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The nine identified human homologues of E . coli AlkB 2-oxoglutarate (2OG) and Fe(II)-dependent dioxygenase, ALKBH1-8 and FTO, display different substrate specificities and diverse biological functions. Here we discovered the combined overexpression of members of the ALKBH family in head and neck squamous cell carcinomas (HNSCC). We found direct correlation of ALKBH3 and FTO expression with primary HNSCC tumor size. We observed unidentified thus far cytoplasmic localization of ALKBH2 and 5 in HNSCC, suggesting abnormal role(s) of ALKBH proteins in cancer. Further, high expression of ALKBHs was observed not only in HNSCC, but also in several cancerous cell lines and silencing ALKBH expression in HeLa cancer cells resulted in dramatically decreased survival. Considering the discovered impact of high expression of ALKBH proteins on HNSCC development, we screened for ALKBH blockers among newly synthetized anthraquinone derivatives and demonstrated their potential to support standard anticancer therapy.

The inert chemical property of RNA modification N -methyladenosine (m A) makes it very challenging to detect. Most m A sequencing methods rely on m A-antibody immunoprecipitation and cannot distinguish m A and N ,2'-O-dimethyladenosine modification at the cap +1 position (cap m A ). Although the two antibody-free methods (m A-REF-seq/MAZTER-seq and DART-seq) have been developed recently, they are dependent on m A sequence or cellular transfection. Here, we present an antibody-free, FTO-assisted chemical labeling method termed m A-SEAL for specific m A detection. We applied m A-SEAL to profile m A landscapes in humans and plants, which displayed the known m A distribution features in transcriptome. By doing a comparison with all available m A sequencing methods and specific m A sites validation by SELECT, we demonstrated that m A-SEAL has good sensitivity, specificity and reliability for transcriptome-wide detection of m A. Given its tagging ability and FTO's oxidation property, m A-SEAL enables many applications such as enrichment, imaging and sequencing to drive future functional studies of m A and other modifications.

Fat mass and obesity-associated protein (FTO) is the first reported RNA N6-methyladenosine (m6A) demethylase in eukaryotic cells. m6A is considered as the most abundant mRNA internal modification, which modulates several cellular processes including alternative splicing, stability, and expression. Genome-wide association studies (GWAS) identified single-nucleotide polymorphisms (SNPs) within FTO to be associated with obesity, as well as cancer including endometrial cancer, breast cancer, pancreatic cancer, and melanoma. Since the initial classification of FTO as an m6A demethylase, various studies started to unravel a connection between FTO’s demethylase activity and the susceptibility to obesity on the molecular level. FTO was found to facilitate adipogenesis, by regulating adipogenic pathways and inducing pre-adipocyte differentiation. FTO has also been investigated in tumorigenesis, where emerging studies suggest m6A and FTO levels are dysregulated in various cancers, including acute myeloid leukemia (AML), glioblastoma, cervical squamous cell carcinoma (CSCC), breast cancer, and melanoma. Here we review the molecular bases of m6A in tumorigenesis and adipogenesis while highlighting the controversial role of FTO in obesity. We provide recent findings confirming FTO’s causative link to obesity and discuss novel approaches using RNA demethylase inhibitors as targeted oncotherapies. Our review aims to confirm m6A demethylation as a risk factor in obesity and provoke new research in FTO and human disorders.

Nonalcoholic fatty liver disease (NAFLD) is common and partially heritable and has no effective treatments. We carried out a genome-wide association study (GWAS) meta-analysis of imaging ( n  = 66,814) and diagnostic code (3,584 cases versus 621,081 controls) measured NAFLD across diverse ancestries. We identified NAFLD-associated variants at torsin family 1 member B ( TOR1B ), fat mass and obesity associated ( FTO ), cordon-bleu WH2 repeat protein like 1 ( COBLL1 )/growth factor receptor-bound protein 14 ( GRB14 ), insulin receptor ( INSR ), sterol regulatory element-binding transcription factor 1 ( SREBF1 ) and patatin-like phospholipase domain-containing protein 2 ( PNPLA2 ), as well as validated NAFLD-associated variants at patatin-like phospholipase domain-containing protein 3 ( PNPLA3 ), transmembrane 6 superfamily 2 ( TM6SF2 ), apolipoprotein E ( APOE ), glucokinase regulator ( GCKR ), tribbles homolog 1 ( TRIB1 ), glycerol-3-phosphate acyltransferase ( GPAM ), mitochondrial amidoxime-reducing component 1 ( MARC1 ), microsomal triglyceride transfer protein large subunit ( MTTP ) , alcohol dehydrogenase 1B ( ADH1B ), transmembrane channel like 4 (TMC4)/membrane-bound O-acyltransferase domain containing 7 ( MBOAT7 ) and receptor-type tyrosine-protein phosphatase δ ( PTPRD ). Implicated genes highlight mitochondrial, cholesterol and de novo lipogenesis as causally contributing to NAFLD predisposition. Phenome-wide association study (PheWAS) analyses suggest at least seven subtypes of NAFLD. Individuals in the top 10% and 1% of genetic risk have a 2.5-fold to 6-fold increased risk of NAFLD, cirrhosis and hepatocellular carcinoma. These genetic variants identify subtypes of NAFLD, improve estimates of disease risk and can guide the development of targeted therapeutics. Genome-wide association meta-analysis across individuals of diverse ancestries identifies risk loci for nonalcoholic fatty liver disease. The associated variants implicate plausible biological pathways and improve estimates of disease risk.

Diabetic retinopathy (DR) is a leading cause of irreversible vision loss in working-age populations. Fat mass and obesity-associated protein (FTO) is an N 6 -methyladenosine (m 6 A) demethylase that demethylates RNAs involved in energy homeostasis, though its influence on DR is not well studied. Herein, we detected elevated FTO expression in vitreous fibrovascular membranes of patients with proliferative DR. FTO promoted cell cycle progression and tip cell formation of endothelial cells (ECs) to facilitate angiogenesis in vitro, in mice, and in zebrafish. FTO also regulated EC-pericyte crosstalk to trigger diabetic microvascular leakage, and mediated EC–microglia interactions to induce retinal inflammation and neurodegeneration in vivo and in vitro. Mechanistically, FTO affected EC features via modulating CDK2 mRNA stability in an m 6 A-YTHDF2-dependent manner. FTO up-regulation under diabetic conditions was driven by lactate-mediated histone lactylation. FB23-2, an inhibitor to FTO’s m 6 A demethylase activity, suppressed angiogenic phenotypes in vitro. To allow for systemic administration, we developed a nanoplatform encapsulating FB23-2 and confirmed its targeting and therapeutic efficiency in mice. Collectively, our study demonstrates that FTO is important for EC function and retinal homeostasis in DR, and warrants further investigation as a therapeutic target for DR patients. Synopsis The fat mass and obesity-associated (FTO) protein, an N 6 -methyladenosine (m 6 A) demethylase, influences endothelial cell (EC) function and retinal homeostasis in diabetic retinopathy (DR), thus providing a promising nanotherapeutic approach for DR. FTO expression is elevated in diabetic mice retinas and vitreous fibrovascular membranes of patients with proliferative DR. FTO triggers diabetes-induced microvascular dysfunction by facilitating neovascularization and regulating EC–pericyte/microglia crosstalk. FTO regulates CDK2 mRNA stability with the YTHDF2 reader in an m 6 A-dependent manner. Lactic acid regulates FTO expression via histone lactylation. FB23-2 suppresses demethylation activity of FTO to inhibit diabetes-induced EC phenotypes, showing therapeutic potential in DR in mice. The fat mass and obesity-associated (FTO) protein, an N 6 -methyladenosine (m 6 A) demethylase, influences endothelial cell (EC) function and retinal homeostasis in diabetic retinopathy (DR), thus providing a promising nanotherapeutic approach for DR.

ObjectiveFat mass and obesity-associated protein (FTO), an eraser of N 6-methyadenosine (m6A), plays oncogenic roles in various cancers. However, its role in hepatocellular carcinoma (HCC) is unclear. Furthermore, small extracellular vesicles (sEVs, or exosomes) are critical mediators of tumourigenesis and metastasis, but the relationship between FTO-mediated m6A modification and sEVs in HCC is unknown.DesignThe functions and mechanisms of FTO and glycoprotein non-metastatic melanoma protein B (GPNMB) in HCC progression were investigated in vitro and in vivo. Neutralising antibody of syndecan-4 (SDC4) was used to assess the significance of sEV-GPNMB. FTO inhibitor CS2 was used to examine the effects on anti-PD-1 and sorafenib treatment.ResultsFTO expression was upregulated in patient HCC tumours. Functionally, FTO promoted HCC cell proliferation, migration and invasion in vitro, and tumour growth and metastasis in vivo. FTO knockdown enhanced the activation and recruitment of tumour-infiltrating CD8+ T cells. Furthermore, we identified GPNMB to be a downstream target of FTO, which reduced the m6A abundance of GPNMB, hence, stabilising it from degradation by YTH N 6-methyladenosine RNA binding protein F2. Of note, GPNMB was packaged into sEVs derived from HCC cells and bound to the surface receptor SDC4 of CD8+ T cells, resulting in the inhibition of CD8+ T cell activation. A potential FTO inhibitor, CS2, suppresses the oncogenic functions of HCC cells and enhances the sensitivity of anti-PD-1 and sorafenib treatment.ConclusionTargeting the FTO/m6A/GPNMB axis could significantly suppress tumour growth and metastasis, and enhance immune activation, highlighting the potential of targeting FTO signalling with effective inhibitors for HCC therapy.

Background N6-methyladenosine (m6A) modification is the most pervasive modification in mRNA, and has been considered as a new layer of epigenetic regulation on mRNA processing, stability and translation. Despite its functional significance in various physiological processes, the role of the m6A modification involved in breast cancer is yet fully understood. Methods We used the m6A-RNA immunoprecipitation sequencing to identify the potential targets in breast cancer. To determine the underlying mechanism for the axis of FTO-BNIP3, we performed a series of in vitro and in vivo assays in 3 breast cancer cell lines and 36 primary breast tumor tissues and 12 adjunct tissues. Results We showed that FTO, a key m6A demethylase, was up-regulated in human breast cancer. High level of FTO was significantly associated with lower survival rates in patients with breast cancer. FTO promoted breast cancer cell proliferation, colony formation and metastasis in vitro and in vivo. We identified BNIP3, a pro-apoptosis gene, as a downstream target of FTO-mediated m6A modification. Epigenetically, FTO mediated m6A demethylation in the 3’UTR of BNIP3 mRNA and induced its degradation via an YTHDF2 independent mechanism. BNIP3 acts as a tumor suppressor and is negatively correlated with FTO expression in clinical breast cancer patients. BNIP3 dramatically alleviated FTO-dependent tumor growth retardation and metastasis. Conclusions Our findings demonstrate the functional significance of the m6A modification in breast cancer, and suggest that FTO may serve as a novel potential therapeutic target for breast cancer.

Cellular senescence is characterized by a tumor-suppressive program as well as a pro-inflammatory secretome. Neutrophils constitute significant compositions of malignancies and play key roles in tumor development. However, the role of senescent neutrophils in cancer progression is presently unexplored. Here, we demonstrate that neutrophils display enhanced senescence in breast cancer patients receiving chemotherapy. The senescent neutrophils produce increased number of exosomes, which confer drug resistance to tumor cells in vitro and in vivo. Mechanistically, senescent neutrophils-derived exosomal piRNA-17560 enhances the expression of fat mass and obesity-associated protein (FTO) in breast cancer cells. The upregulation of FTO further strengthens ZEB1 transcripts stability and expression by decreasing N6-methyladenosine (m6A) RNA methylation, leading to chemoresistance and epithelial-mesenchymal transition (EMT) of tumor cells. Clinically, the level of exosomal piR-17560 correlates with poor chemotherapy response in patients with breast cancer. In addition, YTHDF2 is essential for the posttranscriptional regulation of ZEB1 by piRNA-17560/FTO signaling. Senescent neutrophils secret exosomal piR-17560 in a STAT3-dependent manner. Altogether, this study suggests that senescent neutrophils-derived exosomal piR-17560 confers chemoresistance to tumor cells and senescent neutrophils may serve as a potential therapeutic target in breast cancer.

Alveolar bone resorption (ABR) is a key pathological manifestation in the development of apical periodontitis (AP) and contributes to the AP-associated tooth loss among AP patients in the clinic. However, the underlying mechanism of ABR development is largely unknown. Here we show, the total levels of N6-methyladenosine (m 6 A) were reduced in AP male rat alveolar bone tissues and BMDM-derived osteoclasts (OC), which was associated with the up-regulation of obesity-associated protein (FTO). Subsequently FTO-mediated hexokinase (HK1) demethylation modification enhancing glycolytic pathway that stabilizes receptor activator of NF-κB (RANK) protein via the deubiquitination activity of ubiquitin-specific protease 14 (USP14), which further promotes osteoclastogenesis to participate in the AP-related ABR development. Finally, Dac51 (an FTO inhibitor) and 2-DG (an HK1 inhibitor) both exhibit the inhibitory activity of osteoclastogenesis. Our current study reveals a molecular mechanism on osteoclastogenesis-related ABR and provides a therapeutic target of AP via modulating the FTO/HK1/USP14/RANK axis. Alveolar bone resorption (ABR) is a key pathological manifestation in the development of apical periodontitis (AP) that contributes to the AP-associated tooth loss, and whose underlying mechanism is largely unknown. Here, the authors show a molecular mechanism on osteoclastogenesis-related ABR and provides a therapeutic target of AP via modulating the FTO/HK1/USP14/RANK axis.

Amyotrophic lateral sclerosis (ALS) is a swiftly progressive and fatal neurodegenerative ailment marked by the degenerative motor neurons (MNs). Why MNs are specifically susceptible in predominantly sporadic cases remains enigmatic. Here, we demonstrated N 6 -methyladenosine (m 6 A), an RNA modification catalyzed by the METTL3/METTL14 methyltransferase complex, as a pivotal contributor to ALS pathogenesis. By conditional knockout Mettl14 in murine MNs, we recapitulate almost the full spectrum of ALS disease characteristics. Mechanistically, pervasive m 6 A hypomethylation triggers dysregulated expression of high-risk genes associated with ALS and an unforeseen reduction of chromatin accessibility in MNs. Additionally, we observed diminished m 6 A levels in induced pluripotent stem cell derived MNs (iPSC~MNs) from familial and sporadic ALS patients. Restoring m 6 A equilibrium via a small molecule or gene therapy significantly preserves MNs from degeneration and mitigates motor impairments in ALS iPSC~MNs and murine models. Our study presents a substantial stride towards identifying pioneering efficacious ALS therapies via RNA modifications. Amyotrophic lateral sclerosis (ALS) is a fatal disease characterized by rapid motor neuron (MN) degeneration, with unclear causes in sporadic cases. Here, the authors showed that N6-methyladenosine (m6A) RNA modification drives ALS, and restoring it may protect MNs and lessen motor issues.