Mefatinib, a novel second-generation epidermal growth factor (EGFR) tyrosine kinase inhibitor that has shown promising antitumor activity in targeting non-small cell lung cancer (NSCLC) with common and uncommon EGFR-activating mutations. In this phase III, randomized, double-blind trial in China, 336 eligible patients with advanced nonsquamous NSCLC harboring EGFR L858R or exon 19 deletion (ex19del) were assigned (2:1) to receive either mefatinib (60 mg daily, n = 223) or gefitinib (250 mg daily, n = 113). The primary endpoint was progression-free survival (PFS), assessed by an independent review committee (IRC). The trial is registered with chinadrugtrials.org.cn (CTR20192297). After a median follow-up of 15.9 months for mefatinib and 18.5 months for gefitinib, mefatinib demonstrated a significantly longer median IRC-assessed PFS compared to gefitinib (13.7 vs. 9.7 months; hazard ratio [HR] = 0.68; 95% confidence intervals [CI]: 0.53-0.87; p = 0.002). The 30-month overall survival rate was 60.2% for mefatinib and 54.3% for gefitinib. Patients with EGFR ex19del had comparable PFS for both treatment arms (p > 0.100), whereas patients with EGFR L858R had significantly longer median PFS when treated with mefatinib than gefitinib (13.7 vs 8.3 months HR = 0.55 [95% CI: 0.38-0.78]; p = 0.001). Patients with EGFR L858R had a 30-month overall survival rate of 56.6% with mefatinib and 43.7% with gefitinib. Treatment-related adverse events ≥grade 3 were reported in 45.7% of the mefatinib group and 24.8% of the gefitinib group. No new safety signals were observed for mefatinib. Mefatinib demonstrated superior efficacy to gefitinib with a similar tolerability profile in the first-line treatment of EGFR-mutated advanced NSCLC.

Abstract O-linked β-N-acetylglucosamine (O-GlcNAc) transferase (OGT) is the sole writer for intracellular O-GlcNAcylation. It catalyzes O-GlcNAcylation of thousands of protein substrates, but relatively less is known about the post-translational modifications that occur on OGT itself. Herein, we demonstrate that OGT is S-palmitoylated at Cys-472 and Cys-477, which is mediated by the S-acyltransferase Zinc Finger DHHC-Type Palmitoyl transferase 14 (zDHHC14) and removed by acyl protein thioesterase 2 (APT2). S-Palmitoylation stabilizes OGT by shunting it away from the lysosomal chaperone-mediated autophagy (CMA) pathway, as S-palmitoylation decreases the interaction between OGT and heat shock cognate 70 kDa protein (HSC70), the CMA chaperone. Via label-free quantitative mass spectrometry, we find that S-palmitoylation elevates the affinity between OGT and protein phosphatase 1 catalytic subunit gamma (PPP1CC), but not PPP1CB. We further demonstrate that S-palmitoylation of OGT augments binding with Yes-associated protein-1 (YAP), a protein that associates with PPP1CC, and subsequently enhances YAP O-GlcNAcylation. Our work unearths S-palmitoylation of OGT and CMA-mediated degradation of lysosomal OGT, the orchestration of which finetunes the activity of key OGT complexes, such as OGT-PPP1CC, and contributes to OGT substrate selectivity.

Neuroblastoma is a common and aggressive pediatric sympathetic nervous system tumor. Genomic structural variants (SVs) contribute substantially to neuroblastoma, yet remain under-characterized in high-risk neuroblastomas. We aimed to elucidate neuroblastoma pathogenesis using third-generation whole-genome sequence high-risk cases to identify driver aberrations and explore potential therapeutic strategies. We analyzed third-generation whole-genome sequencing data of 20 high-risk neuroblastoma samples and combined the findings with those obtained from the analysis of clinical samples, in vitro models, and public datasets. The contactin-associated protein-like 2 (CNTNAP2) gene was observed to be frequently aberrated because of structural variants in high-risk neuroblastoma samples. CNTNAP2 expression was significantly correlated with favorable histology and could be used to predict prognosis using clinical samples and neuroblastoma datasets. Overexpression and knockdown experiments and transcriptomic analysis revealed that CNTNAP2 was primarily involved in neuronal differentiation and axon guidance pathways; moreover, CNTNAP2 was required for neuroblastoma differentiation and affected cancer stemness. Immunoprecipitation and mass spectrometry revealed that CNTNAP2 interacted with cytoskeletal proteins like drebrin 1 (DBN1) and myosin-heavy chain 9 (MYH9). CNTNAP2 dynamically reorganises actin and microtubules for DBN1-mediated neuronal differentiation. CNTNAP2 also reduces CTNNB1 transcription and β-catenin pathway activation by inhibiting MYH9 nuclear translocation. CNTNAP2 overexpression in neuroblastoma cell lines resulted in cell cycle arrest, decreased cell proliferation and metastasis. The recurrent loss of CNTNAP2 in neuroblastoma contributes to an aggressive phenotype by impairing neuronal differentiation and increasing cancer stemness. These findings may serve as a foundation for developing therapeutic strategies to overcome barriers to differentiation.

The Cleavage and Polyadenylation Specificity Factor (CPSF) complex, a core regulator of mRNA 3′-end processing, plays an increasingly recognized role in maintaining genomic stability beyond its canonical function in pre-mRNA cleavage and polyadenylation. By facilitating transcription termination, CPSF ensures timely dissociation of RNA Polymerase II (Pol II) from gene termini, thereby preventing transcriptional readthrough, R-loop (three-stranded RNA: DNA hybrids with displaced single-stranded DNA) accumulation, and transcription-replication conflicts (TRCs)—all of which are potent drivers of DNA double-strand breaks and genomic instability. Dysregulation of CPSF subunits (e.g., CPSF3, CPSF4, CPSF6) has been observed in various cancers, including acute myeloid leukemia, glioblastoma, and hepatocellular carcinoma, where aberrant CPSF function disrupts transcriptional fidelity and exacerbates replication stress. Protein interaction analyses further suggest that CPSF associates with DNA replication and repair factors, such as RPA, BARD1, and PARP1, positioning it at the interface of RNA processing and genome maintenance. Given the transcriptional dependency of cancer cells, CPSF—particularly its catalytic subunit CPSF3—has emerged as a promising therapeutic target, with preclinical studies demonstrating that CPSF3 inhibitors can induce transcriptional stress and synthetic lethality. This review comprehensively summarizes the structural and functional roles of the CPSF complex in cancer biology and targeted therapy, and explores its emerging function as a guardian of genomic stability.

Polo-like kinase 1 (PLK1) is a crucial mitotic kinase that is implicated in various aspects of cell cycle. Many post-translational modifications have been identified on PLK1 to regulate its activation, stability, and localization. PLK1 has been shown previously to colocalize with the O-linked β-N-acetylglucosamine (O-GlcNAc) transferase (OGT), and OGT regulates PLK1 stability. In our recent work, we show that PLK1 is O-GlcNAcylated by click chemistry. Using stepped collisional energy/higher energy collision dissociation mass spectrometry, we mapped the PLK1 O-GlcNAc site to be T291. We further utilized fluorescent activated cell sorting and time-lapse microscopy to assess the mitotic defects of PLK1 O-GlcNAc mutants. In vivo studies in mouse xenograft demonstrated that it promoted uterine cancer tumorigenesis. In this chapter, we delineate the methodologies we used in studying PLK1 O-GlcNAcylation, including click chemistry, stepped collisional energy/higher energy collision dissociation mass spectrometry, fluorescent activated cell sorting, time-lapse microscopy, and mouse xenograft assays.

Colorectal cancer (CRC) is one of the most common malignant tumors worldwide, seriously threatening human health. Researchers have revealed that long non-coding RNAs (lncRNAs) are involved in the development of multiple cancers, including CRC. In this study, we explored the expression level, roles, and mechanisms of lncRNA USP2-AS1 in CRC. We discovered that USP2-AS1 was overexpressed in CRC and was relevant to the poor prognosis of CRC patients. Functional experiments clarified that USP2-AS1 facilitated CRC cell growth and migration and reduced apoptosis. Animal experiments demonstrated that USP2-AS1 could promote tumor growth in vivo. Mechanistically, we verified that USP2-AS1 could bind to IGF2BP2, thereby enhancing the stability of PHLDA2 mRNA. Additionally, USP2-AS1 could absorb miR-134-5p to upregulate PHLDA2 expression. Furthermore, our results showed that USP2-AS1 could activate the PI3K/AKT signalling pathway by upregulating the expression of PHLDA2. In conclusion, USP2-AS1 could upregulate PHLDA2 expression by recruiting IGF2BP2 and competitively binding miR-134-5p, thus activating the PI3K/AKT signalling pathway and facilitating CRC malignant progression. Our results prove that USP2-AS1 is a prospective target of CRC.

We aim to identify molecular clusters related to O-GlcNAcylation and establish a novel scoring system for predicting prognosis and immunotherapy efficacy in patients with gastric cancer (GC). The transcriptomic and clinical data are obtained from XENA-UCSC and GEO databases. The O-GlcNAcylation-related genes are obtained from the GSEA database. Consensus clustering analysis is employed to identify O-GlcNAcylation-related molecular clusters, and principal component analysis (PCA) is utilized to develop a novel prognostic scoring system for predicting GC outcomes and immunotherapy efficacy. The prognostic accuracy of the scoring system is assessed across five real-world cohorts. The biological function of actin alpha 2, smooth muscle (ACTA2) in GC is determined through experimental verification. Using 34 O-GlcNAcylation-related genes associated with prognosis in GC patients, these individuals are divided into two distinct subgroups characterized by different outcomes, tumor microenvironment profiles, and clinical case characteristics. The DEGs between the two subgroups are subsequently used to further divide the GC patients into two subgroups by consensus cluster analysis. PCA is used to construct a prognostic scoring system, which reveal that patients in the low-score subgroup have a better prognosis and greater benefit from immunotherapy. The accuracy of the scoring system is confirmed through validation in a cohort of patients receiving immunotherapy in the real world. ACTA2 promotes proliferation and inhibits apoptosis in GC cells. These findings suggest that we successfully establish molecular clusters associated with O-GlcNAcylation and develop a scoring system that demonstrates strong performance in predicting the prognosis of patients with GC and the effect of immunotherapy interventions.

This study presents a rare case of acinar soft tissue sarcoma and provides a detailed analysis of its multi-parameter quantitative functional MRI (fMRI) characteristics. The patient, a 16-year-old male, was diagnosed with acinar soft tissue sarcoma after presenting with a progressively enlarging mass in the right thigh. To determine the extent and nature of the lesion, MRI examinations were performed using conventional plain scans, contrast-enhanced scans, dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI), and intravoxel incoherent motion (IVIM) for quantitative analysis. Functional MRI imaging not only provides crucial diagnostic and differential diagnostic information for acinar soft tissue sarcoma, but also reveals through DCE-MRI and IVIM imaging indicators that ASPS exhibits high cellular density, increased vascular permeability, and abundant neovascularization. The local microcirculation characteristics offer vital insights into the tumor’s biological behavior and prognosis. Furthermore, functional MRI aids in precise diagnosis while providing references for surgical planning, postoperative adjuvant therapy, long-term follow-up evaluation, and recurrence risk prediction.

N6-methyladenosine (m6A) is the most prevalent internal RNA modification, and its regulators include writers, readers and erasers. m6A is under stringent control and takes part in many biological events, but it is not known whether there is an interplay between m6A and glycosylation. Here we investigated an m6A reader, YTHDC1, which has been shown to be recruited to the DNA-RNA hybrid at DNA damage sites and regulate homologous recombination (HR) during DNA damage repair. We found that YTHDC1 is subject to O-linked β-N-acetylglucosamine (O-GlcNAc) modification at Ser396 upon DNA damage, which is pivotal for YTHDC1 chromatin binding and ionization radiation induced focus (IRIF) formation. RNA immunoprecipitation (RIP) and molecular dynamics (MD) simulations indicate that O-GlcNAcylation is vital for YTHDC1 to bind with m6A RNA. Fluorescence recovery after photo bleaching (FRAP) analysis revealed that YTHDC1 O-GlcNAcylation is essential for DNA damage-induced YTHDC1-m6A condensate formation. We further demonstrate that YTHDC1 O-GlcNAcylation promotes HR-mediated DNA damage repair and cell survival, probably through recruitment of Rad51 to the damage sites. We propose that YTHDC1 O-GlcNAcylation is instrumental for HR.

Chemoresistance, the primary cause of mortality among ovarian cancer (OC) patients, is a multifaceted process encompassing numerous biological phenomena. As sequencing technology continues to advance, single-cell sequencing has surfaced as a potent strategy to elucidate the pathogenesis of OC. We examined single-cell sequencing data derived from five OC samples (three resistant and two sensitive) and identified an epithelial subcluster associated with chemotherapy resistance and poor prognosis. Using GSVA and cell communication analysis, we explored the unique biological functions and communication characteristics of this resistant subcluster. We performed high dimensional weighted gene co-expression network analysis and differential expression analysis to identify the hub genes of c3. Lastly, we investigated the correlation between the hub gene, CLIC3, and chemotherapy drug sensitivity. We also validated their involvement in specific pathways using TCGA data. The effects and primary mechanism to chemoresistance of CLIC3 was explored. We identified a cell subcluster, denoted as c3, strongly linked to chemoresistance and poor prognosis in OC. This subcluster demonstrated a correlation with both extracellular matrix (ECM) formation and angiogenesis signature, with CLIC3 identified as its key marker. The expression levels of CLIC3 exhibit a significant association with the sensitivity to various chemotherapeutic drugs in OC. Mechanistically, CLIC3 increases OC resistance to cisplatin by promoting integrin β1 redistribution and PI3K-AKT pathway. This study offers a novel insight into the progression and chemoresistance of OC. Additionally, we identified a specific cell cluster highly associated with chemoresistance. The marker for this cluster, CLIC3, increases OC resistance to cisplatin by promoting integrin β1 redistribution and PI3K-AKT pathway and holds significant potential as a new therapeutic target for OC.

BACKGROUND:The global diabetes epidemic necessitates self-management strategies, and traditional patient education faces challenges in personalization, continuity, and monitoring. This study evaluates the efficacy of the collection-assessment-plan-do-check-aggrandizement (CAPDCA) model, which is a personalized patient education framework for improving glycemic control and self-management. METHODS:A multicenter, two-stage cluster randomized controlled trial was conducted across six community health centers in Beijing. Eligible participants were randomized into intervention (CAPDCA model, n = 90) or control (traditional education, n = 90) groups. Primary outcomes were HbA1c reduction and target achievement; secondary outcomes included fasting blood glucose (FBG) and 2-h postprandial blood glucose (2h-PPG), medication adherence (MMAS-8), and quality of life (SF-36). Follow-ups occurred over 18 months (11 visits). Generalized estimating equations (GEE) analyzed longitudinal trends. RESULTS:178 participants completed the trial (90 in the intervention group and 88 in the control group). Compared to the control group, the intervention group demonstrated ​a significantly lower final HbA1c level​ (t = 6.356, P < .01) and ​a greater reduction in HbA1c​ (t = -6.117, P < .01). Target achievement rate of HbA1c is 83.3% in intervention and 25.0% in controls (risk ratio = 3.33, 95% CI: 2.29-4.84, P < .01). FBG (odds ratio (OR) = 0.663, 95% CI: 0.468-0.938) and 2h-PPG (OR = 0.218, 95% CI: 0.138-0.345) in intervention reductions were significantly greater (P < .05). MMAS-8 scores improved to 7.0 (inter-quartile range: 6.75-7.5) better than control group (Z = 5.912, P < .01). SF-36 scores is higher in the intervention group (t = 9.497, P < .01). CONCLUSIONS:The CAPDCA model enhances glycemic control, medication adherence, and quality of life in patients with diabetes through structured and personalized iterative education. Its scalability and adaptability address critical gaps in personalized diabetes education, offering a feasible framework for global implementation in primary healthcare institutions.

ABSTRACT Background Epidermal growth factor receptor (EGFR)‐mutated lung adenocarcinoma (LUAD) is the most common subtype among non‐small cell lung cancer (NSCLC) and targeted therapies are the primary approach for treatment. However, the development of resistance to therapy and histological transformation into small cell lung cancer (SCLC) present significant challenges. Understanding the mechanisms underlying this transformation is crucial for effective differential diagnosis and the formulation of treatment strategies. Methods In this study, we collected tissue from 5 primary LUAD before SCLC transformation, 12 transformed SCLC after EGFR tyrosine kinase inhibitor (TKI) treatment, and 18 de novo SCLC from lung cancer patients treated at Beijing Chest Hospital, Capital Medical University from January 2015 to December 2021. Whole‐exome sequencing was performed on these samples to compare the genomic alterations of these three tumor types, elucidating their similarities, differences, and connections. Statistical analyses were conducted using the Fisher exact test and performed with R v4.2.1 environment. Results Among 12 transformed SCLC cases, the majority were female (10/12, 83.3%), non‐smokers (10/12, 83.3%) and harbored EGFR 19del mutations (11/12, 91.7%). Four were with limited stage and 8 with extensive stage. TP53 mutations and RB1 loss are important but not necessary for SCLC transformation. The mutation rates of TP53 were 60% (3/5) in primary LUAD, 70% (7/10) in transformed SCLC, and 89% (16/18) in de novo SCLC. RB1 loss rates were 40% (2/5) in primary LUAD, 30% (3/10) in transformed SCLC, and 50% (9/18) in de novo SCLC. Additionally, mutations in COL22A1 and ALMS1 were only observed in transformed SCLC and de novo SCLC. In contrast, mutations in PTCH2, CNGB3, SPTBN5, CROCC, and MYO15A were more common in transformed SCLC, whereas PABPC3 and MUC19 mutations were more frequent in de novo SCLC. Smoking‐related mutations (SBS4) were only found in de novo SCLC, with no changes observed in transformed SCLC. TMB levels were significantly lower in transformed SCLC compared to de novo SCLC (p = 0.01). Genomic instability was significantly higher in transformed SCLC compared to primary LUAD and de novo SCLC. This was supported by higher levels of homologous recombination deficiency (HRD, p = 0.025), uniparental disomy (UPD, p = 0.003), loss of heterozygosity (LOH, p = 0.008), and telomeric allelic imbalance (TAI, p = 0.02). The increased frequency of UPD events in transformed SCLC suggests that UPD may act as a “second hit” in Knudson's model, leading to biallelic inactivation of tumor suppressor genes. High similarity was observed in genetic alterations related to DNA damage repair (DDR) and Notch signaling pathways between transformed SCLC and de novo SCLC. Conclusions The identification of these specific genomic alterations in transformed SCLC contributes to a better understanding of the mechanisms driving this transformation. This knowledge may guide future predicting the transformation of SCLC and the development of personalized treatment strategies for these patients.

BACKGROUND:Our objective was to identify distinct clinical subtypes among critically ill patients with cirrhosis and analyze the clinical features and prognosis of each subtype. METHODS:We extracted routine clinical data within 24 h of ICU admission from the MIMIC-IV database. To determine the number of clinical subtypes, we employed the "elbow method," "cumulative distribution function (CDF) plot," and "consensus matrix." Consensus k-means, k-means, and SOM methods were used to identify different clinical subtypes of critically ill cirrhosis. We validated our findings using patients from the eICU database. The SHapley Additive exPlanations (SHAP) method was used to explore the features of each clinical subtype, and 28-day Kaplan-Meier curves were generated. Survival differences among the clinical subtypes were assessed using the log-rank test. RESULTS:Our study included 2,586 patients from the MIMIC-IV database and 1,670 patients from the eICU database. Based on the clinical routine variables, we identified three clinical subtypes among patients in the MIMIC-IV database. Subtype A (N = 1424, 55.07 %) was labeled the "common subtype" and exhibited the lowest mortality. Subtype B (N = 703, 27.18 %) was classified as the "hyperinflammatory response subtype" and had a relatively high mortality. Subtype C (N = 459, 17.75 %) was identified as the "liver dysfunction subtype" and had the highest mortality. These findings were consistent with the results obtained from both the internal validation set (MIMIC-IV database) and the external validation set (eICU database). CONCLUSIONS:Our study presents a novel and clinically applicable approach for subtyping critically ill cirrhosis.

Purpose:Inflammatory indexes are emerging as sensible prognostic factors for small cell lung cancer (SCLC). However, the prognostic value of dynamic C-reactive protein-to-lymphocyte ratio (CLR) in SCLC patients treated with chemoimmunotherapy remains unclear. Patients and Methods:This retrospective study investigated 88 SCLC patients who underwent chemoimmunotherapy between January 1st, 2020 and December 12th 2022. We examined the association between CLR and prognostic outcomes after chemoimmunotherapy. The associations between objective response rate (ORR), progression-free survival (PFS) with changes in blood indicators were also analyzed. Results:Patients with decreased CLR had significantly higher ORR, with odds ratios of 3.91 (P<0.05) and 3.19 (P<0.05) in univariate and multivariate logistic regression analyses, respectively. Kaplan-Meier analysis showed that decreased CLR was associated with prolonged PFS (P=0.02). Additionally, a CLR higher than 2.47 after treatment was associated with poor survival in both univariate and multivariate analyses. Conclusion:Dynamic CLR can serve as a potential biomarker for predicting the prognosis of SCLC patients treated with chemoimmunotherapy. Reduction of CLR after chemoimmunotherapy is associated with a significantly higher ORR and improved PFS.

Background:High-risk neuroblastoma (NB) still carries a <50% long-term survival despite risk-adapted therapy. Conventional risk metrics (age, stage, MYCN status) fail to capture transcriptional programs that drive tumor aggressiveness and immune escape. We hypothesized that systematic transcription factor (TF) activity profiling would reveal clinically actionable NB subtypes. This study aimed to profile TF activities in NB to develop and validate a TF-based prognostic score and to examine its association with the tumor immune microenvironment. Methods:TF activities for 498 primary NB tumors (GSE49710) were inferred with decoupleR-univariate linear model (ULM) using the OmniPath regulon. TFs that were significantly associated with overall survival (OS; Cox; P<1×10-4; n=146) were subjected to consensus clustering and Boruta-guided principal component analysis (PCA) to create a continuous TF_score. Immune infiltration and immunotherapy surrogates, Immunophenoscore (IPS) and the Tumor Immune Dysfunction and Exclusion (TIDE) framework, were compared across TF_score strata. A nomogram integrating TF_score, stage, and MYCN amplification was developed and validated with an external cohort (E-MTAB-8248; n=223) and single-cell RNA sequencing (RNA-seq) data. Results:Two robust NB subtypes were identified, with cluster 2 associated with worse prognosis, MYCNamplification, older age, distinct pathway activation, and higher stemness indices. The TF_score reliably quantified these clusters, showing superior predictive capability for survival compared to traditional markers (MYCN amplification and tumor stage). TF_score-high patients exhibited reduced immune infiltration and lower predicted responsiveness to immunotherapy. A validated prognostic nomogram effectively stratified risk, highlighting MYC and E2F family activation and FOXO3 and IRF1 suppression in high-risk patients. Single-cell analyses confirmed these bulk RNA-seq findings. Conclusions:TF activity profiling provides robust stratification for NB, integrating clinical prognostication and immune characterization. This study offers novel insights into TF-driven NB biology, with implications for targeted therapy and immunotherapeutic strategies.

OBJECTIVE:The CAPDCA (Collection, Assessment, Plan, Do, Check, Aggrandisement) Model is a structured and individualised health education framework designed for dynamic adjustment and continuous improvement. This study evaluated its efficacy in diabetes management. METHODS:A cluster randomised controlled trial was conducted across 6 community health centres, involving 178 patients with type 2 diabetes. The intervention group (n = 90) received CAPDCA model education, while the control group (n = 88) received traditional education. The intervention spanned 18 months, with HbA1c collected at baseline and study end. Blood glucose was collected at each follow-up. Analysis used Group-Based Trajectory Model (GBTM). RESULTS:Compared with the control group, the intervention group showed: lower HbA1c (t = 6.356, p < 0.01) and greater HbA1c reduction (t = -6.117, p < 0.01). GBTM revealed distinct glucose trajectories: FBG had two trajectories (Steady descent group and rebound group). The 2 h-PG had three trajectories (High BG-high descent group, Medium BG-low descent group, and low BG-high descent group). All trajectories demonstrated that blood glucose levels reached clinically target ranges post-intervention. Baseline HbA1c influenced FBG trajectories, while baseline HbA1c and medication adherence influenced 2 h-PG trajectories. Age, gender, education, and disease duration showed no significant association with trajectories. CONCLUSIONS:The CAPDCA model can effectively improve the control of HbA1c. The analysis of influencing factors of different trajectories suggested that the model was suitable for patients with different ages, genders, education levels, and disease duration. Further studies would be needed in exploring the application to various diseases and integrating the CAPDCA model with technologies such as artificial intelligence.

The intracellular O-linked N-acetylglucosamine (O-GlcNAc) glycosylation mediates many signal transduction events and regulates tumorigenesis. Previously the RNA N6-methyladenosine (m6A) reader, YTH (YT521-B homology) domain 2 (YTHDF2), has been shown to be O-GlcNAcylated on Ser-263 during Hepatitis B virus (HBV) infection and promote HBV-related hepatocellular carcinoma. Herein we mapped YTHDF2 O-GlcNAcylation at Thr-49 via electron-transfer dissociation mass spectrometry under unperturbed conditions. We show that YTHDF2 Thr-49 O-GlcNAcylation antagonizes Extracellular-signal regulated kinase (ERK)-dependent phosphorylation at Ser-39 and promotes YTHDF2 degradation. The downstream signaling pathway of YTHDF2 in lung carcinoma is thus upregulated, which leads to the downregulation of c-Myc. We further used mouse xenograft models to show that YTHDF2-T49A mutants increased lung cancer mass and size. Our work reveals a key role of YTHDF2 O-GlcNAcylation in tumorigenesis and suggests that O-GlcNAcylation exerts distinct functions under different biological stress.

Neuroblastoma (NB) is one of the most common childhood malignancies. Sixty percent of patients present with widely disseminated clinical signs at diagnosis and exhibit poor outcomes. However, the molecular mechanisms triggering NB metastasis remain largely uncharacterized. In this study, we generated a transcriptomic atlas of 15 447 NB cells from eight NB samples, including paired samples of primary tumors and bone marrow metastases. We used time-resolved analysis to chart the evolutionary trajectory of NB cells from the primary tumor to the metastases in the same patient and identified a common ‘starter’ subpopulation that initiates tumor development and metastasis. The ‘starter’ population exhibited high expression levels of multiple cell cycle-related genes, indicating the important role of cell cycle upregulation in NB tumor progression. In addition, our evolutionary trajectory analysis demonstrated the involvement of partial epithelial-to-mesenchymal transition (p-EMT) along the metastatic route from the primary site to the bone marrow. Our study provides insights into the program driving NB metastasis and presents a signature of metastasis-initiating cells as an independent prognostic indicator and potential therapeutic target to inhibit the initiation of NB metastasis.

O-linked GlcNAc (O-GlcNAc) is an emerging post-translation modification that couples metabolism with cellular signal transduction by crosstalk with phosphorylation and ubiquitination to orchestrate various biological processes. The mechanisms underlying the involvement of O-GlcNAc modifications in N6-methyladenosine (m6A) regulation are not fully characterized. Herein, we show that O-GlcNAc modifies the m6A mRNA reader YTH domain family 1 (YTHDF1) and fine-tunes its nuclear translocation by the exportin protein Crm1. First, we present evidence that YTHDF1 interacts with the sole O-GlcNAc transferase (OGT). Second, we verified Ser196/Ser197/Ser198 as the YTHDF1 O-GlcNAcylation sites, as described in numerous chemoproteomic studies. Then we constructed the O-GlcNAc-deficient YTHDF1-S196A/S197F/S198A (AFA) mutant, which significantly attenuated O-GlcNAc signals. Moreover, we revealed that YTHDF1 is a nucleocytoplasmic protein, whose nuclear export is mediated by Crm1. Furthermore, O-GlcNAcylation increases the cytosolic portion of YTHDF1 by enhancing binding with Crm1, thus upregulating downstream target (e.g. c-Myc) expression. Molecular dynamics simulations suggest that O-GlcNAcylation at S197 promotes the binding between the nuclear export signal motif and Crm1 through increasing hydrogen bonding. Mouse xenograft assays further demonstrate that YTHDF1-AFA mutants decreased the colon cancer mass and size via decreasing c-Myc expression. In sum, we found that YTHDF1 is a nucleocytoplasmic protein, whose cytosolic localization is dependent on O-GlcNAc modification. We propose that the OGT-YTHDF1-c-Myc axis underlies colorectal cancer tumorigenesis.

Abstract Background Metastasis, the leading cause of cancer-related death in patients diagnosed with ovarian cancer (OC), is a complex process that involves multiple biological effects. With the continuous development of sequencing technology, single-cell sequence has emerged as a promising strategy to understand the pathogenesis of ovarian cancer. Methods Through integrating 10 × single-cell data from 12 samples, we developed a single-cell map of primary and metastatic OC. By copy-number variations analysis, pseudotime analysis, enrichment analysis, and cell–cell communication analysis, we explored the heterogeneity among OC cells. We performed differential expression analysis and high dimensional weighted gene co-expression network analysis to identify the hub genes of C4. The effects of RAB13 on OC cell lines were validated in vitro. Results We discovered a cell subcluster, referred to as C4, that is closely associated with metastasis and poor prognosis in OC. This subcluster correlated with an epithelial–mesenchymal transition (EMT) and angiogenesis signature and RAB13 was identified as the key marker of it. Downregulation of RAB13 resulted in a reduction of OC cells migration and invasion. Additionally, we predicted several potential drugs that might inhibit RAB13. Conclusions Our study has identified a cell subcluster that is closely linked to metastasis in OC, and we have also identified RAB13 as its hub gene that has great potential to become a new therapeutic target for OC.