N6-Methyladenosine modification mediated by METTL3 promotes DNA-PKcs expression to induce anlotinib resistance in osteosarcoma

IF 6.8 1区医学 Q1 MEDICINE, RESEARCH & EXPERIMENTAL Clinical and Translational Medicine Pub Date : 2025-02-09 DOI:10.1002/ctm2.70228

Yining Zhang, Guohong Shen, Dan Zhang, Tingting Meng, Zhaorui Lv, Lei Chen, Jianmin Li, Ka Li

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Abstract

Background

Acquired anlotinib resistance is still a key challenge in osteosarcoma treatment. Unravelling the mechanisms underlying anlotinib resistance is the key to optimising its efficacy for treating osteosarcoma. Previous studies have explored the pivotal function of the DNA-dependent protein kinase catalytic subunit (DNA-PKcs) with regard to osteosarcoma chemoresistance.

Methods

We used bioinformatics analysis to predict DNA-PKcs and Beclin-1 interactions, confirmed through immunofluorescence (IF) and co-immunoprecipitation (co-IP). Dual-luciferase analyses and Methylated RNA immunoprecipitation (MeRIP) were implemented to detect the detected m⁶A modifications. RNA fluorescence in situ hybridisation (FISH)—IF co-localisation and RNA immunoprecipitation (RIP) were conducted to explore the interplay between PRKDC mRNA and the indicated proteins.

Results

Anlotinib-treated osteosarcoma cells exhibited increased DNA-PKcs levels, and silencing DNA-PKcs augmented osteosarcoma sensitivity to anlotinib. DNA-PKcs affects anlotinib-induced autophagy by interacting with Beclin-1 and regulating its ubiquitination. Notably, PRKDC mRNA, encoding DNA-PKcs, underwent N⁶-Methyladenosine (m⁶A) modification. Methyltransferase-like 3 (METTL3) positively regulated DNA-PKcs expression. Functionally, METTL3 enhances anlotinib resistance in osteosarcoma, which is reversed by PRKDC knockdown. Mechanistically, METTL3 binds to PRKDC mRNA and facilitates m⁶A methylation. Additionally, m⁶A methylated PRKDC mRNA is identified via YTH N⁶-methyladenosine RNA-binding protein 1 (YTHDF1), augmenting its expression.

Conclusion

These findings revealed that DNA-PKcs promotes anlotinib resistance by regulating protective autophagy, while METTL3 induces PRKDC m⁶A modification, enhancing its expression. Thus, targeting METTL3/PRKDC may be a novel strategy for improving therapeutic efficacy in human osteosarcoma.

Key points

DNA-PKcs knockdown heightens osteosarcoma sensitivity to anlotinib.
DNA-PKcs modulates anlotinib-induced protective autophagy through interacts with Beclin-1 and regulates its ubiquitination.
m⁶A modification of OLE_LINK82PRKDC mRNA induced by METTL3 contributes to anlotinib resistance in osteosarcoma.
m⁶A methylation of PRKDC mRNA recognised by YTHDF1 amplifies the expression of DNA-PKcs.

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METTL3介导的n6 -甲基腺苷修饰促进DNA-PKcs表达，诱导骨肉瘤耐安洛替尼

背景获得性anlotinib耐药仍然是骨肉瘤治疗的关键挑战。揭示anlotinib耐药机制是优化其治疗骨肉瘤疗效的关键。先前的研究已经探索了dna依赖性蛋白激酶催化亚基（DNA-PKcs）在骨肉瘤化疗耐药中的关键功能。方法采用生物信息学分析预测DNA-PKcs和Beclin-1的相互作用，并通过免疫荧光（IF）和共免疫沉淀（co-IP）证实。采用双荧光素酶分析和甲基化RNA免疫沉淀（MeRIP）检测检测到的m6A修饰。通过RNA荧光原位杂交(FISH) -IF共定位和RNA免疫沉淀（RIP）来探索PRKDC mRNA与指示蛋白之间的相互作用。结果经安洛替尼处理的骨肉瘤细胞DNA-PKcs水平升高，DNA-PKcs沉默增强了骨肉瘤对安洛替尼的敏感性。DNA-PKcs通过与Beclin-1相互作用并调控其泛素化，影响anlotinib诱导的自噬。值得注意的是，编码DNA-PKcs的PRKDC mRNA经历了n6 -甲基腺苷（m6A）修饰。甲基转移酶样3 （METTL3）正调控DNA-PKcs的表达。功能上，METTL3增强骨肉瘤的anlotinib耐药，这可通过PRKDC敲低逆转。从机制上讲，METTL3与PRKDC mRNA结合并促进m6A甲基化。此外，通过YTH n6 -甲基腺苷rna结合蛋白1 （YTHDF1）鉴定m6A甲基化的PRKDC mRNA，增加其表达。结论DNA-PKcs通过调节保护性自噬促进anlotinib耐药，而METTL3诱导PRKDC m6A修饰，增强其表达。因此，靶向METTL3/PRKDC可能是提高人骨肉瘤治疗效果的新策略。DNA-PKcs敲低可提高骨肉瘤对安洛替尼的敏感性。DNA-PKcs通过与Beclin-1相互作用调控anlotinib诱导的保护性自噬，并调控其泛素化。METTL3诱导OLE_LINK82PRKDC mRNA的m6A修饰有助于骨肉瘤对安洛替尼的耐药。YTHDF1识别的PRKDC mRNA的m6A甲基化可扩增DNA-PKcs的表达。

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来源期刊

Clinical and Translational Medicine Multiple-

CiteScore

15.90

自引率

1.90%

发文量

450

审稿时长

4 weeks

期刊介绍： Clinical and Translational Medicine (CTM) is an international, peer-reviewed, open-access journal dedicated to accelerating the translation of preclinical research into clinical applications and fostering communication between basic and clinical scientists. It highlights the clinical potential and application of various fields including biotechnologies, biomaterials, bioengineering, biomarkers, molecular medicine, omics science, bioinformatics, immunology, molecular imaging, drug discovery, regulation, and health policy. With a focus on the bench-to-bedside approach, CTM prioritizes studies and clinical observations that generate hypotheses relevant to patients and diseases, guiding investigations in cellular and molecular medicine. The journal encourages submissions from clinicians, researchers, policymakers, and industry professionals.