METTL3 inhibitor suppresses the progression of prostate cancer via IGFBP3/AKT pathway and synergizes with PARP inhibitor.

The RNA N6-methyladenosine (m6A) regulator METTL3 is an important regulatory gene in various progressive processes of prostate cancer (PCa). METTL3 inhibitors have been reported to possess potent tumor suppression capacity in some cancer types. Nevertheless, the detailed influence and mechanism of METTL3 inhibitors on PCa progression and their potential synergy with other drugs are poorly understood. In this study, we demonstrated that METTL3 was overexpressed and associated with poor survival in most PCa patients. METTL3 inhibitor STM2457 reduced m6A levels of PCa cells, thus inhibiting their proliferation, colony formation, migration, invasion, and stemness in vitro. Furthermore, STM2457 suppressed PCa progression in both the CDX and PDX models in vivo. MeRIP-seq analysis coupled with biological validation revealed that STM2457 influenced multiple biological processes in PCa cells, mainly through the IGFBP3/AKT pathway. We also proved that STM2457 induced DNA damage and showed synergistic anti-PCa effects with the PARP inhibitor olaparib both in vitro and in vivo. All in all, this work provides a novel therapeutic strategy for targeting RNA m6A modifications for the treatment of PCa and provides a meaningful reference for further clinical trials.

METTL3-Driven m6A Modification of RBPJ Promotes Vascular Remodeling in Pulmonary Hypertension.

The dysregulation of N6-methyladenosine (m6A) RNA modification is widely recognized for its crucial roles in various diseases, including pulmonary hypertension (PH). Prior studies have highlighted the significant role of METTL3 in the pathogenesis of PH. Nevertheless, the potential and underlying mechanisms of METTL3 and its inhibitors as targets for PH treatment require further elucidation. In this study, we observed increased levels of METTL3 in various rodent models of PH. In vitro studies revealed that METTL3 silencing or treatment with STM2457, a specific METTL3 inhibitor, attenuated the proliferation and migration of pulmonary artery smooth muscle cells (PASMCs) stimulated by platelet-derived growth factor-BB (PDGF-BB) or hypoxia. Moreover, in vivo experiments using AAV9-mediated METTL3 silencing or STM2457 inhibition demonstrated improvement in SU5416/hypoxia-induced PH in mice. Additionally, m6A RNA immunoprecipitation analysis identified RBPJ as a gene regulated by METTL3 in rodent models of PH. Loss-of-function studies showed that silencing RBPJ could attenuate the changes in the proliferation and migration of PASMCs induced by PDGF-BB or hypoxia. Further studies indicated that METTL3 and YTHDF1 regulate RBPJ mRNA expression in an m6A-dependent manner. These findings indicated that targeting METTL3 may be a promising therapeutic strategy for treating PH, and modulation of RBPJ could offer a potential intervention mechanism.

METTL3 Inhibition Suppresses Cell Growth and Survival in Colorectal Cancer via ASNS Downregulation.

Background: Colorectal cancer (CRC) presents a significant global health burden, with high rates of incidence and mortality, and an urgent need to improve prognosis. STM2457, a novel small molecule inhibitor specific for N6-methyladenosine (m6A) catalytic enzyme Methyltransferase-like 3 (METTL3) has implicated significant treatment potentials in a few of types of cancer. However, its impact and underlying mechanism are still unclear in CRC cells. Methods: We used CCK-8 and colony formation assay to observe cell growth, flow cytometry and TUNEL approaches to detect cell apoptosis under the treatment of STM2457 on CRC cells in vitro or in vivo. RNA-sequencing, qRT-PCR and western blotting were performed to explore downstream effectors of STM2457. Messenger RNA stability was evaluated by qRT-PCR after treatment with actinomycin D. The methylated RNA immunoprecipitation (MeRIP) qPCR, dual-luciferase reporter analyses and m6A dot blotting were carried out to measure the m6A modification. Associated gene expression pattern and clinical relevance in CRC clinical tissue samples were analyzed using online database. Results: STM2457 exhibited a strong influence on cell growth suppression and apoptosis of CRC cells in vitro and subcutaneous xenograft growth in vivo. Asparagine synthetase (ASNS) was markedly downregulated upon STM2457 treatment or METTL3 knockdown and exogenous overexpression of ASNS could rescue the biological defects induced by STM2457. Mechanistically, the downregulation of ASNS by STM2457 may be due to the decrease of m6A modification level in ASNS mRNA mediated by METTL3. Conclusions: Our findings suggest that STM2457 may serve as a potential therapeutic agent and ASNS may be a new promising therapeutic target for CRC.

Prognostic roles of dysregulated METTL3 protein expression in cancers and potential anticancer value by inhibiting METTL3 function.

BACKGROUND: Many studies have demonstrated the relationship between METTL3 protein expression and clinical outcomes in various cancers and elucidated the mechanism by which METTL3 disrupts the behavior of cancer cells. Here, we attempted to define the prognostic value of METTL3 protein in patients with cancer via systematic analysis and explored the potential effect of inhibiting METTL3 using its specific inhibitor. METHODS: We searched PubMed, Embase, and the Web of Science databases for studies that elucidated the prognostic value of METTL3 protein expression in all cancer types and then calculated the pooled hazard ratios with 95% confidence intervals for the overall survival (OS) of all cancer types and subgroups. Data from The Cancer Genome Atlas dataset were used to study METTL3 mRNA expression in cancers. Further, the effects of a METTL3-specific inhibitor were studied in cancer cells via the colony formation assay, the cell proliferation assay, and apoptosis detection. RESULTS: Meta-analysis of the 33 cohorts in 32 studies (3666 patients in total) revealed that higher METTL3 protein expression indicated poor OS in the majority of cancers. Bioinformatics analysis of METTL3 mRNA expression and cancer prognosis did not show the extremely prominent prognostic value of METTL3 mRNA. Nevertheless, the METTL3-specific inhibitor attenuated cell proliferation and cell cloning formation and promoted apoptosis. CONCLUSIONS: METTL3 protein expression is associated with poor prognosis in most cancer types and could be a biomarker for OS. Further, METTL3 inhibition might be a potential treatment strategy for cancers.

METTL3 inhibitor STM2457 impairs tumor progression and enhances sensitivity to anlotinib in OSCC.

OBJECTIVES: To investigate the inhibitory effects of STM2457, which is a novel METTL3 (m6 A writer) inhibitor, both as a monotherapy and in combination with anlotinib, in the treatment of oral squamous cell carcinoma (OSCC) both in vitro and in vivo. MATERIALS AND METHODS: The efficacy of STM2457 or STM2457 plus anlotinib was evaluated using two OSCC cell lines by CCK8, transwell, colony formation, would-healing, sphere formation, cell cycle, apoptosis assays, and nude mice tumor xenograft techniques. The molecular mechanism study was carried out by western blotting, qRT-PCR, MeRIP-qPCR, immunofluorescence, and immunohistochemistry. RESULTS: STM2457 combined with anlotinib enhanced inhibition of cellular survival/proliferation and promotion of apoptosis in vitro. Moreover, this combinatorial approach exerted a notable reduction in stemness properties and EMT (epithelial-mesenchymal transition) features of OSCC cells. Remarkably, in vivo studies validated the efficacy of the combination treatment. Mechanistically, our investigations revealed that the combined action of STM2457 and anlotinib exerted downregulatory effects on EGFR (epidermal growth factor receptor) expression in OSCC cells. CONCLUSIONS: The combination of STM2457 and anlotinib targeting EGFR exerted a multiple anti-tumor effect. In near future, anlotinib combined with STM2457 may provide a novel insight for the treatment of OSCC.

[METTL3 inhibitor STM2457 improves metabolic dysfunction-associated fatty liver disease by regulating mitochondrial function in mice].

OBJECTIVE: To investigate the effect of methyltransferase-like 3 (METTL3) inhibitor STM2457 in metabolic dysfunction-associated fatty liver disease (MAFLD). METHODS: C57BL/6J mouse models of MAFLD induced by high-fat diet feeding for 16 weeks were treated with intraperitoneal injections of STM2457 (50 mg/kg) for 2 weeks. The changes in m6A modification level in the liver tissue of the mice were determined with dot-blot hybridization, and the hepatic levels of triglyceride (TG), alanine aminotransferase (ALT) and glutathione aminotransferase (AST) were detected. The histological changes of the liver and changes in insulin resistance and metabolic profile of the mice were evaluated using HE staining, insulin tolerance tests and metabolic cages; transmission electron microscopy (TEM) was employed to examine the changes in mitochondrial morphology. In a HepG2 cell model of steatosis induced by treatment with sodium oleate/sodium palmitate for 48 h, the protective effect of STM2457 (1 µmol/L) on mitochondrial function was assessed by measuring mitochondrial membrane potential using a fluorescence probe (JC-1). RESULTS: The mouse models of MAFLD showed significant elevation of m6A modification level in the liver tissues and obviously upregulated mRNA expression of METT3 (P<0.05). Treatment with STM2457 significantly reduced body weight and liver lipid deposition and m6A modification levels, increased glucose tolerance and insulin sensitivity, lowered hepatic TG and serum ALT and AST levels, and increased respiratory entropy (RQ) in the mouse models (all P<0.05). HepG2 cells with steatosis exhibited obvious mitochondrial swelling with decreased mitochondrial membrane potential, but the STM2457-treated cells maintained a normal mitochondrial morphology with a higher membrane potential (P<0.05). CONCLUSION: The METTL3 inhibitor STM2457 improves MAFLD by reducing high-fat diet-induced mitochondrial damage in mice.

METTL3 Affects Spinal Cord Neuronal Apoptosis by Regulating Bcl-2 m6A Modifications After Spinal Cord Injury.

OBJECTIVE: Spinal cord injury (SCI) is a severe type of neurological trauma. N6-methyladenosine (m6A) modification is one of the most common internal modifications of RNA. The role of METTL3, the predominant methylation enzyme of m6A modification, in SCI remains unclear. This study aimed to investigate the role of methyltransferase METTL3 in SCI. METHODS: After establishing the oxygen-glucose deprivation (OGD) model of PC12 cells and rat spinal cord hemisection model, we found that the expression of METTL3 and the overall m6A modification level were significantly increased in neurons. The m6A modification was identified on B-cell lymphoma 2 (Bcl-2) messenger RNA (mRNA) by bioinformatics analysis, and m6A-RNA immunoprecipitation and RNA immunoprecipitation. In addition, METTL3 was blocked by the specific inhibitor STM2457 and gene knockdown, and then apoptosis levels were measured. RESULTS: In different models, we found that the expression of METTL3 and the overall m6A modification level were significantly increased in neurons. After inducing OGD, inhibition of METTL3 activity or expression increased the mRNA and protein levels of Bcl-2, inhibited neuronal apoptosis, and improved neuronal viability in the spinal cord. CONCLUSION: Inhibition of METTL3 activity or expression can inhibit the apoptosis of spinal cord neurons after SCI through the m6A/Bcl-2 signaling pathway.

Effects and translatomics characteristics of a small-molecule inhibitor of METTL3 against non-small cell lung cancer.

In non-small cell lung cancer (NSCLC), the heterogeneity promotes drug resistance, and the restricted expression of programmed death-ligand 1 (PD-L1) limits the immunotherapy benefits. Based on the mechanisms related to translation regulation and the association with PD-L1 of methyltransferase-like 3 (METTL3), the novel small-molecule inhibitor STM2457 is assumed to be useful for the treatment of NSCLC. We evaluated the efficacy of STM2457 in vivo and in vitro and confirmed the effects of its inhibition on disease progression. Next, we explored the effect of STM2457 on METTL3 and revealed its effects on the inhibition of catalytic activity and upregulation of METTL3 protein expression. Importantly, we described the genome-wide characteristics of multiple omics data acquired from RNA sequencing, ribosome profiling, and methylated RNA immunoprecipitation sequencing data under STM2457 treatment or METTL3 knockout. We also constructed a model for the regulation of the translation of METTL3 and PD-L1. Finally, we found PD-L1 upregulation by STM2457 in vivo and in vitro. In conclusion, STM2457 is a potential novel suppressor based on its inhibitory effect on tumor progression and may be able to overcome the heterogeneity based on its impact on the translatome. Furthermore, it can improve the immunotherapy outcomes based on PD-L1 upregulation in NSCLC.

METTL3-m6A-EGFR-axis drives lenvatinib resistance in hepatocellular carcinoma.

Lenvatinib is emerging as the first-line therapeutic option for advanced hepatocellular carcinoma (HCC), but drug resistance remains a major hurdle for its long-term therapy efficiency in clinic. N6-methyladenosine (m6A) is the most abundant mRNA modification. Here, we aimed to investigate the modulatory effects and underlying mechanisms of m6A in lenvatinib resistance in HCC. Our data revealed that m6A mRNA modification was significantly upregulated in the HCC lenvatinib resistance (HCC-LR) cells compared to parental cells. Methyltransferase-like 3 (METTL3) was the most significantly upregulated protein among the m6A regulators. Either genetic or pharmacological inhibition of m6A methylation through METTL3 deactivation in primary resistant cell line MHCC97H and acquired resistant Huh7-LR cells decreased cell proliferation and increased cell apoptosis upon lenvatinib treatment in vitro and in vivo. In addition, the specific METTL3 inhibitor STM2457 improved tumor response to lenvatinib in multiple mouse HCC models, including subcutaneous, orthotopic and hydrodynamic models. The MeRIP-seq results showed that epidermal growth factor receptor (EGFR) was a downstream target of METTL3. EGFR overexpression abrogated the METTL3 knocked down-induced cell growth arrest upon lenvatinib treatment in HCC-LR cells. Thus, we concluded that targeting METTL3 using specific inhibitor STM2457 improved the sensitivity to lenvatinib in vitro and in vivo, indicating that METTL3 may be a potential therapeutic target to overcome lenvatinib resistance in HCC.

METTL3 inhibition reduces N6 -methyladenosine levels and prevents allogeneic CD4+ T-cell responses.

Alloreactive CD4+ T cells play a central role in allograft rejection. However, the post-transcriptional regulation of the effector program in alloreactive CD4+ T cells remains unclear. N6 -methyladenosine (m6 A) RNA modification is involved in various physiological and pathological processes. Herein, we investigated whether m6 A methylation plays a role in the allogeneic T-cell effector program. m6 A levels of CD4+ T cells from spleens, draining lymph nodes and skin allografts were determined in a skin transplantation model. The effects of a METTL3 inhibitor (STM2457) on CD4+ T-cell characteristics including proliferation, cell cycle, cell apoptosis and effector differentiation were determined after stimulation of polyclonal and alloantigen-specific (TEa; CD4+ T cells specific for I-Eα52-68 ) CD4+ T cells with α-CD3/α-CD28 monoclonal antibodies and cognate CB6F1 alloantigen, respectively. We found that graft-infiltrating CD4+ T cells expressed high m6 A levels. Administration of STM2457 reduced m6 A levels, inhibited T-cell proliferation and suppressed effector differentiation of polyclonal CD4+ T cells. Alloreactive TEa cells challenged with 40 µm STM2457 exhibited deficits in T-cell proliferation and T helper type 1 cell differentiation, a cell cycle arrest in the G0 phase and elevated cell apoptosis. Moreover, these impaired T-cell responses were associated with the diminished expression levels of transcription factors Ki-67, c-Myc and T-bet. Therefore, METTL3 inhibition reduces the expression of several key transcriptional factors for the T-cell effector program and suppresses alloreactive CD4+ T-cell effector function and differentiation. Targeting m6 A-related enzymes and molecular machinery in CD4+ T cells represents an attractive therapeutic approach to prevent allograft rejection.