Three AML cell lines, MV-4-11 (human leukemia cells; cat. no. CL-0572), U937 (human monocytic leukemia cells; cat. no. CL-0239) and THP-1 (human histiocytic lymphoma cells; cat. no. CL-0233), and their corresponding cell-specific media were purchased from Procell Life Science & Technology Co., Ltd. All cells were authenticated by short tandem repeat profiling and grown at 37°C under 5% CO₂ and 95% relative humidity. For U937 cells, 5 and 10 µM YY-20394 were used for treatment. For MV-4-11 cells, treatments included 120 nM YY-20394, 30 nM ABT199 and a combination of 120 nM YY-20394 with 30 nM ABT199. The negative control (NC) group received an equal volume of drug-free culture medium.

A CCK-8 assay was performed using the Cell Counting Kit-8 (APeXBIO Technology LLC). A total of 100 µl cell suspension was spread into a 96-well plate (1.5×10⁴ cells per well) and incubated at 37°C in a 5% CO₂ environment. The cells were divided into two groups: The experimental group (As), which received the drug (YY-20394 or ABT199), and the control group (Ac), which received no drug. Additionally, a blank group (Ab), consisting only of culture medium, was included. Following previous studies for reference, the concentration gradients for the drugs were as follows: For YY-20394 (18), the concentrations used in MV4-11 cells were 10, 50, 100, 500, 1,000 and 5,000 nM; in THP-1 cells, they were 500, 1,000, 2,000, 5,000, 1×10⁴ and 2×10⁴ nM; and in U937 cells, the concentrations were 1,000, 5,000, 1×10⁴, 2×10⁴, 5×10⁴ and 1×10⁵ nM. For ABT199, the concentrations used in MV4-11 cells were 5, 25, 100, 200, 300, 400 and 600 nM (19,20); in THP-1 cells, the concentrations used were 1,000, 1×10⁴, 2×10⁴, 3×10⁴, 4×10⁴ and 5×10⁴ nM (21); and in U937, they were 1,000, 5,000, 8,000, 1×10⁴, 1.5×10⁴, 2×10⁴ and 4×10⁴ nM.

Following cell adherence to the plate, the diluted drugs were incubated with the cells for either 24 or 48 h. Subsequently, 10 µl CCK-8 solution was added to each well and incubated for 4 h. The optical density (OD) value at λ=450 nm was measured using a plate reader. The percentage of cell growth inhibition was calculated using the following formula: Cell growth inhibition (%)=1-[(As-Ab)/(Ac-Ab)] ×100%. The IC₅₀ value of the drug was determined through linear regression analysis. Specifically, drug concentrations were transformed to their logarithmic (log) values, and the IC₅₀ values were calculated using the ‘log(inhibitor) vs. normalized response-variable slope’ model in GraphPad software (version 8; Dotmatics). Growth inhibition curves were also plotted based on this analysis.

The experimental groups were divided into the YY-20394, ABT199 and YY-20394+ABT199 combination groups. The drug concentration was a multiple of the IC₅₀ value (1, 0.5, 0.25, 0.125, 0.0625 and 0.03125 times). Cell growth inhibition was measured using a CCK-8 assay as described. Subsequently, CI values were calculated using CompuSyn software (version 1.0; ComboSyn, Inc.), and Fa-CI plots of equivalent dose-effect ratios were drawn based on the obtained data. The strength of drug-drug interactions in the combination of YY-20394 and ABT199 could be quantitatively determined by the magnitude of the CI values: CI >1 was antagonistic, CI=1 was additive, 0.7< CI <1 was weakly synergistic, 0.3< CI <0.7 was synergistic and CI <0.3 was strongly synergistic.

According to the instructions, cell apoptosis was detected using dual AO/EB staining using a normal/apoptotic/necrotic cell detection kit (Jiangsu KeyGen Biotech Co., Ltd.). The results were observed under a fluorescence microscope at 510 nm. According to the cell morphology and staining results, the following four types of cells were counted (the total number of cells >200): i) Normal cells were defined as round cells with uniformly green-stained nucleoplasm and consistent size and shape; ii) necrotic cells were ellipsoidal with uniformly orange-yellow-stained nucleoplasm and consistent size and shape; iii) early apoptotic cells were indicated by green nucleoplasm and cells exhibiting irregular shapes, such as crescent-like morphology; and iv) late apoptotic cells where the nucleoplasm was orange, chromatin was condensed, the nucleus was fragmented into punctate structures of varying sizes and cytoplasmic blebbing was observed. Apoptosis rate=(early apoptotic cells + late apoptotic cells)/total number of cells ×100%. Cell necrosis rate=necrotic cells/total number of cells ×100%.

Drug-exposed cells were washed with PBS and fixed homogeneously in pre-cooled 95% ethanol at 4°C overnight. After washing with PBS, the cells were stained with propidium iodide (PI) solution (Beijing Solarbio Science & Technology Co., Ltd.) containing RNase A and incubated in the dark at 37°C for 30 min. The cell cycle distribution was analyzed using a flow cytometer (NovoCyte; Agilent Biosciences). Flow cytometry data were analyzed using FlowJo software (BD Biosciences). Briefly, target cell populations were first gated based on forward scatter (FSC) and side scatter (SSC) parameters to exclude debris and non-viable fragments. Doublets and cell aggregates were subsequently excluded by gating on FSC-A vs. FSC-H. Cell cycle distribution was then determined based on DNA content in the PE channel. To ensure analytical accuracy and consistency across groups, a gating template was established from NC group and subsequently applied to all other experimental groups.

The mRNA levels of Akt, mTOR, myeloid cell leukemia-1 (Mcl-1), Bcl-2 interacting mediator of cell death (Bim), Bcl-2, B-cell lymphoma-extra large (Bcl-xL), Bcl-2 antagonist killer 1 (Bak) and Bcl-2 associated X (Bax) were assessed using RT-qPCR in drug-exposed cells. GAPDH was used as an internal reference gene. Specific primers are presented in Table SI. Total RNA was extracted from cell samples using TRIzol reagent (Tiangen Biotech Co., Ltd.), and cDNA was synthesized using the FastQuant cDNA First Strand Synthesis Kit (Tiangen Biotech Co., Ltd.). The PCR reaction mixture was prepared by combining SuperReal PreMix Plus (SYBR Green; Tiangen Biotech Co., Ltd.) and specific primers, following the manufacturer's instructions. The PCR amplification protocol was as follows: 95°C for 15 min, followed by 40 cycles of 95°C for 10 sec, 55°C for 30 sec and 72°C for 32 sec. A final extension step was performed at 95°C for 15 sec, 60°C for 60 sec and 95°C for 15 sec. The amplification results were detected using an ABI 7300 fluorescence quantitative PCR instrument (Applied Biosystems; Thermo Fisher Scientific, Inc.), and relative gene expression was analyzed using the 2^−ΔΔCq method (22).

The protein levels of Akt (Akt Polyclonal Antibody; 1:1,000; ImmunoWay Biotechnology Company; cat. no. YT0185), phosphorylated (p)-Akt [Akt (phospho Ser473) Polyclonal Antibody; 1:1,000; ImmunoWay Biotechnology Company; cat. no. YP0006), ERK (Mouse Anti-ERK1/2 antibody; 1:1,000; BIOSS; cat. no. bsm-33337M), p-ERK [Phospho-ERK1/2 (Thr202/Tyr204) Antibody; 1:1,000; Affinity Biosciences; cat. no. AF1015), Mcl-1 [MCL1 Rabbit mAb (hp7u); 1:1,000; Nature Biosciences; cat. no. A57858], Bim [Bim Rabbit mAb (7kqv); 1:1,000; Nature Biosciences; cat. no. A83449], Bcl-2 (Rabbit Anti-Bcl-2 antibody; 1:1,000; BIOSS; cat. no. bs-0032R), Bcl-xL [Bcl-xL Rabbit mAb (kn97); 1:1,000; Nature Biosciences; cat. no. A66923], Bak [Bak Rabbit mAb (RX7I); 1:1,000; Nature Biosciences; cat. no. A53931], Bax (Rabbit Anti-Bax antibody; 1:1,000; BIOSS; cat. no. bs-0127R) and c-Myc [c-Myc Rabbit mAb (paYu); 1:1,000; Nature Biosciences; cat. no. A23647] were assessed using western blot in drug-exposed cells. Actin (1:3,000; BIOSS; cat. no. bs-0061R) was used as an internal reference.

RIPA lysis buffer (Beyotime Biotechnology) was added to the cell samples, and proteins were extracted by ultrasound homogenization on ice. Protein concentration was determined using the BCA protein assay (Beijing Solarbio Science & Technology Co., Ltd.) and adjusted to 1.5 mg/ml (~21 µg/lane). Proteins were separated on 5 and 10% polyacrylamide gels and then transferred to activated PVDF membranes. A color-pre-stained protein marker (10–180 kDa; Biodragon) was used to indicate the molecular weight of the target protein. The membrane was blocked with 5% bovine serum albumin (Wuhan Servicebio Technology Co., Ltd.) at room temperature for 1 h. The membrane was then incubated overnight at 4°C with the primary antibody specific to the target protein. Subsequently, the membrane was incubated with the corresponding secondary antibody (1:5,000; Nature Biosciences; Goat Anti-Mouse, cat. no. M00001; Goat Anti-Rabbit, cat. no. R00001) at room temperature for 1 h, and the results were detected using an ECL reagent kit (Harbin HaiGene Biotech Co., Ltd.) through chemiluminescence.

One-way analysis of variance followed by Tukey's post hoc test was performed using GraphPad Prism for multiple group comparisons. All pairwise comparisons were two-tailed. All data are presented as the mean ± standard deviation. A P-value of <0.05 was considered to indicate a statistically significant difference.

The IC₅₀ values of YY-20394 were evaluated to assess the sensitivity in different AML cell lines. In THP-1 cells, the 24 and 48 h IC₅₀ values of YY-20394 were 6,020 and 2,420 nM, respectively (Fig. 1A). In U937 cells, the 24 and 48 h IC₅₀ values of YY-20394 were 1.42×10⁴ and 7,102 nM, respectively (Fig. 1B). The 24 and 48 h IC₅₀ values of YY-20394 in MV-4-11 cells were 1,127 and 126.9 nM, respectively (Fig. 1C). Comparatively, MV-4-11 cells were more sensitive to YY-20394, while THP-1 and U937 cells were less sensitive. YY-20394 exhibited a smoother dose-response profile in U937 cells, which were selected for further experiments.

Cell apoptosis and the cell cycle are important biological processes that collectively determine the proliferation and survival of malignant cells. The dual AO/EB assay revealed that YY-20394 significantly induced apoptosis in U937 cells (P<0.001), with the apoptosis rate being significantly higher in the 10 µM group compared with the 5 µM group (P<0.0001) (Fig. 2A and B). Flow cytometry analysis was used to assess the effects of YY-20394 on the cell cycle (Fig. 2C and D). Compared with the NC group, treatment with 5 and 10 µM of YY-20394 significantly increased the proportion of cells in the G₁ and S phases (P<0.01) while significantly reducing the population in the G₂ phase (P<0.0001), indicating that YY-20394 interfered with cell cycle progression at the G₁/S transition and early S phase. Overall, YY-20394 could inhibit U937 cell proliferation, promote apoptosis and induce arrest at G₁/S transition and in early S phase.

The percentage of cell proliferation inhibition of ABT199 was assessed to determine its sensitivity in different AML cell lines (Fig. 3A-C). The results revealed that the IC₅₀ values of ABT199 were 2.06×10⁴, 1.36×10⁴ and 161.80 nM for 24 h in THP-1, U937 and MV-4-11 cells, respectively, and 1.17×10⁴, 7.90×10³ and 30.47 nM for 48 h, respectively. To assess the potential synergistic effect of YY-20394 and ABT-199, three AML cell lines were treated with a range of doses based on the respective IC₅₀ values of each drug. No significant synergistic effect was observed in THP-1 cells (CI>1, Fig. 3D), and only a negligible synergistic effect was detected in U937 cells (CI=0.895, Fig. 3E). Notably, synergistic interactions were observed in MV-4-11 cells, with the CI value of the optimal dose combination being 0.17 (Fig. 3F). Based on these results, the concentration with the strongest synergistic effect was selected for further experiments with MV-4-11 cells.

Dual AO/EB staining indicated that YY-20394 and ABT199 alone significantly promoted apoptosis in MV-4-11 cells compared with the NC group (P<0.001) (Fig. 4A and B). Moreover, the combination of the two drugs exhibited a higher apoptosis rate than either drug alone. Flow cytometry analysis further revealed that ABT199 significantly reduced the S phase in MV-4-11 cells (P=0.0006), with YY-20394 showing a similar trend (P=0.0510) (Fig. 4C and D). By contrast, there were no significant differences in G₁ and G₂ phases between the two drugs alone, in combination or compared with the NC group (all P>0.05). These findings suggest that YY-20394 and ABT199 may influence DNA replication in MV-4-11 cells.

ABT199 specifically targets Bcl-2. Mcl-1 and Bcl-2 could inhibit apoptosis by binding to the BH3-specific protein Bim, thereby preventing Bim from activating Bax and Bak (23). Whether YY-20394 and ABT199 synergistically promote apoptosis via the Bcl-2 pathway was investigated (Fig. 5A-E). The results revealed changes in the levels of anti-apoptotic proteins Mcl-1, Bcl-2 and Bcl-xL that were inconsistent with the observed apoptotic phenotype in MV-4-11 cells (Fig. 5A, C and D). Specifically, Mcl-1 transcription (P=0.001) and protein (P=0.0783) levels were increased in YY-20394 alone compared with the NC group. Mcl-1 transcription levels decreased while protein levels increased in the combination group compared with either agent alone. Furthermore, no inhibitory effects on Bcl-2 or Bcl-xL were observed at transcription level with ABT199 alone or in combination (all P>0.05) (Fig. 5B), while significant upregulations were observed in protein levels (P<0.05) (Fig. 5E). Bim and Bak transcription levels increased while protein levels decreased in the YY-20394 group compared with the NC group. Additionally, levels of pro-apoptotic factors Bim, Bak and Bax decreased in the combination group compared with NC or ABT199 alone, which contrasts with the observed pro-apoptotic phenotype (Fig. 5C-E).

Due to the critical role of c-Myc in cancer cell survival and apoptosis regulation (24), the protein levels of c-Myc were evaluated. The results demonstrated that the combination treatment significantly reduced c-Myc protein levels compared with the NC (P=0.001) and ABT199 alone (P=0.0007) (Fig. 5F and G). Therefore, the synergistic pro-apoptotic effects of the combined treatment may be associated with c-Myc suppression.

Previous studies have suggested that the effects of PI3Kδ inhibitors on tumor cell proliferation or apoptotic signaling may be mediated through the regulation of PI3K/Akt and ERK pathways (25,26). Whether the synergistic effects of YY-20394 and ABT199 on MV-4-11 cells are associated with these pathways was investigated (Fig. 6A). Compared with the NC, p-Akt levels were reduced in both the YY-20394 alone (P=0.0074) and in combination (P=0.0149) (Fig. 6B-D). By contrast, p-ERK levels increased in the ABT199 alone and in combination compared to the NC (P=0.0253). Additionally, the p-ERK/ERK ratio was higher in the combination group than in the ABT199 alone (P=0.0291) (Fig. 6E-G).