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Methyltransferase-Like 3 Inhibition-Activated cGAS/STING Axis Enhances Immunotherapy and Poly(ADP-Ribose) Polymerase Inhibitor Sensitivity in Lung Adenocarcinoma
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Jiawang Zhou1, Jiaxin He1, Yunqing Lu1, Cheng Yi1, Xing Chang1, Lijun Tao1, Ke Zhong1, Haisheng Zhang1, 2, Hongxin Niu3, 4, Xiansong Wang1, *, Zhuojia Chen5, *, Hongsheng Wang1, *
Research. Vol 9 Article ID 1265
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Research. Vol 9 Article ID 1265
• RESEARCH ARTICLE •
Methyltransferase-Like 3 Inhibition-Activated cGAS/STING Axis Enhances Immunotherapy and Poly(ADP-Ribose) Polymerase Inhibitor Sensitivity in Lung Adenocarcinoma
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Jiawang Zhou1, Jiaxin He1, Yunqing Lu1, Cheng Yi1, Xing Chang1, Lijun Tao1, Ke Zhong1, Haisheng Zhang1, 2, Hongxin Niu3, 4, Xiansong Wang1, *, Zhuojia Chen5, *, Hongsheng Wang1, *
Affiliations
  • 1Guangdong Provincial Key Laboratory of Chiral Molecule and Drug Discovery, State Key Laboratory of Anti-Infective Drug Discovery and Development, School of Pharmaceutical Sciences, Sun Yat-sen University, Guangzhou 510006, China.
  • 2Department of Physiology, School of Basic Medical Sciences, Gannan Medical University, Ganzhou 341000, China.
  • 3Department of General Practice, Zhujiang Hospital, Southern Medical University, Guangzhou 510282, China.
  • 4Special Medical Service Center, Zhujiang Hospital, Southern Medical University, Guangzhou 510282, China.
  • 5Sun Yat-sen University Cancer Center, State Key Laboratory of Oncology in South China, Collaborative Innovation Center for Cancer Medicine, Guangzhou 510060, China.
About Author:

† These authors contributed equally to this work.

Published: 2026-05-20 doi: 10.34133/research.1265
Outline
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The cyclic GMP-AMP synthase (cGAS)/stimulator of interferon genes (STING)-mediated type I interferon response plays a crucial role in antitumor immunity, yet the regulatory factors involved in this innate immune response remain incompletely understood. Here, we identified methyltransferase-like 3 (METTL3), an RNA N6-methyladenosine methyltransferase, as a key suppressor of the cGAS/STING pathway in lung adenocarcinoma (LUAD) cells. METTL3 was overexpressed in LUAD tissues, and elevated METTL3 expression was associated with decreased CD8+ T-cell infiltration and poor clinical outcomes. Mechanistically, METTL3 deficiency promoted nuclear DNA leakage into the cytoplasm, thereby activating the cGAS pathway and enhancing antitumor immunity. Specifically, METTL3 knockdown decreased the efficiency of homologous recombination repair by down-regulating MutS homolog 5, leading to cytosolic DNA accumulation. On the other hand, N6-methyladenosine methylation of cGAS messenger RNA destabilized the transcript and suppressed its protein expression. Functionally, METTL3 knockdown sensitized LUAD cells to poly(ADP-ribose) polymerase inhibitors. In vivo and clinical data confirmed the beneficial effects of METTL3-inhibition-induced activation of the cGAS/STING axis on tumor growth and LUAD progression. Collectively, our findings reveal that METTL3 inhibition activates cGAS/STING-mediated antitumor immunity by inducing cytosolic DNA accumulation and cGAS up-regulation, thereby modulating poly(ADP-ribose) polymerase inhibitor response and cancer progression in LUAD.

Jiawang Zhou, Jiaxin He, Yunqing Lu, Cheng Yi, Xing Chang, Lijun Tao, Ke Zhong, Haisheng Zhang, Hongxin Niu, Xiansong Wang, Zhuojia Chen, Hongsheng Wang. Methyltransferase-Like 3 Inhibition-Activated cGAS/STING Axis Enhances Immunotherapy and Poly(ADP-Ribose) Polymerase Inhibitor Sensitivity in Lung Adenocarcinoma[J]. Research, 2026 , 9 (5) : 1265 - . DOI: 10.34133/research.1265
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Introduction
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Lung adenocarcinoma (LUAD), the most prevalent histological subtype of non-small-cell lung cancer, accounts for approximately 40% of all lung cancer cases. Globally, it ranks among the leading causes of cancer-related morbidity and mortality, posing a notable public health concern [1]. In recent years, immunotherapy has emerged as a groundbreaking modality for LUAD treatment, leading to major progress in cancer care [2,3]. However, the heterogeneous pathologies, drug resistance, and recurrence of LUAD present formidable challenges for effective treatment. Among these obstacles, immunosuppression and resistance to immunotherapy are particularly prominent [4]. These limitations highlight the need to integrate immunotherapy into a comprehensive treatment approach, leveraging combined effects to improve clinical efficacy and target tumors more effectively, which represents the current frontier of cancer therapy research [5]. Consequently, developing innovative strategies that synergistically augment immunotherapy for LUAD has become a critical and urgent priority for scientific investigation.
N6-methyladenosine (m6A) is the most abundant internal chemical modification in eukaryotic messenger RNAs (mRNAs) [6–8]. Accumulating evidence suggests that m6A, along with its regulatory enzymes and reader proteins, plays critical roles in posttranscriptional gene regulation, and that the dysregulation of these genes is associated with various diseases, especially cancer [9–11]. Methyltransferase-like 3 (METTL3), a well-characterized m6A writer, serves as a key component of the methyltransferase complex and is crucial for establishing m6A modification [12]. Notably, METTL3 has been implicated in immunotherapy responses in multiple cancers [13,14], and its inhibition has been shown to reprogram the tumor microenvironment and enhance the efficacy of anti-PD-1 therapy [15]. A deeper understanding of the role and the underlying molecular mechanisms of METTL3 in anticancer immunity will accelerate the development of effective METTL3-targeted therapeutics, offering promising avenues for improving cancer immunotherapy.
DNA damage induces the accumulation of cytosolic DNA fragments within cells [16], which stimulate type I interferon (IFN) production through the cyclic GMP-AMP synthase (cGAS)/stimulator of interferon genes (STING) signaling pathway. Specifically, cytosolic DNA binds to cGAS, which then catalyzes the production of the second messenger cyclic guanosine monophosphate (cGAMP)[17]. Subsequently, cGAMP interacts with and activates STING, leading to the recruitment and activation of TANK-binding kinase 1, initiating the interferon regulatory factor 3 and nuclear factor kappa-B signaling cascades and cytokine production [18]. Therapeutic reactivation of the cGAS/STING signaling pathway has emerged as a promising strategy for cancer treatment. For instance, combining STING agonists with carboplatin has been shown to reduce tumor burden and prolong survival in mouse models [19]. Thus, activating the cGAS/STING pathway represents a pivotal strategy for potentiating tumor immunotherapy.
The posttranslational regulation of cGAS/STING signaling has been extensively studied [20]. Previous studies have demonstrated that TRIM14 recruits USP14 to cleave the lysine 48 (K48)-linked ubiquitin chain of cGAS at K414, thereby inhibiting p62-mediated autophagic degradation of cGAS and increasing type I interferon signal activation [21]. USP35 can directly deubiquitinate and inactivate STING through phosphorylation of STING at Ser366 [22]. However, evidence connecting the cGAS/STING signaling pathway and epigenetic regulatory factors is limited.
Although posttranscriptional regulators are potent and promising targets for tumor immunotherapy, whether RNA modifications, such as m6A, directly regulate the cGAS/STING pathway and affect antitumor immunity remains unknown. In the present study, we investigated the potential effects of m6A on the activation of cGAS-mediated antitumor immunity in LUAD cells. Our data reveal that METTL3 inhibition activates cGAS-mediated antitumor immunity via the accumulation of cytosolic DNA and up-regulation of cGAS expression, thereby regulating poly(ADP-ribose) polymerase (PARP) inhibitor response and cancer progression in LUAD.
Results
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METTL3 expression is correlated with immune cell infiltration and cancer progression
To elucidate the potential pathological roles of METTL3 in the antitumor immune response within LUAD, we initially investigated the correlation between METTL3 expression and immune cell infiltration using the TIMER2.0 web server (http://timer.comp-genomics.org/) [23]. The results revealed that METTL3 expression was negatively correlated with the infiltration of various antitumor immune cells, including CD8+ T cells (r = −0.125, P = 5.58e−03), CD4+ T cells (r = −0.122, P = 6.53e−03), M1 macrophages (r = −0.139, P = 2.01e−03), monocytes (r = −0.123, P = 6.30e−03), and neutrophils (r = −0.172, P = 1.24e−03) (Fig. 1A and Fig. S1A). Conversely, the expression of the m6A demethylase fat mass and obesity-associated protein (FTO) was positively correlated with the infiltration of these antitumor immune cells (CD8+ T cells, r = 0.146, P = 1.12e−03; CD4+ T cells, r = 0.246, P = 3.18e−08) (Fig. 1B). Furthermore, METTL3 expression was positively correlated with the infiltration of diverse protumor immune cells, including M2 macrophages (r = 0.12, P = 7.85e−03) and regulatory T cells in LUAD tissues (r = 0.199, P = 8.53e−03) (Fig. 1C). Notably, similar correlations were observed in several other types of cancers (Fig. S1B), suggesting that METTL3 is broadly involved in modulating immune-cell infiltration within tumor microenvironments.
Using a previously established method for calculating the interferon-stimulated gene (ISG) score based on the expression profile of a 38-gene signature [24], we observed that elevated METTL3 expression in LUAD was associated with lower ISG scores (Fig. 1D). Additionally, the expression of effector T-cell markers (including CX3CR1, FGFBP2, and FCGR3A) [25], which mediate immune effector functions, was negatively correlated with METTL3 expression (r = −0.17, P = 1.8e−04) (Fig. 1E) but positively correlated with FTO expression (r = 0.29, P = 1.7e−10) (Fig. S1C) in LUAD. Furthermore, immunohistochemical (IHC) staining revealed markedly reduced intratumoral CD8+ T-cell infiltration in LUAD samples with high METTL3 expression (Fig. 1F and Fig. S1D), and METTL3 protein levels were negatively correlated with CD8+ cell infiltration (Fig. 1G). Moreover, analysis of 2 independent single-cell RNA sequencing datasets (GSE277742 and GSE281740 [26]) consistently demonstrated a significant negative correlation between METTL3 expression and CD8+ T-cell infiltration (r = −0.505, P = 3.9e−02; r = −0.546, P = 2.9e−02, respectively) (Fig. 1H). Collectively, these findings indicate that METTL3 plays a critical role in immune regulation within LUAD.
Beyond its up-regulation in LUAD tissues, METTL3 expression was found to be elevated in several other cancer types, including colon adenocarcinoma, esophageal carcinoma, and prostate adenocarcinoma, compared to corresponding normal adjacent tissues (Fig. S1E). Consistent with this, METTL3 protein levels were significantly higher in LUAD patients as demonstrated by IHC staining (Fig. 1I) and the Clinical Proteomic Tumor Analysis Consortium (CPTAC) database (Fig. 1J). Moreover, elevated METTL3 expression was associated with poorer overall survival in LUAD patients (Fig. 1K). Taken together, these findings support an inverse relationship between METTL3 expression and the infiltration of antitumor immune cells while highlighting the oncogenic role of METTL3 in LUAD progression.
METTL3 knockdown promotes the innate immune response and activates the cGAS–STING pathway
To elucidate the mechanism underlying by which METTL3 regulates immune cell infiltration, we established sh-control and sh-METTL3 A549 and H1299 cell lines using lentiviruses (Fig. S2A). Given that cytoplasmic DNA is a key activator of the innate immune response [27], we investigated DNA damage and cytosolic DNA accumulation. To quantify cytosolic DNA, we utilized PicoGreen dye, a fluorescent stain that specifically binds to double-stranded DNA. Notably, under basal conditions, sh-METTL3 A549 cells exhibited substantially higher cytosolic DNA accumulation compared to sh-control cells (Fig. 2A). This accumulation was substantially increased following treatment with Zeocin, a radiomimetic agent that predominantly induces double-strand breaks (DSBs) [28] (Fig. 2B), or exposure to 10 Gy of ionizing radiation (Fig. S2B). To confirm the essential roles of m6A in DNA damage and cytosolic DNA accumulation, A549 cells were transfected with wild-type (WT) METTL3 and the catalytically inactive METTL3 mutant (DA, D395A) [29] (Fig. S2C). The results demonstrated that overexpression of WT METTL3, but not the METTL3 DA mutant, reversed the cytosolic DNA accumulation in A549 cells (Fig. 2C). Collectively, these findings suggest that METTL3 deficiency triggers the release of DNA from the nucleus to the cytoplasm in an m6A-dependent manner.
To determine whether the increase in cytosolic DNA in sh-METTL3 cells activates the cGAS pathway, we quantified cGAMP levels in sh-control and sh-METTL3 LUAD cells. The results showed that sh-METTL3 LUAD cells exhibited significantly higher cGAMP levels compared to sh-control cells, regardless of Zeocin treatment. Moreover, Zeocin treatment further stimulated cGAMP production (Fig. 2D and Fig. S2D). Given that cGAMP activates STING and its downstream factors, the elevated cGAMP levels in sh-METTL3 A549 cells were accompanied by increased phosphorylation of STING and STAT1 (Fig. 2E). Since the interferon pathway is another critical downstream effector of cGAS activation, we assessed the expression of related genes. We found that the herring testes DNA (HT-DNA)-induced expression of IFNB1, as well as that of CXCL10, was significantly up-regulated in sh-METTL3 A549 cells compared to control cells (Fig. 2F). Consistent with these findings, ELISA results confirmed the increased secretion of IFN-β protein (Fig. 2G). Furthermore, the luciferase reporter assay using pGL3-Basic-IFNB1-luc demonstrated that IFNB1 promoter activity was markedly enhanced in sh-METTL3 A549 cells (Fig. 2H). Similar results were obtained following treatment with STM2457, a potent and selective inhibitor of METTL3/14 [30] (Fig. S2E and F). Collectively, these findings demonstrate that METTL3 knockdown activates the cGAS–STING pathway and interferon responses.
The innate immune signaling triggered by METTL3 deficiency is dependent on cGAS and STING, as the METTL3-deficiency-induced up-regulation of interferon genes and IFN-β protein levels were abolished by treatment with either the cGAS inhibitor G140 [31] or the STING inhibitor H151 [32] (Fig. 2I and J). To validate these findings in vivo, we established syngeneic tumor models using sh-control and sh-METTL3 Lewis lung carcinoma (LLC) cells. The results showed that METTL3 knockdown significantly inhibited the growth of LLC syngeneic tumors, an effect that was rescued by G140 treatment (Fig. 2K). At the experimental endpoint, both tumor volume (Fig. 2L) and weight (Fig. 2M) were pronouncedly lower in the sh-METTL3 group compared to the sh-control group, and these reductions were reversed by G140 treatment. No significant change in body weight was observed among the groups (Fig. S2G). Furthermore, flow cytometry analysis (Fig. S2H) demonstrated that METTL3 knockdown significantly increased the proportions of CD45+ cells and CD8+ T cells within the CD45+ population, whereas G140 treatment reversed these effects (Fig. 2N and O). Additionally, IHC analysis revealed that sh-METTL3 LLC syngeneic tumors exhibited high expression levels of CD8 and Granzyme B (GZMB) compared to control tumors (Fig. 2P). In summary, these data indicate that METTL3 deficiency promotes nuclear DNA leakage into the cytoplasm, thereby triggering antitumor immunity through the activation of the cGAS pathway.
METTL3 regulates cytosolic DNA levels by modulating homologous recombination repair efficiency
Given that the accumulation of cytoplasmic DNA can result from nuclear DNA damage, we assessed the phosphorylation of histone H2AX (γH2AX), a surrogate marker for DNA DSBs, in LUAD cells. Western blotting analysis revealed that METTL3 knockdown significantly increased γH2AX level in A549 and H1299 cells (Fig. 3A). Furthermore, γH2AX levels remained elevated in sh-METTL3 A549 cells and were markedly enhanced following Zeocin treatment (Fig. 3B). Consistently, confocal microscopy confirmed elevated γH2AX levels in Zeocin-treated sh-METTL3 A549 cells (Fig. 3C). These results suggest that METTL3 deficiency leads to delayed repair and/or persistent generation of DSBs.
DSBs are primarily repaired by error-free homologous recombination (HR) or error-prone nonhomologous end joining (NHEJ) repair pathways [33]. To determine whether METTL3 influences one or both pathways, we utilized the HR and NHEJ repair reporter systems (Fig. 3D). The results indicated that METTL3 deficiency impaired HR-mediated DSB repair (DSBR) but did not affect NHEJ-mediated DSBR (Fig. 3E). Moreover, overexpression of WT METTL3, but not the METTL3 DA mutant, significantly enhanced HR-mediated DSBR (Fig. 3F). These findings suggest that METTL3 regulates HR efficiency in a methyltransferase-activity-dependent manner. Furthermore, in the presence of B02, a specific inhibitor of RAD51 and HR repair [34], overexpression of METTL3 failed to stimulate the repair of Zeocin-induced DSBs. Conversely, NU7441, a specific inhibitor of DNA ligase IV and NHEJ repair [35], did not impair METTL3-stimulated repair of Zeocin-induced DSBs (Fig. 3G and H). Collectively, these findings indicate that METTL3 knockdown promotes cytosolic DNA accumulation by reducing the efficiency of HR-mediated DSBR.
MSH5 mediates METTL3-regulated HR repair efficiency and cytosolic DNA accumulation
To identify potential targets involved in m6A-regulated HR repair efficiency and cytosolic DNA accumulation, we performed RNA sequencing on sh-control and sh-METTL3 A549 cells. We identified 562 differentially expressed genes in sh-METTL3 A549 cells, including 333 up-regulated and 229 down-regulated genes (Fig. S3A; Table S1). Among 64 HR-related genes (Table S2), MSH5 (mutS homolog 5) was the only gene to exhibit a greater than 2.0-fold change in expression (P < 0.05) in sh-METTL3 A549 cells compared to sh-control cells. Notably, MSH5 was also identified as an m6A-modified gene in A549 cells via m6A-seq (Table S3 and Fig. 4A). Consistent with this, RT-qPCR confirmed that METTL3 knockdown decreased MSH5 mRNA levels in both A549 and H1299 cells (Fig. 4B). Furthermore, Western blot analysis demonstrated that METTL3 knockdown reduced MSH5 protein expression in both cell lines (Fig. 4C).
MSH5 encodes a member of the mutS family, which plays a critical role in DNA mismatch repair and meiotic recombination [36]. To investigate the involvement of MSH5 in METTL3-regulated HR repair efficiency and cytosolic DNA accumulation in LUAD cells, we performed rescue experiments. Our results demonstrated that MSH5 overexpression (Fig. S3B) reversed the reduction in HR repair efficiency (Fig. 4D) and attenuated the increase in cytosolic DNA accumulation (Fig. 4E and F) in sh-METTL3 A549 cells. Collectively, these findings indicate that MSH5 mediates the effects of METTL3 on HR repair efficiency and cytosolic DNA accumulation in LUAD cells.
We further investigated whether m6A regulates MSH5 expression through direct mRNA methylation. m6A-RIP-seq data revealed that the coding sequence (CDS) region of MSH5 mRNA undergoes m6A modification (Fig. 4G). Additionally, m6A-RIP-qPCR confirmed a 10-fold enrichment of MSH5 mRNA using the m6A antibody in MSH5 mRNA in A549 cells, which was significantly reduced in sh-METTL3 A549 and H1299 (Fig. 4H) cells. Furthermore, overexpression of WT METTL3, but not the METTL3 DA mutant, rescued MSH5 level in A549 cells (Fig. 4I). Collectively, these results demonstrate that MSH5 was modified by m6A and that m6A positively regulates MSH5 expression in LUAD cells.
To investigate the underlying mechanisms of m6A-regulated MSH5 expression, we first examined whether METTL3 modulates MSH5 transcription. The luciferase reporter assay by transfecting the promoter reporter plasmid pGL3-Basic-MSH5-luc into A549 cells revealed no significant difference in MSH5 promoter activity between sh-control and sh-METTL3 LUAD cells (Fig. 4J), suggesting that METTL3 does not affect MSH5 transcription. This finding was further corroborated by qRT-PCR analysis, which showed comparable levels of MSH5 precursor mRNA in sh-control and sh-METTL3 LUAD cells (Fig. S3C). Additionally, subcellular fractionation assays revealed no difference in the distribution of MSH5 mRNA between sh-control and sh-METTL3 A549 cells (Fig. S3D). These findings indicated that m6A modification does not influence the subcellular localization of MSH5 mRNA.
However, METTL3 knockdown (Fig. 4B) significantly reduced MSH5 mRNA levels in LUAD cells. Given that m6A did not affect promoter activity but did regulateMSH5 mRNA abundance, we investigated its effect on mRNA stability. Our results showed that METTL3 knockdown substantially decreased the stability of MSH5 mRNA in both A549 (Fig. 4K) and H1299 (Fig. 4L) cells. Thus, MSH5 expression is positively regulated by m6A through the modulation of mRNA stability.
Regarding the translation efficiency of endogenous MSH5 mRNA, which is defined as the ratio of protein production (MSH5/GAPDH) to mRNA abundance [37], METTL3 knockdown had no significant impact on MSH5 translation efficiency in A549 cells (Fig. S3E). To determine whether m6A influences MSH5 posttranslationally, sh-control and sh-METTL3 A549 cells were treated with cycloheximide to block protein synthesis. Our data revealed that the half-life of the MSH5 protein was comparable between sh-control and sh-METTL3 cells (Fig. S3F). Collectively, these data indicated that METTL3 positively regulates MSH5 mRNA stability without affecting its transcription, nuclear export, translation efficiency, or protein stability.
METTL3 stabilizes MSH5 mRNA by recruiting IGF2BP2 to the m6A site A2521
We further investigated the methylation site and the reader protein responsible for m6A-mediated stabilization of MSH5 mRNA. Based on the m6A-seq results (Fig. 4F) and MSH5 mRNA m6A site predictions using SRAMP (http://www.cuilab.cn/sramp) (Fig. S4A), we identified 3 GGAC motifs in the MSH5 CDS (Fig. 5A). m6A-RIP-PCR performed on fragmented poly(A)+ RNA indicated that the relative enrichment of the MSH5 CDS-3 was significantly higher than that of the other 2 CDSs in A549 cells (Fig. 5B). To determine whether m6A modification of CDS-3 contributes to the regulation of MSH5 mRNA stability, we constructed CDS reporters by inserting the WT MSH5 CDS downstream of the firefly luciferase reporter gene in the pmirGLO vector (Fig. 5C). Luciferase assay revealed that F-Luc levels were markedly decreased in sh-METTL3 A549 cells due to the down-regulation of F-Luc mRNA, rather than a decrease in translation efficiency (Fig. 5D). Furthermore, the presence of the MSH5 CDS reduced the half-life of F-Luc mRNA in sh-METTL3 A549 cells (Fig. 5E), confirming that the m6A-methylated CDS mediated the METTL3-regulated MSH5 mRNA stability.
The m6A-binding proteins, insulin-like growth factor 2 mRNA-binding proteins (IGF2BPs; including IGF2BP1/2/3), but not the YTH domain family proteins YTHDF1/2/3, recognize and stabilize m6A-modified cellular RNAs [38,39]. RIP-qPCR revealed that IGF2BP2, but not IGF2BP1/3 or YTHDF1/2/3, binds to MSH5 mRNA in A549 cells (Fig. 5F). Furthermore, the interaction between IGF2BP2 and MSH5 mRNA was diminished in sh-METTL3 A549 cells (Fig. 5G). Knockdown of IGF2BP2 expression suppressed both the mRNA (Fig. 5H) and protein (Fig. 5I) levels of MSH5 in A549 and H1299 cells. This effect was attributed to decreased MSH5 mRNA stability following IGF2BP2 knockdown (Fig. 5J). Additionally, we found that si-IGF2BP2 reduced MSH5 expression and attenuated the reduction in MSH5 expression mediated by METTL3 deletion in A549 cells (Fig. 5K). Collectively, these data suggest that IGF2BP2 mediates the m6A-dependent regulation of MSH5 expression.
Our data suggested that A2521 within CDS-3 might be a potential m6A site on MSH5 mRNA. We further validated this site by the “SELECT” method [40] in A549 cells. The results revealed that METTL3 knockdown significantly reduced the methylation level of A2521 (Fig. 5L). In contrast, the nearby non-m6A-modified nucleotide A2526 exhibited a notably lower threshold cycle value compared to A2521 (Fig. S4B). To investigate the role of A2521 in m6A-regulated MSH5 mRNA stability, we mutated this site within the pmirGLO-MSH5 CDS reporter (Fig. S4C). The A2521 mutation resulted in decreased F-Luc activity (Fig. 5M), whereas the reduction was reversed in sh-METTL3 cells. Furthermore, mutation of A2521 decreased the stability of F-Luc mRNA (Fig. 5N). Collectively, these data confirm that A2521 within the MSH5 CDS mediates the m6A-regulated stability of MSH5 mRNA.
METTL3 destabilizes cGAS mRNA by recruiting YTHDF2 and methylating A1545
To further elucidate the role of METTL3 in modulating the innate immune response, we examined its effects on key components of the cGAS–STING pathway. RT-qPCR analysis revealed that METTL3 knockdown significantly up-regulated cGAS mRNA expression in both A549 (Fig. 6A) and H1299 (Fig. S5A) cells. Consistent with these findings, Western blot analysis demonstrated a corresponding increase in cGAS protein levels upon METTL3 knockdown in LUAD cells (Fig. 6B). These results suggest that METTL3 knockdown may positively regulate the innate immune response by modulating cGAS expression.
We further investigated whether m6A regulates cGAS expression through direct mRNA methylation. m6A-RIP-qPCR confirmed that the enrichment of cGAS mRNA by the m6A antibody was 8-fold higher in A549 cells, whereas this enrichment was significantly reduced in sh-METTL3 A549 (Fig. 6C) and H1299 (Fig. S5B) cells. These results indicated that cGAS mRNA undergoes m6A modification. Moreover, transfection of WT METTL3, but not the METTL3 DA mutant, decreased cGAS expression in A549 cells (Fig. 6D). Additionally, treatment with STM2457 pronouncedly increased cGAS protein levels in A549 cells (Fig. S5C). Collectively, these findings demonstrate that METTL3 negatively regulates cGAS expression in LUAD cells in an m6A-dependent manner.
We further investigated the mechanisms underlying the m6A-regulated expression of cGAS. Our data indicated that the transcription (Fig. S5D), subcellular localization (Fig. S5E), and translation efficiency (Fig. S5F) of cGAS mRNA, as well as the half-life of the cGAS protein (Fig. S5G), were comparable between sh-control and sh-METTL3 cells. We then examined mRNA stability. The results revealed that METTL3 knockdown significantly increased the mRNA stability of cGAS in both A549 (Fig. 6E) and H1299 (Fig. 6F) cells. Collectively, these data suggest that METTL3 negatively regulates cGAS mRNA stability without affecting its transcription, nuclear export, translation efficiency, or protein stability.
Furthermore, we investigated the specific methylation site and reader protein responsible for the m6A-mediated regulation of cGAS mRNA stability. As shown in Fig. S5H, 3 GGAC motifs were identified in the cGAS mRNA sequence by the m6A site predictor. To determine whether m6A modifications within the CDS or 3′ untranslated region (UTR) contribute to the regulation of cGAS, we constructed CDS and 3′UTR reporters by inserting the WT cGAS CDS or 3′UTR downstream of the firefly luciferase gene in the pmirGLO vector (Fig. S5I). Luciferase assays revealed a substantial increase in F-Luc activity from the pmirGLO-cGAS-CDS reporter, but not from the pmirGLO-cGAS-3′UTR reporter, in sh-METTL3 A549 cells (Fig. 6G). Additionally, m6A of A1545 within the GGAC motif in the cGAS CDS was validated by the “SELECT” method in A549 cells, whereas METTL3 knockdown markedly reduced the methylation level at A1545 (Fig. 6H). The nearby non-m6A-modified nucleotide A1542 exhibited a pronouncedly lower threshold cycle value compared to A1545 (Fig. S5J). Moreover, mutation of A1545 in the CDS increased F-Luc activity, which was abolished in sh-METTL3 cells (Fig. 6I). Collectively, these data confirm that methylation of A1545 within the cGAS CDS is critical for the m6A-dependent regulation of cGAS.
We further investigated the reader protein responsible for the m6A-regulated expression of cGAS. RIP-qPCR revealed that YTHDF2, but not IGF2BP1/2/3 or YTHDF1/3, binds to cGAS mRNA (Fig. 6J). Furthermore, the interaction between YTHDF2 and cGAS mRNA was diminished in sh-METTL3 A549 cells (Fig. 6K). Knockdown of YTHDF2 increased both the mRNA (Fig. 6L) and protein (Fig. 6M) expression of cGAS in A549 cells. This effect was attributed to the increased stability of cGAS mRNA following YTHDF2 depletion (Fig. 6N). Additionally, we found that si-YTHDF2 increased cGAS expression and attenuated the up-regulation in sh-METTL3-induced cGAS expression in A549 cells (Fig. 6O). Collectively, these data suggest that YTHDF2 mediates the m6A-regulated expression of cGAS.
METTL3 inhibition enhances LUAD immunotherapy efficacy and suppresses cancer progression
To evaluate the therapeutic potential of combining METTL3 inhibition with immunotherapy, we established syngeneic tumor models using LA795 cells treated with STM2457, anti-PD-1 antibody, or their combination. The results demonstrated that METTL3 inhibition significantly improved the efficacy of anti-PD-1 antibody therapy, resulting in marked reductions in tumor volume and weight (Fig. 7A to C). Subsequently, flow cytometry analysis demonstrated that the combination of METTL3 inhibition and anti-PD-1 antibody treatment notably increased the proportion of CD8+ T cells within the tumor tissue (Fig. 7D). IHC analysis confirmed elevated expression of CD8 and GZMB following the combination treatment (Fig. 7E). Collectively, these findings indicate that METTL3 inhibition enhances tumor immune infiltration and improves the efficacy of immunotherapy. Similar results were observed in LLC syngeneic tumor models (Fig. S6A to E).
To further investigate the potential link between METTL3-inhibition-induced activation of the cGAS/STING axis and LUAD progression, we analyzed clinical datasets from public databases. TIMER2.0 data analysis demonstrated that MSH5 expression was significantly negatively correlated with the infiltration of multiple antitumor immune cell types, including CD8+ T cells (r = −0.161, P = 3.36e−04), CD4+ T cells (r = −0.126, P = 5.21e−03), monocytes (r = −0.264, P = 2.51e−09), and neutrophils (r = −0.113, P = 1.20e−03) (Fig. 7F and Fig. S6A). In contrast, cGAS expression was positively correlated with the infiltration of these immune cell subsets (CD8+ T cells, r = 0.348, P = 1.68e−15; CD4+ T cells, r = 0.278, P = 3.51e−10; monocytes, r = 0.335, P = 2.01e−14; and neutrophils, r = 0.469, P = 2.75e−28, respectively) (Fig. 7G and Fig. S6B). Moreover, the expression levels of effector T-cell markers were inversely correlated with MSH5 expression (r = −0.24, P = 8.4e−08) (Fig. 7H) but positively correlated with cGAS expression (r = 0.097, P = 3.4e−02) (Fig. 7I) in LUAD tissues. Compared with normal tissues, MSH5 expression was up-regulated in LUAD tumor tissues within the The Cancer Genome Atlas (TCGA) cohort (Fig. S6C). Furthermore, Gene Expression Profiling Interactive Analysis data revealed a positive correlation between METTL3 and MSH5 expression (Fig. 7J) in LUAD patients. Similarly, METTL3 protein expression was negatively correlated with cGAS (Fig. 7K), CXCL10, and ISG15 (Fig. S6D) in LUAD patient samples from the CPTAC database. Collectively, these data suggest that METTL3-inhibition-induced activation of the cGAS/STING axis modulates the immune microenvironment and suppresses LUAD progression.
METTL3 inhibition combined with PARP inhibitor enhances the antitumor effects of an anti-PD-1 antibody
Poly(ADP-ribose) polymerase 1 is responsible for initiating the repair of single-strand breaks, and unrepaired single-strand breaks can lead to the formation of detrimental DSBs [41]. Recent studies have shown that PARP inhibitors can induce micronuclei formation in cancer cell lines and trigger the expression of ISGs in a cGAS- and STING-dependent manner [42,43]. Given that we previously observed that METTL3 deficiency impairs HR repair efficacy, thereby activating cGAS/STING-mediated antitumor immunity, we investigated whether METTL3 regulates the response to PARP inhibitors in LUAD cells. Cell viability assays revealed that METTL3 knockdown significantly sensitized A549 and H1299 cells to olaparib, a PARP inhibitor (Fig. 8A). Additionally, colony-formation assay demonstrated that METTL3 knockdown markedly inhibited the clonogenicity of both A549 and H1299 cells (Fig. 8B). Conversely, METTL3 overexpression reduced sensitivity and enhanced colony formation in A549 cells treated with olaparib (Fig. 8C and D). Collectively, these data suggest that METTL3 loss sensitizes tumor cells to PARP inhibitors and impairs oncogenic transformation.
Subsequently, we treated sh-METTL3 LLC syngeneic tumor models with olaparib, an anti-PD-1 antibody, or a combination of both and monitored tumor growth (Fig. 8E). Notably, monotherapy with either the PARP inhibitor or the anti-PD-1 antibody restricted tumor growth and reduced tumor weight, whereas their combination substantially improved the therapeutic effects (Fig. 8F and G). Additionally, flow cytometry analysis revealed that each treatment increased the intratumoral infiltration of CD8+ T cells and that this effect was further enhanced by combined treatment (Fig. 8H). IHC analysis demonstrated that CD8 and GZMB expression levels were elevated following the combination treatment (Fig. 8I). Collectively, our results demonstrate that combining METTL3 targeting with PARP inhibitor enhances the antitumor effects of an anti-PD-1 antibody.
Discussion
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While immune checkpoint blockade therapies targeting the PD-1/PD-L1 axis have revolutionized cancer treatment across multiple malignancies [44], their clinical efficacy in LUAD remains limited by immunosuppressive mechanisms and acquired resistance [45]. Thus, there is an urgent need to explore new strategies to improve the efficacy of LUAD immunotherapy. Our study reveals that METTL3-inhibition-induced activation of the cGAS/STING axis enhances immunotherapy efficacy and suppresses LUAD progression by regulating HR repair efficiency and cGAS expression. Mechanistically, IGF2BP2 binds to A2521 in the MSH5 CDS to increase its mRNA stability, whereas YTHDF2 binds to A1545 in the cGAS CDS to decrease its mRNA stability. Overall, METTL3 inhibition activates the cGAS/STING axis, thereby enhancing immunotherapy and PARP-inhibitor sensitivity via the induction of cytosolic DNA and cGAS expression, which in turn regulates LUAD progression (Fig. 9). While previous studies have demonstrated that METTL3 inhibition potentiates immunotherapy responses by enhancing IFN signaling and up-regulating ISGs [46–48], our study elucidates a novel and distinct mechanism by which METTL3 regulates tumor immunogenicity through the cGAS–STING pathway. Importantly, we provide compelling evidence linking METTL3-mediated m6A modification to both innate and adaptive immune responses in LUAD, offering new therapeutic opportunities for combination immunotherapy strategies. Collectively, these findings not only substantially advance our mechanistic understanding of METTL3’s role in cancer immunology but also establish a strong preclinical foundation for developing METTL3/cGAS-targeted therapies for LUAD patients.
Extensive research has established the pivotal role of m6A modification in regulating cancer development, chemoresistance, and immunotherapy response. The profound influence of m6A on immunotherapy efficacy is primarily mediated through its ability to orchestrate the physiological behaviors of immune cells, such as infiltration, survival, differentiation, or polarization, within the tumor microenvironment [7]. For instance, the absence of YTHDF1 enhances the translation efficiency of lysosomal histone proteases in dendritic cells, thereby potentiating the antitumor activity of CD8+ T cells [49]. While these findings underscore the importance of m6A in adaptive immunity, its role in regulating tumor-intrinsic innate immune signaling remains incompletely understood. Emerging evidence suggests that METTL3 inhibition stimulates an endogenous interferon response in cancer cells through the generation of double-stranded RNA, thereby enhancing antitumor effects [50]. In the present study, we reveal that the knockdown of METTL3 in cancer cells induces cytosolic DNA accumulation and up-regulates cGAS expression, thereby activating the cGAS–STING pathway and amplifying antitumor immunity. Together with prior work, our findings solidify the regulatory role of m6A in the innate immune response and provide new mechanistic insights into m6A-mediated immune modulation.
METTL3 regulates cytosolic DNA levels by modulating the efficiency of HR repair. Mechanistic investigations revealed that MSH5 mediates METTL3-regulated HR repair efficiency and cytosolic DNA accumulation. MSH5 encodes a member of the mutS family involved in DNA mismatch repair and meiotic recombination [36] and is also considered a crucial component in tumor progression [51,52]. Our data suggest that targeting m6A/MSH5-regulated HR repair efficiency could be a promising LUAD therapeutic approach. Additionally, m6A modification at A1545 in cGAS decreases its mRNA stability and contributes to m6A-regulated cGAS pathway activation in LUAD cells. As the key sensor of accumulated cytosolic DNA, cGAS has emerged as an intriguing candidate for targeted cancer therapy [53,54]. While the posttranscriptional regulation of the cGAS/STING pathway has been extensively studied [20], our study reveals another layer of regulatory control over cGAS expression. Notably, the m6A sites on MSH5 and cGAS mRNA are located within their CDS regions but exert opposing effects. A2521 in the MSH5 CDS binds to IGF2BP2 to increase its mRNA stability, whereas A1545 in the cGAS CDS binds to YTHDF2 to reduce its mRNA stability. This divergence can be attributed to the distinct reader proteins involved, which serve as the functional executors of m6A modifications [55].
Our study proposes a combination therapy involving METTL3 inhibitors, PARP inhibitors, and immunotherapy. Nevertheless, it is important to note that targeting METTL3 presents several biological and pharmacological challenges. Given the essential role of m6A modification in normal physiological processes, including hematopoiesis and immune cell homeostasis, systemic inhibition could lead to hematologic toxicity or immune dysregulation [56]. While STM2457 demonstrates high biochemical selectivity, potential off-target effects on other methyltransferases (e.g., METTL1 and METTL16) and demethylases (e.g., FTO) remain a concern [57]. Regarding pharmacokinetics, STM2457 is limited by poor tumor penetration and variable bioavailability, particularly in tumors with dense stromal barriers. New-generation inhibitors such as EP652 exhibit improved oral bioavailability and metabolic stability [58]. Furthermore, peptide-based inhibitors that disrupt the METTL3–METTL14 interaction offer a promising strategy with potentially higher specificity and reduced off-target profiles [59]. Thus, while preclinical results are promising, METTL3-targeted therapies have yet to enter clinical evaluation, underscoring the urgent need for early-phase trials to establish the safety, efficacy, and potential of these combination regimens.
In conclusion, our study highlights a novel relationship between the innate immune response and m6A modification. Specifically, we demonstrate that METTL3 inhibition activates cGAS/STING-mediated antitumor immunity in LUAD cells by modulating HR repair efficiency and up-regulating cGAS expression. Given the numerous genes involved in the innate immune response, it is plausible that m6A modification indirectly regulates the innate immune response by affecting other genes. Additionally, our study indicates that combining METTL3 inhibition with PARP inhibition enhances immunotherapy efficacy, thereby expanding the therapeutic landscape for LUAD patients.
Materials and Methods
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Human tissue microarray and IHC analysis
This study was approved by the Institutional Ethics Committee of Sun Yat-sen University (No. YBL2024157). Paired tumor and adjacent nontumor paraffin tissue microarray for human LUAD was obtained from YEPCOME Biotechnology Co., Ltd (Shanghai, China). The microarray comprised 50 pairs of LUAD and corresponding nontumor paraffin tissue samples. IHC analysis was performed as previously described [60]. The H-Score was calculated as follows: (percentage of weak intensity cells × 1) + (percentage of moderate intensity cells × 2) + (percentage of strong intensity cells × 3).
Flow cytometric analysis
Cell-surface-marker staining and flow cytometric analysis for CD3, CD4, CD8, and CD45 expression were performed as described previously [61]. The following antibodies were used: Cy5.5-anti-Mouse CD3e (65-0031-U025; TONBO Bioscience, USA), APC-anti-Mouse CD4 (25-0042-U025; TONBO Bioscience), Cy7-anti-Mouse CD8a (60-0081-U025; TONBO Bioscience), and FITC-anti-Mouse CD45 (35-0451-U025; TONBO Bioscience).
RNA extraction and real-time PCR
Total RNA was isolated using TRIzol Reagent (Agbio, AG21102, China) and reverse transcribed by Evo M-MLV (Agbio, AG11706). qRT-PCR was assessed with SYBR Green II (Agbio, AG11701) on a CFX Manager 3.1 (Bio-Rad, USA) as recommended by the manufacturer’s protocol. The primers used are listed in Table S4. GAPDH was used as a control for normalization. Relative gene expression levels were calculated using the 2−ΔΔCT method.
Statistical analyses
Data were presented as mean ± SD from a minimum of 3 independent experiments. Statistical analysis was performed using a 2-tailed unpaired Student’s t test for comparing 2 groups, while 1-way or 2-way analysis of variance followed by the Bonferroni test was used for multiple comparisons. All statistical tests were 2-sided. Analysis was performed using SPSS 16.0 for Windows. A P value of <0.05 was considered to be statistically significant. *P < 0.05, **P < 0.01, and ***P< 0.001; ns, no significant.
Full materials and methods are presented in Supplementary Materials and Methods.
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Year 2026 volume 9 Issue 5
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doi: 10.34133/research.1265
  • Receive Date：2025-08-21
  • Online Date：2026-10-16
  • Published：2026-05-20
Article Data
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  • Received：2025-08-21
  • Revised：2026-01-19
  • Accepted：2026-04-02
Affiliations
    1Guangdong Provincial Key Laboratory of Chiral Molecule and Drug Discovery, State Key Laboratory of Anti-Infective Drug Discovery and Development, School of Pharmaceutical Sciences, Sun Yat-sen University, Guangzhou 510006, China.
    2Department of Physiology, School of Basic Medical Sciences, Gannan Medical University, Ganzhou 341000, China.
    3Department of General Practice, Zhujiang Hospital, Southern Medical University, Guangzhou 510282, China.
    4Special Medical Service Center, Zhujiang Hospital, Southern Medical University, Guangzhou 510282, China.
    5Sun Yat-sen University Cancer Center, State Key Laboratory of Oncology in South China, Collaborative Innovation Center for Cancer Medicine, Guangzhou 510060, China.

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* Address correspondence to: (X.W.);
* Address correspondence to: (Z.C.);
* Address correspondence to: (H.W.)
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表12种不同金属材料的力学参数
科
Family		 属数
Number of
genus		 种数
Number of
species		 占总种数比例
Percentage of
total species (%)		 属
Genus		 种数
Number of
species		 占总种数比例
Percentage of total
species (%)
鹅膏菌科Amanitaceae 2 11 5.26 鹅膏菌属 Amanita 10 4.78
小菇科 Mycenaceae 2 12 5.74 丝盖伞属 Inocybe 5 2.39
多孔菌科 Polyporaceae 8 14 6.70 蜡蘑属 Laccaria 5 2.39
红菇科 Russulaceae 3 23 11.00 小皮伞属 Marasmius 6 2.87
小菇属 Mycena 11 5.26
光柄菇属 Pluteus 5 2.39
红菇属 Russula 17 8.13
栓菌属 Trametes 5 2.39
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