Lymphangiogenesis plays a pivotal role in the pathogenesis of various vascular disorders, including ocular vascular diseases and cancers. Deregulation of N6‐methyladenosine (m6A) modification has been identified as a key contributor to human diseases. However, the specific involvement of m6A modification in lymphatic remodeling remains poorly understood. In this study, we demonstrate that inflammatory stimulation and corneal sutures induce elevated levels of methyltransferase‐like 3 (METTL3)‐mediated m6A modification. METTL3 knockdown inhibits lymphatic endothelial viability, proliferation, migration, and tube formation in vitro. METTL3 knockdown attenuates corneal sutures‐induced lymphangiogenesis and intratumoral lymphangiogenesis initiated by subcutaneous grafts, consequently restraining corneal neovascularization, tumor growth, and tumor neovascularization in vivo. Mechanistically, METTL3 knockdown upregulates prostaglandin–endoperoxide synthase 2 expression through an m6A–YTHDF2‐dependent pathway, enhancing the synthesis of cyclopentenone prostaglandins (CyPGs). Aberrant CyPG production in lymphatic endothelial cells impairs mitochondrial oxidative phosphorylation, contributing to pathological lymphangiogenesis. Moreover, selective inhibition of METTL3 with STM2457 reduces m6A levels in lymphatic endothelial cells, effectively suppressing pathological lymphangiogenesis. This study provides compelling evidence that lymphatic‐specific METTL3 plays a critical role in vascular patterning through prostaglandin metabolism reprogramming. Thus, METTL3 emerges as a promising target for treating lymphangiogenesis‐related diseases.

2024-10-01·BIOMEDICINE & PHARMACOTHERAPY

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

Article

作者: Yang, Jingxin ; Zhang, Rongyu ; Wang, Miaomiao ; Ding, Xin ; Zhou, Jinming ; Wang, Haoran ; Hou, Huimin ; Wu, Meng ; Chen, Xin ; Li, Xiaoxin

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.

2024-10-01·EXPERIMENTAL CELL RESEARCH

Increased m6A RNA methylation and METTL3 expression may contribute to the synovitis progression of rheumatoid arthritis

Article

作者: Kang, Jie ; Su, Yazhen ; Zhang, Liyun ; Jia, Junqing ; Wu, Zewen ; Liu, Xinling ; Liu, Yang

OBJECTIVE:

Rheumatoid arthritis (RA) is an autoimmune disease characterized by synovial hyperplasia and progressive bone destruction. The tumor-like growth of fibroblast-like synoviocytes (FLSs) plays a crucial role in the pathogenesis of RA. The N6 methyladenine (m6A) mRNA methylation modification, regulated by methyltransferases (METTL3) and demethylation enzymes, is a novel epigenetic regulator in the development of RA. However, there is limited research on m6A methylation modifications in RA synovitis and a lack of mechanistic studies on their impact on the function of RA-FLSs.

METHODS:

This study utilized clinical synovial tissue specimens and FLSs as research subjects. The m6A methylation level and the expression of methyltransferases and demethylation enzymes were detected. RNA interference and gene overexpression methods were employed to investigate the mechanism of METTL3 in RA-FLSs. The study also examined the proliferation, apoptosis, migration, invasion, and cytokine levels of RA-FLSs, as well as the expression of METTL3 in RA animal models.

RESULTS:

In this study, we found that m6A methylation levels were elevated in synovial tissues and FLSs of RA patients. Immunohistochemical staining showed that METTL3 and METTL14 levels were up-regulated in synovial tissues of RA, the mRNA levels of METTL3, METTL14, WTAP, FTO, and ALKBH5 were significantly higher in synovial tissues and FLSs of RA patients. Overexpression of METTL3 could promote the proliferation, migration, and secretion of IL-6, RANKL of RA-FLSs; inhibition of METTL3 expression could inhibit the abnormal proliferation, migration, invasion, and secretion of IL-6, RANKL, at the same time promoted the apoptosis and secretion of OPG, thus inhibited RA-FLSs tumor-like growth. In CIA mice, the use of MTX and STM2457 reduced METTL3 expression, synovial hyperplasia and bone destruction.

CONCLUSION:

Abnormal modification of m6A methylation exists in synovial tissues and FLSs of RA patients, and inhibition of METTL3 can reduce synovitis and bone destruction. Our findings suggest that m6A methylation might control FLS-mediated tumor-like phenotype, and be a novel target for RA treatment.

项与 STM-2457 相关的新闻（医药）

2024-06-12

·精准药物

【Angew】靶向METTL3-METTL14的抑制剂，用于癌症治疗

N6-甲基腺苷（m6A）是常见于信使RNA中的一种修饰，其调节异常与各种疾病的发生发展有着十分紧密的关系。METTL3-METTL14甲基转移酶复合物是负责催化m6A的关键元件，其中METTL3起主要催化功能，METTL14维持结构完整与RNA结合。研究表明METTL3在多种癌症中异常表达，是表观遗传最热门的靶点之一。自2016年METTL-METTL14晶体结构被解析以来，科学家一直尝试开发针对METTL3的抑制剂，直到2021年英国STORM Therapeutic开发首个METTL3小分子抑制剂STM2457用于白血病的治疗，虽已进入临床一期，但其在实体瘤上的效果未知。与小分子药物相比，多肽药物具有高度特异性和良好的安全性，且能作用于被认为不具有成药性的蛋白-蛋白相互作用（PPI）区域，具有极大的研究与应用潜力。 2024年4月12日，湖南大学生命交叉研究院史俊峰和张定校团队合作在Angew Chem Int Ed Engl（IF=16.6）上发表题为“A Stapled Peptide Inhibitor Targeting the Binding Interface of N6-Adenosine-Methyltransferase Subunits METTL3 and METTL14 for Cancer Therapy”的文章，该研究基于METTL3-METTL14蛋白结构，设计了靶向METTL3的多肽抑制剂RM3。进一步优化获得更高活性的订书肽抑制剂RSM3，该多肽具有广谱的抑癌特性，能够杀伤多种METTL3高表达的肿瘤细胞，同时在多种肿瘤模型中显示出优异的抗肿瘤效果，且具有高度特异性和良好的安全性。 1、肽抑制剂设计与验证使用先前报道的METTL3-METTL14复合物（PDB：5IL1）的晶体结构，作者确定了复合物形成所需的蛋白质-蛋白质相互作用（PPI）可能必不可少的两个不同结构域。基于这些结构信息设计了M1、M2和M3候选肽，并将每个肽融合到经典的细胞穿透肽R9中以增强细胞渗透，形成了RM1、RM2和RM3候选肽（图1）。图1 肽抑制剂的设计考虑到METTL3在前列腺癌细胞中高度上调并在癌症发展中起重要作用，作者使用PC3和DU145细胞系开展MTT测定评估了肽抑制剂的能力，发现与RM1和RM2相比，RM3表现出更大的抗癌作用。克隆形成测定同样表明RM3比RM1或RM2具有更强的抑制作用。这些结果表明RM3作为一种有前途的候选抗癌药物，值得进一步优化（图2）。图2 肽抑制剂的评价 2、体外M3衍生物的理化性质和活性为了增强 RM3肽的作用，作者采用了肽吻合技术从RM3生成RSM3吻合肽。订书位点的选择和设计旨在最大限度地减少潜在的空间冲突，这些空间冲突可能会干扰结合事件，同时保留肽构象。通过MST和SPR检测发现RSM3与METTL3–METTL14复合物表现出高亲和力结合，而非吻合肽RM3的亲和力较低。为了评估肽与METTL3的细胞互作，作者通过pulldown成功地拉下了PC3细胞中的METTL3和METTL14。圆二色谱（CD）测量进一步验证了RSM3在溶液中可以采用α螺旋构象，而非钉合的RM3肽片段主要存在于随机螺旋构象中。功能分析表明RSM3可有效抑制METTL3-METTL14复合物转移酶活性，且强于RM。此外，RSM3表现出更高的生物稳定性，没有明显的毒性（图3）。图3 肽抑制剂的物理化学表征 3、RSM3体外抗癌效果的评估 METTL3在许多癌症类型中作为促癌因子，细胞实验发现RSM3对PC3、HeLa、HepG2等多种癌细胞均表现出广泛的毒性，且细胞毒性显著高于非吻合RM3肽。细胞迁移和集落形成实验显示RSM3和RM3对有效抑制癌细胞侵袭性，EdU标记荧光检测和流式细胞术分析显示RSM3有效地诱导细胞周期停滞，膜联蛋白V-FITC和TUNEL染色、流式细胞术表明RSM3可以比RM3诱导更高水平的细胞凋亡。WB测定证实，裂解的caspase-3在肽处理的细胞中积累。这些结果说明RSM3通过诱导细胞凋亡和细胞周期停滞的抗癌活性（图4）。图4 RSM3抗癌效果的评估 4、RSM3与METTL3–METTL14的相互作用会降低复合物的稳定性和功能为了探索肽抑制剂与METTL3之间的相互作用，免疫染色证实RSM3与METTL3的荧光信号共定位。RSM3处理METTL3和METTL14信号强度均降低，表明RSM3可能导致功能改变和METTL3-METTL14复合物的降解。WB分析进一步证实，RSM3处理后PC3细胞中的METTL3和METTL14水平降低，支持RSM3既可以抑制催化活性，也可以触发METTL3-METTL14复合物的降解。随后使用 m6A ELISA表明RSM3处理后m6A修饰显著减少。此外，RSM3处理可以通过蛋白酶体途径激活METTL3降解。CO-IP实验发现RSM3 处理后METTL3和METTL14的互作减少，这些结果表明RSM3有效破坏了METTL3-METTL14复合物的稳定性，随后通过蛋白酶体途径促进降解（图5）。图5 RSM3与METTL3–METTL14的互作会降低复合物的稳定性和功能 5、PCa细胞对肽处理的转录组为了进一步揭示RSM3抑制癌细胞的分子机制，作者用载体或RSM3处理的PC3细胞中进行RNA-seq，GO分析发现上调的基因富集在参与程序性细胞死亡、p53 肿瘤抑制因子信号传导、分化和免疫的通路中，下调基因主要富集在促进癌症侵袭性、干性和增殖的通路，GSEA进一步证实了GO结果，qPCR分析验证了RNA-seq结果，即与炎症和细胞凋亡相关的NF-κB反应基因上调，与细胞周期和增殖相关的基因下调。此外，结合M6 A2Target数据库分析发现RSM3 处理的PCa细胞中检测到的差异基因确实显著富集了受m6A调节的基因，说明了RSM3抑制METTL3的靶向效应。此外，在RSM3处理的细胞中，GO显示MAPK信号级联基因显著富集，WB验证RSM3组中p42/44的磷酸化水平较低，表明RSM3处理导致ERK/MAPK活性降低。结果表明RSM3抑制METTL3可以以m6A依赖性方式通过抑制MAPK有丝分裂途径以及诱导分化来延缓癌细胞生长并诱发细胞凋亡（图6）。图6 PCa细胞对RSM3处理的转录组 6、RSM3 在体内的安全性和抗肿瘤功效接着通过单次腹腔内注射RSM3到小鼠体内检查其体内急性毒性，血液常规分析和主要器官的组织病理学检查结果显示RSM3没有明显的毒性迹象。然后，作者评估了RSM3在PC3异种移植肿瘤模型中的抗癌活性，发现RSM3治疗可显著抑制肿瘤生长，且在在整个治疗过程中，所有组的体重均未受到影响，多种器官的组织病理学检查未发现明显异常或病变。肿瘤样本的免疫组织化学分析表明RSM3治疗导致METTL3表达和m6A水平降低。此外，caspase-3表达增加，而Ki67降低，表明RSM3诱导细胞凋亡和抑制增殖（图7）。图7 RSM3抑制剂的毒性和抗肿瘤疗效研究总结该论文基于METTL3-METTL14的晶体结构，在蛋白之间的两个相互作用界面(interface loop，residues 462-479), helix a2，residues 435-447)和一个底物催化中心（residues 534-541）中鉴定和设计了一系列多肽序列，并结合穿膜肽R9用于细胞内递送。通过体外酶活力测定、细胞存活、亲和力测定以及多肽Pulldown等实验筛选获得一段有生物活性的多肽RM3（源于residues 435-447），具有靶向METTL3、抑制METTL3酶催化活性和抑制癌细胞生长迁移等功能。同时，为了保持多肽的a二级结构和提高稳定性，进一步构建了订书肽化的多肽抑制剂RSM3。进一步细胞实验发现，该多肽可有效的降低细胞的mRNA甲基化水平，还能抑制多种癌细胞的生长和引起细胞凋亡，显示出较强的广谱抑癌性。机制研究发现多肽抑制剂处理的细胞中METTL3表达量显著下降，推测其可引起METTL3的降解。进一步研究发现，该多肽主要通过蛋白酶体途径引发METTL3降解，而小分子抑制剂STM2457并未引起METTL3的蛋白含量下降，说明多肽抑制剂RSM3具有不同的作用机制。最终，在小鼠前列腺癌和胰腺癌等肿瘤模型中，RMS3等多肽抑制剂展现出较好的抑瘤能力，并具有较好的安全性。此外，在活体水平RSM3的效果也略优于STM2457。上述结果说明该多肽具有较好的应用潜力。图8 订书肽抑制剂（RSM3）的设计及作用机制参考文献 Li Z, Feng Y, Han H, Jiang X, Chen W, Ma X, Mei Y, Yuan D, Zhang D, Shi J. A Stapled Peptide Inhibitor Targeting the Binding Interface of N6-Adenosine-Methyltransferase Subunits METTL3 and METTL14 for Cancer Therapy. Angew Chem Int Ed Engl. 2024 Apr 12:e202402611. doi: 10.1002/anie.202402611. Epub ahead of print. PMID: 38607929. 声明：发表/转载本文仅仅是出于传播信息的需要，并不意味着代表本公众号观点或证实其内容的真实性。据此内容作出的任何判断，后果自负。若有侵权，告知必删！长按关注本公众号粉丝群/投稿/授权/广告等请联系公众号助手觉得本文好看，请点这里↓

临床1期临床2期

2023-09-28

·生物谷

Cell子刊：张金方/魏文毅团队通过靶向METTL3让冷肿瘤变热，改善癌症免疫治疗

总的来说，这项研究强调了靶向METTL3可作为改善非小细胞肺癌这种冷肿瘤的免疫治疗效果的有前途的新策略。肺癌是全球癌症相关死亡的主要原因，每年约有180万人死于肺癌。非小细胞肺癌（NSCLC）是最常见的亚型，约占所有肺癌的85%，其中肺腺癌（LUAD）和肺鳞状细胞癌（LUSC）是NSCLC的两个主要亚型。虽然PD-1/PD-L1免疫检查点阻断疗法在局部或转移性晚期NSCLC患者中显示出前所未有的疗效，但只有约20%的患者对这些免疫疗法有反应并受益。NSCLC患者对抗PD-1/PD-L1免疫疗法的潜在耐药机制尚不清楚。新的研究证据表明，肿瘤微环境（TME）在调控肿瘤进展和对免疫治疗的反应中发挥关键作用。根据肿瘤中浸润淋巴细胞（TIL）的数量、PD-L1的表达水平，可以将肿瘤分为两种类型——非炎症性肿瘤（也叫做冷肿瘤）和炎症性肿瘤（也叫做热肿瘤）。冷肿瘤对免疫疗法具有耐药性，而热肿瘤对免疫疗法敏感。将冷肿瘤重编程为热肿瘤，可以显著改善对免疫疗法的反应，从而可能使更多癌症患者受益。 2023年9月25日，武汉大学中南医院张金方教授、哈佛大学医学院魏文毅教授及Gordon Freeman教授等在 Cell 子刊 Cell Chemical Biology 上发表了题为：Targeting METTL3 reprograms the tumor microenvironment to improve cancer immunotherapy 的研究论文。该研究发现，靶向抑制METTL3，能够将冷肿瘤微环境重编程为热肿瘤，让非小细胞肺癌对抗PD-1免疫疗法重新变得敏感。而临床上表现出低METTL3表达的非小细胞肺癌患者接受抗PD-1免疫疗法的预后也更好。这些结果提示我们，靶向METTL3可作为改善非小细胞肺癌这种冷肿瘤的免疫治疗效果的有前途的新策略。冷肿瘤以TIL数量少、PD-L1低表达、MHC-I低/缺陷、低肿瘤突变负担和大量免疫抑制细胞（例如骨髓源性抑制细胞和调节性T细胞）为特征，其对免疫疗法具有耐药性。而热肿瘤通常表现为TIL数量多、PD-L1高表达、MHC-I高/完整、高肿瘤突变负担和低免疫抑制细胞浸润，其对免疫疗法敏感。将冷肿瘤重编程为热肿瘤，可以显著改善对免疫疗法的反应，从而可能使更多癌症患者受益。 METTL3/ METTL14介导的m6A修饰，在转录后水平调控基因表达中发挥着至关重要的作用，并影响包括肿瘤发生在内的各种生理病理过程。 METTL3在多种癌症类型中起促肿瘤作用，但也有报道指出METTL3具有抑制肿瘤功能，这提示了METTL3在肿瘤发生中的作用仍有待进一步阐明。之前有研究显示，在体外培养细胞和免疫缺陷小鼠的异种移植肿瘤模型中，METTL3是促进肺癌发展的致癌基因。近期一项研究利用生物信息学方法对NSCLC中m6A调控因子与肿瘤微环境的相关性进行了分析，但并没有实验证据。METTL3是否以及如何调控肺癌的肿瘤微环境和对免疫治疗反应，仍然是未知的。在这项最新研究中，研究团队利用肺癌皮下移植小鼠模型、肺癌原位小鼠模型和基因工程小鼠模型来表征METTL3对肺癌肿瘤微环境的调控和对免疫疗法的反应。研究团队发现，在非小细胞肺癌中，METTL3通过提高促肿瘤趋化因子来招募免疫抑制细胞，并以m6A依赖的方式降低PD-L1表达，从而形成冷肿瘤的肿瘤微环境。而抑制METTL3，能够将冷肿瘤免疫微环境重编程为热肿瘤状态，从而使NSCLC对PD-1免疫疗法重新变得敏感。具体来说，METTL3提高了促癌趋化因子（包括CXCL1、CXCL5和CCL20）的表达，并以m6A依赖的方式破坏PD-L1的mRNA，从而形成一个冷肿瘤微环境。而使用STM2457（METTL3的特异性小分子抑制剂）抑制METTL3蛋白，或使用shRNA降低METTL3表达水平，均能够将冷肿瘤免疫微环境重编程为热肿瘤状态，使抗PD-1免疫治疗在多种非小细胞肺癌小鼠模型中更有效。此外，该研究还发现，临床上表现出低METTL3表达的NSCLC患者接受抗PD-1治疗的预后更好。总的来说，这项研究强调了靶向METTL3可作为改善非小细胞肺癌这种冷肿瘤的免疫治疗效果的有前途的新策略。武汉大学中南医院张金方教授、哈佛大学医学院魏文毅教授及Gordon Freeman教授为论文共同通讯作者，张金方教授课题组博士后俞海生，哈佛大学 Liu Jing、Bai Xia，解放军总医院第五医学中心马志强为论文第一作者。

免疫疗法

2023-08-09

·PRNewswire

New Journal of Pharmaceutical Analysis Articles Showcase Promising Therapeutic Candidates for Treating Cancer and Hearing Loss

Researchers report innovative techniques to treat challenging cancers and manage the side-effects of therapy XI'AN, China, Aug. 9, 2023 /PRNewswire/ -- Although cancer treatment has advanced significantly over the last decade, some fairly common cancers remain extremely difficult to treat. Besides, some chemotherapeutics or anti-cancer drugs can have serious, irreversible side-effects. The latest issue of the JPA features three articles that highlight novel treatment strategies based on cutting-edge research on cancer and chemotherapeutic drug-induced adverse events. Continue Reading Highlights of the Journal of Pharmaceutical Analysis June 2023 issue The first study reports a new approach to prevent hearing loss induced by the widely used chemotherapeutic drug "cisplatin". The article was available online in April 2023 and published in Volume 13, Issue 6 in June 2023. The researchers focused on an inhibitor of protein arginine methyltransferase 5 (PRMT5), a histone-modifying enzyme and epigenetic regulator. In vitro and in vivo experiments revealed that inhibition of PRMT5 alleviated cisplatin-induced mitochondrial dysfunction and apoptosis, and thus ameliorated hearing loss by protecting cochlear hair cells and spiral ganglion neurons. A better understanding of the role of PRMT5 in hearing loss could help scientists develop effective hearing protection drugs and prevent this unwanted side-effect. In this regard, corresponding author Yingzi He states that "Our findings suggest that PRMT5 inhibitors have strong potential as agents against cisplatin-induced ototoxicity and can lay the foundation for further research on treatment strategies for hearing loss." In the second study, scientists addressed the problem of inconsistent kinetics of multiple drugs given in a combination therapy. They developed a super-assembled nano-pill that can encapsulate and deliver two different drugs to liver cancer cells. They engineered a supramolecular assembly combining an exportin 1 (XPO1)-inhibiting peptide and a small-molecule inhibitor of ataxia telangiectasia mutated-Rad3-related (ATR). These compounds act synergistically in liver tumors by promoting DNA damage, preventing DNA repair, and arresting the cell cycle. The nano-pills were highly biocompatible with a remarkable therapeutic effect. "This work not only provides a co-delivery strategy for intensive liver cancer treatment with potential for clinical translation, but also extends the scope of drugs for developing advanced combination therapies," highlights the corresponding author Suxia Han. Finally, the third article by Han Xiao et al. presents a comprehensive analysis of the compound STM2457, the novel small-molecule inhibitor of methyltransferase-like 3 (METTL3), which has been reported to promote the disease progression in non-small-cell lung cancer (NSCLC). The authors used a combination of experimental and bioinformatic data to show that STM2457 could significantly inhibit pulmonary colonization and halt NSCLC progression. "The idea of targeting METTL3 is like a "black swan", and its silhouette across the sky was first captured by leukemia and pharmaceutical experts, who synthesized STM2457, the first bioavailable small-molecule inhibitor of METTL3. The cheer of the "black swan" discovery inspired our follow-up work. In the future, targeting the components of translation machinery will become a new therapeutic strategy for NSCLC," states the corresponding author Han Xiao. These studies will pave the way to safer and more effective treatment options for difficult cancers and help improve the quality of life of cancer patients. Reference Authors: Zhiwei Zheng1, Benyu Nan2, Chang Liu3, Dongmei Tang1, Wen Li1, Liping Zhao1, Guohui Nie4, and Yingzi He1 Title of original paper: Inhibition of histone methyltransferase PRMT5 attenuates cisplatin-induced hearing loss through the PI3K/Akt-mediated mitochondrial apoptotic pathway Journal: Journal of Pharmaceutical Analysis DOI: Contact: Mengjie Wang +86-(0)29-82657423 [email protected].com SOURCE Journal of Pharmaceutical Analysis

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适应症	最高研发状态	国家/地区	公司	日期
非小细胞肺癌	临床前	中国	华中科技大学	2023-06-01
冠状病毒感染	临床前	美国	STORM Therapeutics Ltd.初创企业	-
冠状病毒感染	临床前	美国	NYU Langone Medical Center	-
冠状病毒感染	临床前	英国	NYU Langone Medical Center	-