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. 2021 Sep 1;106(9):2527-2532.
doi: 10.3324/haematol.2020.271627.

Targeting BRD4 in acute myeloid leukemia with partial tandem duplication of the MLL gene

Marius Bill  1 , Chinmayee Goda  2 , Felice Pepe  2 , Hatice Gulcin Ozer  3 , Betina McNeil  2 , Xiaoli Zhang  3 , Malith Karunasiri  2 , Rohan Kulkarni  2 , Sonu Kalyan  2 , Dimitrios Papaioannou  4 , Gregory Ferenchak  2 , Ramiro Garzon  4 , James E Bradner  5 , Guido Marcucci  6 , Michael A Caligiuri  6 , Adrienne M Dorrance  7
Affiliations

Targeting BRD4 in acute myeloid leukemia with partial tandem duplication of the MLL gene

Marius Bill et al. Haematologica. .
No abstract available

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Figures

Figure 1.
Figure 1.
Targeting BRD4/Brd4 using the inhibitor JQ1 has an effect on MLL-partial tandem duplication (MLL-PTD)/Mll-PTD acute myeloid leukemia cells. (A) WST-1 assay on EOL-1 and K562 cells treated with the indicated concentrations of JQ1 for 48 hours (h). (B) EOL-1 cells were treated for 24 h with the indicated concentration of JQ1. Cells were then assessed for apoptosis using Annexin V+ staining and flow cytometry at 24 h post treatment; *P<0.05, ***P<0.001. (C) Colony forming unit assays were performed on three CD34+ cord blood and three MLL-partial tandem duplication (MLL-PTD) acute myeloid leukemia (AML) patients’ samples, cells were plated in triplicates, normalized results are shown. Cells were treated with JQ1 at a concentration of 9 nM or 12.5 nM or with vehicle control (dimethyl sulfoxide [DMSO]); **P<0.01. (D) Primary murine MllPTD/WT Flt3ITD/WT blasts were treated for 24 h with the indicated concentration of JQ1. Cells were then assessed for apoptosis using Annexin V+ staining and flow cytometry at 24 h post treatment; *P<0.05, **P<0.01. (E) MllPTD/WT Flt3ITD/WT blasts were transplanted into sub-lethally irradiated BoyJ mice. Starting at 2 weeks post transplantation, mice were treated with 50 mg/kg body weight of JQ1 or vehicle control for 6 days each week for the entire duration of the study. Mice that died early without any signs of leukemia were excluded. The experiment was stopped after 120 days post treatment initiation. Treatment with JQ1 prolonged survival compared to controls (P=0.001) and (F) also led to a reduced weight of the spleen.
Figure 2.
Figure 2.
Aberrant BRD4 binding in MLL-partial tandem duplication cells drives a distinct gene expression profile that can be restored by JQ1 treatment. (A) Venn diagram showing overlap of BRD4 binding sites between CD34+ selected cord blood (CB) samples primary samples from MLL-partial tandem duplication (MLL-PTD) acute myeloid leukemia (AML) patients. (B) Scatter plots showing changes in BRD4 binding peak scores upon JQ1 treatment in healthy CB and MLLPTD AML samples. (C) Schematic overview over the bioinformatic approach used to identify the genes deregulated by BRD4 in MLL-PTD cells. A peak and gene pair was classified as MLL-PTD specific positive response when peak score and gene expression were in first quartile (0-25%) for CB samples, peak score and gene expression were in the top quartile (75-100%) for MLL-PTD dimethyl sulfoxide (DMSO) sample, and a decreased expression was observed in both peak score and gene expression for MLL-PTD sample upon JQ1 treatment (25% or more). Similarly, we identified potential oncosuppressor genes downregulated by MLL-PTD but restored by JQ1 treatment. Genes whose expressions were positively or negatively correlated with changes in BRD4 binding with at least 25% change under JQ1 treatment are shown in the Online Supplementary Table S1.(D) Heat map of genes that were found to be deregulated in MLL-PTD cells and which also showed a response to JQ1 treatment. (E) Example of one of 130 genes that fulfilled all criteria. ADAMDEC1 expression is increased in MLL-PTD AML cells by a binding of BRD4, and is downregulated upon JQ1 treatment.
Figure 3.
Figure 3.
MLL-partial tandem duplication drives the aberrant expression profile through BRD4. (A) Cell lysate of EOL-1 cells, either treated with vehicle (dimethyl sulfoxide [DMSO]) or with the indicated concentration of JQ1, were subjected to a chromatin immunoprecipitation (ChIP) assay. Antibodies against BRD4 or POL1RA (control) or an unspecific immunoglobulin G (IgG) were used for the pulldown. DNA of ADAMDEC1 and SLAMF8 were quantified using quantitative realtime polymerase chain reaction (qRT-PCR). The enriched binding of BRD4 to both genes, i.e., ADAMDEC1 and SLAMF8, was decreased after treatment of JQ1; **P<0.01, ***P<0.001. (B) Relative expression of ADAMDEC1 and SLAMF8 relative to GAPDH. qRT-PCR was performed on CD34+ selected cord blood (CB) cell samples (n=3, pooled) and three primary cell samples from MLL-partial tandem duplication (MLL-PTD) acute myeloid leukemia (AML) patients treated with JQ1 at 50 nM for 24 hours. Treatment with JQ1 leads to significantly lower expression of the genes in MLL-PTD patients’ cells but not in CB cells; ns = not significant, *P<0.05, **P<0.01, ***P<0.001. (C) Normalized expression of BRD4 downstream targets, BCL2, CDK6 and MYC, relative to GAPDH in EOL1 cells. Cells treated with JQ1 for 48 hours showed significantly reduced expression of the genes; *P<0.05, **P<0.01, ***P<0.001. (D) Western Blot analysis of EOL1 cells, which were previously treated with JQ1 or vehicle (DMSO) as control, validated the downregulation of these genes. Similar results were found when cells were transfected with a short hairpin RNA (shRNA) against MLL-PTD (shMLL-PTD) but not when transfected with a scramble control. Data of the densitometry are shown. RNA, cDNA, real-time PCR, ChIP and western blots were performed using previously published methods.,
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