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. 2026 Jun 25;23:229. doi: 10.1186/s12985-026-03229-8

oHSV-1-modulated m6A methylation reprogramming through ALKBH5-mediated suppression of CARM1 induced apoptosis in glioblastoma

Xiaoyang Qin 1,4,#, Yan Jia 2,#, Peiwen Wang 1,4,#, Hongwei Yang 2, Rui Wu 1,4, Fusheng Liu 1,3,4,✉, Junwen Zhang 1,4,5,✉
PMCID: PMC13625249  PMID: 42343346

Abstract

Background

Oncolytic virus therapy is a highly promising approach for the treatment of glioblastoma (GBM). The RNA alteration N6-methyladenosine (m6A) is essential in GBM progression; however its specific function in oncolytic virotherapy remains unclear.

Methods

We employed a new oncolytic herpes simplex virus type 1 (HSV-1), designated oHSV-1, which we had previously engineered. Mechanistic studies were performed to elucidate the involved pathways.

Results

We demonstrate that oHSV-1 specifically downregulates the m6A demethylase ALKBH5, leading to GBM cell death. Mechanistically, ALKBH5 downregulation elevates m6A modification on the transcript of the arginine methyltransferase CARM1, which promotes mRNA degradation and reduces protein expression. As CARM1 is a vital regulator of apoptosis, its loss triggers tumor cell apoptosis. Thus, our study identifies the ALKBH5–m6A–CARM1 axis as a critical mechanism underlying oHSV-1-induced apoptosis in GBM cells. Moreover, the combination of oHSV-1 and CARM1 inhibitor shows a markedly enhanced effect on suppressing tumor cell proliferation in vitro.

Conclusions

These findings reveal a novel epigenetic mechanism of oHSV-1-induced apoptosis and support the therapeutic potential of combining oHSV-1 with epigenetic inhibitors for GBM treatment.

Supplementary Information

The online version contains supplementary material available at https://doi.org/10.1186/s12985-026-03229-8.

Keywords: Oncolytic virus, Glioblastoma, N6-methyladenosine (m6A), ALKBH5, CARM1, Apoptosis

Introduction

Glioblastoma (GBM) is a prevalent malignant neoplasm in the cranial cavity and exhibits significant invasiveness. Notwithstanding existing standard treatment protocols that incorporate extensive surgical resection, adjuvant radiotherapy, and concurrent or adjuvant chemotherapy, patient prognosis remains exceedingly grim, with a median survival of approximately 15 months and a five-year survival rate under 5% [1, 2]. Consequently, the development of innovative methods is of urgent scientific importance and considerable clinical relevance to improving outcomes in GBM patients [3].

Among various emerging treatments, oncolytic virotherapy offers significant potential owing to its distinctive dual-killing mechanism [4–6]. Oncolytic viruses are genetically modified attenuated strains designed to selectively proliferate within tumor cells, leading to their lysis. Concurrently, the liberation of tumor-associated antigens (TAAs) and damage-associated molecular patterns (DAMPs) during viral replication significantly alters the immunosuppressive tumor microenvironment and stimulates a systemic anti-tumor immune response [7, 8]. Moreover, in addition to the direct oncolytic action, the induction of apoptosis in tumor cells constitutes a vital mechanism by which oncolytic viruses eradicate both primary and metastatic cancers [9, 10].

Clinical trials have demonstrated that various oncolytic viruses, especially those derived from herpes simplex virus type 1 (HSV-1), possess favorable safety profiles and initial efficacy in early-stage trials for recurrent or refractory gliomas, presenting a promising therapeutic avenue for GBM [11, 12]. Our research group has created a new oncolytic virus utilizing the HSV-1 viral backbone, referred to as oHSV-1 [13, 14]. Preclinical studies confirm that oHSV-1 markedly impedes tumor progression in in-situ glioma models, while preliminary clinical trials in patients with recurrent malignant gliomas suggest its prospective therapeutic efficacy [15]. Nonetheless, the exact molecular pathways by which oncolytic viruses confer therapeutic benefits, especially in their regulation of malignant cell fate—such as the induction of apoptosis—remain inadequately understood.

Recent studies highlight that, in addition to traditional DNA methylation and histone alterations, RNA epitranscriptomic modifications—especially N6-methyladenosine (m6A)—are imperative in carcinogenesis, progression, and therapeutic response. m6A represents the most prevalent reversible chemical alteration in eukaryotic RNAs, with its dynamic equilibrium meticulously governed by “writers” (e.g., the METTL3/METTL14 complexes, WTAP), “erasers” (e.g., FTO, ALKBH5), and “readers” (e.g., YTH family proteins) [16–18]. Current evidence suggests that m6A-recognizing proteins affect tumor cell sensitivity to oncolytic virotherapy [19]; however, the functions of other m6A-related proteins, particularly erasers, in oncolytic virotherapy remain ambiguous. Our prior study has shown that ALKBH5 plays a crucial role in GBM by modulating paraspeckle formation [20–22]. Nonetheless, the influence of m6A alterations and associated regulatory proteins—especially demethylases like ALKBH5—on virus-induced apoptotic processes in tumor cells remains largely under investigated.

This study demonstrates that oHSV-1 treatment induces death in glioma cells by specifically downregulating the expression of the m6A demethylase ALKBH5. Mechanistically, decreased ALKBH5 expression following oHSV-1 treatment elevates m6A on the mRNA of its downstream target, the arginine methyltransferase CARM1, which subsequently hastens CARM1 mRNA degradation, ultimately decreasing CARM1 protein levels and inducing apoptosis in GBM cells. In conclusion, these findings delineate the ALKBH5-m6A-CARM1 axis as a crucial mechanism facilitating oHSV-1-induced apoptosis in neoplastic cells. This mechanism demonstrates that the combination of CARM1 inhibitor with oHSV-1 therapy enhances therapeutic efficacy against GBM, establishing a theoretical basis for the development of combination tactics to improve oncolytic virotherapy.

Materials and methods

Plasmid construction

EGFP cDNA was amplified and cloned into the pCDH-CMV-MCS-EF1-Puro vector. Human ALKBH5 cDNA was amplified and cloned into the pCDH-CMV-EGFP-MCS-EF1-Puro according to the manufacturer’s protocol and verified by sequencing. Human CARM1 was introduced into the pCDH-EF1-MCS-3xFlag-T2A-PURO vector according to the protocol recommended by the Clone Express II One-Step Cloning Kit (Vazyme, C112). Specific oligonucleotides targeting ALKBH5 or CARM1 were cloned into the pLKO.1 TRC cloning vector. The sequences for primers or oligos are listed in Table S1.

Cell culture

The human glioblastoma cell lines U87 and U251 were authenticated as described previously [23]. All cells were grown in DMEM containing 10% FBS and maintained at 37℃ with 5% CO2. All cells were regularly authenticated by PCR and tested for Mycoplasma.

Viruses

The oHSV-1 used in this study was derived from the wild-type HSV-1 strain HL-1 (isolated in China from a patient with herpes) by deleting the ICP34.5 and ICP47 genes. This virus was constructed similarly to that described previously [13].

Cell viability assays

Cell viability was assessed via the CCK-8 assay (CCK-8, Servicebio, G4103). Cells (3000/well) were treated with oHSV-1 (1 × 108 PFU), ALKBH5, or CARM1 inhibitor for 24–48 h. After incubation, CCK-8 solution was added and incubated for 2 h. Absorbance at 450 nm was measured. Each sample was tested in at least 6 wells in each experiment, and the experiment was repeated at least 3 times.

Flow cytometry

Cells were collected and washed twice with PBS. We then used the Annexin V-FITC/PI Apoptosis Kit (Yeasen Biotechnology, 40302ES60) according to the manufacturer’s instructions for double staining. After collection, we evaluated the samples using a flow cytometer. Data acquisition was done on a FACS Fortessa, and analysis was performed using FlowJo 10.8.1.

TUNEL staining

Apoptosis was assessed using the TUNEL assay kit (Servicebio, G1504). After tissue/cell preparation and permeabilization, samples were equilibrated and incubated with the TdT reaction mix (37 °C, 1 h, dark). Nuclei were counterstained with DAPI. Apoptotic nuclei (green) were visualized alongside total nuclei (blue) by fluorescence microscopy. TUNEL staining was quantified by analyzing three independent fields (200× magnification) per group using ImageJ. Total cell number was determined by counting DAPI-stained nuclei, and TUNEL-positive cells were quantified following automatic thresholding and watershed separation. The percentage of apoptotic cells was calculated as (number of TUNEL-positive cells / number of DAPI-positive nuclei) × 100%.

Western blot

Treated cells were lysed with RIPA and boiled in SDS loading buffer and analyzed by Western blot assays. The following antibodies were used: anti-ALKBH5 (1:5000, Abcam, ab195377), anti-GFP (1:10000, Abcam, ab183734), anti-CARM1 (1:1000, Abcam, ab243638), anti-β-Actin (1:2000, Servicebio, ZB15001-HRP-100). Direct-load Color Prestained Protein Marker (Abclonal, RM19001) was used in all assays.

RNA dot blot

2 µL RNA was immobilized via UV crosslinking for 2 min, then incubated with the m6A antibody (Abclonal, A3800) overnight at 4℃. Following TBST washes, the membrane was incubated with a rabbit secondary antibody, washed again, and finally exposed for signal detection.

Real-time quantitative PCR

Total RNA was isolated using the Super FastPure Cell RNA Isolation Kit (Vazyme, RC102-01) and subjected to reverse transcription with RevertAid Reverse Transcriptase (Thermo Scientific, EP0442). The RT-qPCR used the Taq Pro Universal SYBR qPCR Master Mix (Vazyme, Q712-02). The RT-qPCR primer sequences are in Table S1.

MeRIP (m6A-IP)-qPCR

The MeRIP (m6A-IP)-qPCR was performed as described [24]. Fragmented RNA was immunoprecipitated with anti-m6A antibody (Abclonal, A9841)-bound beads. Then, the RNA reaction mixture was washed in the low/high-salt washing buffer. The m6A-enriched RNA was eluted with 14 µL DEPC H2O according to the instructions of the RNeasy Mini Kit (QIAGEN, 74106). Primers for MeRIP (m6A-IP)-qPCR are listed in Table S1.

IHC staining

The IHC staining was performed as described previously [25]. Briefly, specimen’s tissue slides were deparaffinized, rehydrated, antigen retrieved, blocked, and incubated with appropriate primary antibodies. The antibodies included anti-ALKBH5 (1:2000, Abcam, ab195377), anti-CARM1 (1:500, Abcam, ab243638).

RNA-seq and m6A-seq analysis

RNA-seq-related work, including library preparation, sequencing, and bioinformatics analysis, was performed by Beijing Novogene. The criteria for screening differentially expressed genes were as follows: for volcano plots, |Fold Change| > 2 and p-value < 0.05; for heatmaps, |Fold Change| > 2 and p-value < 0.01 (including adjusted p-values after multiple testing correction). Heatmaps were generated using Log2(FPKM+1) transformed expression values and subjected to two-way hierarchical clustering. Some plots were created using the Wei Sheng Xin online platform (https://www.bioinformatics.com.cn/). The m6A-seq data were obtained from the GEO database (Accession: GSE171500) and visualized using the Integrative Genomics Viewer (IGV) software.

Statistical analysis

Statistical analyses selection was based on the experimental design, data type, and predefined comparison strategy [26–28]. Data were presented as mean ± standard deviation (SD). For comparisons between two groups, unpaired Student t-test was used. When multiple prespecified two-group comparisons were performed within the same panel, p values were adjusted using the Bonferroni correction according to the number of comparisons. For comparisons involving three or more groups with a common control, one-way analysis of variance (ANOVA) followed by Dunnett’s multiple comparisons test was used. For multiple group experiments with prespecified pairwise comparisons, one-way ANOVA followed by Sidak’s multiple comparisons test was used. Adjusted P values from the corresponding multiple comparisons tests were reported. Dose-response curves for oHSV-1 infection were fitted using a quadratic log-dose model based on log10(MOI), and differences between curves were assessed using an extra sum-of-squares F test with Bonferroni adjustment for multiple curve comparisons. Statistical analyses were performed using GraphPad Prism 9 and R version 4.3.1. All statistical tests were two-sided. Statistical significance was set at P value < 0.05, using adjusted P values when multiple comparisons were performed. Significance levels were denoted as ns, p ≥ 0.05; *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.001.

Result

oHSV-1 induces apoptosis in GBM cells

To determine whether oHSV-1 induces apoptosis in GBM cell lines, we selected the classic GBM cell lines U87 and U251 for experimentation. First, we identified the optimal multiplicity of infection (MOI) via a half-maximal inhibitory concentration (IC50) assay. U87 cells showed an IC50 of 0.2313, whereas U251 cells showed an IC50 of 1.447, indicating that U87 cells are more sensitive to oHSV-1 (Fig. 1a-b). Based on the optimal viral MOI and cellular status, we used MOI 0.1 for U87 cells and MOI 0.3 for U251 cells in subsequent experiments.

Fig. 1.

Fig. 1

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oHSV-1 induces apoptosis in GBM cells. (a, b) Dose-response curves of oHSV-1 in U87 (a) and U251 (b) cells at 48 h post-infection (hpi) (n = 6). (c, d) CCK-8 assay of U87 and U251 cells at 48 hpi with oHSV-1. (c) MOI = 0.1; (d) MOI = 0.3. Two-tailed unpaired Student t test with Bonferroni correction for two comparisons; n = 6. ****p < 0.0001.(e, f) TUNEL assay (green) showing apoptosis in U87 (e) and U251 (f) cells at 48 hpi or uninfected (Ctrl),. Nuclei stained with DAPI (blue). Scale bars, 50 μm. n = 3. (g, h) Flow cytometry analysis of apoptosis (Annexin V/PI staining) in U87 (g) and U251 (h) cells at 48 hpi. Representative plots and quantification of Annexin V⁺/PI⁺ cells are shown. Two-tailed unpaired Student t test; n = 3. ****p < 0.0001

To directly compare the sensitivity of the two cell lines under identical conditions, we treated both U87 and U251 cells with the same MOIs (0.1 and 0.3) for 48 h. Under these conditions, U87 cells consistently exhibited a greater reduction in cell viability than U251 cells. Increasing the MOI to 0.3 enhanced the effect in U251 cells, but the reduction remained significantly lower than that in U87 cells (Fig. 1c-d), indicating that U251 cells are intrinsically less sensitive to oHSV-1.

To determine whether oHSV-1 promotes tumor cell apoptosis, we first performed a TUNEL assay, which revealed that oHSV-1 induces nuclear DNA fragmentation, a hallmark of apoptosis (Figs. 1e-f and S1a-b). To further confirm this, we detected phosphatidylserine exposure and cell membrane permeability using Annexin V and propidium iodide (PI) staining. Results showed enhanced Annexin V and PI staining in both U87 and U251 cells after oHSV-1 treatment, which confirmed that oHSV-1 induces apoptosis in GBM cells (Fig. 1g–h). Collectively, these results show that oHSV-1 induces apoptosis in GBM cells.

oHSV-1 inhibits the expression of the m6A demethylase ALKBH5

Recent studies have shown that, in addition to traditional DNA methylation and histone modifications [29], RNA epitranscriptomic modifications—particularly N6-methyladenosine (m6A)—play a crucial role in tumor occurrence, progression, and treatment response [16]. To further investigate whether m6A plays a significant role in oHSV-1-induced apoptosis of tumor cells, we examined the global m6A level in U87 and U251 cells after oHSV-1 treatment, and the results showed that the global m6A level is significantly elevated in GBM cells after oHSV-1 treatment (Fig. 2a).

Fig. 2.

Fig. 2

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oHSV-1 inhibits the expression of the m6A demethylase ALKBH5. Dot blot analysis of total RNA m6A levels in U87 and U251 cells at 24 or 48 hpi or Ctrl, with RNA loadings of 100, 200, and 400 ng. MB, methylene blue staining (loading control). n = 3. (b, c) Volcano plot (b) and heatmap (c) showing mRNA levels of m6A-related proteins in U87 cells at 48 hpi vs. Ctrl. (d, e) qRT-PCR analysis of METTL3, METTL14, WTAP, FTO, and ALKBH5 mRNA in U87 (d) and U251 (e) cells at 48 hpi. Two-tailed unpaired Student t test with Bonferroni correction for five comparisons; n = 3. ns, p ≥ 0.05; *p < 0.05. (f) Western blot analysis of METTL3, METTL14, WTAP, FTO, and ALKBH5 protein levels in U87 and U251 cells at 0, 24 or 48 hpi, with actin as the loading control. (g) Quantification of the Western blot data shown in (f). Protein levels were normalized to actin. One-way ANOVA followed by Dunnett’s multiple comparisons test; n = 3. ns, p ≥ 0.05; *p < 0.05. ***p < 0.001. (h, i) Dose-response curves of oHSV-1 in U87 (g) and U251 (h) cells expressing Ctrl, ALKBH5-WT (overexpression) or FTO-WT (overexpression), assessed at 48 hpi. Dose–response curves were fitted with a quadratic log-dose model and compared using an extra sum-of-squares F test with Bonferroni correction; n = 3. ns, p ≥ 0.05; *p < 0.05; ***p < 0.001; ****p < 0.0001

To identify the key m6A regulator responsible for this change, we performed RNA-seq analysis. The results revealed that upon oHSV-1 treatment, the m6A methyltransferases METTL3 and METTL14 are slightly upregulated at the transcriptional level, while WTAP remains almost unchanged, the demethylases ALKBH5 and FTO are significantly inhibited. Furthermore, the m6A reader protein YTHDF2 shows no significant change, and YTHDF1 and YTHDF3 are slightly downregulated (Fig. 2b-c). To validate these findings, RT-qPCR was performed to examine the expression of METTL3, METTL14, WTAP, ALKBH5, and FTO in U87 and U251 cells. The results showed that only FTO and ALKBH5 were significantly downregulated, whereas METTL3, METTL14, and WTAP were not markedly altered (Fig. 2d-e).

Western blot analysis showed that after 48 h of oHSV-1 treatment, both ALKBH5 and FTO exhibited a downward trend in U87 and U251 cells. Quantitative analysis revealed that ALKBH5 was significantly downregulated in both cell lines. In contrast, FTO showed a significant decrease only in U251 cells, while its downregulation in U87 cells did not reach statistical significance (Fig. 2f-g). Among all the m6A regulatory proteins examined, ALKBH5 was the only one significantly downregulated in both U87 and U251 cells. These results suggest that ALKBH5 plays a critical role in mediating the effects of oHSV-1.

To further identify the dominant factor involved in viral treatment, we overexpressed ALKBH5 or FTO in U251 and U87 cells and examined their effects on the oncolytic virus-mediated killing of tumor cells. The results showed that overexpression of ALKBH5 significantly enhanced the resistance of tumor cells to oHSV-1 treatment compared with FTO overexpression (Fig. 2h-i). The findings indicate that the oHSV-1 primarily achieves its function by inhibiting ALKBH5 expression. In summary, these findings demonstrate that oHSV-1 elevates overall m6A levels in tumor cells primarily by downregulating ALKBH5 expression.

oHSV-1 induces apoptosis in tumor cells by suppressing ALKBH5 expression

To further verify that oHSV-1 induces tumor cell apoptosis by suppressing ALKBH5, we first established ALKBH5-knockdown (shALKBH5) and ALKBH5-WT (wild-type) overexpressing cells in the U87 and U251 lines (Figure S1c-d), and observed that knockdown of ALKBH5 enhanced global m6A levels, while ALKBH5 overexpression decreased global m6A levels (Figure S1e).

Furthermore, ALKBH5 knockdown alone mimicked the effects of oHSV-1, significantly inhibiting GBM cells proliferation (Fig. 3a). TUNEL (Fig. 3b-c and S1f-g) and flow cytometry analyses (Fig. 3d-e) further confirmed that ALKBH5 knockdown promotes GBM cell apoptosis. Rescue assay results showed that compared to the control group, overexpression of ALKBH5-WT partially reverses the inhibition of tumor cell proliferation by the oHSV-1 (Fig. 3f-g). Furthermore, ALKBH5-WT overexpression significantly reduces the proportion of apoptotic cells (Fig. 3h-i). These results conclusively demonstrate that oHSV-1 induces tumor cell apoptosis by suppressing ALKBH5 expression.

Fig. 3.

Fig. 3

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oHSV-1 induces apoptosis in tumor cells by suppressing ALKBH5 expression. CCK-8 assay of scrambled shRNA (shScr) or ALKBH5-deficient (shALKBH5) U87 and U251 cells. One-way ANOVA followed by Dunnett’s multiple comparisons test; n = 6. ****p < 0.0001. (b, c) TUNEL staining (green) of apoptotic U87 (b) and U251 (c) cells expressing shScr or shALKBH5; nuclei stained with DAPI (blue). Scale bars, 50 μm. n = 3. (d, e) Flow cytometry analysis (Annexin V/PI) of U87 (d) and U251 (e) cells expressing shScr or shALKBH5. Representative plots and quantification of Annexin V⁺/PI⁺ cells are shown. One-way ANOVA followed by Dunnett’s multiple comparisons test; n = 3. **p < 0.01;***p < 0.001. (f, g) CCK-8 assay of Ctrl or ALKBH5-WT-expressing U87 (f) and U251 (g) cells at 0, 24, and 48 hpi with oHSV-1. One-way ANOVA followed by Sidak’s multiple comparisons test; n = 6. ***p < 0.001;****p < 0.0001. (h, i) Flow cytometry analysis (Annexin V/PI) of Ctrl or ALKBH5-WT-expressing U87 (h) and U251 (i) cells at 48 hpi with oHSV-1. Representative plots and quantification of Annexin V⁺/PI⁺ cells are shown. One-way ANOVA followed by Sidak’s multiple comparisons test; n = 3. ****p < 0.0001

oHSV-1 suppresses CARM1 expression through the ALKBH5-m6A-CARM1 axis

To further investigate the molecular mechanism by which oHSV-1 promotes tumor cell apoptosis through ALKBH5 inhibition, we first performed RNA-seq analysis in U87 cells. We initially identified differentially expressed genes that were significantly up- or down-regulated following oHSV-1 treatment. Subsequently, from these genes, we further selected those whose expression levels were either up- or down-regulated upon ALKBH5 overexpression. The heatmap (Figure S2) displays all genes that simultaneously satisfied both dynamic criteria.

Because oHSV-1 treatment led to reduced ALKBH5 protein levels, we adapted a screening strategy from our previous work [20]. In this study, we defined genes that were down-regulated upon oHSV-1 treatment but up-regulated upon ALKBH5 overexpression as “ALKBH5-responsive transcripts”. This specific expression pattern indicates that ALKBH5 can reverse the inhibitory effect of oHSV-1 on these genes.

Given that oHSV-1 induces apoptosis and that ALKBH5 expression is regulated by oHSV-1, we hypothesized that the downstream targets of ALKBH5 might include apoptosis-related genes. Therefore, we specifically screened for apoptosis-related genes among the differentially expressed genes and found that CARM1 ranked high in the screening results. Moreover, it has been reported that CARM1 functions as an inhibitor of tumor cell apoptosis [30, 31]. Further RT-qPCR (Fig. 4a-b) and Western blot analyses (Fig. 4c) confirmed that ALKBH5-WT overexpression reverses the oHSV-1-induced downregulation of CARM1, indicating that oHSV-1 suppresses CARM1 expression in an ALKBH5-dependent manner.

Fig. 4.

Fig. 4

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oHSV-1 suppresses CARM1 expression through the ALKBH5-m6A-CARM1 axis. (a, b) qRT-PCR analysis of CARM1 mRNA in Ctrl or ALKBH5-WT-expressing U87 (a) and U251 (b) cells at 0 or 48 hpi with oHSV-1. One-way ANOVA followed by Sidak’s multiple comparisons test; n = 3. ****p < 0.0001. (c) Western blot analysis of CARM1 protein in Ctrl or ALKBH5-WT-expressing U87 and U251 cells at 0 or 48 hpi; actin as loading control. (d, e) qRT-PCR analysis of CARM1 mRNA in U87 (d) and U251 (e) cells expressing shScr or shALKBH5. One-way ANOVA followed by Sidak’s multiple comparisons test; n = 3. ****p < 0.0001.(f) Western blot analysis of CARM1 protein in U87 and U251 cells expressing shScr or shALKBH5; actin as loading control. (g) Public m6A-seq data (GSE171500) showing m6A peaks on CARM1 transcripts under indicated conditions. shScr-H, Ctrl under hypoxia; shA5-H, ALKBH5-deficient under hypoxia. (h, i) MeRIP-qPCR analysis of the CARM1 m6A level in U87 (h) and U251 (i) cells expressing shScr or shALKBH5. Two-tailed unpaired Student t test; n = 2. *p < 0.05. (j, k) mRNA decay assay of CARM1 in shScr or shALKBH5-expressing U87 (j) and U251 (k) cells treated with actinomycin D (Act D, 1 mg/mL) at indicated time points. One-way ANOVA followed by Dunnett’s multiple comparisons test; n = 2. ns, p ≥ 0.05; *p < 0.05; **p < 0.01; ***p < 0.001

To further elucidate the specific molecular mechanism by which ALKBH5 regulates CARM1, we first examined CARM1 expression in ALKBH5-knockdown tumor cells. The results demonstrated that ALKBH5 knockdown significantly suppresses CARM1 expression at both mRNA (Fig. 4d–e) and protein levels (Fig. 4f). Given the role of ALKBH5 as an m6A demethylase, we hypothesized that ALKBH5 might influence m6A modification on CARM1 RNA. Analysis of publicly available data from our previous study (GSE171500) revealed that under hypoxic conditions, inhibition of ALKBH5 significantly enhances m6A modification on CARM1 RNA (Fig. 4g). Additionally, m6A-qPCR detection confirmed that inhibition of ALKBH5 significantly increases the level of m6A modification on CARM1 RNA (Fig. 4h-i). To further explore whether the reduced CARM1 expression in ALKBH5-deficient cells is due to m6A-mediated RNA decay, we measured the half-life of CARM1 RNA in cells treated with the transcriptional inhibitor actinomycin D (Act D) for 0, 3, and 6 h. The half-life of CARM1 is significantly shortened upon ALKBH5 depletion in GBM cells (Fig. 4j-k).

These findings indicate that ALKBH5 directly demethylates and stabilizes CARM1. Taken together, these results demonstrate that oHSV-1 inhibits CARM1 expression via the ALKBH5-m6A-CARM1 axis.

CARM1 is a key molecule in oHSV-1-induced apoptosis

To determine whether CARM1 is a critical downstream regulator of oHSV-1-induced apoptosis, we first established CARM1-knockdown (shCARM1) tumor cells (Figure S3a). Assessment of tumor cell proliferation revealed that CARM1 knockdown significantly suppresses GBM cell proliferation (Fig. 5a and S3b). Flow cytometry (Fig. 5b and S3c) and TUNEL analyses (Fig. 5c and S3d–f) further confirmed that CARM1 inhibition promotes GBM cell apoptosis. To validate the specific role of CARM1 in this process, we first overexpressed the wild-type CARM1 (CARM1-WT) in U87 and U251 cells (Figure S4a). Then, the rescue experiment results showed that compared to the control group, CARM1-WT overexpression partially reverses the tumor cells’ proliferation that is inhibited by oHSV-1 (Fig. 5d and S4b), and significantly reduces the proportion of apoptotic tumor cells (Fig. 5e-f and S4c-f). Collectively, these data indicate that inhibition of CARM1 is a key mechanism through which oHSV-1 promotes apoptosis in GBM cells.

Fig. 5.

Fig. 5

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CARM1 is a key molecule in oHSV-1-induced apoptosis. (a) CCK-8 assay of shScr or CARM1-deficient (shCARM1) U87 cells. One-way ANOVA followed by Dunnett’s multiple comparisons test; n = 6. ****p < 0.0001. (b) Flow cytometry analysis (Annexin V/PI) of shScr or shCARM1-expressing U87 cells. Representative plots and quantification of Annexin V⁺/PI⁺ cells are shown. One-way ANOVA followed by Dunnett’s multiple comparisons test; n = 3. ****p < 0.0001. (c) TUNEL staining (green) of apoptotic shScr or shCARM1-expressing U87 cells; nuclei stained with DAPI (blue). Scale bars, 50 μm. n = 3. (d) CCK-8 assay of Ctrl or CARM1-WT-expressing U87 cells at 0, 24, and 48 hpi with oHSV-1. One-way ANOVA followed by Sidak’s multiple comparisons test; n = 6. *p < 0.05;****p < 0.0001.(e) Flow cytometry analysis (Annexin V/PI) of Ctrl or CARM1-WT-expressing U87 cells at 0 or 48 hpi with oHSV-1; Representative plots and quantification of Annexin V⁺/PI⁺ cells are shown. One-way ANOVA followed by Sidak’s multiple comparisons test; n = 3. ***p < 0.001. (f) TUNEL staining (green) of apoptotic Ctrl or CARM1-WT-expressing U87 cells at 0 or 48 hpi with oHSV-1; nuclei stained with DAPI (blue). Scale bars, 50 μm. n = 3

CARM1 inhibitor can enhance the therapeutic efficacy of oHSV-1

To further validate our findings, we analyzed glioma tissue samples from a previously published GBM mouse model treated with oHSV-1 [13]. TUNEL assay results showed a significant increase in tumor cell apoptosis following oHSV-1 treatment (Fig. 6a), consistent with our in vitro observations. Additionally, immunohistochemical analysis further revealed that the expression levels of both ALKBH5 and CARM1 are significantly downregulated in the oHSV-1-treated tumor tissues (Fig. 6b). Moreover, analysis based on the CGGA database (http://cgga-cns.org.cn) revealed a positive correlation between the RNA expression levels of ALKBH5 and CARM1 in both primary and recurrent gliomas (Fig. 6c-d). Additionally, low expression of either gene was associated with prolonged survival in patients with primary or recurrent GBM (Fig. 6e-h), further supporting the trends observed in our in vitro experiments.

Fig. 6.

Fig. 6

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CARM1 inhibitor can enhance the therapeutic efficacy of oHSV-1. (a) TUNEL staining (green) of tumor sections from Ctrl or oHSV-1-treated mice; nuclei stained with DAPI (blue). Scale bars, 100 μm and 1000 μm. (b) IHC staining of ALKBH5 or CARM1 in tumor sections from Ctrl or oHSV-1-treated mice. Scale bars, 100 μm and 1000 μm. (c, d) Correlation analysis of ALKBH5 and CARM1 expression levels in the CGGA database. (e–h) Kaplan–Meier survival curves for GBM patients (CGGA database) stratified by high vs. low expression of ALKBH5 (e, f) and CARM1 (g, h). (i, j) CCK-8 assay of U87 cells infected with oHSV-1 (MOI = 0.1) and treated with or without ALKBH5 inhibitor (200 µM) or CARM1 inhibitor (40 µM) for 48 h. One-way ANOVA followed by Dunnett’s multiple comparisons testOne-way ANOVA followed by Dunnett’s multiple comparisons test;; n = 3. ns, p ≥ 0.05; ****p < 0.0001

Small molecule inhibitors targeting ALKBH5 and CARM1 have been confirmed by multiple studies to effectively suppress tumor progression in various tumor models, demonstrating significant antitumor activity [32–34]. Therefore, we sought to investigate whether targeting ALKBH5 or CARM1 could suppress GBM cell viability and whether their combination with oHSV-1 could enhance antitumor efficacy. To test this hypothesis, first, we determined the optimal concentrations of ALKBH5 and CAMR1 inhibitors in U87 and U251 cells (Figure S4a-d). Subsequently, we evaluated the effects of various treatments on U87 cell viability using the CCK-8 assay. Compared with the control group (Ctrl), oHSV-1 alone, ALKBH5 inhibitor alone (iALKBH5), CARM1 inhibitor alone (iCARM1), and their combinations (oHSV-1 + iALKBH5, oHSV-1 + iCARM1, and oHSV-1 + iALKBH5 + iCARM1) all significantly reduced cell viability (Fig. 6i). Further comparison (Fig. 6j) showed that, relative to oHSV-1 alone, the addition of the ALKBH5 inhibitor did not enhance the inhibitory effect; however, combining oHSV-1 with the CARM1 inhibitor, or with both inhibitors, significantly enhanced the effect. These findings suggest that although both inhibitors are effective individually, only the CARM1 inhibitor combines with oHSV-1 to achieve stronger antitumor activity. Thus, the combination of oHSV-1 and a CARM1 small-molecule inhibitor may represent a more promising strategy for clinical therapy.

Taken together, we found that oHSV-1 selectively downregulates the expression of the m6A demethylase ALKBH5 in GBM cells, increases m6A modification levels on CARM1 transcripts, promotes degradation of CARM1 mRNA, and reduces CARM1 protein expression, ultimately leading to tumor cell apoptosis (Fig. 7).

Fig. 7.

Fig. 7

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A working model for oHSV-1-induced apoptosis in GBM cells. oHSV-1 infection downregulates ALKBH5, increasing m⁶A modification on CARM1 mRNA and accelerating its degradation. Reduced CARM1 protein levels promote apoptosis

Discussion

Oncolytic viruses represent a novel therapeutic approach for GBM, capable of lysing tumor cells while activating anti-tumor immune responses, thereby inhibiting GBM progression [35, 36]. Oncolytic virus has been found to regulate epigenetic modifications such as DNA methylation and histone acetylation [29]. m6A methylation, a widespread RNA epitranscriptomic modification, has been demonstrated to play critical roles in GBM through its associated proteins [37]. The m6A reader protein IGF2BP3 has been shown to influence resistance to oncolytic virus therapy [19]. However, the specific role of m6A in oncolytic virotherapy of GBM remains incompletely understood.

In this study, we investigated the role of m6A in oncolytic virotherapy using oHSV-1, a novel HSV-1-based oncolytic virus previously developed by our research group. We have identified the ALKBH5–m6A–CARM1 axis as a key pathway mediating oHSV-1-induced apoptosis in GBM cells. Building on this molecular mechanism, we further demonstrated that combining oHSV-1 with a CARM1 inhibitor produces significantly enhanced antitumor effects, markedly inhibiting tumor cell growth in vitro. These findings provide strong rationale for developing combination therapies involving oncolytic viruses and specific epigenetic inhibitors for GBM treatment.

The findings of this study demonstrate that infection with the HSV-1-based oHSV-1 significantly enhances the global m6A modification level in tumor cells, consistent with prior observations in HSV-1-infected oral epithelial cells [38]. However, the mechanism through which oHSV-1 specifically regulates the expression of the demethylase ALKBH5 in GBM remains unclear. Previous studies have shown that the HSV-1 immediate early protein ICP0 functions as an E3 ubiquitin ligase, mediating the ubiquitination and subsequent proteasomal degradation of METTL14 at defined lysine residues [39]. We therefore hypothesize that oHSV-1 may similarly promote ALKBH5 degradation via ubiquitination, or alternatively, modulate transcriptional regulators bound to the ALKBH5 promoter to suppress its expression. These possibilities warrant further investigation.

Additionally, HSV-1 infection has been reported to induce nuclear-to-cytoplasmic redistribution of ALKBH5 [40]. Whether oHSV-1 also alters the subcellular localization of ALKBH5 in GBM, and what functional implications such relocalization might have for viral replication, host antiviral responses, and overall oncolytic efficacy, remain to be explored.

We also observed that U251 cells were less susceptible to oHSV-1-induced viability reduction than U87 cells. Under identical MOI conditions, U87 cells consistently showed greater responses. Although increasing the MOI enhanced the effect in U251 cells, the reduction remained significantly lower than that in U87 cells at the same MOI (Fig. 1c-d), suggesting an inherent cell line-dependent difference in viral susceptibility.

This study has several limitations. Most experiments were performed in vitro; future work should employ more physiologically relevant models, such as patient-derived glioma organoids, to validate the proposed mechanisms. The lack of a significantly enhanced oncolytic effect with the ALKBH5 inhibitor may reflect suboptimal dosing ratios. Moreover, our combination studies tested without a systematic dose-response or combination matrix analysis, the further optimization of drug-virus combinations is needed to fully assess the potential for enhanced effects between ALKBH5 or CARM1 inhibitors and oHSV-1.

Conclusion

In this study, our data demonstrate that oHSV-1 specifically reduced the m6A demethylase -ALKBH5. This downregulation enhances m6A modification on the transcript of arginine methyltransferase CARM1, leading to mRNA degradation and reduced protein expression. As CARM1 is a key regulator of apoptosis, its loss triggers apoptosis in GBM cells. Our study identifies the ALKBH5–m6A–CARM1 axis as a critical mechanism underlying oHSV-1-induced apoptosis in GBM. Furthermore, combining oHSV-1 with the CARM1 inhibitor exhibits a markedly enhanced effect, significantly suppressing tumor cell proliferation in vitro.

Supplementary Information

Supplementary material 1. (508.7KB, pdf)
Supplementary material 2. (10.4KB, xlsx)
Supplementary material 3. (15.2KB, xlsx)
Supplementary material 4. (13.2KB, xlsx)
Supplementary material 5. (9.3MB, docx)
Supplementary material 6. (23.7MB, zip)

Acknowledgements

We thank the Brain Tumor Research Center (Beijing Laboratory of Biomedical Materials) at Beijing Neurosurgical Institute for the glioma cell line, oncolytic virus, and technical support.

Abbreviations

TUNEL

TdT-mediated dUTP nick-end labeling

H

Hypoxia

ALKBH5

AlkB homolog 5

CARM1

Coactivator-associated arginine methyltransferase 1

FTO

Fat mass and obesity-associated protein

IHC

Immunohistochemistry

METTL3

Methyltransferase-like 3

METTL14

Methyltransferase-like 14

WTAP

Wilms’ tumor 1-associated protein

Author contributions

XYQ conceived and designed the project. XYQ, YJ and PWW performed the experiments, collected data, and prepared the figures. HWY and RW contributed to the literature search and collection. XYQ and JWZ wrote the manuscript. FSL and JWZ reviewed and edited the manuscript and secured funding and supervision. The authors discuss the results and their implications. All the authors have read and approved the manuscript.

Funding

This work was supported by grants from the National Natural Science Foundation of China (No. 82503943) and the Youth Innovation Fund of Beijing Neurosurgical Institute (2060354-24) and the Beijing Laboratory of Biomedical Materials.

Data availability

All relevant data supporting the findings of this study are available in the article, additional files, or from the corresponding authors upon reasonable request.

Declarations

Ethics approval and consent to participate

All animal procedures were conducted following the institutional, regional, and national laboratory animal research guidelines and granted through the Experimental Animal Welfare and Ethics Committee of Beijing Neurosurgical Institute (No. 201902015).

Consent for publication

All authors agree to submit the research article for publication.

Competing interests

The authors declare no competing interests.

Footnotes

Publisher’s note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Xiaoyang Qin, Yan Jia and Peiwen Wang contributed equally to this work.

Contributor Information

Fusheng Liu, Email: liufusheng@ccmu.edu.cn.

Junwen Zhang, Email: jewzhang@hotmail.com.

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Associated Data

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Supplementary Materials

Supplementary material 1. (508.7KB, pdf)
Supplementary material 2. (10.4KB, xlsx)
Supplementary material 3. (15.2KB, xlsx)
Supplementary material 4. (13.2KB, xlsx)
Supplementary material 5. (9.3MB, docx)
Supplementary material 6. (23.7MB, zip)

Data Availability Statement

All relevant data supporting the findings of this study are available in the article, additional files, or from the corresponding authors upon reasonable request.

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