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Discovery of a novel selective and cell-active N6-methyladenosine RNA demethylase ALKBH5 inhibitor

ABSTRACT: 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.

ORGANISM(S): Homo sapiens

PROVIDER: GSE255973 | GEO | 2025/07/19

REPOSITORIES: GEO

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BRD4 regulates N6-methyladenosine modification of ESPL1 mRNA via interaction with RNA demethylase ALKBH5 to modulate breast cancer progression

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

ALKBH5 is a New Mammalian RNA Demethylase

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

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Project description:Since m6A demethylases (FTO and ALKBH5) have been reported to be involved in pre-mRNA splicing regulation, we hypothesized that dynamic m6A distribution during mRNA maturation might involve removal of m6A in internal exons by FTO or ALKBH5 accompanied by splicing factors. To explore this we performed pull-down assays coupled with protein mass spectrometry.

2022-12-09 | PXD038705 | Pride

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Mapping the targeting transcripts and binding sites of ALKBH5 by individual-nucleotide resolution UV crosslinking and immunoprecipitation-based sequencing (iCLIP-seq)

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Transcriptome-wide m6A Methylation Profiling of Wild Type and ALKBH5-/- Peritoneal Macrophages by m6A-seq

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2019-08-22 | GSE127732 | GEO

ALKBH5 Induces Fibroblast-to-Myofibroblast Transformation During Hypoxia to Protect Against Cardiac Rupture After Myocardial Infarction

Project description:Objectives: To investigate the role of ALKBH5 in fibroblasts during post-myocardial infarction (MI) repair. Background: N6-methyladenosine (m6A) mRNA modification has been shown to play an important role in cardiovascular diseases. The RNA demethylase, AlkB homolog 5 (ALKBH5), is an m6A "eraser" that is responsible for decreased m6A methylation. However, its role in cardiac fibroblasts during the post-MI healing process remains elusive. Methods: MI was mimicked by permanent left anterior descending artery ligation in global ALKBH5-knockout, ALKBH5-knockin, and fibroblast-specific ALKBH5-knockout mice to study the function of ALKBH5 during post-MI collagen repair. Methylated RNA immunoprecipitation sequencing was performed to explore potential ALKBH5 targets. Results: Dramatic alterations in ALKBH5 expression were observed during the early stage post-MI and in hypoxic fibroblasts. Global ALKBH5 knockin reduced infarct size and improved cardiac function after MI. The global and fibroblast-specific ALKBH5-knockout mice both exhibited low survival rates along with poor collagen repair, impaired cardiac function, and cardiac rupture. Both in vivo and in vitro ALKBH5 loss led to impaired fibroblast activation and decreased collagen deposition. Additionally, hypoxia, but not TGF-β1 or Ang II, upregulated ALKBH5 expression in myofibroblasts in a HIF-1α-dependent transcriptional manner. Mechanistically, ALKBH5 promoted the stability of ErbB4 mRNA and the degradation of ST14 mRNA via m6A demethylation. Fibroblast-specific ErbB4 overexpression ameliorated the impaired fibroblast-to-myofibroblast transformation and poor post-MI repair due to ALKBH5 knockout. Conclusions: Fibroblast ALKBH5 positively regulates post-MI healing via post-transcriptional modification and stabilization of ErbB4 mRNA in an m6A-dependent manner. Targeting ALKBH5/ERBB4 may be a potential therapeutic option for post-MI cardiac rupture.

2023-10-01 | GSE226415 | GEO

Sequencing of mRNA with N6-methyladenosine (m6A) modification in AML cells upon ALKBH5 knockdown or overexpression

Project description:To identify the potential mRNA targets of ALKBH5, we conducted m6A-seq with mRNA samples enriched from AML cells with ALKBH5 knockdown or overexpression

2020-05-26 | GSE134763 | GEO

m6A Demethylase ALKBH5 attenuates doxorubicin-induced cardiotoxicity via posttranscriptional stabilization of Rasal3

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

ALKBH5 facilitates the progression of skin cutaneous melanoma through promoting ABCA1 demethylation and inhibiting autophagy in an m6A-dependent manner

Project description:Background: N6-methyladenosine (m6A) is the most common and abundant mRNA modification, playing an essential role in biological processes and tumor development. However, the role of m6A methylation in skin cutaneous melanoma (SKCM) is not yet clear. This study analyzed the expression of m6A-related functional genes in SKCM and aimed to explore the key demethylase ALKBH5 mediated m6A modification and its potential mechanism in human SKCM. Methods: Based on public databases, the m6A-related gene expression landscape in SKCM was portrayed. MeRIP-Seq and RNA-Seq were used to recognize the downstream target of ALKBH5. In vivo and in vitro functional phenotype and rescue functional experiments were performed to explore the mechanism of the ALKBH5-ABCA1 axis in SKCM. Results: We found ALKBH5 upregulated in SKCM, associated with poor prognosis. ALKBH5 can promote melanoma cell proliferation, colony formation, migration, and invasion and inhibit autophagy in vitro, facilitating tumor growth and metastasis in vivo. We identified ABCA1, a membrane protein that assists cholesterol efflux, as a downstream target of ALKBH5-mediated m6A demethylation. Finally, our data demonstrated that ALKBH5 promoted SKCM via mediating ABCA1 downregulation by reducing ABCA1 mRNA stability in an m6A-dependent manner. Conclusion: Our findings exhibited the functional value of the key demethylase ALKBH5 mediated m6A modification in the progression of SKCM, suggesting ALKBH5 and its target ABCA1 as potential therapeutic targets in SKCM.

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