Discovery of a novel selective and cell-active N6-methyladenosine RNA demethylase ALKBH5 inhibitor
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PROVIDER: PRJNA1077332 | ENA |
REPOSITORIES: ENA
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Project description:N6-methyladenosine (m6A), the most abundant methylation on mRNA, plays pivotal roles in regulating mRNA biological functions, resulting in affecting cell functions. ALKBH5, an m6A demethylase, has been found to be an oncogene in several cancer types, including triple negative breast cancer (TNBC). Here we reported a novel ALKBH5 inhibitor. Firstly, we utilized virtual screening to obtain ALKBH5 inhibitor candidates. Biochemical tests demonstrated that compound AG-690/34437009 could inhibit ALKBH5 with an IC50 value of 11.76 μM. After structure optimization, we acquired a more potent ALKBH5-specific inhibitor, compound W23-1006. Its IC50 value against ALKBH5 came down to 3.848 μM, representing a tripled increase in its inhibitory effect on ALKBH5 as well as roughly 30-fold and 8-fold stronger inhibitory activity than that against FTO and ALKBH3, respectively. Furthermore, we proved that W23-1006 formed a covalent bond with ALKBH5 C200. Cellular experiments demonstrated that W23-1006 could efficiently enhance the m6A level on fibronectin 1 (FN1) mRNA, leading to the strong suppression of TNBC cell migration. Moreover, it could significantly suppress TNBC tumor growth and metastasis in vivo. Collectively, our study developed the novel, selective and cell-active ALKBH5 inhibitor, W23-1006, which could be a potential therapeutic option for cancer such as TNBC treatment.
2025-07-19 | GSE255973 | GEO
Project description:N6-methyladenosine (m6A) is the most prevalent internal modification of messenger RNA (mRNA) in higher eukaryotes. Here we report ALKBH5 as a new mammalian demethylase that oxidatively removes the m6A modification in mRNA in vitro and inside cells. This demethylation activity of ALKBH5 significantly affects mRNA export and RNA metabolism as well as the assembly of mRNA processing factors in nuclear speckles. Alkbh5-deficient male mice are characterized by impaired fertility resulting from apoptosis that affects meiotic metaphase-stage spermatocytes. In accordance with this defect, we have identified in mouse testes 1552 differentially expressed genes which cover broad functional categories and include spermatogenesis-related mRNAs involved in the p53 functional interaction network. We show that Alkbh5-deficiency impacts the expression levels of some of these mRNAs, supporting the observed phenotype. The discovery of this new RNA demethylase strongly suggests that the reversible m6A modification plays fundamental and broad functions in mammalian cells. RNA-seq in two cell types
2013-02-15 | E-GEOD-40132 | biostudies-arrayexpress
Project description:N6-methyladenosine (m6A) is important for RNA metabolism including stability and translation of messenger RNA (mRNA). Recent studies indicate that m6A-methylated RNA can modulate dynamic chromatin accessibility and gene transcription, while the potential link of chromatin to mRNA m6A remains largely unknown. We found that targeted inhibition of bromodomain-containing protein 4 (BRD4) by siRNA or a small compound inhibitor (JQ1) significantly decreases mRNA m6A levels and suppresses the malignancy of breast cancer (BC) cells via increased expression of ALKBH5, a demethylase for mRNA methylation. Mechanistically, inhibition of BRD4 increases the mRNA stability of ALKBH5 via enhanced binding between ALKBH5 3’UTR with RNA-binding protein RALY. Further, BRD4 serves as a scaffold for ubiquitin enzymes TRIM21 and ALKBH5, resulting in the ubiquitination and degradation of ALKBH5 protein. JQ1-increased ALKBH5 then demethylates mRNA of ESPL1, a critical oncogenic driver for breast cancer, and reduces binding between ESPL1 mRNA and m6A reader IGF2BP3, leading to decay of ESPL1 mRNA. Animal and clinical studies confirm a critical role of the BRD4/ALKBH5/ESPL1 pathway in BC progression. Overall, our study sheds light on another layer of gene regulation involving crosstalks between histone modification and RNA methylation.
2021-12-11 | GSE190541 | GEO
Project description:N6-methyladenosine (m6A) is the most prevalent internal modification of messenger RNA (mRNA) in higher eukaryotes. Here we report ALKBH5 as a new mammalian demethylase that oxidatively removes the m6A modification in mRNA in vitro and inside cells. This demethylation activity of ALKBH5 significantly affects mRNA export and RNA metabolism as well as the assembly of mRNA processing factors in nuclear speckles. Alkbh5-deficient male mice are characterized by impaired fertility resulting from apoptosis that affects meiotic metaphase-stage spermatocytes. In accordance with this defect, we have identified in mouse testes 1552 differentially expressed genes which cover broad functional categories and include spermatogenesis-related mRNAs involved in the p53 functional interaction network. We show that Alkbh5-deficiency impacts the expression levels of some of these mRNAs, supporting the observed phenotype. The discovery of this new RNA demethylase strongly suggests that the reversible m6A modification plays fundamental and broad functions in mammalian cells.
2013-02-15 | GSE40132 | GEO
Project description:N6-methyladenosine (m6A) is the most abundant internal modification in the messenger RNA (mRNA) of all higher eukaryotes. This modification has been shown to be reversible in mammals; it is installed by a methyltransferase heterodimer complex of METTL3 and METTL14 bound with WTAP, and reversed by iron(II)- and α-ketoglutarate-dependent demethylases FTO and ALKBH5. This modification exhibits significant functional roles in various biological processes. The m6A modification as a RNA mark is recognized by reader proteins, such as YTH domain family proteins and HNRNPA2B1; m6A can also act as a structure switch to affect RNA-protein interactions for biological regulation. In Arabidopsis thaliana, the methyltransferase subunit MTA (the plant orthologue of human METTL3, encoded by At4g10760) was well characterized and FIP37 (the plant orthologue of human WTAP) was first identified as the interacting partner of MTA. Here we report the discovery and characterization of reversible m6A methylation mediated by AtALKBH10B (encoded by At4g02940) in A. thaliana, and noticeable roles of this RNA demethylase in affecting plant development and floral transition. Our findings reveal potential broad functions of reversible mRNA methylation in plants. m6A peaks were identified from wild type Columbia-0 and atalkbh10b-1 mutant in two biological replicates
2016-03-24 | E-GEOD-79523 | biostudies-arrayexpress
Project description:ALKBH5 (AlkB homolog 5) has been identified as a eukaryotic demethylase, which catalyzes N6 adenosine. N6-methyladenosine (m6A) is oxidatively demethylated by ALKBH5 with α-ketoglutarate as a substrate and Fe (II) as a coenzyme. We used microarrays to detail the differential gene expression by ALKBH5 knockdown in lung cancer cell lines.
2022-06-01 | GSE165454 | GEO
Project description:Faithful genome integrity maintenance plays an essential role in cell survival. Here, we identify the RNA demethylase ALKBH5 as a key regulator that protects cells from DNA damage and apoptosis during reactive oxygen species (ROS)-induced stress. We find that ROS significantly induces global mRNA N6-methyladenosine (m6A) levels by modulating ALKBH5 post-translational modifications (PTMs), leading to the rapid and efficient induction of thousands of genes involved in a variety of biological processes including DNA damage repair. Mechanistically, ROS promotes ALKBH5 SUMOylation through activating ERK/JNK signaling, leading to inhibition of ALKBH5 m6A demethylase activity by blocking substrate accessibility. Moreover, ERK/JNK/ALKBH5-PTMs/m6A axis is activated by ROS in hematopoietic stem/progenitor cells (HSPCs) in vivo in mice, suggesting a physiological role of this molecular pathway in the maintenance of genome stability in HSPCs. Together, our study uncovers a molecular mechanism involving ALKBH5 PTMs and increased mRNA m6A levels that protect genomic integrity of cells in response to ROS.
2021-04-29 | GSE144620 | GEO
Project description:ALKBH5 is the RNA N(6)-methyladenosine (m6A) demethylase. To under sthand the function and mechnism of ALKBH5 in human acute myeloid leukemia, we compared the m6A profiling in wild-type, ALKBH5-knock-down, and ALKBH5 rescue THP1 cells.
2020-05-27 | GSE128574 | GEO
Project description:ALKBH5 is the RNA N(6)-methyladenosine (m6A) demethylase. To under sthand the function and mechnism of ALKBH5 in human acute myeloid leukemia, we compared the m6A profiling in wild-type, ALKBH5-knock-down, and ALKBH5 rescue THP1 cells.
2020-05-27 | GSE128520 | GEO
Project description:The clinical application of anthracyclines such as doxorubicin (DOX) is limited due to their cardiotoxicity. N6-methyladenosine (m6A) plays an essential role in numerous biological processes. However, the roles of m6A and m6A demethylase ALKBH5 in DOX-induced cardiotoxicity (DIC) remain unclear. In this research, DIC models were constructed using Alkbh5-knockout (KO), Alkbh5-knockin (KI), and Alkbh5-myocardial-specific knockout (ALKBH5flox/flox, αMyHC-Cre) mice. Cardiac function and DOX-mediated signal transduction were investigated. As a result, both Alkbh5 whole-body KO and myocardial-specific KO mice had increased mortality, decreased cardiac function, and aggravated DIC injury with severe myocardial mitochondrial damage. Conversely, ALKBH5 overexpression alleviated DOX-mediated mitochondrial injury, increased survival, and improved myocardial function. Mechanistically, ALKBH5 regulated the expression of Rasal3 in an m6A-dependent manner through posttranscriptional mRNA regulation and reduced Rasal3 mRNA stability, thus activating RAS3, inhibiting apoptosis through the RAS/RAF/ERK signaling pathway, and alleviating DIC injury. These findings indicate the potential therapeutic effect of ALKBH5 on DIC.
2023-03-08 | GSE224215 | GEO