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

Despite advances in targeted agent development, effective treatment of acute myeloid leukemia (AML) remains a major clinical challenge. The B-cell lymphoma-2 (BCL-2) inhibitor exhibited promising clinical activity in AML, acute lymphoblastic leukemia (ALL) and diffuse large B-cell lymphoma (DLBCL) treatment. APG-2575 is a novel BCL-2 selective inhibitor, which has demonstrated anti-tumor activity in hematologic malignancies. Homoharringtonine (HHT), an alkaloid, exhibited anti-AML activity. The synergistic effects of APG-2575 and HHT were studied in AML cell lines and primary samples. MTS was used to measure the cell viability. Annexin V/propidium iodide staining was used to measure the apoptosis rate by flow cytometry. AML cell xenografted mouse models were established to evaluate the anti-leukemic effect of BCL-2 inhibitor, HHT and their combination in vivo. Western blot was used to determine the expression of related proteins. APG-2575 showed comparable anti-leukemic effect to the FDA-approved BCL-2 inhibitor ABT-199 in vitro and in vivo. Combined treatment of HHT with APG-2575 synergistically inhibited AML cell growth and engraftment. Mechanistically, HHT promoted degradation of myeloid cell leukemia-1 (MCL-1), which was reported to induce BCL-2 inhibitor resistant, through the PI3K/AKT/GSK3β signaling pathway. Our results provide an effective AML treatment strategy through combination of APG-2575 and HHT, which is worthy of further clinical research.


Background
Acute myeloid leukemia (AML) is a clonal aggressive hematological malignant disease with clinical and genetic heterogeneity [1,2]. Although treatment of AML has made a significant achievement, the majority of patients experience a relapse of primary AML and develop resistance to chemotherapy [3]. In particular, elderly patients with AML are ineligible for intensive chemotherapy and their 5-year overall survival rate is only 5-15% [4]. It remains a major challenge in AML management due to refractoriness, relapse and drug resistance.
Homoharringtonine (HHT) is an alkaloid obtained from the Cephalotaxus hainanensis. It is successfully applied in the treatment of hematological diseases due to its effective anti-tumor activity [20,21]. In our previous study, we found that HHT down-regulated the BCL-2 and MCL-1 expression [22], making us hypothesize combination of HHT with APG-2575 may enhance the response and reverse the APG-2575 resistance in AML cells.
In the present study, we evaluated the synergistic anti-AML effect of combined treatment of APG-2575 and HHT in AML. Our results revealed that the addition of HHT potentiated the anti-leukemic effect of APG-2575 in AML cell lines and primary AML cells in vitro and in vivo. Mechanistically, HHT inhibited the Phosphoinositide-3-Kinase/Protein kinase B/Glycogen synthase kinase-3 (PI3K/AKT/GSK3β) signaling pathway, leading to MCL-1 dual phosphorylated and degraded, which overcame the resistance of APG-2575 mediated by overexpressed MCL-1. In conclusion, our results support further exploring the effect of the combination regimen of HHT and APG-2575 in AML treatment.
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.

Flow-cytometric analysis of apoptosis
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.

Western blot analysis
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). 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.

AML xenograft model
All animal experiments were reviewed and approved by the Institutional Animal Care and Use Committee

Statistical analyses
The one-way ANOVA test were used to assess statistical significance. P value < 0.05 was considered

APG-2575 shows comparable anti-leukemic effect to ABT-199
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.

HHT and APG-2575 synergistically inhibit AML cells growth
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.

HHT and APG-2575 combination exerts synergistically anti-leukemic effect in primary AML samples
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:// syner gyfin der. 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 doseresponse 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 antileukemic effect in primary AML samples. The characteristics of the AML patients were presented in Table 1.

APG-2575 synergized with HHT suppresses AML xenograft tumor growth
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 FDAapproved 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 antileukemic 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).

HHT reverses the resistance of APG-2575 via inhibiting the PI3K/AKT/GSK3β pathway
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][27][28][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).

Discussion
AML is a genetically heterogeneous disorder characterized by a poor survival rate. Regardless of whether regimens combine conventional therapies with multiple novel target drugs or hematopoietic stem cell transplantation (HSCT), AML remains an incurable malignancy of the bone marrow. New regimens with better tolerance and efficacy, less toxicity and fewer side effects are urgently needed. On June 7, 2021, Ascentage Pharma Ascent Pharmaceuticals presented the latest clinical progress of the new generation of Bcl-2 inhibitor APG-2575 at the ASCO meeting. Among 15 patients with R/R CLL/SLL, 12 cases achieved partial remission (PR) and the overall remission rate (ORR) was as high as 80.0%. No dose-limiting toxicity (DLT) was observed at 1200 mg and no tumor lysis syndrome occurred [15]. It means that there are great potentials for clinical development of single drug and combination therapy of APG-2575, which will bring better options for patients. MCL-1 plays a vital role in the development of hematological malignancies and high expression of MCL-1 is associated with BCL-2 inhibitor resistance. Our previous studies identified that HHT could reduce MCL-1 and exert significant anti-leukemic activity [26,27]. Therefore, we propose the combined application of APG-2575 and HHT for the treatment of AML in order to reverse the resistance of APG-2575.
In this study, we first compared the anti-leukemic effect of APG-2575 and ABT-199 and the results suggested that the two agents were comparable in inhibiting AML cells growth. Next, we found HHT in combination with APG-2575 displayed an excellent anti-leukemic effect. APG-2575 showed dose-dependent growth inhibition and accelerate apoptosis when administrated as a monotherapy, and the combined regimen exhibited more obvious synergistic activity, as evidenced by the activation of PARP and caspase family members. Studies had shown that tumor cells with high expression of anti-apoptotic protein MCL-1 have primary resistance to the BCL-2 selective inhibitor [28]. Interestingly, the two agents exerted more significant growth inhibition and apoptosis in OCI-AML3 cells (inherent resistance to APG-2575) and AML patient samples (De novo or R/R). These results demonstrated that HHT enhanced the anti-leukemic effect of APG-2575, suggesting HHT as a potential drug to overcome the resistance of APG-2575.
According to the cell experiments in vitro, MV4-11 cells are moderately sensitive to APG-2575. Therefore, we first used human MV4-11 cells to establish a mouse xenograft tumor model to evaluate the anti-tumor effect of the combination of APG-2575 and HHT. The results suggested that APG-2575 and ABT-199 have equally anti-leukemic effects in vivo. The data proved that APG-2575 combined with HHT had a synergistic effect in vivo. And we further confirmed co-administration of APG-2575/HHT also exerting synergistic anti-leukemic effect in the OCI-AML3 xenograft model. Together, these above results verified that the combination of APG-2575 and HHT has a significant synergistic anti-AML effect in vitro and in vivo, indicating that this combined regimen could bring clinical benefits to AML patients.

Conclusion
Some studies have reported that high expression of MCL-1 was related to non-remission following ABT-199 treatment, and the original resistance in AML cells could reverse by co-administration of MCL-1 inhibitor. In this study, HHT combined with APG-2575 treatment could reduce the level of MCL-1. Overall, HHT combined with APG-2575 could overcome the resistance to APG-2575 in AML, and these preclinical results provided strong evidence for the treatment of AML patients with APG-2575 combined with HHT.