N6-methyladenosine RNA Modification Research Articles

Single-nucleus multiomic mapping of m6A methylomes and transcriptomes in native populations of cells with sn-m6A-CT

N6-methyladenosine (m6A) RNA modification plays important roles in the governance of gene expression and is temporally regulated in different cell states. In contrast to global m6A profiling in bulk sequencing, single-cell technologies for analyzing m6A heterogeneity are not extensively established. Here, we developed single-nucleus m6A-CUT&Tag (sn-m6A-CT) for simultaneous profiling of m6A methylomes and transcriptomes within a single nucleus using mouse embryonic stem cells (mESCs). m6A-CT is capable of enriching m6A-marked RNA molecules in situ, without isolating RNAs from cells. We adapted m6A-CT to the droplet-based single-cell omics platform and demonstrated high-throughput performance in analyzing nuclei isolated from thousands of cells from various cell types. We show that sn-m6A-CT profiling is sufficient to determine cell identity and allows the generation of cell-type-specific m6A methylome landscapes from heterogeneous populations. These indicate that sn-m6A-CT provides additional dimensions to multimodal datasets and insights into epitranscriptomic landscape in defining cell fate identity and states.

Global hypermethylation of the N6-methyladenosine RNA modification associated with apple heterografting.

Grafting can facilitate better scion performance and is widely used in plants. Numerous studies have studied the involvement of mRNAs, small RNAs, and epigenetic regulations in the grafting process. However, it remains unclear whether the mRNA N6-methyladenosine (m6A) modification participates in the apple (Malus x domestica Borkh.) grafting process. Here, we decoded the landscape of m6A modification profiles in 'Golden delicious' (a cultivar, Gd) and Malus prunifolia 'Fupingqiuzi' (a unique rootstock with resistance to environmental stresses, Mp), as well as their heterografted and self-grafted plants. Interestingly, global hypermethylation of m6A occurred in both heterografted scion and rootstock compared with their self-grafting controls. Gene Ontology (GO) term enrichment analysis showed that grafting-induced differentially m6A-modified genes were mainly involved in RNA processing, epigenetic regulation, stress response, and development. Differentially m6A-modified genes harboring expression alterations were mainly involved in various stress responses and fatty acid metabolism. Furthermore, grafting-induced mobile mRNAs with m6A and gene expression alterations mainly participated in ABA synthesis and transport (e.g. carotenoid cleavage dioxygenase 1 [CCD1] and ATP-binding cassette G22 [ABCG22]) and abiotic and biotic stress responses, which might contribute to the better performance of heterografted plants. Additionally, the DNA methylome analysis also demonstrated the DNA methylation alterations during grafting. Downregulated expression of m6A methyltransferase gene MdMTA (ortholog of METTL3) in apples induced the global m6A hypomethylation and distinctly activated the expression level of DNA demethylase gene MdROS1 (REPRESSOR OF SILENCING 1) showing the possible association between m6A and 5mC methylation in apples. Our results reveal the m6A modification profiles in the apple grafting process and enhance our understanding of the m6A regulatory mechanism in plant biological processes.

ALKBH5 promotes hypopharyngeal squamous cell carcinoma apoptosis by targeting TLR2 in a YTHDF1/IGF2BP2-mediated manner

Hypopharyngeal squamous cell carcinoma (HPSCC) is one of the most aggressive cancers and is notorious for its extremely poor prognosis. However, very few molecular biological studies have been performed. As a novel method of epigenetic gene modulation, N6-methyladenosine (m6A) RNA modification occurs in HPSCC. The expression of the m6A demethylase AlkB homolog 5 (ALKBH5) is frequently downregulated in human HPSCC. Furthermore, we found that ALKBH5 impaired cell proliferation by regulating human Toll-like receptor 2 (TLR2) in an m6A-dependent manner in HPSCC cells. ALKBH5 decreased TLR2 m6A modification, which could be recognized by the m6A readers IGF2BP2 and YTHDF1. IGF2BP2 facilitates TLR2 mRNA stability, whereas YTHDF1 promotes TLR2 mRNA translation. The current work uncovered a critical function of ALKBH5 in TLR2 regulation and provides a novel role for m6A demethylation of mRNA in HPSCC. The inhibition of m6A modification of ALKBH5 in HPSCC deserves further clinical investigation.

Comprehensive analysis of m6A regulators and relationship with tumor microenvironment, immunotherapy strategies in colorectal adenocarcinoma

BackgroundThe N6-methyladenosine (m6A) RNA modification is the most prevalent and abundant type found in eukaryotic cells. It plays a crucial role in the initiation and progression of cancers. In this study, we aimed to comprehensively investigate the landscape of m6A regulators and their association with tumor microenvironment (TME), immunotherapeutic strategies in colon adenocarcinoma (COAD).ResultsThe differential expression, mutation, CNV frequency and prognostic value of 27 m6A regulators were systematically analyzed in COAD. Patients were classified into two clusters based on m6A regulators through consistent clustering analysis, with cluster A showing significant survival benefits. Most of the m6A regulators were negatively correlated with immune cells, except for WTAP, IGF2BP3, FTO, ALKBH5, which showed a positive correlation. We developed an m6A scoring system to calculate the m6Ascore for each patient. Patients with a high-m6Ascore had a better outcome, with the AUC of 0.775. An independent cohort of 416 COAD patients acquired from GSE38832 database was used to validate the prognosis prediction ability of m6Ascore. Moreover, the m6Ascore was negatively correlated with infiltration of anti-tumor immune cells. Additionally, patients with a high-m6Ascore responded better to anti-PD1 and anti-CTLA4 therapies, and those with MSI-H had a higher m6Ascore. Finally, we investigated the value of m6Ascore in predicting the response of patients to 15 commonly used drugs.ConclusionsWe comprehensively analyzed m6A regulators in COAD, including RNA expression, CNV changes, mutations and their correlation with TME. Our results showed that the m6A scoring system had significant predictive power for the prognosis of COAD patients, potentially leading to new personalized immunotherapy strategies.

N6-methyladenosine RNA modifications: a potential therapeutic target for AML.

N6-methyladenosine (m6A) RNA modification has recently emerged as an essential regulator of normal and malignant hematopoiesis. As a reversible epigenetic modification found in messenger RNAs and non-coding RNAs, m6A affects the fate of the modified RNA molecules. It is essential in most vital bioprocesses, contributing to cancer development. Here, we review the up-to-date knowledge of the pathological functions and underlying molecular mechanism of m6A modifications in normal hematopoiesis, leukemia pathogenesis, and drug response/resistance. At last, we discuss the critical role of m6A in immune response, the therapeutic potential of targeting m6A regulators, and the possible combination therapy for AML.

ALKBH5-mediated m6A demethylation fuels cutaneous wound re-epithelialization by enhancing PELI2 mRNA stability

BackgroundImpaired wound re-epithelialization contributes to cutaneous barrier reconstruction dysfunction. Recently, N6-methyladenosine (m6A) RNA modification has been shown to participate in the determination of RNA fate, and its aberration triggers the pathogenesis of numerous diseases. Howbeit, the function of m6A in wound re-epithelialization remains enigmatic.MethodsAlkbh5‒/‒ mouse was constructed to study the rate of wound re-epithelialization after ALKBH5 ablation. Integrated high-throughput analysis combining methylated RNA immunoprecipitation sequencing (MeRIP-seq) and RNA-seq was used to identify the downstream target of ALKBH5. In vitro and in vivo rescue experiments were conducted to verify the role of the downstream target on the functional phenotype of ALKBH5-deficient cells or animals. Furthermore, the interacting reader protein and regulatory mechanisms were determined through RIP-qPCR, RNA pull–down, and RNA stability assays.ResultsALKBH5 was specifically upregulated in the wound edge epidermis. Ablation of ALKBH5 suppressed keratinocyte migration and resulted in delayed wound re-epithelialization in Alkbh5‒/‒ mouse. Integrated high-throughput analysis revealed that PELI2, an E3 ubiquitin protein ligase, serves as the downstream target of ALKBH5. Concordantly, exogenous PELI2 supplementation partially rescued keratinocyte migration and accelerated re-epithelialization in ALKBH5-deficient cells, both in vitro and in vivo. In terms of its mechanism, ALKBH5 promoted PELI2 expression by removing the m6A modification from PELI2 mRNA and enhancing its stability in a YTHDF2-dependent manner.ConclusionsThis study identifies ALKBH5 as an endogenous accelerator of wound re-epithelialization, thereby benefiting the development of a reprogrammed m6A targeted therapy for refractory wounds.Graphical

Cinnamic acid mitigates left ventricular hypertrophy and heart failure in part through modulating FTO-dependent N6-methyladenosine RNA modification in cardiomyocytes

Left ventricular hypertrophy leads to heart failure, a serious medical condition associated with high rates of hospitalization and mortality. Limited success with the existing pharmacological treatments necessitates the development of mechanisms-based new therapies to better control the progression from left ventricular hypertrophy to heart failure. The current work investigated the pharmacological potentials and mechanisms of naturally occurring cinnamic acid in the treatment of left ventricular hypertrophy and heart failure. The in vitro findings reveal that cinnamic acid attenuates the hypertrophic responses and mitochondrial dysfunction in the phenylephrine (PE)-stimulated cardiomyocytes. Furthermore, cinnamic acid offsets PE-induced increases in N6-methyladenosine (m6A) RNA modification and reductions in the expression of the key m6A demethylase FTO in cardiomyocytes. Most importantly, FTO knockdown abrogates anti-hypertrophic and mitochondrial protective effects of cinnamic acid in the PE-stimulated cardiomyocytes. The in vivo results further demonstrate that cinnamic acid mitigates left ventricular hypertrophy, left ventricular systolic dysfunction and ultrastructural impairment of cardiomyocyte mitochondria and myofibrils in the mice subjected to transverse aortic constriction (TAC)-induced pressure overload. Moreover, FTO knockdown abolishes these beneficial effects of cinnamic acid in the TAC mice. In conclusion, the work here demonstrates for the first time that cinnamic acid is effective at mitigating pressure overload-induced left ventricular hypertrophy and heart failure in part by modulating the expression of FTO and the level of FTO-dependent m6A RNA modification in cardiomyocytes. These novel findings warrant further evaluation of cinnamic acid as a pharmacological agent/component to complement the existing treatment of pressure overload-mediated left ventricular hypertrophy and heart failure.

Aberrant activation of estrogen receptor-α signaling in Mettl14-deficient uteri impairs embryo implantation.

The precise control of endometrial receptivity is crucial for successful embryo implantation, which is strictly regulated by the ovarian steroid hormones estrogen and progesterone. Despite our improved understanding of the genetic regulation of implantation downstream of the action of hormones, we do not know much about the epigenetic regulation that occurs during early pregnancy. To investigate the role of the N6-methyladenosine (m6A) RNA modification in embryo implantation, we generated mice with conditional deletion of Mettl14, a core component of the m6A writer complex, in the uterus. These mice were infertile due to implantation failure. We showed that Mettl14-deficient uteri had aberrant upregulation of estrogen receptor α (ERα) signaling and ERα phosphorylation, but progesterone receptor (PGR) signaling was largely unaffected. Additionally, Mettl14 deletion led to abnormal activation of the innate immune pathway in Mettl14-deficient uteri. This effect was accompanied by the infiltration of immune cells, such as macrophages and dendritic cells, into the basal region of the endometrial epithelium. Methylated RNA immunoprecipitation sequencing (MeRIP-seq) showed that genes involved in the innate immune response had decreased m6A peaks in Mettl14-deficient mice. These results suggest that Mettl14 plays a crucial role in successful implantation by precisely regulating both ERα signaling and innate immunity in the uterus.

N6-methyladenosine RNA modification: an emerging molecule in type 2 diabetes metabolism.

Type 2 diabetes (T2D) is a metabolic disease with an increasing rate of incidence worldwide. Despite the considerable progress in the prevention and intervention, T2D and its complications cannot be reversed easily after diagnosis, thereby necessitating an in-depth investigation of the pathophysiology. In recent years, the role of epigenetics has been increasingly demonstrated in the disease, of which N6-methyladenosine (m6A) is one of the most common post-transcriptional modifications. Interestingly, patients with T2D show a low m6A abundance. Thus, a comprehensive analysis and understanding of this phenomenon would improve our understanding of the pathophysiology, as well as the search for new biomarkers and therapeutic approaches for T2D. In this review, we systematically introduced the metabolic roles of m6A modification in organs, the metabolic signaling pathways involved, and the effects of clinical drugs on T2D.

Altered m6A modification is involved YAP-mediated apoptosis response in 4-vinylcyclohexene diepoxide induced ovotoxicity

4-Vinylcyclohexene diepoxide (VCD), an industrial occupational health hazard chemical associated with premature ovarian insufficiency (POI) and reproductive failure. Recently, investigators have paid an increasing attention on VCD model of menopause recapitulates the natural, physiological transition through perimenopause to menopause. The current study sought to examining the mechanisms of follicular loss and exploring the effect of the model on systems outside of the ovaries. In this study, 28 days female SD rats were injected with VCD (160 mg/kg) vehicle for 15 consecutive days, euthanized in the diestrus phase approximately 100 days after the onset of treatment. Reproductive system injury, Neuroendocrine, sex hormone levels and receptor were observed, the levels of N6-methyladenosine (m6A) RNA modification and the expression of modulator genes were first measured. The VCD treated rats showing irregular estrous cycles, significantly reduced in the number of primordial follicles, the preantral and antral follicles also decreased significantly, accompanied by the plasma level of FSH increased and anti-Mullerian hormone (AMH) were decreased. The total m6A level was significantly decreased after exposure to VCD. Moreover, ALKBH5-mediated YAP m6A modification changed in VCD - induced premature ovarian insufficiency. These present work provides a new perspective on m6A modification in the VCD-induced POI rat model, which could provide valuable insights into the mechanisms underlying follicle development and finding new therapeutic targets for follicle prematurely exhausted. Also provide novel methodological guidance and endocrine basis to guide research and extend the applications in premature ovarian insufficiency model.

The mRNA methyltransferase Mettl3 modulates cytokine mRNA stability and limits functional responses in mast cells

Mast cells are central players in allergy and asthma, and their dysregulated responses lead to reduced quality of life and life-threatening conditions such as anaphylaxis. The RNA modification N6-methyladenosine (m6A) has a prominent impact on immune cell functions, but its role in mast cells remains unexplored. Here, by optimizing tools to genetically manipulate primary mast cells, we reveal that the m6A mRNA methyltransferase complex modulates mast cell proliferation and survival. Depletion of the catalytic component Mettl3 exacerbates effector functions in response to IgE and antigen complexes, both in vitro and in vivo. Mechanistically, deletion of Mettl3 or Mettl14, another component of the methyltransferase complex, lead to the enhanced expression of inflammatory cytokines. By focusing on one of the most affected mRNAs, namely the one encoding the cytokine IL-13, we find that it is methylated in activated mast cells, and that Mettl3 affects its transcript stability in an enzymatic activity-dependent manner, requiring consensus m6A sites in the Il13 3’-untranslated region. Overall, we reveal that the m6A machinery is essential in mast cells to sustain growth and to restrain inflammatory responses.

Microenvironment modulation by key regulators of RNA N6-methyladenosine modification in respiratory allergic diseases

BackgroundRNA N6-methyladenosine (m6A) regulators are considered post-transcriptional regulators that affect several biological functions, and their role in immunity, in particular, is emerging. However, the role of m6A regulators in respiratory allergic diseases remains unclear. Therefore, we aimed to investigate the role of key m6A regulators in mediating respiratory allergic diseases and immune microenvironment infiltration characteristics.MethodsWe downloaded gene expression profiles of respiratory allergies from the Gene Expression Omnibus (GEO) database and we performed hierarchical clustering, difference analysis, and construction of predictive models to identify hub m6A regulators that affect respiratory allergies. Next, we investigate the underlying biological mechanisms of key m6A regulators by performing PPI network analysis, functional enrichment analysis, and immune microenvironment infiltration analysis. In addition, we performed a drug sensitivity analysis on the key m6A regulator, hoping to be able to provide some implications for clinical medication.ResultsIn this study, we identified four hub m6A regulators that affect the respiratory allergy and investigated the underlying biological mechanisms. In addition, studies on the characteristics of immune microenvironment infiltration revealed that the expression of METTL14, METTL16, and RBM15B correlated with the infiltration of the mast and Th2 cells in respiratory allergy, and METTL16 expression was found to be significantly negatively correlated with macrophages for the first time (R = -0.53, P < 0.01). Finally, a key m6A regulator, METTL14, was screened by combining multiple algorithms. In addition, by performing a drug sensitivity analysis on METTL14, we hypothesized that it may play an important role in the improvement of allergic symptoms in the upper and lower airways with topical nasal glucocorticoids.ConclusionsOur findings suggest that m6A regulators, particularly METTL14, play a crucial role in the development of respiratory allergic diseases and the infiltration of immune cells. These results may provide insight into the mechanism of action of methylprednisolone in treating respiratory allergic diseases.

Cross-Regulation Between Redox and Epigenetic Systems in Tumorigenesis: Molecular Mechanisms and Clinical Applications.

Significance: Redox and epigenetics are two important regulatory processes of cell physiological functions. The cross-regulation between these processes has critical effects on the occurrence and development of various types of tumors. Recent Advances: The core factor that influences redox balance is reactive oxygen species (ROS) generation. The ROS functions as a double-edged sword in tumors: Low levels of ROS promote tumors, whereas excessive ROS induces various forms of tumor cell death, including apoptosis and ferroptosis as well as necroptosis and pyroptosis. Many studies have shown that the redox balance is influenced by epigenetic mechanisms such as DNA methylation, histone modification, chromatin remodeling, non-coding RNAs (microRNA, long non-coding RNA, and circular RNA), and RNA N6-methyladenosine modification. Several oxidizing or reducing substances also affect the epigenetic state. Critical Issues: In this review, we summarize research on the cross-regulation between redox and epigenetics in cancer and discuss the relevant molecular mechanisms. We also discuss the current research on the clinical applications. Future Directions: Future research can use high-throughput methods to analyze the molecular mechanisms of the cross-regulation between redox and epigenetics using both in vitro and in vivo models in more detail, elucidate regulatory mechanisms, and provide guidance for clinical treatment. Antioxid. Redox Signal. 39, 445-471.

IGF2BP3/HIF1A/YAP signaling plays a role in driving acute-on-chronic liver failure through activating hepatocyte reprogramming

BackgroundAcute-on-chronic liver failure (ACLF) is a syndrome with both high prevalence and mortality. However, the underlying mechanisms remain elusive and there is no effective therapeutic approach available. Here we aim to uncover novel molecular mechanisms of ACLF and identify potential therapeutic targets. MethodWe performed integrative analysis of 3 transcriptomic datasets and subsequent bioinformatic analysis aiming for potential genes of significance in ACLF development, identifying a critical role of IGF2BP3/HIF1A signaling in development of ACLF. Expression of molecules in IGF2BP3/HIF1A pathway and hepatocyte reprogramming markers in clinical samples were then determined by western blot and quantitative PCR. N6-methyladenosine (m6A) RNA modification of HIF1A was analyzed by m6A dot assay and PCR following m6A-antibody precipitation. The molecular mechanisms among IGFBP3, HIF1α and YAP1 were further validated by gene overexpression and knockdown experiments in HepG2 and Hep3B cells. Cell phenotypes of hepatocyte reprogramming were determined by EdU staining, sphere formation assay and immunoblotting of relevant markers. ResultsOur data demonstrated that IGF2BP3 recognized m6A modification in HIF1A mRNA as an m6A reader, thereby promoting expression of HIF1A by increasing RNA stability. HIF1A activated Rho GTPases (RhoA) and suppressed phosphorylation of YAP via inhibiting LATS1/2, promoting translocation of non-phosphorylated YAP into the nucleus, resulting in fetal liver programme and ultimate hepatic injury in ACLF patients. ConclusionWe reveal a novel molecular mechanism that IGF2BP3/HIF1A/YAP signaling promotes hepatocyte reprogramming, causing hepatic injury in ACLF. Our study provides potential targets for treatment of ACLF.

N6-methyladenosine RNA modification in PD-1/PD-L1: Novel implications for immunotherapy.

Cancer immunotherapy has been shown to achieve significant antitumor effects in a variety of malignancies. Out of all the immune checkpoint molecules, PD-1/PD-L1 inhibitor therapy has achieved great success. However, only some cancer patients benefit from this treatment strategy owing to drug resistance. Therefore, identifying the underlying modulators of the PD-1/PD-L1 pathway to completely comprehend the mechanisms of anti-PD-1/PD-L1 treatment is crucially important. Recent research has validated that m6A modification plays a critical role in the PD-1/PD-L1 axis, thus regulating the immune response and immunotherapy strategies. In this review, we summarized the latest research on the regulation of m6A modification in PD-1/PD-L1 pathways in cancer proliferation, invasion, and prognosis based on different kinds of cancers and discussed the possible mechanisms. We also reviewed m6A-associated lncRNAs in the regulation of the PD-1/PD-L1 pathway. More importantly, we outlined the influence of m6A modulation on anti-PD-1 therapy and m6A-related molecules that could predict the curative effect of anti-PD-1/PD-L1 therapy. Further studies exploring the definitive regulation of m6A on the PD1/PD-1 pathway and immunotherapy are needed, which may address some of the current limitations in immunotherapy.

ALKBH5-mediated m 6A RNA demethylation promotes antibacterial innate defense by intrinsically driving neutrophil generation and recruitment

Abstract Neutrophil generation and effective recruitment into the site of infection are essential for innate immune defense against bacterial infection. These processes that directed by extracellular signals such as growth factors or chemokines have been extensively studied, yet the intrinsic and new mechanism for determining neutrophil generation and recruitment needs further investigation. N6-methyladenosine (m 6A) RNA modification and its demethylase alkB homolog 5 (ALKBH5) are key epigenetic regulators of immunity and inflammation, whereas their role in neutrophil generation and recruitment remains unclear. Here, using cecal ligation and puncture-induced sepsis to model systemic bacterial infection, we found that ALKBH5 is required for antibacterial innate defense by intrinsically inducing neutrophil generation and recruitment. Indeed, Alkbh5-deficient mice exhibited increased mortality, higher bacterial burden and proinflammatory cytokine production than wild-type littermates undergoing sepsis. Meanwhile, ALKBH5 depletion significantly impaired the generation of immature neutrophils in bone marrow and also reduced neutrophil recruitment into the infected site of sepsis mice. Mechanistically, ALKBH5 imprinted generation- and recruitment-promoting transcriptome signatures in both mouse and human neutrophils during bacterial infection, specially modulated the protein expression of SOCS3 and CXCR2 through altering m 6A methylation-mediated decay of their mRNAs. Together, our findings reveal a previously unknown role of ALKBH5 in intrinsically driving neutrophil generation and recruitment, indicating neutrophil m 6A RNA modification as potential target for controlling bacterial infections. Supported by grants from the National Natural Science Foundation of China (81788101, 82071793), the Chinese Academy of Medical Sciences Innovation Fund for Medical Sciences (2021-I2M-1-017), the Beijing Natural Science Foundation (7212069), and the Beijing Nova Program (20220484065).

Profiling of N6-methyladenosine methylation in porcine longissimus dorsi muscle and unravelling the hub gene ADIPOQ promotes adipogenesis in an m6A-YTHDF1–dependent manner

BackgroundIntramuscular fat (IMF) content is a critical indicator of pork quality, and abnormal IMF is also relevant to human disease as well as aging. Although N6-methyladenosine (m6A) RNA modification was recently found to regulate adipogenesis in porcine intramuscular fat, however, the underlying molecular mechanisms was still unclear.ResultsIn this work, we collected 20 longissimus dorsi muscle samples with high (average 3.95%) or low IMF content (average 1.22%) from a unique heterogenous swine population for m6A sequencing (m6A-seq). We discovered 70 genes show both differential RNA expression and m6A modification from high and low IMF group, including ADIPOQ and SFRP1, two hub genes inferred through gene co-expression analysis. Particularly, we observed ADIPOQ, which contains three m6A modification sites within 3′ untranslated and protein coding region, could promote porcine intramuscular preadipocyte differentiation in an m6A-dependent manner. Furthermore, we found the YT521‑B homology domain family protein 1 (YTHDF1) could target and promote ADIPOQ mRNA translation.ConclusionsOur study provided a comprehensive profiling of m6A methylation in porcine longissimus dorsi muscle and characterized the involvement of m6A epigenetic modification in the regulation of ADIPOQ mRNA on IMF deposition through an m6A-YTHDF1-dependent manner.

Abstract 3908: M6A RNA modifications regulate expression of transcripts that promote transition to cisplatin resistance in bladder cancer

Abstract BACKGROUND: Chemotherapy resistance is recognized to occur not only through selection of pre-existing genetically resistant clones, but also through rapid phenotypic plasticity mechanisms. We previously reported that bladder cancer cells can rapidly transition to and from a chemo-resistant phenotype. Recently, N6-methyladenosine (m6A) RNA modifications were shown to dynamically and reversibly regulate mRNA processing, differentiation, and cell fate. Here, we tested the hypothesis that m6A modifications regulate phenotypic plasticity and transition to cisplatin resistance in bladder cancer (BC). METHODS: We utilized methyl-RNA-immunoprecipitation followed by sequencing (MeRIP-seq) and by RNA-seq to identify transcripts that were both differentially methylated and differentially expressed between cisplatin-sensitive and cisplatin-resistant BC cell lines. Candidate transcripts were annotated using Gene Ontology (GO) enrichment analysis and Gene Set Enrichment Analysis (GSEA). Clinical relevance was evaluated using databases such as The Cancer Genome Atlas (TCGA) and the Oncology Research Information Network (ORIEN) avatar. Cancer-relevant genes were then validated in vitro by targeted immunoprecipitation and PCR (MeRIP-PCR) and qPCR. Candidate genes were then functionally validated in cell lines and in patient derived organoids (PDOs) by siRNA-mediated knockdown and cisplatin treatment, as well as by m6A measurement after short-term cisplatin treatment. RESULTS: MeRIP-seq and RNA-seq revealed that cisplatin-sensitive and cisplatin-resistant BC cells have distinct m6A profiles, with 130 transcripts that were both differentially methylated and differentially expressed. Using clinical and functional database tools, we filtered this list to 37 transcripts and ranked them based on clinical relevance in BC. Of the top 15 candidates, eight were successfully validated in vitro via repeat qPCR and MeRIP-PCR. In cisplatin-resistant cells, one candidate, SLC7A11, was found to have decreased m6A level, which was associated with increased RNA stability and elevated transcript and protein levels. Consistent with this, 48 hr cisplatin treatment of cisplatin-sensitive cells led to SLC7A11 hypomethylation, increased RNA stability, and elevated transcript and protein levels. Furthermore, depletion of SLC7A11 by siRNA-knockdown re-sensitized BC cell lines and PDOs to cisplatin. CONCLUSIONS: Using unbiased transcriptome-wide m6A profiling followed by targeted validation, we demonstrated that m6A modifications regulate expression of clinically relevant gene transcripts in BC, and that a subset of these modifications may promote resistance to chemotherapy Collectively, these results implicate epitranscriptomic plasticity as a driver in potentiating cisplatin resistance in bladder cancer. Citation Format: Emmanuelle Hodara, Aubree Mades, Tong Xu, Amir Goldkorn, Suhn Rhie. M6A RNA modifications regulate expression of transcripts that promote transition to cisplatin resistance in bladder cancer. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3908.

The m6A methylation landscape, molecular characterization and clinical relevance in prostate adenocarcinoma.

Despite the recent progress of therapeutic strategies in treating prostate cancer (PCa), the majority of patients still eventually relapse, experiencing dismal outcomes. Therefore, it is of utmost importance to identify novel viable targets to increase the effectiveness of treatment. The present study aimed to investigate the potential relationship between N6-methyladenosine (m6A) RNA modification and PCa development and determine its clinical relevance. Through systematic analysis of the TCGA database and other datasets, we analyzed the gene expression correlation and mutation profiles of m6A-related genes between PCa and normal tissues. Patient samples were divided into high- and low-risk groups based on the results of Least Absolute Shrinkage and Selection Operator (LASSO) Cox analysis. Subsequently, differences in biological processes and genomic characteristics of the two risk groups were determined, followed by functional enrichment analysis and gene set enrichment (GSEA) analysis. Next, we constructed the protein-protein interaction (PPI) network of differentially expressed genes between patients in high- and low-risk groups, along with the mRNA-miRNA-lncRNA network. The correlation analysis of tumor-infiltrating immune cells was further conducted to reveal the differences in immune characteristics between the two groups. A variety of m6A-related genes were identified to be differentially expressed in PCa tissues as compared with normal tissues. In addition, the PPI network contained 278 interaction relationships and 34 m6A-related genes, and the mRNA-miRNA-lncRNA network contained 17 relationships, including 91 miRNAs. Finally, the immune characteristics analysis showed that compared with the low-risk group, the levels of M1 and M2 macrophages in the high-risk group significantly increased, while the levels of mast cells resting and T cells CD4 memory resting significantly decreased. This study provides novel findings that can further the understanding of the role of m6A methylation during the progression of PCa, which may facilitate the invention of targeted therapeutic drugs.

A novel MYCN-YTHDF1 cascade contributes to retinoblastoma tumor growth by eliciting m6A -dependent activation of multiple oncogenes.

Retinoblastoma, the most prevalent primary intraocular tumor in children, leads to vision impairment, disability and even death. In addition to RB1 inactivation, MYCN activation has been documented as another common oncogenic alteration in retinoblastoma and represents one of the high-risk molecular subtypes of retinoblastoma. However, how MYCN contributes to the progression of retinoblastoma is still incompletely understood. Here, we report that MYCN upregulates YTHDF1, which encodes one of the reader proteins for N6-methyladenosine (m6A) RNA modification, in retinoblastoma. We further found that this MYCN-upregulated m6A reader functions to promote retinoblastoma cell proliferation and tumor growth in an m6A binding-dependent manner. Mechanistically, YTHDF1 promotes the expression of multiple oncogenes by binding to their mRNAs and enhancing mRNA stability and translation in retinoblastoma cells. Taken together, our findings reveal a novel MYCN-YTHDF1 regulatory cascade in controlling retinoblastoma cell proliferation and tumor growth, pinpointing an unprecedented mechanism for MYCN amplification and/or activation to promote retinoblastoma progression.