Venetoclax efficacy on acute myeloid leukemia is enhanced by the combination with butyrate

Abstract

Venetoclax has been approved recently for treatment of Acute myeloid leukemia (AML). Venetoclax is a BH3-mimetic and induces apoptosis via Bcl-2 inhibition. However, venetoclax's effect is still restrictive and a novel strategy is needed. In the present study, we demonstrate that sodium butyrate (NaB) facilitates the venetoclax's efficacy of cell death in AML cells. As a single agent, NaB or venetoclax exerted just a weak effect on cell death induction for AML cell line KG-1. The combination with NaB and venetoclax drastically induced cell death. NaB upregulated pro-apoptotic factors, Bax and Bak, indicating the synergistic effect by the collaboration with Bcl-2 inhibition by venetoclax. The combined treatment with NaB and venetoclax strongly cleaved a caspase substrate poly (ADP-ribose) polymerase (PARP) and a potent pan-caspase inhibitor Q-VD-OPh almost completely blocked the cell death induced by the combination, meaning that the combination mainly induced apoptosis. The combination with NaB and venetoclax also strongly induced cell death in another AML cell line SKNO-1 but did not affect chronic myeloid leukemia (CML) cell line K562, indicating that the effect was specific for AML cells. Our results provide a novel strategy to strengthen the effect of venetoclax for AML treatment.

Figure 1

Venetoclax rarely affected for cell growth of AML cells. KG-1 cells which were derived from AML patient were treated with venetoclax for 48 h at the indicated concentrations. (A) Cell morphologies were observed using a microscope. (B) Trypan blue dye exclusion assay to examine the rate of live or dead cells. (C,D) Cell cycle analyses of cells stained with PI using flow cytometry. A bar graph with triplicate data was shown in (C). Representative histograms were shown in (D). The data was n = 3 ± S.D.

Figure 2

Sodium butyrate (NaB) enhanced venetoclax’s effect on cell growth inhibition. KG-1 cells were treated with venetoclax with or without NaB for 48 h at the indicated concentrations. (A) Trypan blue dye exclusion assay to examine the rate of live or dead cells. (B) Cell morphologies were observed using a microscope. The data was n = 3 ± S.D.. Vene; venetoclax.

Figure 3

The combined treatment of venetoclax with NaB drastically induced cell death. AML cells (A,B KG-1 cells; C,D SKNO-1 cells) were treated with venetoclax with or without NaB for 48 h at the indicated concentrations. Cell cycle analyses of cells stained with PI using flow cytometry. Bar graphs (A,C) and representative histograms (B,D) were shown. The data was n = 3 ± S.D.. Vene; venetoclax.

Figure 4

NaB upregulated Bax and Bak expressions. (A) BloodSpot analysis of Bcl-2 expression in AML patients. *P < 0.05, ***P < 0.001 and NS = non significant. (B–D). KG-1 cells were treated with NaB at the indicated concentrations and total RNA was extracted from cells. Bcl-2 (B), Bax (C) and Bak (D) expressions were examined by RT-qPCR with specific primers. The data was standardized by Actin expression. The data was n = 3 ± S.D.. (E) and (F). ChIP-Atlas analysis of HDAC1 on Bax (E) and Bak (F) gene locus. ChIP data were derived from SRX17989497, SRX17989501, SRX17989501 and SRX2423955.

Figure 5

Caspase activation was induced by the combination of venetoclax and NaB. KG-1 was treated with venetoclax alone or the combination with venetoclax and NaB for 48 h. (A) Western blotting of PARP. Cell lysates were extracted and western blotting was performed. Upper panel; PARP, Lower panel; actin. Molecular weight marker was shown at the left side. The PARP-cleaved form generated by caspase activation was indicated by an arrow. Original blots are presented in Supplemental Fig. 3. Quantification of band intensity was shown in Supplemental Fig. 4. (B) Trypan blue dye exclusion assay to examine the rate of live or dead cells. (C) Cell morphologies were observed using a microscope. (D,E) Cell cycle analyses of cells stained with PI using a flow cytometry. A bar graph with triplicate data (D) and representative histograms (E) were shown. The data was n = 3 ± S.D.. Vene; venetoclax.

Figure 6

The combination of venetoclax with NaB did not affect on chronic myeloid leukemia K562 cells. K562 CML cell line was treated with the combination of venetoclax and NaB for 48 h. (A) Trypan blue dye exclusion assay to examine the rate of live or dead cells. (B) Cell morphologies were observed using a microscope. (C,D) Cell cycle analyses of cells stained with PI using flow cytometry. A bar graph with triplicate data (C) and representative histograms (D) were shown. The data was n = 3 ± S.D..