Targeting METTL14 and RNA methylation to treat osteosarcoma.

Abstract

Osteosarcoma (OS) is one of the most common and aggressive primary bone malignancies in both adults and children. During the past 4 decades, the substantial improvements in surgery and neoadjuvant chemotherapy have significantly increased the survival rate of OS patients. However, with currently available treatment regimens, more than 80% of the OS patients with metastasis and/or relapse do not survive over 5 years,1Gill J Gorlick R. Advancing therapy for osteosarcoma.Nat Rev Clin Oncol. 2021; 18: 609-624https://doi.org/10.1038/s41571-021-00519-8Crossref PubMed Scopus (81) Google Scholar which underscores the unmet medical need to discover novel effective therapeutic target(s). The poor understanding of cellular and molecular mechanisms during osteosarcomagenesis has seriously prevented the development of new treatment approaches.In eukaryotes, the gene expression is strictly regulated at multiple layers, including genetics, epigenetics, and epitranscriptomics, to maintain the normal bioprocess, while dysregulation of gene expression may lead to pathogenesis and tumorigenesis. The covalent chemical modifications in RNA have been revealed since 1950s and emerged as a crucial layer to manipulate gene expression at post-transcriptional level. To date, although more than 170 distinct chemical marks have been identified in RNAs, N6-methyladenosine (m6A), methylated adenosine at the N6 position, is the most abundant internal modification in messenger RNA (mRNA). Since the initial discovery in 1974, the studies focusing on m6A modification have been stuck for several decades due to the lack of quantification and sequencing approaches. Until 2011, identification of the first m6A demethylase, fat mass and obesity-associated protein (FTO), indicates that m6A is dynamic and reversable and resurged the broad interest in RNA modifications. The m6A is always enriched in the RRACH (R=A or G; H = A, C, or U) context around stop codon region, and its machinery includes “writers”, “erasers” and “readers” that deposit, remove, and recognize it, respectively.2Shi H Wei J He C. Where, when, and how: context-dependentfundtions of RNA methylation writers, readers, and erasers.Mol Cell. 2019; 16: 640-650https://doi.org/10.1016/j.molcel.2019.04.025Summary Full Text Full Text PDF Scopus (573) Google Scholar Specifically, the m6A methylation is co-transcriptionally installed in mRNA by a multicomponent complex composed of a stable core subunit, the methyltransferase-like 3 (METTL3)/METTL14 heterodimer, and additional regulators. The m6A modification can be reversed by a-ketoglutarate (a-KG)-dependent and Fe(II)-dependent demethylases, FTO and AlkB homolog 5 (ALKBH5). The characterization of m6A readers, such as YT521-B homology (YTH) domain family 1-3 (YTHDF1-3), YTH domain containing 1-2 (YTHDC1-2), and insulin-like growth factor 2 mRNA-binding proteins 1-3 (IGF2BP1-3), has provided insightful knowledge into the underlying mechanisms of m6A-mediated gene expression regulation, including nuclear export, mRNA stability, alternative splicing, and translation efficiency. Therefore, m6A represents the best characterized RNA modification thus far. Recently, accumulating evidence have suggested that both the m6A abundance and its machinery are usually dysregulated in various cancers and such dysregulation is responsible for tumor initiation, progression, drug resistance, metastasis, and refractory.3Weng H Huang H Wu H et al.METTL14 inhibits hematopoietic stem/progenitor differentiation and promotes leukemogenesis via mRNA m6A Modification.Cell Stem Cell. 2018; 191: 191-205.e9https://doi.org/10.1016/j.stem.2017.11.016Summary Full Text Full Text PDF Scopus (527) Google Scholar More exciting, the novel small compounds targeting m6A machinery have been developed and utilized to treat tumors, especially leukemia.4Su R Dong L Li Y et al.Targeting FTO suppresses cancer stem cell maintenance and immune evasion.Cancer Cell. 2020; 38 (79-96.e11): 79-96.e11https://doi.org/10.1016/j.ccell.2020.04.017Summary Full Text Full Text PDF PubMed Scopus (205) Google Scholar However, the biological functions and molecular mechanisms of m6A and its regulators remain poorly understood in OS. It's still elusive about the role of m6A machinery during osteosarcomagenesis.In a recent issue of eBioMedicine, Li and colleagues determined the global m6A abundance in OS tumors, systemically revealed the crucial tumor-promoting role of METTL14 during OS progression and metastasis, and highlighted that METTL14 might act as a promising therapeutic target for OS treatment.5Li Hong-Bo Huang Gang Tu Jian et al.METTL14-mediated epitranscriptome modification of MN1 mRNA promote tumorigenicity and all-trans-retinoic acid resistance in osteosarcoma.EBiomedicine. 2022; 82104142Summary Full Text Full Text PDF Scopus (2) Google Scholar Here, the authors observed that the overall m6A abundance was significantly up-regulated in OS tumors in contrast to their corresponding controls, and such increased m6A modification in mRNA was mainly attributed to the robust upregulation of METTL14, but not other m6A regulators. Furthermore, the higher expression of METTL14 predicted the more adverse survival of OS patients. Via both in vitro and in vivo studies, the authors demonstrated that METTL14 played a critical oncogenic role in OS via facilitating cell growth/proliferation, promoting lung metastasis, and maintaining the stemness of cancer stem cells (CSCs). To further clarify the underlying cellular/molecular mechanism, they conducted transcriptome-wide m6A-modified RNA immunoprecipitation sequencing (MeRIP-seq) and RNA sequencing (RNA-seq) in OS cells upon METTL14 depletion, demonstrated Meningioma 1 (MN1) as a functionally essential and m6A-dependent downstream target of METTL14. Mechanistically, METTL14 could directly install m6A modification in MN1 mRNA, and IGF2BP2 specifically recognized the m6A marks and potentiated the mRNA stability and translation efficiency of MN1 in OS. Additionally, previous studies have supported that forced expression of MN1 conferred resistance to all-trans-retinoic acid (ATRA)-mediated anti-tumor activity in acute promyelocytic leukemia (APL),6Lo-Coco F Avvisati G Vignetti M et al.Retinoic acid and arsenic trioxide for acute promyelocytic leukemia.N Engl J Med. 2013; 369: 111-121https://doi.org/10.1056/NEJMoa1300874Crossref PubMed Scopus (970) Google Scholar while it's still underdetermined whether such effect is involved in OS. In this study, the authors also demonstrated that METTL14/m6A/IGF2BP2 axis-induced upregulation of MN could lead to the chemotherapy resistance to ATRA in OS, and suggested the potent therapeutic efficiency of ATRA in treating OS patients with endogenous low expression of MN1.Overall, this proof-of-concept article has comprehensively investigated the oncogenic role of m6A writer METTL14, elucidated its molecular mechanism via targeting MN1, and indicated that METTL14 might function as a prognostic predictor and a promising therapeutic target for OS patients. For further bench-to-bedside research, there are still a series of fundamental and clinical questions to be answered: (1) Given that several previous studies have reported the opposite (tumor-suppressor) role of METTL14 in OS,7Li Z Liu N Huang Z Wang W. METTL14 overexpression promotes osteosarcoma cell apoptosis and slows tumor progression via caspase 3 activation.Caner Manag Res. 2020; 12: 12759-12767https://doi.org/10.2147/CMAR.S284273Crossref Scopus (14) Google Scholar,8Li J Rao B Yang J et al.Dysregulated m6A-related regulators are associated with tumor metastasis and poor prognosis in osteosarcoma.Front Oncol. 2020; 10769https://doi.org/10.3389/fonc.2020.00769Crossref Scopus (39) Google Scholar further studies will be warranted to elucidate the divergent functions of m6A modification and its writer in distinct OS subtypes; (2) It's also necessary to evaluate whether METTL14 is required for the survival and growth of normal human osteoblasts; (3) ATRA is not the standard first-line chemotherapy agent to treat OS and the IC50 values of ATRA in killing OS cells are pretty high (> 50 μM), indicating ATRA might be not the ideal drug for OS therapy. It will be of significance to assess whether genetic depletion and/or pharmacological inhibition of METTL14 can sensitize OS cells to standard chemotherapy, including doxorubicin, cisplatin, and methotrexate.ContributorsLiterature search: R.L. and X.L.; Data collection R.L., X.L., and R.S.; Data interpretation: X.L. and R.S.; Writing: R.L., X.L., and R.S. All authors read and approved the final manuscript. Osteosarcoma (OS) is one of the most common and aggressive primary bone malignancies in both adults and children. During the past 4 decades, the substantial improvements in surgery and neoadjuvant chemotherapy have significantly increased the survival rate of OS patients. However, with currently available treatment regimens, more than 80% of the OS patients with metastasis and/or relapse do not survive over 5 years,1Gill J Gorlick R. Advancing therapy for osteosarcoma.Nat Rev Clin Oncol. 2021; 18: 609-624https://doi.org/10.1038/s41571-021-00519-8Crossref PubMed Scopus (81) Google Scholar which underscores the unmet medical need to discover novel effective therapeutic target(s). The poor understanding of cellular and molecular mechanisms during osteosarcomagenesis has seriously prevented the development of new treatment approaches. In eukaryotes, the gene expression is strictly regulated at multiple layers, including genetics, epigenetics, and epitranscriptomics, to maintain the normal bioprocess, while dysregulation of gene expression may lead to pathogenesis and tumorigenesis. The covalent chemical modifications in RNA have been revealed since 1950s and emerged as a crucial layer to manipulate gene expression at post-transcriptional level. To date, although more than 170 distinct chemical marks have been identified in RNAs, N6-methyladenosine (m6A), methylated adenosine at the N6 position, is the most abundant internal modification in messenger RNA (mRNA). Since the initial discovery in 1974, the studies focusing on m6A modification have been stuck for several decades due to the lack of quantification and sequencing approaches. Until 2011, identification of the first m6A demethylase, fat mass and obesity-associated protein (FTO), indicates that m6A is dynamic and reversable and resurged the broad interest in RNA modifications. The m6A is always enriched in the RRACH (R=A or G; H = A, C, or U) context around stop codon region, and its machinery includes “writers”, “erasers” and “readers” that deposit, remove, and recognize it, respectively.2Shi H Wei J He C. Where, when, and how: context-dependentfundtions of RNA methylation writers, readers, and erasers.Mol Cell. 2019; 16: 640-650https://doi.org/10.1016/j.molcel.2019.04.025Summary Full Text Full Text PDF Scopus (573) Google Scholar Specifically, the m6A methylation is co-transcriptionally installed in mRNA by a multicomponent complex composed of a stable core subunit, the methyltransferase-like 3 (METTL3)/METTL14 heterodimer, and additional regulators. The m6A modification can be reversed by a-ketoglutarate (a-KG)-dependent and Fe(II)-dependent demethylases, FTO and AlkB homolog 5 (ALKBH5). The characterization of m6A readers, such as YT521-B homology (YTH) domain family 1-3 (YTHDF1-3), YTH domain containing 1-2 (YTHDC1-2), and insulin-like growth factor 2 mRNA-binding proteins 1-3 (IGF2BP1-3), has provided insightful knowledge into the underlying mechanisms of m6A-mediated gene expression regulation, including nuclear export, mRNA stability, alternative splicing, and translation efficiency. Therefore, m6A represents the best characterized RNA modification thus far. Recently, accumulating evidence have suggested that both the m6A abundance and its machinery are usually dysregulated in various cancers and such dysregulation is responsible for tumor initiation, progression, drug resistance, metastasis, and refractory.3Weng H Huang H Wu H et al.METTL14 inhibits hematopoietic stem/progenitor differentiation and promotes leukemogenesis via mRNA m6A Modification.Cell Stem Cell. 2018; 191: 191-205.e9https://doi.org/10.1016/j.stem.2017.11.016Summary Full Text Full Text PDF Scopus (527) Google Scholar More exciting, the novel small compounds targeting m6A machinery have been developed and utilized to treat tumors, especially leukemia.4Su R Dong L Li Y et al.Targeting FTO suppresses cancer stem cell maintenance and immune evasion.Cancer Cell. 2020; 38 (79-96.e11): 79-96.e11https://doi.org/10.1016/j.ccell.2020.04.017Summary Full Text Full Text PDF PubMed Scopus (205) Google Scholar However, the biological functions and molecular mechanisms of m6A and its regulators remain poorly understood in OS. It's still elusive about the role of m6A machinery during osteosarcomagenesis. In a recent issue of eBioMedicine, Li and colleagues determined the global m6A abundance in OS tumors, systemically revealed the crucial tumor-promoting role of METTL14 during OS progression and metastasis, and highlighted that METTL14 might act as a promising therapeutic target for OS treatment.5Li Hong-Bo Huang Gang Tu Jian et al.METTL14-mediated epitranscriptome modification of MN1 mRNA promote tumorigenicity and all-trans-retinoic acid resistance in osteosarcoma.EBiomedicine. 2022; 82104142Summary Full Text Full Text PDF Scopus (2) Google Scholar Here, the authors observed that the overall m6A abundance was significantly up-regulated in OS tumors in contrast to their corresponding controls, and such increased m6A modification in mRNA was mainly attributed to the robust upregulation of METTL14, but not other m6A regulators. Furthermore, the higher expression of METTL14 predicted the more adverse survival of OS patients. Via both in vitro and in vivo studies, the authors demonstrated that METTL14 played a critical oncogenic role in OS via facilitating cell growth/proliferation, promoting lung metastasis, and maintaining the stemness of cancer stem cells (CSCs). To further clarify the underlying cellular/molecular mechanism, they conducted transcriptome-wide m6A-modified RNA immunoprecipitation sequencing (MeRIP-seq) and RNA sequencing (RNA-seq) in OS cells upon METTL14 depletion, demonstrated Meningioma 1 (MN1) as a functionally essential and m6A-dependent downstream target of METTL14. Mechanistically, METTL14 could directly install m6A modification in MN1 mRNA, and IGF2BP2 specifically recognized the m6A marks and potentiated the mRNA stability and translation efficiency of MN1 in OS. Additionally, previous studies have supported that forced expression of MN1 conferred resistance to all-trans-retinoic acid (ATRA)-mediated anti-tumor activity in acute promyelocytic leukemia (APL),6Lo-Coco F Avvisati G Vignetti M et al.Retinoic acid and arsenic trioxide for acute promyelocytic leukemia.N Engl J Med. 2013; 369: 111-121https://doi.org/10.1056/NEJMoa1300874Crossref PubMed Scopus (970) Google Scholar while it's still underdetermined whether such effect is involved in OS. In this study, the authors also demonstrated that METTL14/m6A/IGF2BP2 axis-induced upregulation of MN could lead to the chemotherapy resistance to ATRA in OS, and suggested the potent therapeutic efficiency of ATRA in treating OS patients with endogenous low expression of MN1. Overall, this proof-of-concept article has comprehensively investigated the oncogenic role of m6A writer METTL14, elucidated its molecular mechanism via targeting MN1, and indicated that METTL14 might function as a prognostic predictor and a promising therapeutic target for OS patients. For further bench-to-bedside research, there are still a series of fundamental and clinical questions to be answered: (1) Given that several previous studies have reported the opposite (tumor-suppressor) role of METTL14 in OS,7Li Z Liu N Huang Z Wang W. METTL14 overexpression promotes osteosarcoma cell apoptosis and slows tumor progression via caspase 3 activation.Caner Manag Res. 2020; 12: 12759-12767https://doi.org/10.2147/CMAR.S284273Crossref Scopus (14) Google Scholar,8Li J Rao B Yang J et al.Dysregulated m6A-related regulators are associated with tumor metastasis and poor prognosis in osteosarcoma.Front Oncol. 2020; 10769https://doi.org/10.3389/fonc.2020.00769Crossref Scopus (39) Google Scholar further studies will be warranted to elucidate the divergent functions of m6A modification and its writer in distinct OS subtypes; (2) It's also necessary to evaluate whether METTL14 is required for the survival and growth of normal human osteoblasts; (3) ATRA is not the standard first-line chemotherapy agent to treat OS and the IC50 values of ATRA in killing OS cells are pretty high (> 50 μM), indicating ATRA might be not the ideal drug for OS therapy. It will be of significance to assess whether genetic depletion and/or pharmacological inhibition of METTL14 can sensitize OS cells to standard chemotherapy, including doxorubicin, cisplatin, and methotrexate. ContributorsLiterature search: R.L. and X.L.; Data collection R.L., X.L., and R.S.; Data interpretation: X.L. and R.S.; Writing: R.L., X.L., and R.S. All authors read and approved the final manuscript. Literature search: R.L. and X.L.; Data collection R.L., X.L., and R.S.; Data interpretation: X.L. and R.S.; Writing: R.L., X.L., and R.S. All authors read and approved the final manuscript. The authors declare no conflict of interest. This study was supported by the HMU Marshal Initiative Funding (HMUMIF-21001, X.L.), Leukemia Research Foundation New Investigator Research Grant (R. S.), The Margaret E. Early Medical Research Trust (R.S.), and American Association for the Study of Liver Diseases (AASLD) Foundation PNC22-261362 (R.S.). METTL14-mediated epitranscriptome modification of MN1 mRNA promote tumorigenicity and all-trans-retinoic acid resistance in osteosarcomaOur study revealed that METTL14 contributes to OS progression and ATRA resistance as an m6A RNA methylase by regulating the stability and translation efficiency of MN1 and thus provides both an underlying biomarker panel for prognosis prediction in OS patients. Full-Text PDF Open Access