Homoharringtonine is synergistically lethal with BCL-2 inhibitor APG-2575 in acute myeloid leukemia

APG-2575 was provided by Ascentage Pharma (Suzhou) Co., Ltd and ABT-199 was purchased from Selleck Chemicals (Houston, TX, USA). HHT was purchased from Sigma-Aldrich (St. Louis, MO, USA). The antibodies GAPDH (#5174), PARP (#9532), Caspase3 and Cleaved-Caspase3 (Cleaved-C3) (#9662), Caspase7 and Cleaved-Caspase7 (Cleaved-C7) (#9494), PI3K (p85α) (#4257), AKT (#4691), AKT (Ser473) (#4060), GSK3β (#9315), GSK3β (Ser9) (#9323), MCL-1 (#94296), MCL-1 (Thr163) (#14765) and MCL-1 (Thr163/Ser159) (#4579) were purchased from Cell Signaling Technology (CST, Beverly, MA, USA).

MV4-11 cell line was kindly endowed by Professor R. Bhatia (City of Hope National Medical Center, Duarte, CA, USA). THP-1, HL-60, U937 and OCI-AML3 cell lines were purchased from the Shanghai Cell Bank of the Chinese Academy of Sciences. Kasumi-1 cell line was gifted by Professor Chen Saijuan (Shanghai Institute of Hematology, Shanghai, China). MV4-11 cells were maintained in Iscove’s Modified Dulbecco’s medium (IMDM, Gibco, Billings, MT, USA) supplemented with 10% fetal bovine serum (FBS, Gibco), and THP-1, HL-60, U937 and OCI-AML3 cells were cultured in Roswell Park Memorial Institute 1640 (RPMI 1640, Gibco) medium supplemented with 10% FBS at 37 °C in a humidified incubator containing 5% CO₂.

Bone marrow samples were obtained from AML patients following written informed consent. Mononuclear cells were isolated by Ficoll-Hypaque (Sigma-Aldrich, St. Louis, MO, USA) density gradient centrifugation. The study protocol was approved by the Ethics Committee of the First Affiliated Hospital of Zhejiang University, China.

20,000 AML cells (cell density of 2.0 × 10⁵ cells/ml) or 100,000 primary AML cells (cell density of 1.0 × 10⁶ cells/ml) were seeded in a 96-well plate and treated with designated drugs for the 24 or 48 h. Then 10 ul MTS solution (Promega CellTitre 96, Promega Corporation, Madison, WI, USA (5 mg/ml) was added by incubation for additional 3–4 h at 37℃. The plates were measured at an absorbance of 490 nm. IC50 values and combination index (CI) were calculated by CalcuSyn Software (Biosoft, Cambridge, UK) (CI: < 1, synergism; > 1, antagonism; = 1, additivity). That program was based on the following equation: q = E_A+B / (E_A + E_B—E_A × E_B), where E_A and E_B are the inhibition rate of group A and group B, respectively, and E_A+B is the inhibition rate of group A combined with B (q: > 1.15, synergism; < 0.85, antagonism; 0.85–1.15, additivity) [23, 24].

The extent of apoptosis was assessed through an apoptosis detection kit (Beyotime Institute of Biotechnology, Hangzhou, China). Cells were treated with drugs for 24 h, and then harvested and washed with phosphate buffered saline (PBS). According to the manufacturer’s instruction, after resuspending in binding buffer and staining with Annexin V-FITC (Annexin-V) and propidium iodide (PI) in dark at room temperature for 15 min, cells were subjected to FACScan™ flow cytometer (Becton Dickinson, San Diego, CA, USA). Data were presented as percentages of Annexin V + cells.

Cells were harvested and washed in PBS, and then lysed in radioimmunoprecipitation (RIPA) buffer in the presence of protease and phosphatase inhibitor (Cell Signaling Technology (CST), Beverly, MA, USA)) on ice for 30 min. Cells were centrifuged at 12,000g for 15 min at 4 °C to pellet cell debris. The protein concentration was determined using BCA reagent. Equal protein was electrophoresed in 10% SDS-PAGE (Life Technologies, Carlsbad, CA, USA) and transferred to a PVDF membrane (Millipore, Billerica, MA, USA). The transblotted membranes were blocked with 5% non-fat milk in TBS-T for 1 h and then incubated with primary antibodies overnight at 4 ℃. Then the membranes were washed thrice with TBS-T buffer, 15 min each time, followed by secondary HRP-conjugated antibody (CST, Beverly, MA, USA) for 1 h at room temperature. The target proteins were visualized using an ECL detection kit (Amersham, Little Chalfont, UK) and analyzed using IMAGE LAB^TM software (Bio-Rad Laboratories, Hercules, CA, USA).

BALB/c Nude mice (female, 4–6 weeks of age, weight 17–24 g) were purchased from Shanghai Lingchang Biotechnology Co., Ltd. (Shanghai, China) and Hangzhou Medical College Laboratory animal center. MV4-11 cells were resuspended in PBS (1 × 10⁶ cells/mouse), and then subcutaneously injected into the right back of BALB/c Nude mice to establish a subcutaneous xenograft tumor model. When the tumor volume reached to 50–150 mm³, the mice were randomly divided into 6 groups (vehicle control, 50 mg/kg APG-2575, 50 mg/kg ABT-199, 1 mg/kg HHT, 50 mg/kg APG-2575 + 1 mg/kg HHT, 50 mg/kg ABT-199 + 1 mg/kg HHT). The administration was started on the day of grouping (ie D1; APG-2575/1BT-199, PO, QDx21D; HHT, IP, QDx14D). The mouse body weight and tumor size were measured twice a week during the experiment. After the administration, all mice were killed by cervical dislocation. In addition, OCI-AML3 cells were injected subcutaneously on the left back of BALB/c Nude mice, and the other operations referred to the above.

Tumor volume (TV) was calculated as: TV = L × W²/2, Where L and W represent the length and width of the tumor, respectively. The relative tumor volume (RTV) was: RTV = Vt/V1. V1 is the TV on the first day of administration (Day1) and Vt is the TV at the time of measurement (Day t). The evaluation index of anti-leukemic effect was the relative tumor proliferation rate: (T/C (%) = (T_RTV/C_RTV) × 100%) [T_RTV: the RTV of the treatment group, C_RTV: the RTV of the vehicle control group]. Synergy analysis was measured by using the formula [25]: expected value = (A/C) × (B/C) [actual value = (AB)/C; A: RTV of A drug; B: RTV of B drug; C = The RTV of the vehicle control group; AB = the RTV of the combined administration group]. The synergy index = expected value/actual value; ratio > 1, indicating that the two drugs have a synergistic effect; ratio = 1, indicating that the two drugs have an additive effect; ratio < 1, indicating that the two drugs have a weaker than additive effect. Change of body weight (%) = (measured weight-weight at grouping)/weight at grouping × 100%. In addition, complete regression (CR), partial regression (PR) and stable disease (SD) among the number of animals in each group represented the remission rate. T/C (%) ≤ 42% and statistical analysis P < 0.05 is effective. If the weight of the mouse drops by more than 20% or the number of drug-related death exceeds 20%, the drug dose is considered to be severely toxic.

All animal experiments were reviewed and approved by the Institutional Animal Care and Use Committee (IACUC) of Suzhou GenePharma (Approval No: IACUC-2021003) and Hangzhou Medical College Laboratory animal center (Approval No: ZJCLA-IACUC-20120003).

The one-way ANOVA test were used to assess statistical significance. P value < 0.05 was considered statistically significant. Experiments were presented as the means ± standard deviation (SD) in three independent experiments. The results of animal experiments and cell inhibition assay in primary AML samples were presented as the means ± standard error of the mean (SEM). Statistical analyses were conducted using Prism software v8.3.0 (GraphPad Software, La Jolla, CA, USA) and IBM SPSS Statistics 24 (Armonk, NY, USA).

HL-60, Kasumi-1, MV4-11 and OCI-AML3 cells were treated with APG-2575 or ABT-199 for 24 h. The results showed that APG-2575 or ABT-199 treatment inhibited AML cells viability in a dose-dependent manner. The four AML cell lines showed similar response to APG-2575 and ABT-199 (Fig. 1A). The IC50 values of six cell lines (HL-60, Kasumi-1, MV4-11, THP-1, U937 and OCI-AML3) were determined after treatment with APG-2575 or ABT-199 for 24 h. The anti-leukemic activity of APG-2575 in AML cells was equal or even better than that of ABT-199 (Additional file 1: Fig. S1, Additional file 2: Table S1). Additionally, similar anti-leukemic effect of APG-2575 and ABT-199 was also found when they were used to treat primary AML samples from 5 newly diagnosed patient and 2 relapse/ refractory patients (Fig. 1B, C). The clinical characteristics of the patient samples were presented in Table 1.

To test the synergistic anti-leukemic activity between HHT and APG-2575, we treated several AML cells lines with HHT, APG-2575 and their combination at various concentrations and monitored cell viability. Remarkably, combine treatment of HHT with APG-2575 augmented the inhibitory effect to a greater extent than either of the two agents alone in HL-60, Kasumi-1, MV4-11 and OCI-AML3 cell lines (Fig. 2A, Additional file 2: Table S2).

Considering that APG-2575 is a BCL-2 inhibitor and exerts an anti-leukemic effect through the apoptotic pathway, we tested whether the combination treatment could synergistically trigger apoptosis in AML cells. HL-60, Kasumi-1, MV4-11 and OCI-AML3 cell lines were exposed to APG-2575 and HHT alone or in combination for 24 h. The Annexin-V/PI staining results indicated that APG-2575 induced AML cells apoptosis, and combination treatment resulted in superior induction of apoptosis compared with either single agent (Fig. 2B). Moreover, we analyzed the level of proteins involving apoptosis signaling pathway by western blot. Cleaved PARP, cleaved caspase-3 and cleaved caspase-7 were more significantly increased in the combined treatment group compared with the single-agent group (Fig. 2C), indicating HHT potentiates APG-2575 induced apoptosis in AML cells.

We further validated the synergistic effect of HHT and APG-2575 combination in primary AML patient samples. The results revealed that APG-2575 or HHT can inhibit the viability of primary AML cells, and indeed, synergies (CI < 1.0) were also observed in AML specimens (Fig. 3A, Additional file 2: Table S3). The Annexin-V/PI staining results suggested that APG-2575 combined with HHT could induce significant primary AML cells (De novo or R/R) apoptosis compared with the single agent (Fig. 3B). Furthermore, inhibition rate was presented for every dose combination of HHT and APG-2575 using the Synergy Finder application (https://​synergyfinder.​fimm.​fi). Synergy score was calculated based on the Loewe model. Dose-dependent inhibition of proliferation was observed upon HHT or APG-2575 treatment for 48 h, according to the overall drug combination dose–response matrix pattern (Fig. 3C). The average synergy score was 16.18 and the most synergistic area score was 20.41 in AML patient 08. These results indicated HHT combined with APG-2575 exerted a synergistically anti-leukemic effect in primary AML samples. The characteristics of the AML patients were presented in Table 1.

To evaluate the anti-leukemic effect of the combination of BCL-2 inhibitor and HHT in vivo, we established AML xenograft mouse model (Fig. 4). The APG-2575 and HHT monotherapy group showed some anti-leukemic effect in MV4-11 xenograft mouse model, with T/C values of 62.76% and 32.43%, respectively, however, the APG-2575/HHT co-treatment group presented a significant synergistic anti-leukemic effect, with a T/C value of 3.31%. The synergy index was 6.14 and tumor remission rate was 100% (6/6 PR). In parallel comparison, another FDA-approved BCL-2 inhibitor ABT-199 combined with HHT also showed an obvious synergistic anti-AML effect, with a T/C value of 2.59%. The synergy index was 11.07 and the tumor remission rate was 100% (6/6 PR) (Fig. 4A, Additional file 2: Table S4). These results suggested that APG-2575 combined with HHT had a significant synergistic anti-AML effect, and the synergistic effect was comparable to that of ABT-199 combined with HHT. No significant body weight loss was observed in the animals in each administration group (Fig. 4B), indicating that the animals were well tolerated the compounds.

OCI-AML3 cells were used to establish a mouse subcutaneous xenograft tumor model as well. OCI-AML3 cell was characterized by a rapidly growing xenograft tumor. On the 15th day after administration, the animals in the vehicle control group were obviously anxious and restless. Therefore, the experiment was terminated on the 15th day. APG-2575 and HHT single agent did not show significant antitumor activity in this model, with a T/C value of 75.44% and 41.95%, respectively (Fig. 4C, E, Additional file 2: Table S5). Notably, the co-treatment group had significant synergistic anti-leukemic effect with T/C value of 8.52% (compared with the vehicle control group, P < 0.001). The synergy index was 3.71 (Additional file 2: Table S5). Furthermore, the tumor weights of the APG-2575 and HHT single agent treated groups were lower than the vehicle control group, while the co-treatment group showed the lowest tumor burden among four groups (Fig. 4D). The q value (q = 1.30) indicated HHT combined with APG-2575 exerted a synergistically anti-leukemic effect. No significant body weight loss was observed in the animals in each group (Fig. 4F).

As MCL‐1 is a vital player in the resistance to BCL-2 inhibitor in AML cells. We examined the expression of MCL-1 protein after exposure to APG-2575 or HHT alone or combined regimen. Western blot analysis revealed that, while exposure to APG-2575 lead to increase MCL-1 expression, treatment with HHT alone or the combination results in a significant reduction in the protein level of MCL-1 in MV4-11, OCI-AML3 cells. It was reported that GSK3β was involved in MCL-1 degradation [26‐29]. We next examined whether the combination of APG-2575 and HHT affects the PI3K/AKT/GSK3β signaling pathway. As shown in Fig. 5 A, HHT in the presence or absence of APG-2575 led to a reduction of PI3K, p-AKT (Ser473), and p-GSK3β (Ser9) expression, but has no obvious effect on the total protein level of AKT and GSK3β. Of note, our study indicated that APG-2575 showed no effects on PI3K/AKT/GSK3β signaling pathway. In addition, monophosphorylated and dual phosphorylated MCL-1 were detected by western blot. The results suggested that HHT could reduce p-MCL-1 (Thr163) and up-regulate p-MCL-1 (Thr163/Ser159), and notably, this effect of HHT was enhanced in the combination treated cells. We also verified that APG-2575 combined with HHT inhibited the PI3K/AKT/GSK3β signaling pathway and promoted MCL-1 degradation in vivo (Fig. 5A). Treatment with APG-2575 increased MCL-1 level and HHT promoted the phosphorylation and degradation of MCL-1 via inhibiting the PI3K/AKT/GSK3β signaling pathway, thereby reversing the resistance of APG-2575 (Fig. 5B).