Fat Mass And Obesity-associated Protein Research Articles

Abstract 179: M6A demethylation enzyme FTO upregulates LDHA and promotes glycolysis and invasion and migration in oral squamous cell carcinoma

Abstract Objective: To investigate the role of lactate dehydrogenase A (LDHA) in the glycolysis and invasion and metastasis of oral squamous cell carcinoma (OSCC), and to explore the molecular mechanism of fat mass and obesity-associated protein (FTO) regulating LDHA on the glycolysis and invasion and metastasis of OSCC. Methods: Immunohistochemistry and RT-qPCR were performed to detect the expression of LDHA protein and gene in OSCC and adjacent non-cancerous tissues, and their correlation with clinical prognosis and pathological parameters were analyzed. Lentivirus-mediated shRNA silencing of LDHA and lactate dehydrogenase inhibitor (oxamate) were used to establish stable OSCC Cell lines HSC3 and SCC15, Cell Counting Kit-8 and colony formation assay were applied to evaluate cell proliferation, scratch test and Transwell assay were performed to detect cell migration and invasion, Glucose utilization assay and Lactate production assay were carried out to assess cell uptake of glucose and lactate production respectively. The molecular mechanism of FTO regulating LDHA on glycolysis, invasion and metastasis of oral squamous cell carcinoma was further explored, OSCC cell lines HSC3 and SCC15 transfected with siRNAs to silence FTO expression, EpiQuik™ m6A RNA Methylation Quantification Kit was applied to assess the m6A% in total RNA, the effect of FTO on LDHA expression was detected by Western blot and RT-qPCR. The biological function of FTO on oral squamous cell carcinoma cells was demonstrated by cell experiments in vitro. Results: Compared with adjacent non-cancerous tissues, LDHA was highly expressed in OSCC (P<0.001), which was closely related to the differentiation, clinical stage and T stage of OSCC (P<0.05). Silencing LDHA, FTO or treating oxamate in HSC3 and SCC15 cells reduced glucose consumption, lactate production and cell proliferation, invasion and migration (P<0.05). In addition, after FTO silencing, the m6A level of total RNA increased (P<0.05), while the expression level of LDHA protein and mRNA decreased (P<0.01). Conclusion: FTO may promote the expression of LDHA protein and mRNA in a m6A-dependent manner and enhance the ability of glycolysis, proliferation, invasion and metastasis of OSCC. Note: This abstract was not presented at the meeting. Citation Format: Jinsong Hou. M6A demethylation enzyme FTO upregulates LDHA and promotes glycolysis and invasion and migration in oral squamous cell carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 179.

Abstract 4022: Novel evidence for m6A methylation regulators as prognostic biomarkers and potential therapeutic targets in gastric cancer

Abstract Purpose: N6-methyladenosine (m6A) post-transcriptional RNA modification represents the most abundant epigenetic alteration in eukaryotic cells. These m6A modifications are regulated by a cascade of enzymes and cofactors, categorized as ‘writers’ [methyltransferase-like 3 (METTL3), METTL14, and Wilms’ tumor 1-associating protein (WTAP1)], ‘erasers’ [fat mass and obesity-associated protein (FTO) and alkylated DNA repair protein AlkB homolog 5 (ALKBH5)] and ‘readers’ [YTHDF1, and YTHDF2 (YTH N6-Methyladenosine RNA Binding Protein)]. While emerging evidence indicates that these enzymes play a central role in RNA metabolism (e.g. RNA stability, translation, splicing, transport and localization), their clinical significance, if any, remains unclear. Herein, we for the first time, systematically unraveled the functional role, as well as the clinical significance of m6a regulators in gastric cancer (GC). Experimental design: First, we investigated the expression of the seven m6A regulator genes (FTO, METTL3, METTL14, WTAP, ALKBH5, YTHDF1, and YTHDF2) in a large cohort of 171 GC patients by qRT-PCR assays. Subsequently, a multivariate Cox-regression model was developed using the 7-gene panel to evaluate its predictive potential, followed by Kaplan Meier analyses for survival outcomes. In addition, we undertook a series of functional studies to investigate the oncogenic role of FTO in GC cells, and subsequent validation of our findings in an animal model. Results: Gastric cancer patients with low-expression of METTL3, METTL14, ALKBH5, WTAP and YTHDF1 demonstrated significantly poor OS (p=0.02, 0.001, 0.01, 0.02 and 0.02, respectively), while patients with high FTO expression exhibited markedly worse OS (p<0.0001). Furthermore, the cumulative risk-score derived from these gene panel also significantly associated with poor OS, with a corresponding hazard ratio of 5.47 (95% CI: 3.18-9.41, p<0.0001). We observed that FTO expression was frequently up-regulated in GC cell lines, particularly in those with epithelial-mesenchymal-transition (EMT) features. Functional studies with FTO knockdown in HGC27 and AGS cells inhibited cell proliferation (p=0.005, and <0.0001, respectively) and migratory potential (p<0.0001, respectively); while its overexpression in MKN28 cells resulted in enhanced proliferation and migration (p<0.0001, and 0.007, respectively). Finally, confirming our in-vitro findings, FTO suppression led to significant tumor growth inhibition in a xenograft animal model (p<0.0001). Conclusions: We provide first evidence that m6A regulators may serve as important prognostic biomarkers in GC patients. Our functional studies reveal that FTO is an important oncogene, and may be a novel therapeutic target in patients with gastric cancer. Citation Format: Tadanobu Shimura, Raju Kandimalla, Yuji Toiyama, Yoshinaga Okugawa, Masato Kusunoki, Ajay Goel. Novel evidence for m6A methylation regulators as prognostic biomarkers and potential therapeutic targets in gastric cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4022.

M6A mRNA demethylase FTO regulates melanoma tumorigenicity and response to anti-PD-1 blockade

Melanoma is one of the most deadly and therapy-resistant cancers. Here we show that N6-methyladenosine (m6A) mRNA demethylation by fat mass and obesity-associated protein (FTO) increases melanoma growth and decreases response to anti-PD-1 blockade immunotherapy. FTO level is increased in human melanoma and enhances melanoma tumorigenesis in mice. FTO is induced by metabolic starvation stress through the autophagy and NF-κB pathway. Knockdown of FTO increases m6A methylation in the critical protumorigenic melanoma cell-intrinsic genes including PD-1 (PDCD1), CXCR4, and SOX10, leading to increased RNA decay through the m6A reader YTHDF2. Knockdown of FTO sensitizes melanoma cells to interferon gamma (IFNγ) and sensitizes melanoma to anti-PD-1 treatment in mice, depending on adaptive immunity. Our findings demonstrate a crucial role of FTO as an m6A demethylase in promoting melanoma tumorigenesis and anti-PD-1 resistance, and suggest that the combination of FTO inhibition with anti-PD-1 blockade may reduce the resistance to immunotherapy in melanoma.

The Fat Mass and Obesity-Associated Protein (FTO) Regulates Locomotor Responses to Novelty via D2R Medium Spiny Neurons

Variations in the human FTO gene have been linked to obesity and altered connectivity of the dopaminergic neurocircuitry. Here, we report that fat mass and obesity-associated protein (FTO) in dopamine D2 receptor-expressing medium spiny neurons (D2 MSNs) of mice regulate the excitability of these cells and control their striatopallidal globus pallidus external (GPe) projections. Lack of FTO in D2 MSNs translates into increased locomotor activity to novelty, associated with altered timing behavior, without impairing the ability to control actions or affecting reward-driven and conditioned behavior. Pharmacological manipulations of dopamine D1 receptor (D1R)- or D2R-dependent pathways in these animals reveal altered responses to D1- and D2-MSN-mediated control of motor output. These findings reveal a critical role for FTO to control D2 MSN excitability, their projections to the GPe, and behavioral responses to novelty.

The role of N6-methyladenosine RNA methylation in the heat stress response of sheep (Ovis aries).

With the intensive development of the sheep industry and increasing global temperatures, heat stress in sheep has become an increasingly severe and important issue in recent years. The level of N6-methyladenosine (m6A) RNA methylation changes in response to stress plays important roles in stress responses. However, the role of m6A in the heat stress response of sheep remains unclear. To explore this issue, we measured heat stress protein (HSP) expression, liver function indexes, m6A on RNA, m6A-related enzyme expression, and tissue damage in sheep that had been subjected to heat stress. At the transcriptome level, our results showed significant increases in m6A on RNA and increased mRNA levels of HSPs (HSP70, HSP90, and HSP110) and m6A-related enzymes [METTL3 (methyltransferase-like 3), METTL14 (methyltransferase-like 14), WTAP (wilms tumor 1-associated protein), FTO (fat mass and obesity-associated protein), ALKBH5 (alkB homologue 5), YTHDF1-3 (YTH domain family proteins), and YTHDC1-2 (YTH domain-containing proteins)] following heat stress. At the protein level, the expression of METTL3, YTHDF1-2, and YTHDC2 showed no significant differences following heat stress. However, in contrast to its mRNA level after heat stress, the protein expression of YTHDF3 was reduced, while the expression of HSPs (HSP70, HSP90, and HSP110), METTL14, WTAP, FTO, ALKBH5, YTHDF3, and YTHDC1 increased in line with their measured mRNA levels. Histological experiments revealed that heat stress caused varying degrees of damage to sheep liver tissue. Moreover, immunohistochemical staining indicated that the m6A-related enzymes were expressed in sheep hepatocytes, and differences in expression patterns were observed between the control and heat stress groups. In summary, differences in the level of m6A and the expression of m6A-related enzymes in the liver of sheep were observed after heat stress. This indicates that m6A is involved in the regulation of heat stress in sheep. Our findings provide a new avenue for studying the responses to heat stress in sheep.

FTO-Dependent N6-Methyladenosine Regulates Cardiac Function During Remodeling and Repair.

Despite its functional importance in various fundamental bioprocesses, studies of N6-methyladenosine (m6A) in the heart are lacking. Here, we show that the FTO (fat mass and obesity-associated protein), an m6A demethylase, plays a critical role in cardiac contractile function during homeostasis, remodeling, and regeneration. We used clinical human samples, preclinical pig and mouse models, and primary cardiomyocyte cell cultures to study the functional role of m6A and FTO in the heart and in cardiomyocytes. We modulated expression of FTO by using adeno-associated virus serotype 9 (in vivo), adenovirus (both in vivo and in vitro), and small interfering RNAs (in vitro) to study its function in regulating cardiomyocyte m6A, calcium dynamics and contractility, and cardiac function postischemia. We performed methylated (m6A) RNA immunoprecipitation sequencing to map transcriptome-wide m6A, and methylated (m6A) RNA immunoprecipitation quantitative polymerase chain reaction assays to map and validate m6A in individual transcripts, in healthy and failing hearts, and in myocytes. We discovered that FTO has decreased expression in failing mammalian hearts and hypoxic cardiomyocytes, thereby increasing m6A in RNA and decreasing cardiomyocyte contractile function. Improving expression of FTO in failing mouse hearts attenuated the ischemia-induced increase in m6A and decrease in cardiac contractile function. This is performed by the demethylation activity of FTO, which selectively demethylates cardiac contractile transcripts, thus preventing their degradation and improving their protein expression under ischemia. In addition, we demonstrate that FTO overexpression in mouse models of myocardial infarction decreased fibrosis and enhanced angiogenesis. Collectively, our study demonstrates the functional importance of the FTO-dependent cardiac m6A methylome in cardiac contraction during heart failure and provides a novel mechanistic insight into the therapeutic mechanisms of FTO.

FTO Knockout Causes Chromosome Instability and G2/M Arrest in Mouse GC-1 Cells.

N6-methyladenosine (m6A) is the most abundant modification on eukaryotic mRNA. m6A plays important roles in the regulation of post-transcriptional RNA splicing, translation, and degradation. Increasing studies have uncovered the significance of m6A in various biological processes such as stem cell fate determination, carcinogenesis, adipogenesis, stress response, etc, which put forwards a novel conception called epitranscriptome. However, functions of the fat mass and obesity-associated protein (FTO), the first characterized m6A demethylase, in spermatogenesis remains obscure. Here we reported that depletion of FTO by CRISPR/Cas9 induces chromosome instability and G2/M arrest in mouse spermatogonia, which was partially rescued by expression of wild type FTO but not demethylase inactivated FTO. FTO depletion significantly decreased the expression of mitotic checkpoint complex and G2/M regulators. We further demonstrated that the m6A modification on Mad1, Mad2, Bub1b, Cdk1, and Ccnb2 were directly targeted by FTO. Therefore, FTO regulates cell cycle and mitosis checkpoint in spermatogonia because of its m6A demethylase activity. The findings give novel insights into the role of RNA methylation in spermatogenesis.

The partial inhibition of hypothalamic IRX3 exacerbates obesity.

BackgroundThe Iroquois homeobox 3 (Irx3) gene has been identified as a functional long-range target of obesity-associated variants within the fat mass and obesity-associated protein (FTO) gene. It is highly expressed in the hypothalamus, and both whole-body knockout and hypothalamic restricted abrogation of its expression results in a lean phenotype, which is mostly explained by the resulting increased energy expenditure in the brown adipose tissue. Because of its potential implication in the pathogenesis of obesity, we evaluated the hypothalamic cell distribution of Irx3 and the outcomes of inhibiting its expression in a rodent model of diet-induced obesity.MethodsBioinformatics tools were used to evaluate the correlations between hypothalamic Irx3 and neurotransmitters, markers of thermogenesis and obesity related phenotypes. Droplet-sequencing analysis in >20,000 hypothalamic cells was used to explore the types of hypothalamic cells expressing Irx3. Lentivirus was used to inhibit hypothalamic Irx3 and the resulting phenotype was studied.FindingsIRX3 is expressed predominantly in POMC neurons. Its expression is inhibited during prolonged fasting, as well as when mice are fed a high-fat diet. The partial inhibition of hypothalamic Irx3 using a lentivirus resulted in increased diet-induced body mass gain and adiposity due to increased caloric intake and reduced energy expenditure.InterpretationContrary to the results obtained when lean mice are submitted to complete inhibition of Irx3, partial inhibition of hypothalamic Irx3 in obese mice causes an exacerbation of the obese phenotype. These data suggest that at least some of the Irx3 functions in the hypothalamus are regulated according to a hormetic pattern, and modulation of its expression can be a novel approach to modifying the body's energy-handling regulation.FundSao Paulo Research Foundation grants 2013/07607-8 (LAV) and 2017/02983-2 (JDJ); NIH grants R01DK083567 (YBK).

Identification of nafamostat mesilate as an inhibitor of the fat mass and obesity-associated protein (FTO) demethylase activity

Nafamostat mesilate is a serine protease inhibitor and used for the treatment of pancreatitis and cancers. The fat mass and obesity-associated protein (FTO) was identified to be a demethylase and played an important role in physiological processes. The aim of this study was to examine the inhibition of Nafamostat mesilate on FTO demethylase activity. A combination of thermodynamic and enzymatic activity studies provides an insight into the recognition of Nafamostat mesilate by FTO. The binding of Nafamostat mesilate to FTO was driven by higher positive entropy changes and smaller negative enthalpy changes. The structural and thermodynamic information provides the basis for the design of more effective inhibitors for FTO. Our results might provide a novel target protein for Nafamostat mesilate.

The Fat Mass- and Obesity-Associated (FTO) Gene to Obesity: Lessons from Mouse Models.

Genetic variants at the fat mass- and obesity-associated (FTO) locus are strongly associated with obesity-related traits by regulating neighboring genes. Nevertheless, it is possible that FTO protein is directly involved in mechanisms regulating body composition and adiposity. Here, the in vivo biological functions of FTO in the risk for obesity were studied by reviewing murine models. The effects of the locus-specific manipulations of the murine Fto gene on metabolic-related phenotypes in genetically modified mouse models were reviewed and summarized into the following three categories: growth and body composition, eating behaviors, and metabolic homeostasis. The mouse models showed different phenotypes depending on target tissues and methods for gene manipulation. Mice harboring deletions or point mutations at the Fto locus had high metabolic rates, while FTO-overexpressing mice showed dyslipidemia. Both deletion and overexpression of the Fto gene led to abnormal eating behaviors. Intriguingly, several phenotypes were differently expressed depending on developmental timing of the genetic manipulations. For instance, a germ line deletion decreased total body fat mass, while the deletion in adult mice increased it. The results highlight that FTO is critical not only for body composition but also normal development, and its function might differ depending on the stage of development.

PO-294 Effects of HIIT on FTO protein expression and its relationship with glucose and fat metabolism

Objective FTO (Fat mass and obesity-associated) is associated with increased risk of obesity and type 2 diabetes incurrence. Studies have shown that the expression of FTO protein in skeletal muscle and adipose tissue is related to the oxidation rate of whole body substrate. With the increase of age, the body's carbohydrate oxidation rate decreases, the fat oxidation rate increases, and at the meanwhile the expression of FTO protein in skeletal muscle decreases and that in adipose increases. HIIT is very helpful for inhibiting obesity, insulin resistance and type 2 diabetes. So the purpose of this study is to investigate the effect of HIIT exercise on the expression of FTO protein in rats and its relationship to glucose and fat metabolism.
 Methods 20 Male, 3-week-old SD rats were randomly divided into two groups, each group has 10 rats. C group: sedentary; HIIT group: high-intensity intermittent training group (85% ~ 90% VO2max exercise for 6min, 50% VO2max exercise interval 4min, repeated 6 times. 5 times/week ,4 weeks). All subjects were maintained in a free facility with constant temperature of 25°C, light-dark cycle of 12/12 h and free access to water. 48 hours after the last exercise, all samples were taken with an overnight fast. The expression of FTO protein in skeletal muscle and adipose tissue was measured by Western Blot. Serum insulin was tested by ELISA; Estimation of blood glucose was tested by Glucose oxidase method.
 Results 1.The expression of FTO protein in skeletal muscle was significantly higher than that of group C (P <0.01); The expression of FTO protein in adipose tissue of HIIT group was significantly lower than that of group C (P <0.05); 2. Serum insulin levels of group HIIT was significantly lower than that of group C (p <0.01); And the blood glucose of group HIIT was significantly lower than that of group C (p <0.01). 3. Serum LDL-C of group HIIT was significantly lower than that of group C (p <0.01), and serum HDL-C of group HIIT was significantly higher than that of group C (p <0.01);4. Correlation analysis showed that serum insulin level was negatively correlated with skeletal muscle FTO protein expression (R = -0.454, p < 0.05). Correlation analysis showed that serum LDL-C levels was positively correlated with adipose tissue FTO protein expression (R=0.559, p < 0.05) and serum HDL-C levels was negatively correlated with adipose tissue FTO protein expression (R=-0.474, p < 0.05).
 Conclusions 1. HIIT can increase the protein expression of FTO in rat skeletal muscle and decrease the expression of FTO protein in adipose tissue; 2. HIIT can regulate glucose metabolism and lipid metabolism in rats; 3. The regulation of glucose metabolism by HIIT may be related to the increase of FTO protein expression in skeletal muscle. The regulation of lipid metabolism may be related to the reduction of FTO protein expression in adipose tissue.

The GDF11-FTO-PPARγ axis controls the shift of osteoporotic MSC fate to adipocyte and inhibits bone formation during osteoporosis

During osteoporosis, the shift of bone mesenchymal stem cell (BMSC) lineage commitment to adipocyte leads to the imbalance between bone mass and fat, which increases the risk of fracture. The mechanism underlying this process is not fully understood. Fat mass and obesity-associated protein (FTO) is an RNA demethylase that demethylates various methylated nucleic acids and participates in various physiological and pathological processes. Here we identified FTO as a regulator for BMSC fate determination during osteoporosis. FTO was up-regulated in bone marrow during aging or osteoporosis in human and mice in a GDF11(growth differentiation factor 11)-C/EBPα-dependent mechanism. The expression of FTO was also up-regulated during adipocyte differentiation of BMSCs whereas its expression was down-regulated during osteoblast differentiation. Gain-of-function and loss-of-function experiments showed that FTO favored the BMSCs to differentiate to adipocytes rather than osteoblasts. Further mechanism study demonstrated that FTO bound and demethylated the mRNA of the Peroxisome proliferator-activated receptor gamma (Pparg), leading to the increase in the expression of Pparg mRNA. Reversely, Pparg knockdown blocked the function of GDF11-FTO during osteoblast differentiation of BMSCs. Furthermore, conditionally genetic knockout of Fto in osteoblasts inhibited the development of osteopenia in mice. Collectively, our findings demonstrated that GDF11-FTO-Pparg axis promoted the shift of osteoporotic BMSC fate to adipocyte and inhibited bone formation during osteoporosis.

Development of formaldehyde dehydrogenase-coupled assay and antibody-based assays for ALKBH5 activity evaluation

N6-methyladenosine (m6A) is the most prevalent internal modification of eukaryotic messenger RNA (mRNA). Until now, two RNA demethylases have been identified, including FTO (fat mass and obesity-associated protein) and ALKBH5 (α-ketoglutarate-dependent dioxygenase alkB homologue 5). As a mammalian m6A demethylase, ALKBH5 significantly affects mRNA export and RNA metabolism as well as the assembly of mRNA processing factors in nuclear speckles, and ALKBH5 may play a significant role in these biological processes. Nevertheless, no modulator of ALKBH5 has been reported. The reason for that may be the lack of in vitro assays for ALKBH5 inhibitor screening. Herein, we describe the development of two homogeneous assays for ALKBH5 using N6-methyladenosine as substrate with different principles. Using ALKBH5 recombinant, we developed a formaldehyde dehydrogenase coupled fluorescence based assay and an antibody based assay for the activity evaluation of ALKBH5. These robust coupled assays are suitable for screening ALKBH5 inhibitors in 384-well format (Z’ factors of 0.74), facilitating the discovery of modulators in the quest for the regulation of biological processes.

Identification of Natural Compound Radicicol as a Potent FTO Inhibitor.

The fat mass and obesity-associated protein (FTO), as an m6A demethylase, is involved in many human diseases. Virtual screening and similarity search in combination with bioactivity assay lead to the identification of the natural compound radicicol as a potent FTO inhibitor, which exhibits a dose-dependent inhibition of FTO demethylation activity with an IC50 value of 16.04 μM. Further ITC experiments show that the binding between radicicol and FTO was mainly entropy-driven. Crystal structure analysis reveals that radicicol adopts an L-shaped conformation in the FTO binding site and occupies the same position as N-CDPCB, a previously identified small molecular inhibitor of FTO. Unexpectedly, however, the 1,3-diol group conserved in radicicol and N-CDPCB assumes strikingly different orientations for interaction with FTO. The identification of radicicol as an FTO inhibitor and revelation of its recognition mechanism not only opens the possibility of developing new therapeutic strategies for treatment of leukemia but also provide clues for elucidation of the acting mechanisms of radicicol, which is a possible clinical candidate worth in-depth study.

Critical Enzymatic Functions of FTO in Obesity and Cancer.

Fat mass and obesity-associated protein (FTO) single-nucleotide polymorphisms (SNPs) have been linked to increased body mass and obesity in humans by genome-wide association studies (GWAS) since 2007. Although some recent studies suggest that the obesity-related SNPs in FTO influence obesity susceptibility likely through altering the expression of the adjacent genes such as IRX3 and RPGRIP1L, rather than FTO itself, a solid link between the SNP risk genotype and the increased FTO expression in both human blood cells and fibroblasts has been reported. Moreover, multiple lines of evidence have demonstrated that FTO does play a critical role in the regulation of fat mass, adipogenesis, and body weight. Epidemiology studies also showed a strong association of FTO SNPs and overweight/obesity with increased risk of various types of cancers. As the first identified messenger RNA N6-methyladenosine (m6A) demethylase, FTO has been shown recently to play m6A-dependent roles in adipogenesis and tumorigenesis (especially in the development of leukemia and glioblastoma). Given the critical roles of FTO in cancers, the development of selective and effective inhibitors targeting FTO holds potential to treat cancers. This mini review discusses the roles and underlying molecular mechanisms of FTO in both obesity and cancers, and also summarizes recent advances in the development of FTO inhibitors.

Abstract 3321: Dysregulation of m6A RNA methylation regulators in colorectal cancer: Clinical implication as prognostic biomarkers and potential therapeutic targets

Abstract Purpose Among more than 100 known post-transcriptional modifications, N6-Methyladenosine (m6A) represents the most prevalent internal modification in mammalian mRNAs. This epigenomic modification is orchestrated by a distinct group of enzymes and cofactors, categorized as writers, erasers and readers. Writers consist of a multicomponent complex consisting of at least three proteins, namely methyltransferase-like 3 (METTL3), METTL14, and Wilms' tumor 1-associating protein (WTAP1). These m6A marks are erased by two RNA demethylases, fat mass and obesity-associated protein (FTO) and alkylated DNA repair protein alkB homolog 5 (ALKBH5). Lastly, YTHDF1/2/3 family genes constitute the ‘readers' that preferentially bind to RNA harboring m6A modification. Accumulating evidence suggests that m6a RNA methylation plays a crucial role in cancer progression. Herein, we for the first time elucidate the role of m6a regulators in colorectal cancer (CRC), in order to gain insights into their functional role and potential clinical significance in this malignancy. Experimental design Two large independent publicly available genome-wide mRNA expression datasets (N=509, TCGA and N=566, CIT) were used to study the expression of m6a RNA methylation regulators and their association with prognosis and gene expression based Consensus Molecular Subtypes (CMS) in CRC. Furthermore, we studied the tumorigenic properties of the RNA demethylase, FTO, by knockdown and overexpression strategies in CRC cells. Results The risk-score derived from a seven gene mRNA expression classifier consisting of METTL3, METTL14, WTAP, YTHDF1, YTHDF2, FTO and ALKBH5 was associated with poor disease-free survival in the training (TCGA) and validation (CIT) cohorts, with corresponding hazard ratios of 1.90 (95% CI: 1.24-2.92, p<0.002) and 1.86 (95% CI: 1.25-2.78, p<0.001) respectively. Furthermore, a high-m6a risk-score was significantly associated with a mesenchymal, CMS4 subtype, with FTO being the most overexpressed gene in CMS4 subtype of CRCs. Functionally, knockdown of the FTO gene in CMS4 cell lines (MDST8 and NCIH716) dramatically inhibited cellular proliferation, invasion and colony formation abilities. In contrast, overexpression of FTO in RKO cells resulted in enhanced proliferation, invasion and colony formation. Conclusions In summary, we for the first time report the dysregulation of m6a RNA methylation regulators in CRC, wherein we discovered their significant association with poor prognosis and a CMS4 CRC subtype, as well as elucidated the functional role of FTO in CRC cell lines. Besides the potential of utilizing this seven gene m6a regulator gene expression classifier to predict prognosis, our data suggest that targeting FTO might represent an attractive strategy for therapeutic intervention in patients with a mesenchymal CRC subtype with poor prognosis. Citation Format: Raju Kandimalla, Feng Gao, Chun Ruan, Michael Hsieh, Xin Wang, Ajay Goel. Dysregulation of m6A RNA methylation regulators in colorectal cancer: Clinical implication as prognostic biomarkers and potential therapeutic targets [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3321.

Glycogen synthase kinase-3 (GSK-3) activity regulates mRNA methylation in mouse embryonic stem cells

Glycogen synthase kinase-3 (GSK-3) activity regulates multiple signal transduction pathways and is also a key component of the network responsible for maintaining stem cell pluripotency. Genetic deletion of Gsk-3α and Gsk-3β or inhibition of GSK-3 activity via small molecules promotes stem cell pluripotency, yet the mechanism underlying the role for GSK-3 in this process remains ambiguous. Another cellular process that has been shown to affect stem cell pluripotency is mRNA methylation (m6A). Here, we describe an intersection between these components, the regulation of m6A by GSK-3. We find that protein levels for the RNA demethylase, FTO (fat mass and obesity-associated protein), are elevated in Gsk-3α;Gsk-3β-deficient mouse embryonic stem cells (ESCs). FTO is normally phosphorylated by GSK-3, and MS identified the sites on FTO that are phosphorylated in a GSK-3-dependent fashion. GSK-3 phosphorylation of FTO leads to polyubiquitination, but in Gsk-3 knockout ESCs, that process is impaired, resulting in elevated levels of FTO protein. As a consequence of altered FTO protein levels, mRNAs in Gsk-3 knockout ESCs have 50% less m6A than WT ESCs, and m6A-Seq analysis reveals the specific mRNAs that have reduced m6A modifications. Taken together, we provide the first evidence for how m6A demethylation is regulated in mammalian cells and identify a putative novel mechanism by which GSK-3 activity regulates stem cell pluripotency.

Abstract SY43-02: RNA modifications and cell identity: The importance of forgetting the past to embrace the future

Abstract Impaired gene regulation lies at the heart of many disorders, including cancer. Gene expression is controlled through multiple mechanisms that are coordinated to ensure the proper and timely expression of each gene. From transcription to translation, every step of mRNA processing is controlled by interaction between the mRNA transcript and regulatory factors, such as RNA binding proteins or small noncoding RNAs. We now understand that these regulatory interactions can be modulated by RNA modifications. Presence of a modified nucleotide can create, or abrogate, sites for regulatory interactions by at least two distinct mechanisms: by inducing changes in RNA structure or through direct recognition by the RNA binding protein of the modified nucleotide. The RNA modification N6-methyladenosine (m6A) is present in several classes of RNAs, including mRNAs and lncRNAs, where it is the most abundant internal RNA modification. In mRNAs and lncRNAs, the m6A modification is present in a well-defined RNA motif, RRACH (R = A or G, H = A, C or U), and is enriched at specific transcript landmarks such as the last transcribed exon, near the STOP codon. The METTL3/METTL14 heterodimer is at the core of the large protein complex that catalyzes addition of m6A to mRNAs. To date, two enzymes capable of removing m6A have been identified: fat mass and obesity-associated protein (FTO) and alkB homolog 5 (ALKBH5). Our studies on the role of m6A in ESCs revealed that thousands of messenger and long noncoding RNAs are m6A-modified, including transcripts encoding core pluripotency transcription factors such as Nanog, Sox2, and Myc. Our observations suggest that m6A marks unstable transcripts, including transcripts that need to be turned over upon differentiation. Loss of Mettl3 did not affect self-renewal, but resulted in impaired ESC exit from self-renewal towards differentiation to several lineages. Mettl3 KO cells did not differentiate into cardiomyocites or neurons in vitro, and subcutaneous injection of Mettl3 KO cells formed tumors consistent in morphology with teratomas that tended to be larger than tumors derived from wild-type cells. Furthermore, teratomas derived from Mettl3 KO cells were predominantly composed of poorly differentiated cells with very high mitotic indices and numerous apoptotic bodies, whereas wild-type cells differentiated predominantly into neuroectoderm. The role of m6A in facilitating transition between cell states is not limited to embryonic stem cells. The m6A interacting protein YTHDC2 is required for a clean transition from mitosis to meiosis in male and female germ cells. Ythdc2-/- male germ cells attempt to enter meiotic prophase but appear to have a mixed identity, maintaining expression of mitotic Cyclin A2 and failing to properly express many meiotic markers. Instead of executing meiotic prophase, the cells attempt an abnormal mitotic-like division, then quickly die. YTHDC2 is required during the early stages of meiotic entry in mice to both downregulate the previous mitotic program and facilitate proper expression of meiotic and differentiation genes. We propose that m6A makes the transition between cell states possible by facilitating a reset mechanism between stages, as occurs in ESCs and during gametogenesis. In contrast to epigenetic mechanisms that provide cellular memory of gene expression states, m6A enforces the transience of genetic information, helping cells to forget the past and thereby embrace the future. Citation Format: Pedro J. Batista. RNA modifications and cell identity: The importance of forgetting the past to embrace the future [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr SY43-02.

M6A RNA Methylation Controls Neural Development and Is Involved in Human Diseases.

RNA modifications are involved in many aspects of biological functions. N6-methyladenosine (m6A) is one of the most important forms of RNA methylation and plays a vital role in regulating gene expression, protein translation, cell behaviors, and physiological conditions in many species, including humans. The dynamic and reversible modification of m6A is conducted by three elements: methyltransferases ("writers"), such as methyltransferase-like protein 3 (METTL3) and METTL14; m6A-binding proteins ("readers"), such as the YTH domain family proteins (YTHDFs) and YTH domain-containing protein 1 (YTHDC1); and demethylases ("erasers"), such as fat mass and obesity-associated protein (FTO) and AlkB homolog 5 (ALKBH5). In this review, we summarize the current knowledge on mapping mRNA positions of m6A modification and revealing molecular processes of m6A. We further highlight the biological significance of m6A modification in neural cells during development of the nervous system and its association with human diseases. m6A RNA methylation is becoming a new frontier in neuroscience and should help us better understand neural development and neurological diseases from a novel point of view.

FTO, m6 Am , and the hypothesis of reversible epitranscriptomic mRNA modifications.

The fate of mRNA is regulated by epitranscriptomic nucleotide modifications, the most abundant of which is N6 -methyladenosine (m6 A). Although the pattern and distribution of m6 A in mRNA is mediated by specific methyltransferases, a recent hypothesis is that specific demethylases or 'erasers' allow m6 A to be dynamically reversed by signaling pathways. In this Review, we discuss the data in support and against this model. New insights into the function of fat mass and obesity-associated protein (FTO), the original enzyme thought to be an m6 A eraser, reveal that its physiologic target is not m6 A, but instead is N6 ,2'-O-dimethyladenosine (m6 Am ). Another m6 A demethylase, ALKBH5, appears to have functions limited to sperm development in normal mice. Overall, the majority of the data suggest that m6 A is generally not reversible, although m6 A may be susceptible to demethylation in pathophysiological states such as cancer.