This study was approved by the Affiliated Hospital of Qingdao University, Qingdao, China (QY202386). All animal experiments were conducted following the guidelines of the institutional animal committee of Qingdao University

The human melanoma cell line A375 was obtained from the American Type Culture Collection (ATCC) (Manassas, VA, USA). Cells were cultured in Dulbecco’s Modified Eagle Medium (DMEM) (Thermo Fisher Scientific, Waltham, MA, USA) supplemented with 10% fetal bovine serum (Thermo Fisher Scientific) and 1% penicillin–streptomycin (Sigma Aldrich, St. Louis, MO, USA). Cultures were maintained at 37°C in a humidified atmosphere containing 5% CO₂. All cell lines were verified to be mycoplasma-free before the experiment. The identities of the cell lines were confirmed through short tandem repeat validation analysis performed by Biosynthetic Inc. (Lewisville, TX, USA) before use. Cells were maintained at about 50%–60% confluence, and the culture media were changed twice a week. Once thawed, the cells were used for a maximum of 4 months in continuous culture to ensure genomic stability and reproducibility.

VEM-resistant cell lines (A375R) were generated by continuous exposure of parental A375 cells to increasing concentrations of VEM (Selleck Chemicals, Houston, TX, USA), from 0.05 to 10 µM over 6 months. The resulting A375R cells were maintained in 2 µM VEM. Before sensitivity assays, A375R cells were cultured in VEM-free media for 10–14 days to eliminate residual drug effects.

Rocaglates (RocA), selective inhibitors of the eIF4F complex, dissolved in DMSO at a concentration of 100 mM, were purchased from MedChemExpress (Monmouth Junction, NJ, USA). RocA (Cat No. HY-19356, purity: 96.43%) was dissolved in DMSO to a stock concentration of 100 µM, and CR-1-31-B (Cat. No. HY-136453) was dissolved in DMSO at a concentration of 10 μM. The EZH2 inhibitor tazemetostat was purchased from ApexBio Technology (Houston, TX, USA). The AKT inhibitor MK-2206 and ERK1/2 inhibitor U0126, as well as VEM, were purchased from Selleck Chemicals. Gene-specific siRNAs were purchased from Santa Cruz Biotechnology (Santa Cruz, Dallas, TX, USA), including: eIF4E siRNA (sc-35284), BMF siRNA(sc-45930), c-Myc siRNA(sc-29226), c-Jun siRNA (sc-29223), c-Fos siRNA (sc-44200), and EGR1 siRNA (sc-44203). EZH2 siRNA (#6509) was purchased from Cell Signaling Technology (CST, Danvers, MA, USA). All negative control siRNAs were purchased from Santa Cruz Biotechnology (sc-44231). siRNAs were transfected at a final concentration of 100 nM, and cells were harvested 48 or 72 h after transfection for analysis. Primary antibodies (dilution, 1:1000) against eIF4E (#9742), p-eIF4E (#9741), bcl-2 (#15071), c-Myc (# 9402), Mcl-1 (#4572), c-Jun (#3270), c-Fos (#5348), EGR1 (#4153), t-AKT1 (#2938), p-AKT1 (#9018), p-ERK1/2 (#9101), ERK1/2 (#9102), EZH2 (#4905), BMF (#50542), PUMA (#4976), Bax (#2772), Noxa(#14766), β-actin(#4967),Bad (#9292), and cleaved-caspase-3 (#9661) were purchased from Cell Signaling Technology. Primary antibodies (dilution, 1:1000) against Bim (sc-374358) were purchased from Santa Cruz Biotechnology.

For siRNA transfection, A375 and A375R cells were seeded at approximately 40% confluence in OPTI-MEM serum-free medium (Gibco, Waltham, MA, USA) in 6-cm tissue culture plates and transfected with different small interfering RNAs using Lipofectamine RNAiMAX (Invitrogen, Carlsbad, CA, USA). Cells were harvested 48 or 72 h post-transfection. Each transfection experiment was performed at least three times, and the blocking efficiency of the siRNA clones was evaluated by western blotting.

Cell viability was evaluated using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay (Sigma-Aldrich, St. Louis, MO, USA), according to the manufacturer’s instructions. Briefly, A375 and A375R cells were seeded in 96-well plates at a density of 2 × 10³ cells per well and allowed to adhere overnight. Cells were then treated with different concentrations (0, 10, 30, 50, 100, 200, and 300 nM) of RocA and cultured in 96-well plates for 72 h or exposed to VEM (1 and 10 μM) in combination with RocA (50, 100, and 200 nM) for 48 or 72 h. Additionally, cells were transfected with different siRNAs or treated with specific inhibitors for 16 h before exposure to different concentrations of RocA for 72 h. Subsequently, the medium was replaced with 100 μL of fresh medium containing 10 µL of 5 mg/mL MTT reagent, and the plates were incubated at 37°C for 4 h. The medium was then aspirated, and formazan crystals were dissolved in isopropanol containing 0.04 N HCl. Absorbance was measured at 570 nm with a reference wavelength of 750 nm using a Varioskan LUX multimode microplate reader (Thermo Fisher Scientific).

To determine the effect of EZH2, c-Fos, and EGR1 on RocA-induced cell viability, A375 cells were transfected with EZH2 siRNA, EGR1 siRNA, or c-Fos siRNA or co-transfected with EGR1 siRNA + c-Fos siRNA for 16 h. Cells were then treated with RocA (50 and 100 nM) for 72 h, and cell viability was assessed by the MTT assay, as described above.

To determine whether pharmacological inhibition of EZH2 and AKT1 influences RocA-induced cell viability, A375 and A375R cells (2 × 10³) were exposed to tazemetostat (2.5 and 5 µM) + RocA (50 and 100 nM), MK-2206 (2.5 and 5 µM) + RocA (50 and 100 nM), or tazemetostat (2.5 and 5 µM) + MK-2206 (2.5 and 5 µM) + RocA (50 and 100 nM). Cells were cultured in 96-well plates for 72 h, and cell viability was assessed by the MTT assay, as described above. To determine the effect of EZH2 and AKT1 inhibition on VEM sensitivity, A375 and A375R cells (2 × 10³) were exposed to VEM (1 and 10 µM) + tazemetostat (2.5 µM), VEM (1 and 10 µM) + MK-2206 (2.5 µM), or VEM (1 and 10 µM) + tazemetostat (2.5 µM) + MK-2206 (2.5 µM) and cultured in 96-well plates for 72 h. Cell viability was assessed by the MTT assay, as described above.

Apoptotic cell death was assessed using the TUNEL fluorescence assay kit (Cat. No. C1089, Beyotime, Shanghai, China) according to the manufacturer’s protocol. Briefly, A375 and A375R cells were seeded in 6-well plates at a density of 3 × 10⁵ cells/well and treated with the indicated concentrations of RocA for 48 h. In parallel experiments, cells were transfected with the specified siRNAs (100 nM) for 16 h, followed by exposure to various concentrations of RocA for 48 h. At different experimental time points, cells from each experimental group were seeded onto a 24-well plate containing coverslips. Following incubation with the labeling working solution, 10% phosphate-buffered saline (PBS; pH 7.4, P0149B; Beyotime) wash, addition of DAPI working solution (C1006; Beyotime) and anti-fluorescence quenching agent, the slides were sealed and examined under a fluorescence microscope IX73 (Olympus Corporation, Tokyo, Japan) at 200× magnification, with apoptotic nuclei identified by red fluorescence.

For immunoblotting experiments involving RocA treatment in vitro, A375 and A375R cells were treated with 100 nM RocA for 1, 3, 6, 12, 18, 24, 36, and 48 h, or with 50, 100, or 200 nM RocA for 48 h. Whole-cell lysates were collected at the indicated time points for protein analysis.

For combination treatment assays, A375 cells were pre-treated with 2.5 μM of MK-2206 for 2 h or 10 μM of U0126 for 4 h, followed by treatment with 100 nM RocA for 1, 3, 6, 12, 18, 24, 36, and 48 h.

For siRNA transfection experiments, siRNAs (siRNA1, siRNA2 and siRNA3) were transfected at a final concentration of 100 nM for analysis (We chose siRNA with the highest transfection efficiency as the research object): ① A375 cells were transfected with EZH2 siRNA (100 nM) for 16 h, followed by 100 nM RocA treatment for 1, 3, 6, 12, 18, 24, 36, and 48 h; ② A375 cells were transfected with EGR1 siRNA (100 nM) or c-Fos siRNA (100 nM) for 16 h, and then treated with RocA (50 and 100 nM) for 72 h; ③ Cells were transfected with c-Myc siRNA, c-Jun siRNA, or BMF siRNA (100 nM each) for 16 h and then co-treated with RocA (50, 100, or 200 nM) and MK-2206 (1.25, 2.5, or 5 μM) for 48 h; ④ Cells were transfected with EIF4EsiRNA for 16 h and then treated with RocA (50, 100, and 200 nM) for 48 h.

After treatment, cells were lysed at the indicated time points using standard radioimmunoprecipitation assay (RIPA) buffer (Cat. No. 89901, Thermo Fisher Scientific) containing 1% protease inhibitors (Cat. No. 36978, Thermo Fisher Scientific) and PhosphoStop phosphatase inhibitor cocktails (Cat. Nos. P5726, Sigma-Aldrich). Lysates were vortexed, sonicated, and centrifuged, and supernatants were stored at −80°C until use. Protein concentrations were determined using the BCA protein assay kit (Cat. No. P0012S Beyotime). All experiments were performed in triplicate (n = 3). Equal amounts of proteins (20–40 µg/lane) were separated on a 4%–12% sodium dodecyl sulfate-polyacrylamide gel (SDS-PAGE) under constant voltage of 120 V for approximately 90 min, followed by transfer to nitrocellulose membranes at 100 V for 60 min. Membranes were blocked for 1 h in 5% (w/v) bovine serum albumin (Sigma-Aldrich) and incubated with primary antibodies overnight at 4°C. The primary antibodies included anti-(p)-ERK1/2, anti-(p)-AKT1, anti-EZH2, anti-BMF, anti-c-Myc, anti-c-Jun, anti-c-Fos, anti-EGR1 anti-bcl-2, anti-Mcl-1, anti-PUMA, anti-Bim, anti-Noxa, anti-Bax, anti-Bad, anti-cleaved-caspase-3, and anti-β-actin. The dilution factor for each primary antibody was established based on the manufacturer’s recommendations and validated by preliminary experiments.

Following incubation with primary antibodies, membranes underwent three 10-min washes with TBST buffer solution. They were then incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies (Cat. No. BA1054, Boster, Wuhan, China; 1:10,000) at room temperature for 1 h. Protein bands were visualized using the enhanced chemiluminescence (ECL) detection kit (Cat. No. RPN2209, Amersham International, Amersham, UK), according to the manufacturer’s protocol. Band intensities were quantified using Image J software 3.0 (Bio-Rad Laboratories Inc., Hercules, CA, USA). Original western blot data are reported as WB Original Data. For immunoblots examining combined treatment with VEM + CR-1-31-B (CR-31) + MK-2206 + tazemetostat treatment in vivo, tumor tissues from xenografts were homogenized using a Fisher Scientific homogenizer, lysed in RIPA buffer, and 30 µg/lane of protein lysates were separated by SDS-PAGE followed by immunoblotting with the indicated antibodies, as described above.

All in vivo experiments were approved by the Affiliated Hospital of Qingdao University, Qingdao, China. Female BALB/c-nu mice aged 4–6 weeks (n = 6 per group) were obtained from Doo Yeol Biotech (SLRC Laboratories, Shanghai, China). A375 and A375R cells (5 × 10⁶) suspended in 200 µL of sterile medium were subcutaneously injected into the flank region of each mouse [23]. For long-term therapeutic response analysis, treatment was initiated 5 days after tumor cell injection. Mice received one of the following regimens: 20 mg/kg VEM i.p for 14 days, 0.2 mg kg⁻¹ CR-31/mouse/day i.p for 14 days + 20 mg/kg VEM i.p for 14 days, or a combination of 20 mg/kg VEM i.p for 14 days + 0.2 mg kg⁻¹ CR-31/mouse/day i.p for 14 days + MK-2206 (120 mg/kg, orally, three times weekly for 14 days) + tazemetostat (400 mg/kg oral gavage, bis in die (b.i.d.) for 10 days).

In the short-term therapeutic response group, treatments were administered daily for 5 consecutive days, and animals were sacrificed 6 h after the final treatment. Tumors were excised, lysed in RIPA buffer, and analyzed via western blotting. For long-term therapeutic response assessment, tumor size was measured every two days using a digital caliper. Tumor volume was calculated using the equation V = (W² × L)/2, where W represents the tumor width and L the tumor length.

Statistical analyses were conducted using IBM SPSS Statistics Grad Pack 22.0 BASE DOWNLOAD-Win (IBM, Armonk, NY, USA). Data are presented as the mean ± SEM, with all experiments performed in triplicate. One-way ANOVA followed by Tukey’s post hoc test was used for multiple group comparisons, while two-group comparisons were analyzed using Student’s t-test, where appropriate. Statistical significance was defined as *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001.

The eIF4F complex inhibitor RocA functions by inhibiting the eIF4A helicase subunit of the eIF4F complex and enhancing eIF4A RNA affinity, resulting in loss of eIF4A helicase activity [24]. Previous research demonstrates that RocA effectively inhibits cancer cell growth in vitro and tumor development in vivo [22]. This study initially examined the effect of RocA on cell viability in VEM-sensitive melanoma cells (A375) and VEM-resistant melanoma cells (A375 R). Cells were exposed to varying RocA concentrations (10, 30, 50, 100, 200, and 300 nM) for 72 h. Cell viability assessment via MTT assay revealed significant dose-dependent decreases in both cell lines, with A375 cells showing greater sensitivity to RocA treatment (Fig. 1A).

Following 48-h treatment with RocA at concentrations of 10, 30, 50, 100, 200, and 300 nM, apoptosis was evaluated using the TUNEL assay. The results demonstrated that RocA treatment induced a significant dose-dependent apoptosis in A375 cells (Fig. 1B and Fig S1). In contrast, A375R cells showed minimal apoptotic response to RocA treatment (Fig. 1B).

The study subsequently investigated whether RocA enhanced the sensitivity of A375 and A375R cells to VEM. Both cell lines were treated with VEM (1 and 10 μM) and RocA (50, 100, and 200 nM) for 48 h (for apoptosis detection) and 72 h (for cell viability assessment). The combination treatment significantly increased cell apoptosis (Fig. 1E,F) (Figs. S2 and S3) and reduced cell viability (Fig. 1C,D) in both cell types. Notably, the VEM-sensitive A375 cells exhibited a greater enhancement in drug sensitivity following RocA co-treatment compared with the resistant A375R cells.

Treatment of A375 cells with 30–200 nM RocA for 20 h induced ERK1/2 hyperactivation in a dose-dependent manner [13]. In the present study, A375 cells were treated with 100 nM RocA for 1, 3, 6, 12, 18, 24, 36, and 48 h. The levels of p-ERK1/2 increased progressively from 1 h after RocA administration, peaked at 18–24 h, and subsequently decreased to baseline levels by 48 h (Fig. 2A,B).

EZH2 plays a crucial role in regulating proliferation in basal epidermal cells, with reduced proliferative capacity observed in cells lacking EZH2 [25]. Previous research demonstrated that EIF4E inhibition suppressed EZH2 and CDK1 protein expression, resulting in decreased proliferation of human epidermal keratinocytes [26]. To investigate whether eIF4F regulated EZH2 expression, we first confirmed abundant EZH2 expression in A375 cells through western blot analysis. Treatment of A375 cells with 100 nM RocA led to a significant reduction in EZH2 levels after 12 h, followed by a gradual increase and sustained high expression at 48 h (Fig. 2C,D). Previous studies have shown that ERK1/2 signaling positively regulates EZH2 expression, correlating with aggressive breast cancer subtypes [18]. Additionally, MEK-ERK and PI3K/AKT signaling modulate EZH2 expression in KRAS-mutant colon and pancreatic cancer cell lines [27]. To explore this regulatory mechanism in melanoma, A375 cells were pretreated with U0126 (10 µM) for 4 h or AKT inhibitor MK-2206 (2.5 µM) for 2 h, followed by treatment with 100 nM RocA for various time intervals. U0126 treatment completely suppressed RocA-induced EZH2 upregulation (Fig. 2E,H), while MK-2206 pretreatment did not affect EZH2 reactivation (Fig. 2F,H), indicating that targeting hyperactive ERK1/2 inhibited RocA-induced EZH2 reactivation.

Previous research has shown that eIF4F inhibitor treatment elevated EGR1 and c-Fos levels, correlating with increased ERK activity in BRAFV600E and NRAS-mutant melanoma cell lines [13]. To determine whether EGR1 and c-Fos expression are regulated by EZH2, A375 cells were treated with 100 nM RocA for various time intervals. EGR1 and c-Fos protein levels increased gradually, reaching maximum levels at 48 h (Fig. 2G,H). However, when A375 cells were co-treated with EZH2 siRNA (100 nM) and 100 nM RocA, the reduction in EZH2 expression led to complete inhibition of RocA-induced EGR1 and c-Fos expression (Fig. 2I,J), demonstrating EZH2-dependent regulation of these proteins.

The study examined whether targeting EZH2 enhances RocA sensitivity in A375 cells. A375 and A375R cells were transfected with EZH2 siRNA (100 nM) for 16 h, followed by RocA treatment (50 and 100 nM) for 48 or 72 h. MTT assay demonstrated that the combined treatment significantly inhibited cell viability in both cell lines (Fig. 3A). To further determine whether this combination affected cell apoptosis, A375 cells were treated under the same conditions and analyzed for apoptotic activity. The results indicated that co-treatment with EZH2 siRNA and RocA did not significantly enhance cell apoptosis compared to either treatment alone (Fig. 3B and Fig. S4), indicating that EZH2 targeting primarily enhances RocA sensitivity by inhibiting cell proliferation rather than inducing apoptosis.

Subsequently, the study investigated whether targeting EZH2 sensitized A375 cells to RocA through EGR1 or c-Fos. A375 cells were transfected with EGR1 siRNA or c-Fos siRNA for 16 h, followed by RocA treatment (50 and 100 nM) for 72 h. Western blot analysis confirmed that EGR1 (Fig. 3C) and c-Fos (Fig. 3D) expression decreased significantly following their respective siRNA transfections. Cell viability analysis revealed that silencing EGR1 or c-Fos partially restored cell viability following RocA treatment (Fig. 3E). The combination of RocA treatment with either EGR1 siRNA or c-Fos siRNA significantly increased cell viability, although complete recovery was not achieved (Fig. 3E). These findings suggest that EZH2 influences A375 cell sensitivity to RocA, at least in part, through regulation of EGR1 and c-Fos expression. Similarly, in A375R cells, EZH2 silencing sensitized cells to RocA treatment, and co-transfection with EGR1 or c-Fos siRNA led to a comparable partial restoration of cell viability without complete recovery (Fig. 3E).

Treatment with eIF4F inhibitors has been shown to reduce c-Myc and c-Jun expression, while treatment with the specific MEK inhibitor PD184352 failed to increase pro-apoptotic c-Myc or c-Jun expression [13], indicating that these transcription factors are not regulated by ERK1/2 signaling. Previous research has demonstrated that AKT1 regulates eIF4F complex formation and modulates mRNA translation, resulting in activation of a caspase-dependent apoptotic cascade and growth inhibition [14]. To determine whether AKT1 regulates c-Myc and c-Jun, A375 cells were treated with 100 nM RocA for 1, 3, 6, 12, 18, 24, 36, and 48 h. Western blot analysis revealed that p-AKT1 levels decreased in a time-dependent manner after 6 h of RocA treatment, followed by a gradual increase and peak activation at 24 h, before returning to baseline by 48 h (Fig. 4A).

A375 cells were exposed to 2.5 μM AKT inhibitor MK-2206 for 2 h, followed by treatment with 100 nM RocA for 1, 3, 6, 12, 18, 24, 36, and 48 h. MK-2206 abolished RocA-induced AKT1 reactivation in a time-dependent manner (Fig. 4B). However, MK-2206 did not affect RocA-induced ERK1/2 activation (Fig. 4B) compared to RocA treatment alone (Fig. 2A), suggesting that RocA-induced ERK1/2 reactivation occurs through an AKT1-independent pathway. To further investigate whether AKT1 regulates c-Myc and c-Jun, A375 cells were exposed to 2.5 μM MK-2206 for 2 h, followed by 100 nM RocA treatment for various time points. As shown in Fig. 4B, untreated A375 cells exhibited high basal levels of c-Myc and c-Jun. RocA treatment reactivated p-AKT1 and suppressed c-Myc and c-Jun expression, whereas MK-2206 treatment inhibited p-AKT1 and restored c-Myc and c-Jun expression. In contrast, U0126 treatment did not affect RocA-induced c-Myc and c-Jun downregulation (Fig. 4C), compared to RocA treatment alone (Fig. 4A). These findings indicate that c-Myc and c-Jun are AKT1-dependent regulators.

The study also examined whether c-Myc and c-Jun were regulated in an eIF4E-dependent manner. A375 cells were transfected with eIF4E siRNA for 16 h, followed by treatment with 100 nM RocA for 24 h. The results demonstrated that silencing eIF4E did not affect RocA-induced c-Myc and c-Jun expression (Fig. 4D), compared to RocA treatment alone (Fig. 4A). This evidence suggests that c-Myc and c-Jun are eIF4E-independent regulators.

Previous research demonstrated that eIF4F inhibitor treatment induces cell apoptosis and inhibits cell proliferation through BMF upregulation in melanoma with BRAF and NRAS mutations [14]. In addition, inhibition of the PI3K/AKT pathway or direct pharmacological inhibition of eIF-4E has been shown to increase BMF expression [27]. To investigate whether BMF is regulated by AKT1, A375 cells were treated with 100 nM RocA for 0–48 h. The results showed that BMF expression increased at 3 h, peaked at 12 h, and subsequently returned to basal levels (Fig. 4A).

When A375 cells were exposed to 2.5 μM MK-2206 for 2 h followed by 100 nM RocA treatment for 0–48 h, BMF expression showed a gradual increase over time (Fig. 4B). However, the combination of RocA and U0126 treatment showed no significant effect on BMF expression compared to RocA treatment alone (Fig. 4C). These findings suggest that BMF regulation is AKT1-dependent but not ERK1/2-dependent.

To further examine the apoptotic response induced by RocA, additional pro-apoptotic BH3-only proteins, including PUMA, Bax, and Bad, were examined. Among these, only PUMA showed slight upregulation after 100 nM RocA treatment for 0–48 h (Fig. 4A). Neither U0126 (Fig. 4C) nor MK-2206 (Fig. 4B) treatment affected PUMA expression.

The administration of RocA did not alter Bax and Bad protein levels (Fig. 4A). However, combined MK-2206 and RocA treatment resulted in minimal upregulation of Bax expression (Fig. 4B), while combined U0126 and RocA treatment showed moderate upregulation of Bax expression (Fig. 4C).

Akt functions as an apoptotic regulator that, when activated, contributes to clinical drug resistance by inhibiting apoptosis and activating eIF4E [20]. Preliminary experiments demonstrated that exposure of A375 cells to 1.25–5 μM MK-2206 for 4 h resulted in complete inhibition of p-AKT1 without causing significant cytotoxicity. Therefore, 2.5 μM MK-2206 was selected as the optimal concentration for subsequent studies. The present investigation revealed increased p-eIF4E expression following RocA treatment (Fig. 5A). MK-2206 treatment suppressed both RocA-induced AKT1 reactivation (Fig. 4B) and p-eIF4E expression (Fig. 5B). In contrast, treatment with the ERK1/2 inhibitor U0126 (10 μM) (Fig. 5C) did not affect RocA-induced p-eIF4E expression compared to RocA treatment alone. These findings suggest that RocA-induced p-eIF4E expression occurs in an AKT1-dependent but ERK1/2-independent manner.

RocA treatment effectively inhibited cell proliferation and promoted apoptosis in A375 cells, but failed to induce apoptosis in A375R cells. Previous research indicates that the antiproliferative efficacy of the AKT inhibitor MK-2206 alone is limited in hepatocellular carcinoma cells [28], and it does not affect in vitro and in vivo A375 cell growth or survival. However, while single-agent MK-2206 inhibited phospho-AKT signaling, targeting AKT1 using siRNA for 48 h induced apoptosis in melanoma cells [29]. Based on these findings, the current study investigated whether combined AKT1 inhibition using MK-2206 enhances the antitumor efficacy of RocA by promoting apoptosis in A375 cells. Cells were co-treated with MK-2206 (1.25, 2.5, and 5 μM) and RocA (50, 100, and 200 nM) for 48 h. While MK-2206 alone did not induce apoptosis, the combination treatment significantly induced apoptosis compared to RocA treatment alone (Fig. 6A and Fig. S5).

Since c-Myc, c-Jun, and BMF were regulated by AKT1, this study examined whether these proteins contributed to AKT1-knockdown-induced apoptosis. A375 cells were transfected with c-Myc siRNA, c-Jun siRNA, or BMF siRNA for 16 h, followed by co-treatment with RocA (50, 100, and 200 nM) and MK-2206 (1.25, 2.5, and 5 μM) for 48 h. Western blotting confirmed that siRNA transfection effectively inhibited the expression of their respective proteins (Fig. 6B–D), resulting in decreased apoptosis compared to cells receiving combined RocA and MK-2206 co-treatment (Fig. 6A and Fig. S5).

BMF knockdown significantly inhibited apoptosis induced by combined RocA and MK-2206 treatment (Fig. 6E). In contrast, silencing c-Myc or c-Jun only partially inhibited apoptosis, and these effects did not reach statistical significance (Fig. 6E).

Given that eIF4E expression is regulated by AKT1 and that AKT1 inhibition enhanced RocA-induced apoptosis, this study examined whether targeting eIF4E could similarly increase RocA-induced apoptosis. A375 cells were transfected with eIF4E siRNA for 16 h, followed by RocA treatment (50, 100, and 200 nM) for 48 h. eIF4E knockdown effectively inhibited RocA-induced eIF4E expression (Fig. 6F). While eIF4E siRNA modestly increased RocA-induced apoptosis, the difference was not statistically significant (Fig. 6A). These findings suggest that AKT1 inhibition enhances RocA-induced cell apoptosis primarily through BMF upregulation.

Previous research has shown that silencing eIF4A or eIF4E reduces melanoma cell proliferation and invasion [9]. Additionally, eIF4E has been demonstrated to promote neutrophil survival via regulation of the anti-apoptotic proteins Bcl-2 and Mcl-1 [30]. In the current study, A375 and A375R cells were treated with RocA (50, 100, and 200 nM) for 48 h, resulting in significantly decreased Bcl-2 and Mcl-1 expression in both cell lines (Fig. 7A,B). These findings suggest that RocA-induced inhibition of cell viability may be mediated, at least in part, by the downregulation of Bcl-2 and Mcl-1. Further investigation is required to elucidate this mechanism and examine the direct or indirect regulation of Bcl-2 and Mcl-1 expression by AKT1.

Previous research [31] demonstrated that combined VEM and RocA treatment exhibited modest effects on cell apoptosis and proliferation inhibition in A375R cells, though these effects were temporary owing to acquired resistance [9]. Consistent with this, our earlier findings indicated that combined inhibition of eIF4F with either EZH2i or AKT1i enhanced the sensitivity of A375 cells to eIF4F inhibition. To investigate whether pharmacological inhibition of eIF4F, AKT1, and EZH2 enhances the antitumor effect in vitro, A375 cells were co-treated with RocA (50 and 100 nM), tazemetostat (2.5 and 5 μM), and MK-2206 (2.5 and 5 μM) for 72 h. In line with previous studies indicating that AKT1 and EZH2 inhibitors alone have minimal impact on cell growth or apoptosis [9], the current study revealed that the triple-drug combination of MK-2206 + tazemetostat + RocA significantly enhanced the antitumor effect in A375 cells, as measured by the MTT assay (Fig. 8A). Similar effects were observed in A375R cells in vitro (Fig. 8B). Further investigation examined whether combined RocA, tazemetostat, and MK-2206 treatment enhanced A375R cell sensitivity to VEM. A375R cells were co-treated with VEM (1 and 10 μM), tazemetostat (2.5 μM), and MK-2206 (2.5 μM) for 72 h. This triple-drug combination significantly improved A375 cell sensitivity to VEM (Fig. 8C) and enhanced VEM’s antitumor efficacy in VEM-sensitive A375 cells (Fig. 8D).

Having established that combined inhibition of eIF4A, AKT1, and EZH2 effectively overcomes VEM resistance in vitro, we proceeded to investigate the therapeutic potential of this combination in vivo and its effect on VEM sensitivity.

For the in vivo study, CR-31, a synthetic rocaglate with superior pharmacokinetic properties compared to RocA, was used. A375R cells (5 × 10⁶) were subcutaneously injected into female BALB/c-nu mice (4–6 weeks old, weighing 16–18 g). After 5 days of tumor establishment, mice were randomized into three treatment groups, as previously described. Tumor volumes were measured every 2 days for 19 days. The VEM monotherapy group exhibited rapid tumor growth, while both the combination therapy groups (VEM + CR-31 + MK-2206 + tazemetostat and CR-31 + VEM) demonstrated significant tumor growth reduction compared to the VEM-only group (Fig. 8E).

In A375 xenograft models, the combination treatments exhibited markedly enhanced antitumor efficacy compared to VEM monotherapy. The (VEM + CR-31 + MK-2206 + tazemetostat) group and the (CR-31 + VEM) group demonstrated tumor growth inhibition rates of 81.8% and 60%, respectively, which were substantially higher than the rate of 13.6% observed in the VEM-only group (Fig. 8F). Both combination regimens demonstrated significantly superior efficacy compared to VEM monotherapy. Importantly, no notable toxicity or adverse effects were observed in any treatment groups when compared with the VEM-only group. Furthermore, CR-31 alone demonstrated antitumor activity in VEM-sensitive A375 cells, while the triple combination (CR-31 + MK-2206 + tazemetostat) showed a significantly enhanced sensitivity to VEM treatment compared to the CR-31 + VEM combination.

Subsequently, we examined the molecular mechanisms underlying the effects of CR-31 and the combined treatment on both VEM-sensitive and VEM-resistant A375 xenograft models. After 5 days of tumor establishment, mice in the short-term therapeutic response group were randomized into six treatment groups and treated as previously described for 5 days. Xenograft tumors were harvested 6 h after the final treatment, and protein expression was analyzed by western blotting. The four-drug combination therapy (VEM + CR-31 + MK-2206 + tazemetostat) resulted in a significant reduction in p-AKT1, EZH2, and eIF4E expression compared to other treatment groups (Fig. 8E,F). Additionally, the combination therapy significantly upregulated pro-apoptotic proteins and downregulated growth-promoting proteins in both A375 (Fig. 8G) and A375R (Fig. 8H) tumor tissues. These findings align with the in vitro results.