The venetoclax and ATO combination synergistically promotes apoptosis in AML LSC-like cells
To assess the combined antileukaemic effects of the venetoclax and ATO in AML LSC-like cells, we used the KG1 cell line, which is characterised by the high surface expression of CD34 but lacking CD38 (CD34+CD38-). The KG1a cell line, with similar phenotypic characteristics, was also included in the present study. We first treated these two cell lines with various concentrations of venetoclax (0–1,000 μM) in the presence/absence of ATO (3 µM). After incubation for 48 h, cell viability, apoptosis, and cell-cycle distribution were examined using the respective methods described in Methods. As shown in Fig. 1a, b, the decrease in the level of cell viability was minimal to modest with the single-agent treatment of increasing concentrations of venetoclax in KG1 and KG1a cells. However, venetoclax substantially impaired the viability of these cells in a dose-dependent manner, when treated in combination with ATO (Fig. 1a, b).
We then measured the proportion of apoptotic cells using Annexin V and PI co-staining and flow cytometry analysis in these cells following treatment with increasing concentrations of venetoclax (0–1,000 μM) in the presence/absence of ATO (3 µM). A consistent trend was noted, involving a significant rise in apoptosis rate with the combination of venetoclax and ATO in KG1 and KG1a cells; however, only a minimal to a modest degree of apoptosis was observed following venetoclax treatment alone (Fig. 1c, d). A noteworthy observation was that the cell death-enhancing effects of ATO were evident even with the lower doses of venetoclax (Fig. 1c, d). The combination effect generated by venetoclax and ATO on apoptosis was further examined by quantitative analysis of the dose–effect relationships based on the Chou–Talalay method (31). As shown in Fig. 1e, f, there was a synergistic effect on apoptosis generated by the interaction between venetoclax and ATO; this was further indicated by CI values less than 1 in both KG1 and KG1a cells.
Further analysis of cell cycle distribution revealed that the proportion of cells in the sub-G1 phase was significantly increased after the venetoclax and ATO combination treatment compared with either agent alone, indicating that the disrupted cell cycle caused by the combination treatment may be linked to the induction of apoptosis (Fig. 2a, b).
The venetoclax and ATO combination preferentially induces apoptosis in primary CD34+ AML cells while sparing HSCs from healthy donors
To further examine whether ATO effectively promotes the venetoclax-induced apoptosis in primary AML LSC-like cells, we harvested diagnostic BMMCs from AML patients without any cytogenetic abnormalities. The clinical characteristics of the AML patients are summarised in Supplementary Table 1. BMMCs from AML patients were treated with 100 nM of venetoclax in the presence/absence of 3 μM of ATO. After 48 h of incubation, the fraction of apoptotic cells was measured among the blast gate, CD34+ blasts, and CD34+CD38- cells using Annexin V and 7-AAD staining and flow cytometry analysis. Representative flow cytometric plots of gated CD34+CD38- primary AML cells after the single or combination treatment with venetoclax and ATO are shown in Fig. 3a.
With venetoclax treatment alone, the frequencies of Annexin V+7-AAD+ apoptotic cells in the mononuclear cell blast gate, CD34+ blasts, and CD34+CD38- blast population were 33.93±9.13%, 49.98±9.31%, and 58.51±6.42, respectively (Fig. 3b). After ATO treatment alone, the apoptotic cell fraction was 11.54±7.51%, 7.91±6.79%, and 19.1±12.3% in the mononuclear cell blast gate, CD34+ blast, and CD34+CD38- blast population, respectively (Fig. 3b). However, with the combination treatment of venetoclax and ATO, the apoptotic fraction was significantly increased to 68.00±5.66% in the mononuclear cell blast gate (P = 0.0007 vs venetoclax alone; P < 0.0001 vs ATO alone) (Fig. 3b). The differences in the level of apoptosis remained significant when analyses were performed for gated CD34+ cells and CD34+CD38- LSC-like cells (for CD34+ cells, P = 0.0031 vs venetoclax and P < 0.0001 vs ATO; for CD34+CD38- blasts, P = 0.0002 vs venetoclax and P < 0.0001 vs ATO) (Fig. 3b).
Interestingly, as depicted in the representative flow cytometric plots of gated CD34+CD38- cells after the single or combination treatment with venetoclax and ATO in the BMMCs of healthy donors (Fig. 3c), the combined effect of these agents on apoptosis was minimal in CD34+CD38- cells as well as in the gated blasts, CD34+ cells of healthy BMMCs (Fig. 3d). These findings implied that the combination of venetoclax and ATO promotes induction of apoptosis preferentially in bulk AML cells and LSC-like cells while sparing HSCs.
The venetoclax and ATO combination potentially activates the caspase-dependent mitochondrial apoptotic pathway
We next evaluated the changes in caspase cleavage and MMP to unravel the mechanism of cell death involved in the combination of venetoclax and ATO in LSC-like cells. Compared with a single treatment, the combination of venetoclax and ATO resulted in an increase in the levels of cleaved caspase-9, cleaved caspase-3, and cleaved PARP (Fig. 4a).
To further investigate the cell death mechanism potentially triggered by the combination of venetoclax and ATO, MMP was analysed by flow cytometry using the DiOC6 probe. The venetoclax and ATO combination robustly induced a higher degree of MMP depolarisation compared with the extent of depolarisation induced by a single treatment with either agent in both KG1 and KG1a cells (Fig. 4b). Next, apoptotic status was analysed after the preincubation of these cells with pan-caspase inhibitor z-VAD-fmk (20 μM) for 2 h. As shown in Fig 4c, d, z-VAD-fmk preincubation significantly alleviated apoptosis induction by combination treatment with venetoclax and ATO in both KG1 and KG1a cells. Collectively, these findings indicated that the synergistic increase in the rate of apoptosis by the venetoclax and ATO combination is, at least in part, attributable to the activation of the caspase-dependent mitochondrial apoptotic pathway.
Mcl-1 protein is downregulated by adding ATO to venetoclax treatment in KG1 and KG1a cells
To elucidate the molecular mechanisms involved in the synergistic increase in apoptosis level with the combination of venetoclax and ATO, we next examined the effects of this combination on the Bcl-2 family members, which are critical regulators of apoptosis (7). There were no apparent changes in the protein level of Bcl-2 following treatment with venetoclax in the presence/absence of ATO for 48 h in KG1 and KG1a cells (Fig. 5a). However, cleaved Bcl-2 was generated after treatment with ATO alone or in combination with venetoclax in KG1a cells, which was not observed in KG1 cells (Fig. 5a), suggesting the contextual differences between these cells with respect to the molecular alterations involving the cellular apoptosis machinery.
Next, we evaluated the changes in the Mcl-1 protein level. There was a noticeable elevation in the Mcl-1 protein level with venetoclax single treatment in both KG1 and KG1a cells (Fig. 5a). However, when these cells were treated with the combination of venetoclax and ATO, Mcl-1 protein levels were substantially downregulated in both these cell types (Fig. 5a). Remarkably, alleviation of the venetoclax-induced upregulation of Mcl-1 by ATO addition was conspicuous in KG1a cells (Fig. 5a).
To further elucidate the mechanism of Mcl-1 protein downregulation, we investigated the levels of Mcl-1 phosphorylation at Ser159 (p-Mcl-1 Ser159) as well as at Thr163 residue (p-Mcl-1 Thr163). In parallel with the downregulation of Mcl-1 protein, the level of p-Mcl-1 Ser159 was increased, whereas the level of p-Mcl-1 Thr163 was decreased following the combination treatment of venetoclax and ATO in both KG1 and KG1a cells (Fig. 5a). However, the change in the protein expression level of another anti-apoptotic member, Bcl-xL, was minimal after the combination treatment or either agent alone (Fig. 5a).
Notably, the protein level of Noxa, which is antagonised by Mcl-1 (33), was elevated after venetoclax and ATO combination treatment in both KG1 and KG1a cells (Fig. 5b). Likewise, the increase in the proapoptotic protein level of Bim, which forms a complex with Mcl-1 (7), was highest when these cells were treated with the combination of venetoclax and ATO (Fig. 5b). The protein level of Bak, which is a core regulator of mitochondrial outer membrane permeabilisation and apoptosis that is normally sequestered by Mcl-1 (7), was strongly induced upon combination treatment, especially in KG1a cells, while to a lesser extent with ATO single treatment (Fig. 5b). The change in the protein level of another proapoptotic effector Bax was minimal, implying that Bax may not be a major contributor to enhanced cell death with the venetoclax and ATO combination in these cells (Fig. 5b). Taken together, these findings indicated that Noxa and Bim may be untethered from Mcl-1-mediated binding through downregulation of Mcl-1, leading to the increased induction of apoptosis associated with Bak activation.
The venetoclax and ATO combination activates GSK-3β in KG1 and KG1a cells
Since GSK-3β activation promotes p-Mcl-1 Ser159 leading to proteasomal degradation of Mcl-1 protein (7), we evaluated GSK-3β activity by measuring the level of GSK-3β phosphorylation at Ser9 (p-GSK-3β Ser9). Although the change in the total protein level of GSK-3β was minimal, the level of p-GSK-3β Ser9 was notably decreased after treatment with ATO. This was mediated by enhanced GSK-3β activity induced by adding ATO to venetoclax treatment in KG1 and KG1a cells (Fig. 5c). The phosphorylated form of AKT (Ser473) was diminished without significant changes in the level of total AKT by the combination treatment in both these cell types (Fig. 5c). The level of phosphorylated ERK (Thr202/Tyr204), which phosphorylates Mcl-1 at the Thr163 residue (p- Mcl-1 Thr163) and stabilises Mcl-1, was also reduced in KG1 and KG1a cells in response to treatment with the combination of venetoclax and ATO (Fig. 5c). These results signified that venetoclax-induced Mcl-1 upregulation is mitigated by ATO via GSK-3β activation-mediated increase in the level of p-Mcl-1 Ser159 and decrease in the level of p-Mcl-1 Thr163. This may subsequently lead to the proteasomal degradation and destabilsation of Mcl-1 protein.
Ectopic Mcl-1 overexpression alleviates the venetoclax and ATO combination-induced apoptosis
To further elucidate the role of Mcl-1 downregulation in the venetoclax and ATO combination-induced synergistic cell death, Mcl-1 overexpression was ectopically induced in KG1a cells (Fig. 6a), as described in Methods. Briefly, KG1a cells were transfected with pcDNA3.1 control or pcDNA3.1-Mcl-1 vector, and the level of apoptosis was examined with the Annexin V and PI staining after the single or combined treatment with venetoclax and ATO (Fig. 6b). The proportion of apoptotic cells was significantly diminished after the combination treatment in KG1a cells upon Mcl-1 overexpression (52.80±6.59%), compared with the combination treatment in control KG1a cells without Mcl-1 overexpression (72.50±5.47%, P = 0.0286) (Fig. 6b,c). These findings indicate that downregulation of Mcl-1 is critically involved in the synergistic increase in the level of apoptosis in AML LSC-like cells treated with a combination of venetoclax and ATO.
Venetoclax and ATO combination triggers robust DNA damage response
The findings above led us to explore the changes in the level of proteins involved in the DNA damage response after venetoclax and ATO treatment alone or in combination in KG1 and KG1a cells. As shown in Fig. 7, the combination of venetoclax and ATO robustly increased the levels of p-ATM (Ser1981) and p-H2AX (Ser139) compared with the single treatment of either agent alone. In parallel, Chk2 phosphorylation at Thr68 residue was markedly augmented with the combination treatment (Fig. 7). Phosphorylation of p38 mitogen-activated protein kinase (MAPK) was also augmented with the combined treatment of venetoclax and ATO compared with the treatment of either agent alone (Fig. 7), signifying the robust induction of apoptosis by the combination treatment in these cells. However, compared with the phosphorylation of ATM and Chk2, the phosphorylation of ATR (Ser428) and Chk1(Ser317) were modest following the combination treatment (Fig. 7). Changes in p53 protein expression levels were not apparent in both KG1 and KG1a cells (Fig. 7). Taken together, these findings implied that p53-independent DNA damage response, especially the p-ATM (Ser1981) and p-Chk2 (Thr68) pathway, is associated with enhanced induction of apoptosis by the combination of venetoclax and ATO.