N(6)-methyladenosine (m(6)A) is a modified base that has long been known to be present in non-coding RNAs, ribosomal RNA, polyadenylated RNA and at least one mammalian mRNA. However, our understanding of the prevalence of this modification has been fundamentally redefined by transcriptome-wide m(6)A mapping studies, which have shown that m(6)A is present in a large subset of the transcriptome in specific regions of mRNA. This suggests that mRNA may undergo post-transcriptional methylation to regulate its fate and function, which is analogous to methyl modifications in DNA. Thus, the pattern of methylation constitutes an mRNA 'epitranscriptome'. The identification of adenosine methyltransferases ('writers'), m(6)A demethylating enzymes ('erasers') and m(6)A-binding proteins ('readers') is helping to define cellular pathways for the post-transcriptional regulation of mRNAs.

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The dynamic epitranscriptome: N6-methyladenosine and gene expression control

N6-methyladenosine (m6A), the most abundant internal modification in eukaryotic messenger RNAs (mRNAs), has been shown to play critical roles in various normal bioprocesses such as tissue development, stem cell self-renewal and differentiation, heat shock or DNA damage response, and maternal-to-zygotic transition. The m6A modification is deposited by the m6A methyltransferase complex (MTC; i.e., writer) composed of METTL3, METTL14 and WTAP, and probably also VIRMA and RBM15, and can be removed by m6A demethylases (i.e., erasers) such as FTO and ALKBH5. The fates of m6A-modified mRNAs rely on the functions of distinct proteins that recognize them (i.e., readers), which may affect the stability, splicing, and/or translation of target mRNAs. Given the functional importance of the m6A modification machinery in normal bioprocesses, it is not surprising that evidence is emerging that dysregulation of m6A modification and the associated proteins also contributes to the initiation, progression, and drug response of cancers. In this review, we focus on recent advances in the study of biological functions and the underlying molecular mechanisms of dysregulated m6A modification and the associated machinery in the pathogenesis and drug response of various types of cancers. In addition, we also discuss possible therapeutic interventions against the dysregulated m6A machinery to treat cancers.

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RNA N6-methyladenosine modification in cancers: current status and perspectives

Since the identification of the first RNA demethylase and the establishment of methylated RNA immunoprecipitation-sequencing methodology 6 to 7 years ago, RNA methylation has emerged as a widespread phenomenon and a critical regulator of transcript expression. This new layer of regulation is termed “epitranscriptomics.” The most prevalent RNA methylation, N6-methyladenosine (m6A), occurs in approximately 25% of transcripts at the genome-wide level and is enriched around stop codons, in 5′- and 3′-untranslated regions, and within long internal exons. RNA m6A modification regulates RNA splicing, translocation, stability, and translation into protein. m6A is catalyzed by the RNA methyltransferases METTL3, METTL14, and METTL16 (writers), is removed by the demethylases FTO and ALKBH5 (erasers), and interacts with m6A-binding proteins, such as YTHDF1 and IGF2BP1 (readers). RNA methyltransferases, demethylases, and m6A-binding proteins are frequently upregulated in human cancer tissues from a variety of organ origins, increasing onco-transcript and oncoprotein expression, cancer cell proliferation, survival, tumor initiation, progression, and metastasis. Although RNA methyltransferase inhibitors are not available yet, FTO inhibitors have shown promising anticancer effects in vitro and in animal models of cancer. Further screening for selective and potent RNA methyltransferase, demethylase, or m6A-binding protein inhibitors may lead to compounds suitable for future clinical trials in cancer patients.

https://cancerres.aacrjournals.org/content/canres/79/7/1285.full.pdf

The Critical Role of RNA m6A Methylation in Cancer

Small-Molecule Targeting of Oncogenic FTO Demethylase in Acute Myeloid Leukemia.

Two human demethylases, the fat mass and obesity-associated (FTO) enzyme and ALKBH5, oxidatively demethylate abundant N(6)-methyladenosine (m(6)A) residues in mRNA. Achieving a method for selective inhibition of FTO over ALKBH5 remains a challenge, however. Here, we have identified meclofenamic acid (MA) as a highly selective inhibitor of FTO. MA is a non-steroidal, anti-inflammatory drug that mechanistic studies indicate competes with FTO binding for the m(6)A-containing nucleic acid. The structure of FTO/MA has revealed much about the inhibitory function of FTO. Our newfound understanding, revealed herein, of the part of the nucleotide recognition lid (NRL) in FTO, for example, has helped elucidate the principles behind the selectivity of FTO over ALKBH5. Treatment of HeLa cells with the ethyl ester form of MA (MA2) has led to elevated levels of m(6)A modification in mRNA. Our collective results highlight the development of functional probes of the FTO enzyme that will (i) enable future biological studies and (ii) pave the way for the rational design of potent and specific inhibitors of FTO for use in medicine.

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Meclofenamic acid selectively inhibits FTO demethylation of m6A over ALKBH5

The direct nucleic acid repair dioxygenase FTO is an enzyme that demethylates N6-methyladenosine (m6A) residues in mRNA in vitro and inside cells. FTO is the first RNA demethylase discovered that also serves a major regulatory function in mammals. Together with structure-based virtual screening and biochemical analyses, we report the first identification of several small-molecule inhibitors of human FTO demethylase. The most potent compound, the natural product rhein, which is neither a structural mimic of 2-oxoglutarate nor a chelator of metal ion, competitively binds to the FTO active site in vitro. Rhein also exhibits good inhibitory activity on m6A demethylation inside cells. These studies shed light on the development of powerful probes and new therapies for use in RNA biology and drug discovery.

Development of Cell-Active N6-Methyladenosine RNA Demethylase FTO Inhibitor

Background: Lung cancer has a high incidence and mortality rate worldwide. Vasculogenic mimicry (VM) is a specific modality of tumor angiogenesis that could potentially be a new target for tumor therapy. The purpose of this study was to explore the role of VM-related genes in assessing the prognosis and immune landscape of lung cancer. Methods: VM-related genes were obtained from previous studies, and the expression data and clinical data of lung adenocarcinoma (LUAD) patients were obtained from the TCGA database and GEO database. We performed enrichment analysis of 24 VM-related genes and screened hub genes by constructing a protein–protein interaction network and using Cytoscape software. Subsequently, we developed the VM score based on univariate Cox regression analysis and Lasso analysis and validated the VM score on the GSE72094 dataset. In addition, we constructed a nomogram based on the VM score in the TCGA cohort. Finally, we explored the correlation between the VM score and the tumor microenvironment, immune cell infiltration, immune checkpoints, and drug sensitivity. Results: Enrichment analysis revealed that VM-related genes were associated with the HIF signaling pathway and angiogenic pathway. We developed a VM score based on 3 genes (EPHA2, LAMC2 and LOXL2) in LUAD patients. Kaplan-Meier analysis showed that the VM score was associated with poor prognosis in LUAD patients. The receiver operating characteristic curve suggested that the VM score and nomogram are valid predictors for the overall survival of LUAD patients. The VM score was significantly correlated with immune cell infiltration, such as naïve B cells, neutrophils, and eosinophils, and there was a difference in the TME between the high VM score group and the low VM score group. LUAD patients in the high VM score group were more sensitive to antitumor drugs. Conclusion: In summary, the VM score developed in this study is a valuable indicator for evaluating the prognosis and immune landscape of LUAD patients. VM may be a potential target for antitumor therapy in lung cancer.

Xiaotian Huang

Papers

Development of Cell-Active N6-Methyladenosine RNA Demethylase FTO Inhibitor

Vasculogenic mimicry score identifies the prognosis and immune landscape of lung adenocarcinoma