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

IF 7.9 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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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.