As one amino acid can often be encoded by multiple co-dons,the genetic code is redundant,which accounts for synonymous mutations in protein-coding regions.1 Since synonymous mutations do not cause any alterations in amino acid sequence,it was previously believed that they do not change the structure and function of the proteins and thus are functionally silent.1

Background While liver cancer stem cells (CSCs) play a crucial role in hepatocellular carcinoma (HCC) initiation, progression, recurrence, and treatment resistance, the mechanism underlying liver CSC self-renewal remains elusive. We aim to characterize the role of Methyltransferase 16 (METTL16), a recently identified RNA N 6 -methyladenosine (m 6 A) methyltransferase, in HCC development/maintenance, CSC stemness, as well as normal hepatogenesis. Methods Liver-specific Mettl16 conditional KO (cKO) mice were generated to assess its role in HCC pathogenesis and normal hepatogenesis. Hydrodynamic tail-vein injection (HDTVi)-induced de novo hepatocarcinogenesis and xenograft models were utilized to determine the role of METTL16 in HCC initiation and progression. A limiting dilution assay was utilized to evaluate CSC frequency. Functionally essential targets were revealed via integrative analysis of multi-omics data, including RNA-seq, RNA immunoprecipitation (RIP)-seq, and ribosome profiling. Results METTL16 is highly expressed in liver CSCs and its depletion dramatically decreased CSC frequency in vitro and in vivo. Mettl16 KO significantly attenuated HCC initiation and progression, yet only slightly influenced normal hepatogenesis. Mechanistic studies, including high-throughput sequencing, unveiled METTL16 as a key regulator of ribosomal RNA (rRNA) maturation and mRNA translation and identified eukaryotic translation initiation factor 3 subunit a ( eIF3a ) transcript as a bona-fide target of METTL16 in HCC. In addition, the functionally essential regions of METTL16 were revealed by CRISPR gene tiling scan, which will pave the way for the development of potential inhibitor(s). Conclusions Our findings highlight the crucial oncogenic role of METTL16 in promoting HCC pathogenesis and enhancing liver CSC self-renewal through augmenting mRNA translation efficiency.

ObjectiveThis study aims to assess the association between the non-HDL-c/HDL-c ratio and stroke risk among middle-aged and older adults participating in the China Health and Retirement Longitudinal Study (CHARLS).MethodsThis study conducted a prospective cohort analysis, enrolling a total of 10,183 participants who met the designated criteria from CHARLS between 2011 and 2012. We then used the Cox proportional-hazards regression model to explore the relationship between baseline non-HDL-c/HDL-c ratio and stroke risk. Using a Cox proportional hazards regression with cubic spline function, we were able to identify the non-linear relationship between the non-HDL-c/HDL-c ratio and stroke occurrence. A series of sensitivity analyses were also carried out.ResultsThe average age of the participants included in this study was 59.16 ± 9.35 years, and 4,735 individuals (46.68%) were male. Over a median follow-up period of 7.0 years, a total of 1,191 people (11.70%) experienced a stroke. Using a Cox proportional hazards regression model that was fully adjusted, we found no statistically significant correlation between the non-HDL-c/HDL-c ratio and the risk of stroke (HR=1.022; 95% CI 0.964, 1.083). Nevertheless, we did observe a non-linear relationship and saturation effect between the non-HDL-c/HDL-c ratio and stroke. Employing a two-piece Cox proportional hazards regression model and a recursive algorithm, we determined an inflection point of 2.685 for the non-HDL-c/HDL-c ratio. In instances where the non-HDL-c/HDL-c ratio fell below 2.685, for every 1-unit decrease in the non-HDL-c/HDL-c ratio, the likelihood of stroke decreased by 21.4% (HR=1.214, 95% CI: 1.039-1.418). In contrast, when the non-HDL-c/HDL-c ratio exceeded 2.685, there was no statistically significant change in the risk of stroke for each unit decrease in the non-HDL-c/HDL-c ratio (HR: 0.967, 95% CI: 0.897-1.042). The consistency of these findings across multiple sensitivity analyses suggests their robustness.ConclusionThis study unveils a non-linear relationship between the non-HDL-c/HDL-c ratio and stroke risk in middle-aged and older adults in China. Specifically, when the non-HDL-c/HDL-c ratio was below 2.685, a significant and clearly positive association with stroke risk was observed. Additionally, maintaining the non-HDL-c/HDL-c ratio below 2.685 could potentially lead to a substantial reduction in the risk of stroke.

Epigenetic dysregulation has been reported in multiple cancers including leukemias. Nonetheless, the roles of the epigenetic reader Tudor domains in leukemia progression and therapy remain unexplored. Here, we conducted a Tudor domain–focused CRISPR screen and identified SGF29, a component of SAGA/ATAC acetyltransferase complexes, as a crucial factor for H3K9 acetylation, ribosomal gene expression, and leukemogenesis. To facilitate drug development, we integrated the CRISPR tiling scan with compound docking and molecular dynamics simulation, presenting a generally applicable strategy called CRISPR-Scan Assisted Drug Discovery (CRISPR-SADD). Using this approach, we identified a lead inhibitor that selectively targets SGF29’s Tudor domain and demonstrates efficacy against leukemia. Furthermore, we propose that the structural genetics approach used in our study can be widely applied to diverse fields for de novo drug discovery.

As the most common internal modification of mRNA, N6-methyladenosine (m6A) and its regulators modulate gene expression and play critical roles in various biological and pathological processes including tumorigenesis. It was reported previously that m6A methyltransferase (writer), methyltransferase-like 3 (METTL3) adds m6A in primary microRNAs (pri-miRNAs) and facilitates its processing into precursor miRNAs (pre-miRNAs). However, it is unknown whether m6A modification also plays a role in the maturation process of pre-miRNAs and (if so) whether such a function contributes to tumorigenesis. Here, we found that YTHDF2 is aberrantly overexpressed in acute myeloid leukemia (AML) patients, especially in relapsed patients, and plays an oncogenic role in AML. Moreover, YTHDF2 promotes expression of miR-126-3p (also known as miR-126, as it is the main product of precursor miR-126 (pre-miR-126)), a miRNA that was reported as an oncomiRNA in AML, through facilitating the processing of pre-miR-126 into mature miR-126. Mechanistically, YTHDF2 recognizes m6A modification in pre-miR-126 and recruits AGO2, a regulator of pre-miRNA processing, to promote the maturation of pre-miR-126. YTHDF2 positively and negatively correlates with miR-126 and miR-126's downstream target genes, respectively, in AML patients, and forced expression of miR-126 could largely rescue YTHDF2/Ythdf2 depletion-mediated suppression on AML cell growth/proliferation and leukemogenesis, indicating that miR-126 is a functionally important target of YTHDF2 in AML. Overall, our studies not only reveal a previously unappreciated YTHDF2/miR-126 axis in AML and highlight the therapeutic potential of targeting this axis for AML treatment, but also suggest that m6A plays a role in pre-miRNA processing that contributes to tumorigenesis.

BACKGROUND:Despite the advent of combination antiretroviral therapy, people living with human immunodeficiency virus (PLWH) are at an increased risk for cardiac disease. PURPOSE:To explore the presence and extent of diastolic atrial and left ventricular dysfunction in PLWH using cardiac MRI in correlation with clinical markers of disease activity. STUDY TYPE:Prospective. POPULATION:A total of 163 participants comprising 101 HIV-infected individuals (age: 52 years [42-62 years]; 92% male) and 62 age- and sex-matched healthy volunteers (age: 51 years [30-72 years]; 85% male). FIELD STRENGTH/SEQUENCE:3.0 T, cardiac MRI including balanced steady-state free precession (SSFP) for the short-axis, two-, three-, and four-chamber views were performed. ASSESSMENT:Assessment of cardiac function and strain analysis were accomplished by CVI42 software. Blood samples for CD4+ T cells and cardiac risk factors were also collected before MRI. STATISTICAL TESTS:Independent t tests, Mann-Whitney U test, Pearson's correlation analysis, and multivariate linear analyses (significance level: P < 0.05). RESULTS:PLWH had a significantly larger left atrial volume maximum index (LAVImax: 32.6 ± 8.7 vs. 28.7 ± 8.1 mL/m2), minimum (LAVImin: 14.8 ± 5.5 vs. 11.5 ± 5.4 mL/m2,), and prior to atrial contraction (LAVIpre-a: 23.4 ± 6.7 vs. 19.7 ± 7.2 mL/m2) as compared to healthy volunteers. The LA reservoir (LAtEF: 55.0 ± 10.2 vs. 61.4 ± 10.4; Sls: 29.0 ± 8.1 vs. 33.8 ± 11.8), conduit (LApEF: 28.4 ± 8.2 vs. 32.3 ± 11.3, P = 0.01; Sle: 16.3 ± 6.5 vs. 18.9 ± 8.2), and booster pump function (LAaEF: 37.4 ± 12.4 vs. 42.7 ± 13.1, P = 0.01, Sla: 12.7 ± 5.1 vs. 14.9 ± 5.7) were all significant impaired in PLWH. Global circumferential left ventricular diastolic strain rate (LVGCS-d) was significantly lower in the HIV patients. Multivariate analysis results showed that Nadir CD4+ T cells had a significant adverse association with LVGCS-d (β = 0.51). CONCLUSION:LA structure abnormalities and LV diastolic dysfunction were manifested in PLWH, with Nadir CD4+ T cell counts potentially serving as a risk factor for early cardiac diastolic dysfunction. LEVEL OF EVIDENCE:2 TECHNICAL EFFICACY: Stage 3.

In many ways, circular RNAs (circRNAs) have been demonstrated to be crucial in the onset and advancement of cancer throughout the last ten years and have become a new focus of intense research in the field of RNAs. Accumulating studies have demonstrated that circRNAs can regulate parental gene expression via a variety of biological pathways. Furthermore, research into the complex interactions between circRNAs and their parental genes will shed light on their biological roles and open up new avenues for circRNAs' potential clinical translational uses. However, to date, multi-dimensional cross-talk between circRNAs and parental genes have not been systematically elucidated. Particularly intriguing is circRNA's exploration of tumor targeting, and potential therapeutic uses based on the parental gene regulation perspective. Here, we discuss their biogenesis, take a fresh look at the molecular mechanisms through which circRNAs control the expression of their parental genes in cancer. We further highlight We further highlight the latest circRNA clinical translational applications, including prognostic diagnostic markers, cancer vaccines, gDNA, and so on. Demonstrating the potential benefits and future applications of circRNA therapy.

Focal cortical dysplasia (FCD) is a neurological disorder distinguished by faulty brain cell structure and development. Repetitive and uncontrollable seizures may be linked to FCD's aberrant cortical thickness, gyrification, and sulcal depth. Quantitative cortical surface analysis is a crucial alternative to ineffective visual inspection. This study recruited 42 subjects including 22 FCD patients who underwent surgery and 20 healthy controls (HC). For the FCD patients, T1-weighted and PET images were obtained by a PET-MRI scanner, and the confirmed epileptogenic zone (EZ) was collected from postsurgical follow-up. For the HCs, CT and PET images were obtained by a PET-CT scanner. Cortical thickness, gyrification index, and sulcal depth were calculated using a computational anatomical toolbox (CAT12). A cluster-based analysis is carried out to determine each FCD patient's aberrant cortical surface. After parcellating the cerebral cortex into 68 regions by the Desikan-Killiany atlas, a region of interest (ROI) analysis was conducted to know whether the feature in the FCD group is significantly different from that in the HC group. Finally, the features of all ROIs were utilised to train a support vector machine classifier (SVM). The classification performance is evaluated by the leave-one-out cross-validation. The cluster-based analysis can localize the EZ cluster with the highest accuracy of 54.5 % (12/22) for cortical thickness, 40.9 % (9/22) and 13.6 % (3/22) for sulcal depth and gyrification, respectively. Moderate concordance (Kappa, 0.6) is observed between the confirmed EZs and identified clusters by using the cortical thickness. Fair concordance (Kappa, 0.3) and no concordance (Kappa, 0.1) is found by using sulcal depth and gyrification. Significant differences are found in 46 of 68 regions (67.7 %) for the three measures. The trained SVM classifier achieved a prediction accuracy of 95.5 % for the cortical thickness, while the sulcal depth and the gyrification obtained 86.0 % and 81.5 %. Cortical thickness, as determined by quantitative cortical surface analysis of PET data, has a greater ability than sulcal depth and gyrification to locate aberrant EZ clusters in FCD. Surface measures might be different in many regions for FCD and HC. By integrating machine learning and cortical morphologies features, individual prediction of FCD seems to be feasible.

N7-methylguanosine (m7G) modification, routinely occurring at mRNA 50 cap or within tRNAs/rRNAs, also ex-ists internally in messenger RNAs (mRNAs). Although m7G-cap is essential for pre-mRNA processing and protein synthesis, the exact role of mRNA internal m7G modification remains elusive. Here, we report that mRNA internal m7G is selectively recognized by Quaking proteins (QKIs). By transcriptome-wide profiling/ mapping of internal m7G methylome and QKI-binding sites, we identified more than 1,000 high-confidence m7G-modified and QKI-bound mRNA targets with a conserved "GANGAN (N = A/C/U/G)"motif. Strikingly, QKI7 interacts (via C terminus) with the stress granule (SG) core protein G3BP1 and shuttles internal m7G-modified transcripts into SGs to regulate mRNA stability and translation under stress conditions. Specifically, QKI7 attenuates the translation efficiency of essential genes in Hippo signaling pathways to sensitize cancer cells to chemotherapy. Collectively, we characterized QKIs as mRNA internal m7G-binding proteins that modulate target mRNA metabolism and cellular drug resistance.

Background:This study aimed to explore the value of cardiac magnetic resonance tissue tracking (CMR-TT) technology in evaluating heart failure with preserved ejection fraction (HFpEF) in patients with chronic myocardial infarction (CMI).Methods:Between June 2016 and March 2022, we included a consecutive series of 92 patients with CMI and 40 healthy controls in this retrospective study. The CMI patients enrolled were divided into different subgroups [HFpEF-CMI group (n=54) and non- heart failure (HF)-CMI group (n=38)] according to the Heart Failure Association (HFA)-PEFF (step 1: P, pre-test assessment; step 2: E, echocardiography and natriuretic peptide score; step 3: F1, functional testing; step 4: F2, final aetiology) diagnostic algorithm. CMR scan was performed at the First Hospital of China Medical University. Quantitative measurements of myocardial damage, such as myocardial strain parameters of both ventricles derived by CMR-TT and infarct size and transmurality by late gadolinium enhancement (LGE), were assessed. One-way analysis of variance, independent samples t-test, and rank sum test were used to compare myocardial impairment among groups. Pearson or Spearman correlation coefficient was used to measure correlations between left ventricular (LV) strains and clinical and functional parameters. Logistic regression analysis and receiver operating characteristic (ROC) curve were performed to identify the best parameter for diagnosing HFpEF-CMI.Results:HFpEF-CMI patients demonstrated significantly impaired LV strains and strain rates in all of the three directions (radial, circumferential and longitudinal) compared to non-HF-CMI patients and healthy controls (P<0.001 for all), whereas only global longitudinal strain (GLS) was significantly impaired in HFpEF-CMI patients vs. controls for right ventricular strain parameters (P<0.001). LV strains showed moderate correlation with N-terminal pro-brain natriuretic peptide (radial, circumferential and longitudinal strain, R=-0.401, R=0.408, R=0.407, respectively, P<0.001 for all). LV strains in the three directions (radial, circumferential and longitudinal) [area under ROC curve (AUC) =0.707, 95% confidence interval (CI): 0.603-0.797; AUC =0.708, 95% CI: 0.604-0.798; AUC =0.731, 95% CI: 0.628-0.818; respectively, P<0.01 for all] were discriminators for HFpEF-CMI and non-HF-CMI. LV strains and myocardial infarction volume were independent factors in multi-logistic regression analysis after adjusting for body mass index, age, and sex (P<0.05 for all).Conclusions:CMR-TT provides clinicians with useful additional imaging parameters to facilitate the assessment of CMI patients with HFpEF. LV strain parameters can detect early cardiac insufficiency in patients with HFpEF-CMI and have potential value for discriminating between HFpEF and non-HF patients post-CMI.

ObjectiveEpilepsy is considered as a neural network disorder. Seizure activity in epilepsy may disturb brain networks and damage brain functions. We propose using resting-state functional magnetic resonance imaging (rs-fMRI) data to characterize connectivity patterns in drug-resistant epilepsy.MethodsThis study enrolled 47 participants, including 28 with drug-resistant epilepsy and 19 healthy controls. Functional and effective connectivity was employed to assess drug-resistant epilepsy patients within resting state networks. The resting state functional connectivity (FC) analysis was performed to assess connectivity between each patient and healthy controls within the default mode network (DMN) and the dorsal attention network (DAN). In addition, dynamic causal modeling was used to compute effective connectivity (EC). Finally, a statistical analysis was performed to evaluate our findings.ResultsThe FC analysis revealed significant connectivity changes in patients giving 64.3% (18/28) and 78.6% (22/28) for DMN and DAN, respectively. Statistical analysis of FC was significant between the medial prefrontal cortex, posterior cingulate cortex, and bilateral inferior parietal cortex for DMN. For DAN, it was significant between the left and the right intraparietal sulcus and the frontal eye field. For the DMN, the patient group showed significant EC connectivity in the right inferior parietal cortex and the medial prefrontal cortex for the DMN. There was also bilateral connectivity between the medial prefrontal cortex and the posterior cingulate cortex, as well as between the left and right inferior parietal cortex. For DAN, patients showed significant connectivity in the right frontal eye field and the right intraparietal sulcus. Bilateral connectivity was also found between the left frontal eye field and the left intraparietal sulcus, as well as between the right frontal eye field and the right intraparietal sulcus. The statistical analysis of the EC revealed a significant result in the medial prefrontal cortex and the right intraparietal cortex for the DMN. The DAN was found significant in the left frontal eye field, as well as the left and right intraparietal sulcus.ConclusionOur results provide preliminary evidence to support that the combination of functional and effective connectivity analysis of rs-fMRI can aid in diagnosing epilepsy in the DMN and DAN networks.

TET2 is recurrently mutated in acute myeloid leukemia (AML) and its deficiency promotes leukemogenesis (driven by aggressive oncogenic mutations) and enhances leukemia stem cell (LSC) self-renewal. However, the underlying cellular/molecular mechanisms have yet to be fully understood. Here, we show that Tet2 deficiency significantly facilitates leukemogenesis in various AML models (mediated by aggressive or less aggressive mutations) through promoting homing of LSCs into bone marrow (BM) niche to increase their self-renewal/proliferation. TET2 deficiency in AML blast cells increases expression of Tetraspanin 13 (TSPAN13) and thereby activates the CXCR4/CXCL12 signaling, leading to increased homing/migration of LSCs into BM niche. Mechanistically, TET2 deficiency results in the accumulation of methyl-5-cytosine (m5C) modification in TSPAN13 mRNA; YBX1 specifically recognizes the m5C modification and increases the stability and expression of TSPAN13 transcripts. Collectively, our studies reveal the functional importance of TET2 in leukemogenesis, leukemic blast cell migration/homing, and LSC self-renewal as an mRNA m5C demethylase.

Background: Anthracyclines are known to be associated with chemotherapy-induced cardiotoxicity. Limited data focus on dynamic myocardial injury during the course of chemotherapy in patients with breast cancer.Purpose: To investigate the variation of tissue characterization and myocardial deformation derived by cardiac MRI during anthracycline chemotherapy.Study Type: Prospective.Population: Fifty-eight female breast cancer patients (mean age: 52.82 +/- 2.61 years) were enrolled.Field Strength/Sequence: A 3.0-T, cardiac MRI including cine balanced steady-state free precession, a modified Looker Locker inversion recovery (MOLLI), and a fast spin echo (FSE) T2-weighted sequences were performed.Assessment: Cardiac MRI was performed baseline and after two, four, and six cycles of chemotherapy. Assessment of global longitudinal strain (GLS), global circumstance strain (GCS), global radial strain (GRS), and strain rate (GLS-s, GCS-s, GRS-s) and T1, T2 and T2* were accomplished by CVI42. The anthracycline dose and risk factors were also collected before each cardiac MRI.Statistical Tests: Analysis of variance (ANOVA) for repeated measures was used to compare the changes in LVEF cardiac function, strain and T1/T2/T2* parameters over time. Pearson correlation analyses were performed to estimate the potential associations between differences in myocardial characteristics (delta) and the chemotherapy cycle. A P value < 0.05 was considered statistically significant.Results: LVEF was not significantly different from pretreatment MRI regarding each cycle of chemotherapy (P = 0.54). Compared with baseline, patients had significantly lower GLS (-15.85% +/- 0.83%,-14.50% +/- 0.88%,-12.34% +/- 1.01% vs. -18.82% +/- 0.92%) and GLS-s (-0.71% +/- 0.07%,-0.65% +/- 0.05%,-0.64% +/- 0.04% vs. -0.95 +/- 0.06%) and increased T2 values (57.21 +/- 4.27 msec, 58.60 +/- 3.93 msec, 58.10 +/- 3.17 msec vs. 43.88 +/- 3.28 msec) at two, four and six cycles of chemotherapy treatment. delta GLS and delta GLS-s were significantly associated with the chemotherapy cycle (correlation coefficients for GLS = 0.75, GLS-s = 0.75).Data Conclusion: Cardiac MRI can precisely detect the dynamic changes of anthracycline-induced subclinical myocardial injury that is represented as a gradually decrease in GLS and GLS-s. These parameters may provide new insight for monitoring risk and therapy in patients with breast cancer.

Background: LA function has been recognized as a significant prognostic marker in many cardiovascular diseases. Cardiovascular magnetic resonance feature tracking (CMR-FT) represents a promising technique for left atrial function evaluation. The size and location of myocardial infarction are important factors in the cause of adverse left ventricular remodeling, but the effect on the left atriam is unclear. Purpose: to investigate the effect of location and size of previous myocardial infarction (MI) on LA function using CMR-FT. Study type: retrospective. Population: patients formerly diagnosed with anterior MI (n = 42) or non-anterior MI (n = 40) and healthy controls (n = 47). Field Strength/Sequence: a 3.0T MR, Steady state free precession (SSFP), Phase-sensitive inversion recovery (PSIR). Assessment: infarct location and size were assigned and quantified by late-gadolinium enhancement (LGE) imaging. LA performance was analyzed using CMR-FT in 2- and 4-chamber cine images, including LA reservoir, conduit and booster pump function. Statistics: descriptive statistics, ANOVA with post Bonferroni correction, Kruskal–Wallis H, Spearman’s correlation, intraclass correlation coefficient. Results: Anterior MI patients had impaired LA reservoir function (LATEF, εs, SRs), conduit function (LAPEF, εe, SRs) and booster pump function (LAAEF, εa) compared with controls (p < 0.05). Non-anterior MI patients had impaired LA strain (εs, εe, εa; p < 0.05) but preserved LAEFs (p > 0.05). After adjusting the area of MI, there was no significant difference in the LA morphology and function between the anterior and non-anterior wall groups. Stratification analysis by MI size revealed that LA volumes and LAEFs were unchanged in patients with MI size ≤ 15% compared with controls (p > 0.05); only εs and εe were decreased (p < 0.05). Increased LAVIpre-a, LAVImin and decreased LATEF, and LAAEF were found in patients with MI size > 15% compared with the MI size ≤ 15% group (p < 0.05). LVSVI, εs and MI size were significant correlated with LAVI pre-a in multiple stepwise regression analysis. Data conclusions: The location of myocardial infarction is not a major factor affecting the morphology and function of the left atrium. Patients with MI size > 15% experience more pronounced post-infarction LA remodeling and dysfunction than MI size ≤ 15% patients.

Metastatic triple negative breast cancer (TNBC) patients have a 5-year survival rate of just 12%. Therefore, there is an urgent need to discover novel molecular mechanisms that contribute to TNBC metastasis such as N6-methyladenosine (m6A) RNA modification. To do this, we conducted a selective CRISPR-cas9 screen of 14 m6A-regulators and identified an m6A reader protein, YTHDC1, as an essential gene for the survival of MDA-MB-231 cells. The Cancer Genome Atlas (TCGA) dataset showed that patients with poorer prognosis had elevated YTHDC1 expression, which supports a tumor-promoting role of YTHDC1. In vitro, genetic depletion of YTHDC1 significantly impaired Transwell migration and invasion of human TNBC (MDA-MB-231 and SUM159) cells. In vivo, YTHDC1 overexpressing TNBC cells engrafted into the mammary fat pad of NSG mice formed greater numbers and larger sizes of lung metastatic nodules compared to control. Conversely, YTHDC1 knockout (KO) cells produced fewer and smaller lung metastatic nodules compared to control. Together, our in vitro and in vivo data demonstrate YTHDC1’s role in promoting TNBC metastasis. To identify high confidence YTHDC1 targets, we conducted three layers of transcriptome-wide sequencing with MDA-MB-231 cells: RNA-seq (profiling differential gene expression and nuclear export), m6A-seq (profiling m6A methylation in mRNA), and RIP-seq (profiling YTHDC1 binding sites in mRNA). Pathway analysis of differentially expressed genes in MDA-MB-231 YTHDC1 KO cells identified pathways related to metastasis and TGF-β signaling, which is consistent with our phenotypic observations. Overlapping the three sequencing datasets identified SMAD3 as a YTHDC1 target mRNA. Mechanistically, YTHDC1 KO impairs the nuclear export of SMAD3 mRNA, resulting in reduced protein levels of this critical effector of TGF-β signaling. YTHDC1 KO significantly reduced Transwell migration and invasion of TGF-β induced TNBC cells and the expression of TGF-β responsive genes such as SNAIL, IL11, FN, and CTGF. Overexpression of SMAD3 was able to rescue the impaired migration and invasion of YTHDC1 KO TNBC cells and restore the expression of TGF-β responsive genes following TGF-β treatment. We also demonstrated that the oncogenic role of YTHDC1 is mediated through its recognition of m6A, as m6A-binding defective mutants of YTHDC1 were unable to rescue impaired Transwell migration and invasion of YTHDC1 KO TNBC cells. In summary, our study reveals that TNBC metastasis is mediated at least in part through YTHDC1’s function in promoting SMAD3 expression to augment the TGF-β signaling cascade. Citation Format: Brandon Tan, Wei Liu, Keren Zhou, Emily Prince, Lu Yang, Yangchan Li, Ying Qing, Li Han, Zhicong Zhao, Rui Su, Chun-Wei Chen, Jianjun Chen. The N6-methyladenosine reader YTHDC1 is essential for TGF-beta-mediated metastasis of triple negative breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2188.

In eukaryotes, gene expression is highly orchestrated not only by genomic promoters and enhancers but also by covalent modifications added to either chromatin or RNAs. Traditionally, “epigenetics” refers to the chemical modifications that govern heritable changes in gene expression independent of the DNA sequence; “epitranscriptomics” indicates the covalent decorations in RNA, which plays a central role in posttranscriptional gene regulation. To date, >170 RNA chemical modifications have been characterized. Most of these modifications were originally identified in highly abundant noncoding RNA species, such as ribosomal RNAs (rRNAs), transfer RNAs (tRNAs), and small nuclear RNA (snRNAs). Recently, the substantial advances in high-throughput sequencing and analytical chemistry have enabled the precise detection and characterization of chemical modifications in messenger RNA (mRNA). Indeed, a considerable number of mRNA decorations have been documented, including N6-methyladenosine (m6A); N1-methyladenosine (m1A); N6,2′-O-dimethyladenosine (m6Am); 3-methylcytidine (m3C); 5-methylcytidine (m5C); 5-hydroxymethylcytidine (hm5C); N4-acetylcytidine (ac4C); Adenosine-to-inosine (A-to-I) editing; pseudouridine (Ψ); N7-methylguanosine (m7G) and 2′-O-methylated nucleotides (Nm) (Fig. 1A). The studies from us and other researchers have unveiled that mRNA modifications play important roles in myeloid malignancies.1–11 Here, we highlight recent findings focusing on the functions and regulatory mechanisms of mRNA modifications (with an emphasis on m6A) and provide our insights to better elucidate epitranscriptomics during leukemogenesis.Figure 1.: The covalent chemical modifications in mRNA. (A) A schematic view of chemical structures of RNA modifications (m6Am, m1A, A-to-I, ac4C, m5C, Ψ, m6A, hm5C, m3C, m7G and Nm) in mRNA. (B) The regulation of m6A by “writers,” “erasers,” and “readers.” The m6A modification is installed by writers, multicomponent m6A MTC (composed of METTL3, METTL14, WTAP, RBM15/15B, KIAA1429, and ZC3H13) or METTL16 alone. The two demethylases (eraser), FTO and ALKBH5, remove m6A modifications. The m6A residue is recognized by the three main classes, including the YTH domain family, IGF2BP family and HNRP family. (C) ADAR enzymes catalyze the A-to-I hydrolytic deamination reaction. (D) The NSUN methyltransferases and DNMT2 catalyze methylation of cytosine-5 and TET family function as dioxygenases catalyzing m5C to hm5C. (E) The formation of m7G is catalyzed by methyltransferases complex, which is composed of METTL1 and WDR4. ADAR = adenosine deaminases acting on RNA.m6A, the most abundant and best-characterized mRNA modification, was initially discovered in 1974.12 Owing to the lack of effective and sensitive technologies, recognition of functional significance of m6A has been largely absent over the past decades. Until 2011, the identification of fat mass- and obesity-associated protein (FTO) as the first m6A demethylase13 has profoundly revived the interest in the biological relevance of m6A and RNA chemical modifications. Both FTO and AlkB homolog 5 (ALKBH5) can catalyze the demethylation of m6A, demonstrating the reversible and dynamic posttranscriptional modification in RNA (Fig. 1B). Methyltransferase-like 3 (METTL3) forms a heterodimer with METTL14 to deposit m6A residues in mRNAs. METTL3 acts as the catalytically active methyltransferase with METTL14 serving as an allosteric activator to enhance catalysis.14 The large methyltransferase complex (MTC) contains additional subunits, including Wilms tumor 1-associated protein (WTAP), zinc finger CCCH-type contain 13 (ZC33H13), vir like m6A methyltransferase associated (VIRMA) and RNA-binding motif protein 15/15B (RBM15/15B) (Fig. 1B). More recently, METTL16 has been identified as a new m6A writer, which could exert its enzymatic activity independently and catalyze m6A formation on limited mRNAs and noncoding RNAs15,16 (Fig. 1B). Characterization of m6A “reader” proteins, including YT521-B homology (YTH) domain, insulin-like growth factor 2 mRNA-binding protein (IGF2BP) family, and heterogeneous nuclear ribonucleoprotein (HNRP) family, has provided valuable insights into understanding the underlying mechanism of m6A-mediated posttranscriptional gene regulation. The dysregulation of m6A regulators has been reported to be extensively involved in the pathogenesis of myeloid malignancies. We first reported that FTO is highly expressed in acute myeloid leukemia (AML) with t(11q23)/MLL-rearrangements, t(15;17)/PML-RARA, FLT3-ITD and/or NPM1 mutations, and enhances leukemic malignant transformation and leukemogenesis as an m6A demethylase.3 Subsequently, we showed that R-2-hydroxyglutarate (R-2HG), an oncometabolite of mutant isocitrate dehydrogenase 1/2 (IDH1/2), exerts a broad and intrinsic antileukemic activity through competitively inhibiting the demethylase activity of FTO.1 Such identification indicates that FTO might act as a druggable target for leukemia therapy. Moreover, FTO drives and sustains tyrosine kinase inhibitor (TKI) tolerance in leukemia cells by enhancing mRNA stability or translation efficiency of antiapoptotic genes via m6A decoration.17 Analogous to FTO, the other m6A “eraser” ALKBH5 also exerts a tumor-promoting role in leukemia.4,18 ALKBH5 is highly expressed in AML, especially in leukemia stem/initiating cells (LSCs/LICs), and maintains LSC/LIC frequency by posttranscriptional regulation of its critical targets, such as TACC3 and AXL.4,18 Two independent studies have reported that METTL3 exerts an essential oncogenic role in AML.6,7 Mechanistically, METTL3 could deposit m6A modification on its mRNA targets, including MYC and BCL2, which, in turn, leads to their translational activation to block myeloid differentiation and promote leukemogenesis.7 Another recent study in AML showed that a portion of METTL3 is recruited to the promoter regions of ~80 genes by CAATT enhancers binding protein zeta (CEBPZ) to activate MYC signaling.6 METTL14, another essential component of m6A MTC, is highly expressed in AML and also plays a critical oncogenic role during leukemogenesis.5 Genetic depletion of METTL14 inhibits cell proliferation, induces myeloid differentiation and cell apoptosis, suppresses self-renewal of LSCs/LICs and delays AML progression In vivo via an m6A-deppendent manner.5 Several other studies have revealed the oncogenic role of WTAP and RBM15, another 2 components of m6A MTC, in leukemia pathogenesis.19,20 However, whether such oncogenic roles are attributed to m6A decoration remains to be elucidated. Via genome-wide CRISPR-Cas9 screen, METTL16 has been identified as one of the most essential genes for the survival of leukemia cells.6 Yet further studies are required to address its role in hematological malignancies. In addition, YTHDF2 is highly expressed in a broad spectrum of human AMLs and required for LSC/LIC self-renewal and AML initiation and propagation via shortening the half-life of m6A-modified transcripts.8 Another reader protein YTHDC1 is also required for the survival of AML cells.21,22 YTHDC1 could undergo liquid–liquid phase separation and form nuclear YTHDC1-m6A condensates to protect MYC from the PAXT complex and exosome-associated RNA degradation.21 Moreover, YTHDC1 drives leukemogenesis through controlling stability of MCM4.22 Taken together with these reported studies on m6A regulators, both m6A writers and erasers function as oncoproteins in leukemia, despite their opposite role in mRNA methylation. It might be highly possible that different m6A writers, especially erasers and readers regulate different groups of target genes, and different m6A regulators preferentially bind to distinct regions of the same transcript and result in divergent fates. Actually, IGF2BP preferentially binds to the 3′ UTR region of MYC and increases its stability,23 whereas YTHDF2 prefers to bind to the 5′ UTR and middle exons of MYC and leads to its decay.1 Thus, it will be very important and interesting to systemically characterize the specific targets of each m6A regulator and clarify how a particular m6A site in a given transcript fine-tunes its metabolism and determines its fate. Considering the critical roles of m6A modification and its machinery in hematological malignancies, targeting the dysregulated m6A regulator(s) may represent an attractive strategy for leukemia treatment. Indeed, several small molecular compounds targeting m6A machinery have been discovered. STM2457, a potent and selective inhibitor of METTL3 leads to reduced AML growth and an increase in differentiation and apoptosis.24 We have discovered several specific and highly efficient inhibitors targeting FTO to treat leukemia.25 More promisingly, m6A modification also modulates drug resistance and reprograms immune response.26 Therefore, combining such inhibitors targeting m6A machinery with chemotherapy and/or immunotherapy may lead to the development of more effective therapies to overcome therapy resistance and cure leukemia. Aside from m6A, other RNA epigenetic marks, such as A-to-I editing, m5C and m7G, are also implicated in the pathogenesis of hematological malignancies. The adenosine deaminases acting on RNA 1 (ADAR1)-mediated A-to-I editing is correlated with a poor prognosis of chronic myeloid leukemia (CML) patients,9 and promotes LSC self-renewal and CML progression (Fig. 1C).10 The m5C modification in RNA is deposited by m5C methyltransferases (RCMTs), including NOL1/NOP2/SUN domain (NSUN) family and DNA methyltransferase homologue (DNMT2), oxidized by TET proteins,27 and recognized by Y-box binding protein 1 (YBX1)28 (Fig. 1D). NSUN3 and DNMT2 directly interact with hnRNPK to form a functional complex which is important for the survival and drug response of leukemia cells.11 Although extensive studies have reported the critical roles of TET protein in blood malignancies, it is totally unknown whether TET-induced demethylation of mRNA m5C is involved in leukemogenesis and if so, what the biological function would be. METTL1/WDR4-mediated m7G decoration in tRNA potentiates oncogenic transformation and tumorigenesis.29 METTL1 also acts as the methyltransferase for internal m7G modification in mRNA (Fig. 1E); while it is still underdetermined whether METTL1-induced m7G modification in mRNA is involved in leukemogenesis. Within the past few years, tremendous efforts have been devoted to deciphering the role of RNA modifications in the pathogenesis, leading to rapid expansion of epitranscriptomics. Unlike the well-established methods to investigate m6A decoration, studies on other mRNA chemical modifications, such as m5C, m7G, m1A, and m6Am, are still at their infant stages due to the lack the transcriptome-wide sequencing approaches as well as the characterization of the modification machinery, especially the “eraser” and “reader” proteins. The development of novel tools that can precisely determine the landscape of RNA modifications at single-nucleotide resolution will greatly push the field forward. In addition, further unraveling the fundamental mechanisms of RNA epigenetic modifications and the related machinery may reveal the promising novel therapeutic strategies to treat leukemia and other life-threatening diseases. ACKNOWLEDGMENTS We apologize to colleagues whose work could not be cited due to space limitations.

Introduction: Albeit that over 170 types of chemical modifications have been identified in RNAs, the biological functions of most of those decorations are still elusive. N7-methylguanosine (m7G), routinely occurring at the 5’ cap of mRNA or within tRNA and rRNA, also exists in internal mRNA. However, the “reader” proteins that recognize internal mRNA m7G and regulate the metabolism and fate of target mRNAs have yet to be identified. Here, we aim to identify the reader(s) of internal mRNA m7G and elucidate the biological function. Methods: To identify internal m7G reader(s), we utilized RNA-pull down, mass spectrometry (MS), RNA immunoprecipitation (RIP)-seq, and m7G-seq. To evaluate the interaction between QKI and G3BP1, we used Co-IP, immunofluorescence (IF) and proximity ligation assay (PLA). To investigate the function of QKI in stress granule (SG), we performed polyA & m7G RNA fluorescence in situ hybridization (FISH), triple quadrupole MS (QQQ-MS), and Ribo-seq. To assess the role of QKI in drug resistance, we carried out both in vitro cell survival assays and in vivo xenograft mouse model. Results: Our QQQ-MS results suggest cytoplasmic mRNA, in contrast to nuclei mRNA, are highly enriched with internal m7G modification. Quaking proteins (QKI), especially QKI7, can selectively recognize the internal mRNA m7G decoration in the cytosol of various cell types. Our high-throughput sequence data revealed over 1000 confident m7G-modified and QKI-binding RNA targets with a conserved motif, “GANGAN (N=A/U/G)”. Those transcripts are functionally enriched in the “pathways in cancer” and “Hippo signaling pathway”. More strikingly, internal m7G reader QKI7 directly interacts with the SG core protein G3BP1 and can shuttle a subset of m7G-modified transcripts into SG mRNA pool under oxidative stress condition. Additionally, we identified 314 confident m7G-modified & QKI7-binding & SG-enriched target genes in U2OS cells. Ribo-seq indicated that QKI7 modulates the translation efficiency of a set of m7G-modified transcripts via sequestering and silencing them in SGs. Moreover, in line with the observation that many chemotherapy drugs could trigger the assembly of SGs, QKI7 increases the sensitivity of cancer cells to drug treatment (e.g., doxorubicin) in vitro and in vivo in a m7G-dependent manner. Consistently, by analyzing the hepatocellular carcinoma (HCC) pharmacogenomic landscape study dataset, we found that HCC cell lines with higher QKI expression levels are more sensitive to a variety of chemotherapy or targeted therapeutic drugs. Conclusion: Our study identifies QKI as the first internal mRNA m7G reader, provides insights into the biological function of internal mRNA m7G modification in remodeling stress granules transcriptome, and highlights the therapeutic potential of targeting the QKI7/m7G axis in overcoming drug resistance. Citation Format: Zhicong Zhao, Lei Dong, Li Han, Yangchan Li, Jianhuang Xue, Keren Zhou, Wei Li, Xiaolan Deng, Ying Qing, Brandon Tan, Zhenhua Chen, Chao Shen, Andrew Small, Kitty Wang, Keith Leung, Zheng Zhang, Xi Qin, Qiang Xia, Rui Su, Jianjun Chen. Recognition of internal mRNA N7-methylguanosine by QKI shuttles transcripts into stress granules and modulates drug resistance [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5685.

RNA N 6 -methyladenosine (m 6 A) modification and its regulators fine tune gene expression and contribute to tumorigenesis.This study aims to uncover the essential role and the underlying molecular mechanism(s) of the m 6 A reader YTHDC1 in promoting triple negative breast cancer (TNBC) metastasis.Methods: In vitro and in vivo models were employed to determine the pathological function of YTHDC1 in TNBC metastasis.To identify bona fide YTHDC1 target RNAs, we conducted RNA-seq, m 6 A-seq, and RIP-seq, followed by integrative data analysis and validation assays.Results: By analyzing The Cancer Genome Atlas (TCGA) dataset, we found that elevated expression of YTHDC1 is positively correlated with poor prognosis in breast cancer patients.Using a mammary fat pad mouse model of TNBC, YTHDC1 significantly promoted lung metastasis of TNBC cells.Through multiple transcriptome-wide sequencing and integrative data analysis, we revealed dysregulation of metastasis-related pathways following YTHDC1 depletion and identified SMAD3 as a bona fide YTHDC1 target RNA.Depletion of YTHDC1 caused nuclear retention of SMAD3 mRNA, leading to lower SMAD3 protein levels.Loss of YTHDC1 led to impaired TGF-β-induced gene expression, leading to inhibition of epithelial-mesenchymal transition (EMT) and suppressed TNBC cell migration and invasion.SMAD3 overexpression was able to restore the response to TGF-β in YTHDC1 depleted TNBC cells.Furthermore, we demonstrated that the oncogenic role of YTHDC1 is mediated through its recognition of m 6 A as m 6 A-binding defective mutants of YTHDC1 were unable to rescue the impaired cell migration and invasion of YTHDC1 knockout TNBC cells.Conclusions: We show that YTHDC1 plays a critical oncogenic role in TNBC metastasis through promoting the nuclear export and expression of SMAD3 to augment the TGF-β signaling cascade.Overall, our study demonstrates that YTHDC1 is vital for TNBC progression by enhancing TNBC cell survival and TGF-β-mediated EMT via SMAD3 to enable the formation of distant metastasis and highlights the therapeutic potential of targeting the YTHDC1/m 6 A/SMAD3 axis for TNBC treatment.

Objectives Several reports suggest that epigenetic therapy may be a potential method for treating epilepsy, and circular RNAs (circRNAs) play important roles in mediating the epigenetic mechanisms associated with epilepsy; however, currently there are no effective treatment methods to prevent the progression of epileptogenesis. The circRNA serine/arginine repetitive matrix 4 (circSRRM4) was found to exert regulatory effects in temporal lobe epilepsy (TLE); however, the mechanisms involved are still unknown. Materials and methods To elucidate the molecular mechanism of circSRRM4, we investigated human epileptic brain tissue, epileptic rats, neuron and astrocyte cell lines using RT-qPCR, western blot, fluorescence in situ hybridisation, immunofluorescence staining, Nissl stain, micro-PET-CT, RNA-pulldown, liquid chromatography-mass spectrometry, and RBP immunoprecipitation techniques. Furthermore, we evaluated the pyruvate kinase M1/2 (PKM) expression patterns in the human and rat models of TLE. Results We detected the increased circSRRM4 expression in the hypometabolic lesions of patients with TLE and discovered that circSrrm4 has specific spatiotemporal characteristics in rats with kainic acid-induced epilepsy. The decreased PKM1 expression and increased PKM2 expression were similar to the Warburg effect in tumours. Notably, circSrrm4 silencing reduced the incidence and frequency of epilepsy, improved local hypometabolism, and prevented neuronal loss and astrocyte activation. Conclusion PKM2 promotes lactic acid production in the astrocytes by inducing glycolysis, thereby contributing to the energy source for epileptic seizures. Notably, circSRRM4 combines with and inhibits serine and arginine rich splicing factor 3 (SRSF3) from joining the ubiquitin-proteasome pathway, improving the SRSF3-regulated alternative splicing of PKM, and consequently stimulating glycolysis in cells.

Hypoxic tumor microenvironment (TME) plays critical roles in induction of cancer stem cell-like phenotype in breast cancer and contribute to chemoresistance. However, the mechanism underlying stemness reprogramming of breast cancer cells (BCs) by hypoxic TME remains largely unknown. In the present study, we illustrated that HIF-2α, but not HIF-1α, induces stemness in BCs under hypoxia through SOD2-mtROS-PDI/GRP78-UPR ER pathway, linking mitochondrial metabolic state to endoplasmic reticulum (ER) response via mitochondrial reactive oxygen species (mtROS) level. HIF-2α activates endoplasmic reticulum unfolded protein response (UPR ER ) in drug-sensitive MCF7 and T47D cells to induce drug-resistant stem-like phenotype. Genetic depletion or pharmacological inhibition (YQ-0629) of HIF-2α abolished hypoxia-induced stem-like phenotype in vitro and in vivo. Mechanistically, HIF-2α activates transcription of superoxide dismutase 2 (SOD2) under hypoxia and thereby decreases mtROS level. With less mtROS transported to endoplasmic reticulum, the expression and activity of protein disulfide isomerase (PDI) is suppressed, allowing glucose-regulated protein 78 (GRP78) to dissociate from receptor proteins of UPR ER and bind misfolded protein to activate UPR ER , which eventually confer chemoresistance and stem-like properties to BCs. Moreover, the increase in mtROS and PDI levels caused by HIF-2α knockdown and the subsequent UPR ER inhibition could be substantially rescued by mitoTEMPOL (a mtROS scavenger), 16F16 (a PDI inhibitor), or GRP78 overexpression. Overall, we reported the critical roles of HIF-2α-SOD2-mtROS-PDI/GRP78-UPR ER axis in mediating hypoxia-induced stemness in BCs, highlighting the interaction between organelles and providing evidence for further development of targeted HIF-2α inhibitor as a promising therapeutic strategy for chemoresistant breast cancer.