.05, 10 µM quercetagenin vs combined ** p < 0.01. Purple asterisks indicate Student’s t-test for significance of day 5 for 100 nM pinometostat vs. combination treatment ** p < 0.01. ( I ) Western blots of MV4;11 cell extract from clonal cell lines expressing shRNA to FLT3 (clone 3) or GFP blotted for phosphorylated STAT5 or histone H3 as a loading control. ( J ) Western blots of cell extract from MV4;11 cells treated with 100 nM pinometostat for the indicated number of days and then blotted for H3K79me2 and HNRNPK as a loading control.

Article Snippet: DOT1L inhibitor pinometostat (EPZ5676, Cayman Chemical cat # 16175), EZH2 inhibitor EI1 (Cayman Chemical cat # 19146–1), FLT3 inhibitor tandutinib (MLN518) (Selleckchem cat # S1043), MI-503 (Selleckchem cat # S7817), and PIM1 inhibitor (MedChemExpress cat # HY-15604) were resuspended in DMSO.

Techniques: Quantitative RT-PCR, Expressing, Western Blot, Control, Staining, Quantitative Proteomics, RNA Sequencing, Standard Deviation, Proliferation Assay, shRNA

Journal: eLife

Article Title: Non-canonical H3K79me2-dependent pathways promote the survival of MLL-rearranged leukemia

doi: 10.7554/eLife.64960

Figure Lengend Snippet: ( 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 , 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 log 2 (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).

Article Snippet: DOT1L inhibitor pinometostat (EPZ5676, Cayman Chemical cat # 16175), EZH2 inhibitor EI1 (Cayman Chemical cat # 19146–1), FLT3 inhibitor tandutinib (MLN518) (Selleckchem cat # S1043), MI-503 (Selleckchem cat # S7817), and PIM1 inhibitor (MedChemExpress cat # HY-15604) were resuspended in DMSO.

Techniques: Western Blot, Control, ChIP-qPCR, ChIP-sequencing, Activation Assay, Gene Expression, Expressing

Journal: eLife

Article Title: Non-canonical H3K79me2-dependent pathways promote the survival of MLL-rearranged leukemia

doi: 10.7554/eLife.64960

Figure Lengend Snippet:

Article Snippet: DOT1L inhibitor pinometostat (EPZ5676, Cayman Chemical cat # 16175), EZH2 inhibitor EI1 (Cayman Chemical cat # 19146–1), FLT3 inhibitor tandutinib (MLN518) (Selleckchem cat # S1043), MI-503 (Selleckchem cat # S7817), and PIM1 inhibitor (MedChemExpress cat # HY-15604) were resuspended in DMSO.

Techniques: Recombinant, Plasmid Preparation, Construct, Knockdown, cDNA Library Assay, RNA Extraction, SYBR Green Assay, Reverse Transcription, Proliferation Assay, Software, Sequencing, shRNA

Journal: eLife

Article Title: Non-canonical H3K79me2-dependent pathways promote the survival of MLL-rearranged leukemia

doi: 10.7554/eLife.64960

Figure Lengend Snippet: ( A ) MLL-rearranged leukemia lines with genotypes indicated were treated with 100 nM pinometostat (left panel, DOT1L inhibitor) or 30 nM tandutinib (right panel, FLT3 inhibitor MLN518), and relative growth monitored by CellTiter Glo 2.0 assay on the indicated days. Relative viability presented is the mean fraction of luminescence of treated versus side-by-side mock treated cultures (same volume of DMSO) for three independent replicates ± S.E.M. Student’s t-test (** p ≤ 0.01, *** p ≤ 0.001). ( B ) Western blots of phosphorylated STAT5 (active) or total STAT5A with H3 or HNRNPK as loading controls across the cell lines from panel A treated as indicated; H3K79me2 is monitored in pinometostat-treated lines to confirm inhibition. ( C ) Time course of gene expression by RT-qPCR, presented as mean fold-change of FLT3, PBX3, PIM1 , and MEF2C in MV4;11 cells ± 100 nM pinometostat at each time point indicated ± S.E.M.; n = 3; Student’s t-test (ns p > 0.05, * p ≤ 0.05, **** p ≤ 0.0001, ***** p < 0.00001). ( D-E ) DOT1L and FLT3 inhibition downregulate STAT5A targets in FLT3-ITD . RT-qPCR expression analysis presented as mean fold-change ± S.E.M. for the indicated transcript in MV4;11 cells treated with indicated inhibitor versus mock-treatment for 7 days. Student’s t-test (** p < 0.01, **** p < 0.0001, ***** p < 0.00001). ( F ) Proliferation assay as in panel A, with three clonal populations of MV4;11 cells virally transduced, selected, then induced to express shRNA to FLT3 or a scrambled shRNA control by 1 µg/mL doxycycline. Means of fractional viability relative to uninduced cells ± S.E.M. are shown for three independent experiments; Student’s t-test (** p < 0.01). ( G ) RT-qPCR analysis of PIM1, PIM2 , and ARID3B expression in MV4;11 cells expressing an inducible shRNA targeting FLT3 for 7 days. Results are depicted as fold-change expression of control cells expressing shRNA to GFP . ( H ) Proliferation assay of K562, PL-21, and EOL-1 cells treated with 10 µM or 100 nM pinometostat using CellTiter Glo 2.0 to measure viability, 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 (EOL-1 cells), day 9 (PL-21), or days 9 and 11 (K562 and PL-21) values: ns p > 0.05, * p < 0.05, **** p < 0.0001. ( I ) Gene expression analysis by RT-qPCR in PL-21 cells treated for 9 days with 10 µM pinometostat. Results are displayed as fold-change over DMSO-treated cells with means ± SE for three independent experiments (ND = not detected). Student’s t-test (**** p < 0.0001). ( J ) Western blots of (left) cell extract from PL-21 cells treated with 10 µM pinometostat for 9 days and (right) EOL-1 cells treated with 100 nM pinometostat for 7 days and then blotted for H3K79me2 and p-STAT5 with H2B or HNRNPK as a loading controls. ( K ) Gene expression analysis by RT-qPCR in EOL-1 cells treated for 7 days with 100 nM or 10 µM pinometostat. Results are displayed as fold-change over DMSO-treated cells with means ± SE for three independent experiments. Student’s t-test (ns p > 0.05, **** p < 0.0001).

Article Snippet: chemical compound, drug , Tandutinib (MLN518) , Selleckchem , Cat. #: S1043 , .

Techniques: Western Blot, Inhibition, Gene Expression, Quantitative RT-PCR, Expressing, Proliferation Assay, shRNA, Control

Journal: eLife

Article Title: Non-canonical H3K79me2-dependent pathways promote the survival of MLL-rearranged leukemia

doi: 10.7554/eLife.64960

Figure Lengend Snippet:

Article Snippet: chemical compound, drug , Tandutinib (MLN518) , Selleckchem , Cat. #: S1043 , .

Techniques: Recombinant, Plasmid Preparation, Construct, Knockdown, cDNA Library Assay, RNA Extraction, SYBR Green Assay, Reverse Transcription, Proliferation Assay, Software, Sequencing, shRNA

Journal: eLife

Article Title: Non-canonical H3K79me2-dependent pathways promote the survival of MLL-rearranged leukemia

doi: 10.7554/eLife.64960

Figure Lengend Snippet:

Article Snippet: chemical compound, drug , Tandutinib (MLN518) , Selleckchem , Cat. #: S1043 , .

Techniques: Recombinant, Plasmid Preparation, Construct, Knockdown, cDNA Library Assay, RNA Extraction, SYBR Green Assay, Reverse Transcription, Proliferation Assay, Software, Sequencing, shRNA