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. 2020 Apr 8:26:e923411.
doi: 10.12659/MSM.923411.

Bromodomains and Extra-Terminal (BET) Inhibitor JQ1 Suppresses Proliferation of Acute Lymphocytic Leukemia by Inhibiting c-Myc-Mediated Glycolysis

Meng-Yi Zhang  1   2 , Sheng-Li Liu  1 , Wen-Li Huang  3 , Da-Bin Tang  1 , Wei-Wei Zheng  1 , Neng Zhou  1 , Hang Zhou  1 , Tuersunayi Abudureheman  1 , Zhong-Hua Tang  1 , Bin-Bing S Zhou  1   2 , Cai-Wen Duan  1   2
Affiliations

Bromodomains and Extra-Terminal (BET) Inhibitor JQ1 Suppresses Proliferation of Acute Lymphocytic Leukemia by Inhibiting c-Myc-Mediated Glycolysis

Meng-Yi Zhang et al. Med Sci Monit. .

Abstract

BACKGROUND Acute lymphocytic leukemia (ALL) is a common blood cancer which induces high mortality in children. Bromodomains and extra-terminal (BET) protein inhibitors, such as JQ1 and ARV-825, are promising cancer therapeutic agents that can be used by _targeting c-Myc. A recent work reported that JQ1 effectively attenuates ALL in vitro by suppressing cell proliferation and accelerating apoptosis. The purpose of this research was to probe into the potential mechanism of how JQ1 inhibits ALL cell proliferation in vitro. MATERIAL AND METHODS Cell viability of ALL cells were measured by CTG after treatment by JQ1. Cell cycle analysis was done by EdU and PI staining. Cell apoptosis was assessed by Annexin V/PI staining. Glycolysis was detected using Seahorse and LC-MS kits. The expression of glycolytic rate-limiting enzymes was assessed by RNA-seq, qRT-PCR, and Western blot. RESULTS JQ1 suppressed cell proliferation by arresting the cell cycle and inducing the apoptosis of acute lymphocytic leukemia cells. JQ1 inhibited cell proliferation of B-ALL cells by restraining glycolysis. Conversely, the cell cycle block of B-ALL cells induced by JQ1 was partially abolished after pretreatment with 2-Deoxy-D-glucose (2-DG), an inhibitor of glycolysis. Furthermore, JQ1 restrained the glycolysis of B-ALL cell lines by remarkably downregulating the rate-limiting enzymes of glycolysis, such as hexokinase 2, phosphofructokinase, and lactate dehydrogenase A. Moreover, the cell cycle arrest was reversed in B-ALL cells with overexpressed c-Myc treated by JQ1, which is involved in the enhancement of glycolysis. CONCLUSIONS The BET inhibitor JQ1 suppresses the proliferation of ALL by inhibiting c-Myc-mediated glycolysis, thus providing a new strategy for the treatment of ALL.

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Conflict of interest statement

Conflicts of interest

None.

Figures

Figure 1
Figure 1
JQ1 restrained the proliferation of B-ALL cell lines. (A) Drug sensitivity assay of NALM6, REH, SEM, and RS411 cell lines after treatment with gradient concentrations of JQ1 for 72 h. (B) The cell growth curve of NALM6, REH, SEM, and RS411 cells after treatment with DMSO or JQ1 (1 μM) for 5 days. (C) EdU- and PI-labeled cell cycle of NALM6, REH, SEM, and RS411 cells were analyzed after treatment with DMSO or JQ1 (1 μM) for 48 h. (D) Statistical analysis of cell cycle distributions in C. (E) Heatmap of cell cycle and apoptosis-related genes detected by RNA-seq in NALM6 cells after treatment with DMSO or JQ1 (1 μM) for 48 h. (F) Annexin V- and PI-labeled cell apoptosis of NALM6, REH, SEM, and RS411 cells analyzed by flow cytometry after DMSO or JQ1 (1 μM) treatment for 48 h. (G) Statistical analysis of cell apoptosis rates in F. (H) Expressions of cleaved caspase 9 and BCL-2 detected by Western blot in NALM6, REH, SEM, and RS411 cells after treatment with DMSO or JQ1 (1 μM) for 48 h.* P<0.05; ** P<0.01.
Figure 2
Figure 2
JQ1 suppressed the growth of B-ALL cell lines by inhibiting glycolysis. (A) Bubble diagram of enrichment analysis in NALM6 cells measured by RNA-seq after treatment with DMSO or JQ1 (1 μM) for 48 h. (B) Pathway analysis of downregulated metabolic genes (False Discovery Rate <0.05) in JQ1-treated NALM6 cells. (C) Glucose uptake of NALM6, REH, SEM, and RS411 cells detected by flow cytometry after treatment with DMSO or JQ1 (1 μM) for 48 h. (D) Statistical analysis of NALM6, REH, SEM, and RS411 cells estimated using the related kits after DMSO or JQ1 (1 μM) treatment for 48 h. (E) Quantification of lactate production in NALM6, REH, SEM, and RS411 cells assessed using the related kit after treatment with DMSO or JQ1 (1 μM) for 48 h. (F) Quantification of intracellular ATP in NALM6, REH, SEM, and RS411 cells assessed using the related kits after treatment with DMSO or JQ1 (1 μM) for 48 h. (G) Detection of ECAR in NALM6 cell after treatment with DMSO or JQ1 (1 μM) for 48 h by seahorse. (H) Schematic map of glycolytic metabolism and the metabolic intermediates of glycolysis measured by LC-MS/MS. (I) The cell cycle of JQ1 (2 uM)-treated NALM6 cells analyzed by flow cytometry after pretreatment with 2DG (5mM) for 12 h. * P<0.05; ** P<0.01.
Figure 3
Figure 3
JQ1 restrained the glycolysis of B-ALL cell lines by downregulating the expression of metabolic enzymes. (A) Heatmap of glycolysis-related genes analyzed by RNA-seq in NALM6 cells after treatment with DMSO or JQ1 (1 μM) for 48 h. (B–D) Relative mRNA expression levels of HK2, PKM2, and LDHA measured by qRT-PCR in NALM6, REH, SEM, and RS411 cells after treatment with DMSO or JQ1 (1 μM) for 48 h. (E) Protein expressions of HK2, PFKP, PKM2, and LDHA detected by Western blot in NALM6, REH, SEM, and RS411 cells after DMSO or JQ1 (1 μM) treatment for 48 h.* P<0.05; ** P<0.01.
Figure 4
Figure 4
JQ1 induced cell cycle arrest by inhibiting c-Myc-mediated glycolysis. (A) Protein expression of c-Myc determined by Western blot in NALM6, REH, SEM, and RS411 cells after treatment with DMSO or JQ1 (1 μM) (1, 2, and 4 μM) for 48 h. (B) Overexpression level of c-Myc in NALM6 and REH cells was checked by Western blot. (C, D) The expression level of c-Myc in NALM6 and REH cells tested by Western blot after treatment with DMSO or JQ1 (1 μM) in a time course. (E, F) EdU- and PI-labeled cell cycle of c-Myc-overexpressing NALM6 and REH cells analyzed by flow cytometry after DMSO or JQ1 (2 μM) treatment for 24 h. (G) Quantification of intracellular lactate in vector and c-Myc-overexpressing NALM6 cells after DMSO or JQ1 (2 μM) treatment for 24 h. (H) Quantification of intracellular ATP in vector and c-Myc-overexpressing NALM6 cells after DMSO or JQ1 (2 μM) treatment for 24 h. (I) Glucose uptake of vector and c-Myc-overexpressing NALM6 cells analyzed by flow cytometry after treatment with DMSO or JQ1 (2 μM) for 24 h. (J) Levels of HK2 and LDHA in c-Myc-overexpressing NALM6 and REH cells tested by Western blot after treatment with DMSO or JQ1 (2 μM) for 24 h.* P<0.05.
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