Arsenic trioxide synergistically promotes the antileukaemic activity of venetoclax by downregulating Mcl-1 in acute myeloid leukaemia cells

Abstract

Background: The evasion of apoptosis through dysregulated Bcl-2 family members is a hallmark of leukaemia stem cells (LSCs) in acute myeloid leukaemia (AML). Therefore, targeting Bcl-2 with venetoclax has been suggested as an attractive strategy for inducing apoptosis in AML LSCs. However, the selective inhibition of Bcl-2 in AML often leads to upregulation of Mcl-1, another dominant anti-apoptotic Bcl-2 family protein conferring venetoclax resistance.

Methods: We assessed the combined effect of venetoclax and arsenic trioxide (ATO) on leukaemic cell viability, apoptosis, combination index, and cell cycle in the human LSC-like KG1 and KG1a cells. The synergistic effect of venetoclax and ATO on apoptosis was also examined in primary CD34⁺ and CD34⁺CD38^- LSCs from the bone marrow (BM) of AML patients, and compared with those from healthy donors.

Results: Venetoclax efficiently impaired cell viability and dose-dependently promoted apoptosis when combined with ATO; their synergism was aptly represented by the combination index. The combination of venetoclax and ATO impaired cell cycle progression by restricting cells within the sub-G1 phase and facilitating caspase-dependent apoptotic cell death associated with the loss of mitochondrial membrane potential, while sparing healthy BM haematopoietic stem cells. Mechanistically, ATO mitigated venetoclax-induced upregulation of Mcl-1 by the inhibition of AKT and ERK, along with activation of GSK-3β. This led to the Mcl-1 destabilisation, triggering Noxa and Bim to facilitate apoptosis and the consequent activation of the apoptosis executioner protein Bak. Moreover, the combination promoted phosphorylation of ATM, Chk2, p38, and H2AX, indicating an active DNA damage response.

Conclusions: Our findings demonstrate the synergistic, preferential antileukaemic effects of venetoclax and ATO on LSCs, providing a rationale for preclinical and clinical trials by combining these agents already being used in clinical practice to treat acute leukaemia.

Keywords: Acute myeloid leukaemia; Apoptosis; Arsenic trioxide; Venetoclax.

Fig. 1

Venetoclax dose-dependently impairs cell viability and synergistically promotes apoptosis in KG1 and KG1a cells when in combination with ATO. a, b, Assessment of cell viability in KG1 (a) and KG1a (b) cells treated with the indicated concentration of venetoclax (0–1,000 nM) with or without ATO (3 µM) for 48 h. Cell viability was assessed by measuring the absorbance at 450 nm after incubating the treated cells for 4 h with Cell Counting Kit-8 solution (Dojindo). Values were obtained from three independent experiments, and horizontal bars indicate mean ± s.d. *P < 0.05 versus respective control by two-tailed Mann–Whitney U test. c, d, Summary data of the percentage of apoptotic fraction in KG1 (c) and KG1a (d) cells, as assessed by Annexin V and PI staining and flow cytometric analysis after treatment with the indicated concentration of venetoclax (0–1,000 nM) with or without ATO (3 µM) for 48 h. Values were obtained from three independent experiments, and horizontal bars indicate mean ± s.d. *P < 0.05 versus control treatment by two-tailed Mann–Whitney U test. e, f, Combination index of apoptotic cells after treatment with venetoclax and ATO in KG1 (e) and KG1a (f) cells. Values were obtained by median dose–effect analysis, and each dot indicates the value obtained from six independent experiments

Fig. 2

The combination of venetoclax and ATO disrupts the KG1 and KG1a cell cycles. a, b, Cell cycle analysis by flow cytometry after treatment with control (DMSO 0.1% v/v), venetoclax (200 nM), ATO (3 µM), or the combination of venetoclax (200 nM) and ATO (3 µM) for 48 h. Representative histograms of the cell cycles of the gated live KG1 and KG1a cells after treatment (a) and their summary data (b). Values were obtained from three independent experiments, and horizontal bars indicate mean ± s.d

Fig. 3

The venetoclax and ATO combination preferentially induces apoptosis of primary LSCs from AML patients while sparing healthy donor HSCs. a, b, Representative flow cytometric analysis (a) and summary data (b) of the percentage of Annexin V⁺7-AAD⁺ apoptotic cells after gating for CD34⁺CD38⁻ primary AML cells in the BMMCs of AML patients at diagnosis after treatment with venetoclax (100 nM), ATO (3 µM), or both in combination for 48 h in total mononuclear cells (b, far left), gated CD34⁺ cells (b, middle), or CD34⁺CD38⁻ cells (b, far right). Values were obtained from two independent experiments of n = 4 patients, and horizontal bars indicate mean ± s.d. *P values versus control treatment by two-tailed Mann–Whitney U test. n.s., not significant. c, d, Representative flow cytometric analysis (c) and summary data (d) of the percentage of Annexin V⁺7-AAD⁺ apoptotic cells after gating for CD34⁺CD38⁻ cells in the BMMCs of healthy donors following treatment with venetoclax (100 nM), ATO (3 µM), or both in combination for 48 h in total mononuclear cells (d, far left), gated CD34⁺ cells (d, middle), or CD34⁺CD38⁻ cells (d, far right). Values were obtained from two independent experiments of n = 4 healthy donors, and horizontal bars indicate mean ± s.d. *P values versus control treatment by two-tailed Mann–Whitney U test. n.s., not significant

Fig. 4

The combination of venetoclax and ATO promotes caspase-dependent apoptosis in KG1 and KG1a cells. a, Representative western blot analysis of the indicated proteins in KG1 and KG1a cells treated with venetoclax (200 nM), ATO (3 µM), or both in combination for 48 h. Similar results were obtained from three independent experiments. b, Summary data of MMP disruption, as assessed by flow cytometry, using the DePsipher Kit (Trevigen) in KG1 and KG1a cells treated with venetoclax (200 nM), ATO (3 µM), or both in combination for 48 h. Values were obtained from three independent experiments, and horizontal bars indicate mean ± s.d. *P values versus control treatment by two-tailed Mann–Whitney U test. n.s., not significant. c, d, Summary data of the percentage of apoptotic cells, as assessed by Annexin V/PI staining and flow cytometric analysis after treatment with or without the pan-caspase inhibitor z-VAD-fmk (20 µM) for 2 h prior to the addition of venetoclax (200 nM), ATO (3 µM), or both in combination for 48 h in KG1 (c) and KG1a (d) cells. Values were obtained from three independent experiments, and horizontal bars indicate mean ± s.d. *P values versus control treatment with or without z-VAD-fmk by two-tailed Mann–Whitney U test. n.s., not significant

Fig. 5

The venetoclax and ATO combination downregulates Mcl-1 through GSK-3β activation in KG1 and KG1a cells. a–c, Representative western blot analysis of the indicated proteins in KG1 and KG1a cells treated with venetoclax (200 nM), ATO (3 µM), or both in combination for 48 h. Similar results were obtained from three independent experiments

Fig. 6

Mcl-1 overexpression alleviates apoptosis induced by the combination of venetoclax and ATO in KG1a cells. a, Western blot analysis of indicated proteins in KG1a cells treated with venetoclax (200 nM), ATO (3 µM), or both in combination for 48 h after transfection with pcDNA3.1 control or pcDNA3.1-Mcl-1 vector. b, c, Representative flow cytometric analysis (b) and summary data (c) of the percentage of Annexin V⁺PI⁺ apoptotic cells in KG1a cells transfected with pcDNA3.1 control or pcDNA3.1-Mcl-1 vector after treatment with venetoclax (100 nM), ATO (3 µM), or both in combination for 48 h. Values were obtained from two independent experiments, and horizontal bars indicate mean ± s.d. *P values versus control treatment by two-tailed Mann–Whitney U test. n.s., not significant

Fig. 7

The venetoclax and ATO combination triggers robust DNA damage response. Representative western blot analysis of the indicated proteins in KG1 and KG1a cells treated with venetoclax (200 nM), ATO (3 µM), or both in combination for 48 h. Similar results were obtained from three independent experiments

Fig. 8

Proposed mechanism of synergistic action between venetoclax and ATO. Schematic diagram depicting the synergism between venetoclax and ATO in LSC-like leukaemia cells and primary LSCs. Bcl-2 inhibition by venetoclax in LSC-like leukaemia cells leads to the upregulation of Mcl-1, which confers resistance to venetoclax. However, upon combination treatment with venetoclax and ATO, AKT is attenuated and subsequently impairs GSK-3β phosphorylation at Ser9 and activates GSK-3β. This leads to the phosphorylation of Mcl-1 at Ser159, triggering Mcl-1 degradation. The levels of Noxa and Bim, which are normally sequestered by Mcl-1, are elevated upon Mcl-1 degradation and promote caspase-dependent apoptosis associated with Bak activation. In parallel, ERK downregulation attenuates Mcl-1 phosphorylation at Thr163, leading to Mcl-1 destabilisation. The venetoclax and ATO combination also induces the phosphorylation of ATM at Ser1981 and Chk2 at Thr68, promoting cell cycle arrest associated with a robust DNA damage response and apoptosis