METTL3/16-mediated m6A modification of ZNNT1 promotes hepatocellular carcinoma progression by activating ZNNT1/osteopontin/S100A9 positive feedback loop-mediated crosstalk between macrophages and tumour cells.

Macrophages are the major components of tumour microenvironment, which play critical roles in tumour development. N6-methyladenosine (m6A) also contributes to tumour progression. However, the potential roles of m6A in modulating macrophages in hepatocellular carcinoma (HCC) are poorly understood. Here, we identified ZNNT1 as an HCC-related m6A modification target, which was upregulated and associated with poor prognosis of HCC. METTL3 and METTL16-mediated m6A modification contributed to ZNNT1 upregulation through stabilizing ZNNT1 transcript. ZNNT1 exerted oncogenic roles in HCC. Furthermore, ZNNT1 recruited and induced M2 polarization of macrophages via up-regulating osteopontin (OPN) expression and secretion. M2 Macrophages-recruited by ZNNT1-overexpressed HCC cells secreted S100A9, which further upregulated ZNNT1 expression in HCC cells via AGER/NF-κB signaling. Thus, this study demonstrates that m6A modification activated the ZNNT1/OPN/S100A9 positive feedback loop, which promoted macrophages recruitment and M2 polarization, and enhanced malignant features of HCC cells. m6A modification-triggered ZNNT1/OPN/S100A9 feedback loop represents potential therapeutic target for HCC.

The Effect of Renal Replacement Therapy in a Patient with Mercaptopurine Toxicity: Time to Revise Guidelines.

INTRODUCTION: Mercaptopurine, a thiopurine, is used in various disorders of immune regulation, such as autoimmune hepatitis. Thiopurine metabolism is complex with risk for overdosing, especially when metabolism is impaired by liver dysfunction. Hepatotoxicity may be due to mercaptopurine overdose and is often reversible after prompt cessation of the drug. CASE PRESENTATION: Treatment of thiopurine toxicity is mainly supportive and literature on enhanced elimination by renal replacement therapy is ambiguous. CONCLUSION: In this case of thiopurine toxicity, a patient with autoimmune hepatitis presents with abdominal pain, nausea, vomiting, and diarrhea. We show in this case report that renal replacement therapy had no effect on total body clearance of mercaptopurine.

[Modulation of Expression of Drug Metabolizing Enzymes and Augmentation of Anti-cancer Drug Effects: Through Epigenetics and Three-dimensional Cancer Cell Culture Systems].

Since commencing my role as a professor in a newly established Department of Pharmacodynamics and Molecular Genetics at the School of Pharmacy, Iwate Medical University, on April 1, 2007, my research has focused on modifying gene expression of cytochrome P-450 (CYP) in established human colon cancer cells. Additionally, I have been investigating methods to enhance the anti-tumor effects of irinotecan (CPT-11) and 5-fluorouracil (5-FU) using epigenetic modifying inhibitors of DNA methyltransferase and histone deacetylase. Treating colon cancer cells with a DNA methyltransferase inhibitor, 5-aza-2'-deoxycytidine (DAC), led to elevated expression levels of CYP1B1 and CYP3A4 through demethylation of the promoter regions of related genes. Furthermore, the administration of DAC and the histone deacetylase inhibitor depsipeptide [(DEP), an anti-cancer drug romidepsin] significantly increased the cellular sensitivities of human colon cancer cells to CPT-11 and 5-FU, respectively. Remarkably, DAC treatment also increased colon cancer cell sensitivity to SN-38, an active metabolite of CPT-11, through the suppression of the anti-apoptotic protein Bcl-2. DEP increased colon cancer cell sensitivity to 5-FU in association with increased expressions of tumor-suppressor p21 and major histocompatibility complex class II genes. Another facet of my research is centered around understanding the gene regulatory mechanisms of the CYP1 family through aryl hydrocarbon receptors (AhR)s under glucose-deprivation stress and in three-dimensional (3D) culture systems of human solid tumor cells. In the 3D culture of human liver cancer cells, I found Pregnane X Receptor being implicated in the regulation of CYP1A2, which aligns with the in vivo mode of CYP1A2 expression.

Hsa-LINC02418/mmu-4930573I07Rik regulated by METTL3 dictates anti-PD-L1 immunotherapeutic efficacy via enhancement of Trim21-mediated PD-L1 ubiquitination.

BACKGROUND: Limited response to programmed death ligand-1 (PD-L1)/programmed death 1 (PD-1) immunotherapy is a major hindrance of checkpoint immunotherapy in non-small cell lung cancer (NSCLC). The abundance of PD-L1 on the tumor cell surface is crucial for the responsiveness of PD-1/PD-L1 immunotherapy. However, the negative control of PD-L1 expression and the physiological significance of the PD-L1 inhibition in NSCLC immunotherapy remain obscure. METHODS: Bioinformatics analysis was performed to profile and investigate the long non-coding RNAs that negatively correlated with PD-L1 expression and positively correlated with CD8+T cell infiltration in NSCLC. Immunofluorescence, in vitro PD-1 binding assay, T cell-induced apoptosis assays and in vivo syngeneic mouse models were used to investigate the functional roles of LINC02418 and mmu-4930573I07Rik in regulating anti-PD-L1 therapeutic efficacy in NSCLC. The molecular mechanism of LINC02418-enhanced PD-L1 downregulation was explored by immunoprecipitation, RNA immunoprecipitation (RIP), and ubiquitination assays. RIP, luciferase reporter, and messenger RNA degradation assays were used to investigate the m6A modification of LINC02418 or mmu-4930573I07Rik expression. Bioinformatics analysis and immunohistochemistry (IHC) verification were performed to determine the significance of LINC02418, PD-L1 expression and CD8+T cell infiltration. RESULTS: LINC02418 is a negative regulator of PD-L1 expression that positively correlated with CD8+T cell infiltration, predicting favorable clinical outcomes for patients with NSCLC. LINC02418 downregulates PD-L1 expression by enhancing PD-L1 ubiquitination mediated by E3 ligase Trim21. Both hsa-LINC02418 and mmu-4930573I07Rik (its homologous RNA in mice) regulate PD-L1 therapeutic efficacy in NSCLC via Trim21, inducing T cell-induced apoptosis in vitro and in vivo. Furthermore, METTL3 inhibition via N6-methyladenosine (m6A) modification mediated by YTHDF2 reader upregulates hsa-LINC02418 and mmu-4930573I07Rik. In patients with NSCLC, LINC02418 expression is inversely correlated with PD-L1 expression and positively correlated with CD8+T infiltration. CONCLUSION: LINC02418 functions as a negative regulator of PD-L1 expression in NSCLC cells by promoting the degradation of PD-L1 through the ubiquitin-proteasome pathway. The expression of LINC02418 is regulated by METTL3/YTHDF2-mediated m6A modification. This study illuminates the underlying mechanisms of PD-L1 negative regulation and presents a promising target for improving the effectiveness of anti-PD-L1 therapy in NSCLC.

SUV39H1 Expression as a Guideline for Omitting Radiotherapy in Lymph Node-positive Triple-negative Breast Cancer Patients.

BACKGROUND/AIM: The role of postoperative radiotherapy (RT) combined with chemotherapy (CT) for lymph node-positive (LN+) triple-negative breast cancer (TNBC) remains controversial. SUV39H1-mediated epigenetic regulation is associated with cancer cell migration, invasion, metastasis, and treatment resistance. This study aims to identify the role of SUV39H1 in TNBCs. MATERIALS AND METHODS: Overall, 498 TNBCs with SUV39H1 RNA-seq profiles were retrieved from TCGA-BRCA and analyzed; the X-tile algorithm was used to stratify the population into low, intermediate, and high SUV39H1. Furthermore, we performed an in vitro clonogenic cell survival assay using the MDA-MB-231 cell line to assess the effects of SUV39H1 on cellular responses. RESULTS: The results showed that SUV39H1 was significantly higher in TNBC than normal tissue and luminal subtype breast cancer. Notably, SUV39H1 is significantly expressed in the basal-like 1 (BL1) and immunomodulatory (IM) subgroups, compared to other subtypes. Compared to patients with a low or medium expression of SUV39H1, omitting RT only worsens disease-free survival (DFS) in those with high SUV39H1 expression. The experimental results showed SUV39H1 was suppressed by si-SUV39H1, and SUV39H1 knockdown in MDA-MB-231-IV2-1 cells enhanced the cellular toxicity of doxorubicin and paclitaxel. CONCLUSION: Targeting SUV39H1 may provide a potential guiding indication of omitting RT to avoid over-treatment and chemosensitivity for TNBC.

The m6A modulator-mediated cytarabine sensitivity and immune cell infiltration signature in acute myeloid leukemia.

PURPOSE: The study aims to investigate the impact of m6A modulators on drug resistance and the immune microenvironment in acute myeloid leukemia (AML). The emergence of drug resistance is a significant factor that contributes to relapse and refractory AML, leading to a poor prognosis. METHODS: The AML transcriptome data were retrieved from the TCGA database. The "oncoPredict" R package was utilized to assess the sensitivity of each sample to cytarabine (Ara-C) and classify them into distinct groups. Differential expression analysis was performed to identify m6A modulators differentially expressed between the two groups. Select Random Forest (RF) to build a predictive model. Model performance was evaluated using calibration curve, clinical decision curve, and clinical impact curve. The impacts of METTL3 on Ara-C sensitivity and immune microenvironment in AML were examined using GO, KEGG, CIBERSORT, and GSEA analyses. RESULTS: Seventeen out of 26 m6A modulators exhibited differential expression between the Ara-C-sensitive and resistant groups, with a high degree of correlation. We selected the 5 genes with the highest scores in the RF model to build a reliable and accurate prediction model. METTL3 plays a vital role in m6A modification, and further analysis shows its impact on the sensitivity of AML cells to Ara-C through its interaction with 7 types of immune-infiltrating cells and autophagy. CONCLUSION: This study utilizes m6A modulators to develop a prediction model for the sensitivity of AML patients to Ara-C, which can assist in treating AML drug resistance by targeting mRNA methylation.

Global Shift in Alternative Splicing and Therapeutic Susceptibilities in Leukemia Driven by METTL3 Overexpression.

SUMMARY: Mutations in splicing factors are commonly observed in chronic lymphocytic leukemia (CLL); however, other mechanisms can also contribute to the dysregulation of alternative splicing. One example is the overexpression of the m6A RNA methyltransferase METTL3, that by depositing the epitranscriptomic mark in spliceosome transcripts leads to aberrant splicing, but at the same time creates vulnerability to METTL3 inhibitors. See related article by Wu et al., p. 228 (8) .

Novel treatment strategy of targeting epigenetic dysregulation in pancreatic neuroendocrine tumors.

BACKGROUND: Epigenetic dysregulation is an integral step in the progression of pancreatic neuroendocrine tumors. We hypothesized that tumor suppressor repression by DNA methyltransferase 1 in pancreatic neuroendocrine tumors could be targeted with epigenetic treatment. METHODS: Resected pancreatic neuroendocrine tumors from 32 patients were stained for DNA methyltransferase 1 and scored. Human (BON1) and murine (STC) pancreatic neuroendocrine tumor cells were treated with DNA methyltransferase 1 inhibitor 5-azacytidine and chemotherapeutic agents 5-fluorouracil and temozolomide. Cell proliferation assay and tumor suppressor gene analysis were performed with qRT-PCR and Clarion S microarray. Tumor measurements were compared in a murine treatment model. RESULTS: High DNA methyltransferase scores were associated with high Ki-67 (6.7% vs 70.6% P < .01), mitotic rate (0.0% vs 31.3%), and grade (20.0% vs 80.4%, P < .01). Treatment with 5-azacytidine and chemotherapy resulted in a reduction of cell proliferation compared to chemotherapy alone in BON1 (77.3% vs 53.1%, P < .001) and STC (73.4% vs 34.2%, P < .001). Treatment with 5-azacytidine and chemotherapy resulted in upregulation of tumor suppressors CDKN1A (7.6 rel. fold, P < .001), BRCA2 (4.3 rel. fold, P < .001), and CDH1 (6.0 rel. fold, P = .026) in BON1 and CDKN1a (14.5 rel. fold, P < .001) and CDH (17.5 rel. fold, P < .001) in STC. In microarray, 5-azacytidine drove global genetic changes in combination treatment. In vivo tumors treated with chemotherapy measured 88.6 ± 19.54 mm3 vs 52.89 ± 10.51 mm3 in those treated with combination therapy (P = .009). CONCLUSION: Epigenetic dysregulation with DNA methyltransferase 1 is associated with pancreatic neuroendocrine tumors and is a potential targetable strategy. 5-azacytidine and chemotherapy in combination can reduce cell proliferation, upregulate silenced tumor suppressor genes, and decrease in vivo tumors in pancreatic neuroendocrine tumors.

Fused deep learning paradigm for the prediction of o6-methylguanine-DNA methyltransferase genotype in glioblastoma patients: A neuro-oncological investigation.

BACKGROUND: The O6-methylguanine-DNA methyltransferase (MGMT) is a deoxyribonucleic acid (DNA) repairing enzyme that has been established as an essential clinical brain tumor biomarker for Glioblastoma Multiforme (GBM). Knowing the status of MGMT methylation biomarkers using multi-parametric MRI (mp-MRI) helps neuro-oncologists to analyze GBM and its treatment plan. METHOD: The hand-crafted radiomics feature extraction of GBM's subregions, such as edema(ED), tumor core (TC), and enhancing tumor (ET) in the machine learning (ML) framework, was investigated using support vector machine(SVM), K-Nearest Neighbours (KNN), random forest (RF), LightGBM, and extreme gradient boosting (XGB). For tissue-level analysis of the promotor genes in GBM, we used the deep residual neural network (ResNet-18) with 3D architecture, followed by EfficientNet-based investigation for variants as B0 and B1. Lastly, we analyzed the fused deep learning (FDL) framework that combines ML and DL frameworks. RESULT: Structural mp-MRI consisting of T1, T2, FLAIR, and T1GD having a size of 400 and 185 patients, respectively, for discovery and replication cohorts. Using the CV protocol in the ResNet-3D framework, MGMT methylation status prediction in mp-MRI gave the AUC of 0.753 (p < 0.0001) and 0.72 (p < 0.0001) for the discovery and replication cohort, respectively. We presented that the FDL is ∼7% superior to solo DL and ∼15% to solo ML. CONCLUSION: The proposed study aims to provide solutions for building an efficient predictive model of MGMT for GBM patients using deep radiomics features obtained from mp-MRI with the end-to-end ResNet-18 3D and FDL imaging signatures.

KDM1A-mediated upregulation of METTL3 ameliorates Alzheimer's disease via enhancing autophagic clearance of p-Tau through m6A-dependent regulation of STUB1.

BACKGROUND: Alzheimer's disease (AD) is a severe neurodegenerative disorder that progressively destroys cognitive skills. Exploring the mechanism underlying autophagic clearance of phosphorylated tau (p-Tau) contributes to developing novel therapeutic strategies for AD. METHODS: SH-SY5Y and HT22 cells were treated with Aß1-42 to establish an in vitro model of AD. Cell viability was examined using CCK-8. TUNEL staining was applied to evaluate cell apoptosis. LC3 puncta was examined by IF staining. m6A modification level was evaluated through MeRIP. RNA pull-down and RIP assays were used for analyzing the interaction between IGF2BP1 and STUB1 transcripts. The binding of KDM1A to the promoter of METTL3 was confirmed by ChIP assays. APP/PS1 transgenic mice were used as an in vivo model of AD. Cognitive skills of mice were evaluated with the Morris water maze. Hippocampal damage and Aß deposition were detected through H&E and IHC staining. RESULTS: Dysregulated levels of autophagy, p-Tau and m6A was observed in an in vitro model of AD. Overexpression of METTL3 or STUB1 enhanced autophagy but reduced p-Tau level in Aß1-42-treated cells. METTL3 stabilized STUB1 mRNA through the m6A-IGF2BP1-dependent mechanism and naturally promoted STUB1 expression, thereby enhancing autophagic p-Tau clearance in Aß1-42-treated cells. Overexpression of KDM1A enhanced autophagy, m6A modification and autophagic p-Tau clearance in Aß1-42-treated cells. KDM1A-mediated upregulation of METTL3 promoted autophagic p-Tau clearance and ameliorated Alzheimer's disease both in vitro and in vivo. CONCLUSION: KDM1A-mediated upregulation of METTL3 enhances autophagic clearance of p-Tau through m6A-dependent regulation of STUB1, thus ameliorating Alzheimer's disease. Our study provides novel mechanistic insights into AD pathogenesis and potential drug targets for AD.

Role of Pharmacogenomics in the Efficacy and Safety of Thiopurines in Inflammatory Bowel Disease: A Systematic Review and Meta-analysis.

BACKGROUND: Thiopurines' toxicity often leads to dose reduction or discontinuation. This systematic review aims to synthesize the evidence on the effect of genotype-based dosing of thiopurines on treatment efficacy and safety in inflammatory bowel disease (objective #1), and the association between genotype status and the efficacy and safety profile (objective #2). METHODS: The Cochrane Library, MEDLINE, and EMBASE were searched in August 2021. A total of 80 studies (19,859 individuals) were included. Meta-analyses for mortality, different types of adverse events (AEs), withdrawal due to AE, change in disease activity and clinical remission were performed following mainly a fixed-effects model. PROSPERO registration: CRD42020148130. RESULTS: Genotype-based dosing was associated to a significantly lower incidence of hematologic AEs (risk ratio=0.71; 95% CI: 0.56-0.90; I2 : 47%; 4 randomized controlled trials; moderate quality), which may be attributable to nudix hydrolase 15 (NUDT15) testing more than to thiopurine methyltransferase (TPMT) genotyping. No differences were found in other outcomes. Mutations in TPMT and NUDT15 genes were associated to a higher probability of serious AEs [odds ratio (OR) TPMT=4.98; OR NUDT15=11.44], hematologic AEs (OR TPMT=3.18), and serious hematologic AEs (OR TPMT=7.88; OR NUDT15=12.83). TPMT was also associated with a higher risk of withdrawals due to AEs (OR=3.38), and NUDT15 with gastrointestinal AEs (OR=2.04). Mutations in the ITPA gene did not lead to significant differences. Evidence of an association between other genes and clinical outcomes is still scarce. CONCLUSIONS: Mutations in TPMT and NUDT15 genes predispose patients to suffer thiopurine-induced toxicity, and genotype-guided treatment has been shown to contribute to the prevention of thiopurine-induced toxicity, especially in the case of NUDT15 in Asians.

RNA interference-mediated silencing of DNA methyltransferase 1 attenuates neuropathic pain by accelerating microglia M2 polarization.

BACKGROUND: DNA methyltransferase 1 (DNMT1) exerts imperative functions in neuropathic pain (NP). This study explored the action of RNA interference-mediated DNMT1 silencing in NP by regulating microglial M2 polarization. METHODS: NP rat models were established using chronic constriction injury (CCI) and highly aggressive proliferating immortalized (HAPI) microglia were treated with lipopolysaccharide (LPS) to induce microglia M1 polarization, followed by treatment of DNMT1 siRNA or si-DNMT1/oe-DNMT1, respectively. The pain threshold of CCI rats was assessed by determining mechanical withdrawal threshold (MWT) and thermal withdrawal latency (TWL). Levels of inflammatory factors (TNF-α/IL-1ß/IL-6/IL-10) and DNMT1 in rat L4-L6 spinal cord samples and HAPI cells were measured using ELISA, RT-qPCR, and Western blot. iNOS and Arg-1 mRNA levels were measured via RT-qPCR. DNMT1, M1 marker (iNOS), and M2 marker (Arg-1) levels in microglia of CCI rats were detected by immunofluorescence. Percentages of M1 microglia phenotype (CD16) and M2 microglia phenotype (CD206) were detected by flow cytometry. The phosphorylation of PI3K/Akt pathway-related proteins was determined by Western blot. RESULTS: CCI rats exhibited diminished MWT and TWL values, increased pro-inflammatory cytokines, and decreased anti-inflammatory cytokine IL-10. Additionally, DNMT1 was upregulated in CCI rat microglia. DNMT1 siRNA alleviated CCI-induced NP and facilitated M2 polarization of microglia in CCI rats. DNMT1 knockdown inhibited LPS-induced M1 polarization of HAPI cells and promoted M2 polarization by blocking the PI3K/Akt pathway, but DNMT1 overexpression inhibited the M1-to-M2 polarization of microglia. CONCLUSION: RNA interference-mediated DNMT1 silencing accelerates microglia M2 polarization by impeding the PI3K/Akt pathway, thereby alleviating CCI-induced NP.

Positive epigenetic regulation loop between AR and NSUN2 promotes prostate cancer progression.

BACKGROUND: Prostate cancer (PCa) is a major type of cancer in man worldwide. Androgen deprivation therapy (ADT) and the next-generation androgen receptor (AR) pathway inhibitors have acquired great success in treating PCa. However, patients treated with ADT or AR targeted therapy are inevitably developing into castration-resistant prostate cancer (CRPC) or becoming drug resistance. The role of mRNA 5-methylcytosine (m5C) modification in cancers is largely unknown. This study aimed to explore the role of the m5C methyltransferase NSUN2 in Prostate cancer (PCa). METHODS: The expression of NSUN2 and its clinicopathological impact were evaluated in PCa cohorts. The effect of NSUN2 on the biological characteristics of PCa cells was investigated on the basis of gain-offunction and loss-of-function analyses. Subcutaneous models further uncovered the role of NSUN2 in tumor growth. Epi-transcriptome assays with RNA bisulfite sequencing (RNA-BisSeq) analysis and in vitro enzyme reaction assays were performed to validate the targeted effect of NSUN2 on AR. AR-binding sites in the NSUN2 promoter were investigated by ChIP and luciferase assays to uncover the interplay between NSUN2 and AR signaling. RIP-qPCR and EMSA methods were performed to confirm that YBX1 binds to AR m5 C sites. RESULTS: NSUN2 is highly expressed in PCa and predicts poor outcome. NSUN2 plays roles as a PCa oncogene both in vitro and in vivo. Depletion of NSUN2 results in decreased expression and activities of AR, including AR-V7. Mechanistically, NSUN2 posttranscriptionally stabilized AR by cluster m5 C modification in a m5CYBX1-dependent manner. Strikingly, treatment with enzalutamide, an effective AR inhibitor, reduces NSUN2 expression and decreases the m5C modification level in prostate cancer cells. Finally, we found that AR transcriptionally regulates NSUN2. CONCLUSION: NSUN2 stabilizes AR mRNA through cluster 5-methylcytosine modification and activates a positive feedback loop to promote prostate cancer.

APAF1-Binding Long Noncoding RNA Promotes Tumor Growth and Multidrug Resistance in Gastric Cancer by Blocking Apoptosome Assembly.

Chemotherapeutics remain the first choice for advanced gastric cancers (GCs). However, drug resistance and unavoidable severe toxicity lead to chemotherapy failure and poor prognosis. Long noncoding RNAs (lncRNAs) play critical roles in tumor progression in many cancers, including GC. Here, through RNA screening, an apoptotic protease-activating factor 1 (APAF1)-binding lncRNA (ABL) that is significantly elevated in cancerous GC tissues and an independent prognostic factor for GC patients is identified. Moreover, ABL overexpression inhibits GC cell apoptosis and promotes GC cell survival and multidrug resistance in GC xenograft and organoid models. Mechanistically, ABL directly binds to the RNA-binding protein IGF2BP1 via its KH1/2 domain, and then IGF2BP1 further recognizes the METTL3-mediated m6A modification on ABL, which maintains ABL stability. In addition, ABL can bind to the WD1/WD2 domain of APAF1, which competitively prevent cytochrome c from interacting with APAF1, blocking apoptosome assembly and caspase-9/3 activation; these events lead to resistance to cell death in GC cells. Intriguingly, targeting ABL using encapsulated liposomal siRNA can significantly enhance the sensitivity of GC cells to chemotherapy. Collectively, the results suggest that ABL can be a potential prognostic biomarker and therapeutic target in GC.

Continuing maintenance temozolomide therapy beyond 12 cycles confers no clinical benefit over discontinuation at 12 cycles in patients with IDH1/2-wildtype glioblastoma.

OBJECTIVE: The optimal duration of maintenance temozolomide therapy is controversial. We aimed to examine the clinical benefits of continuing temozolomide therapy beyond 12 cycles in patients with glioblastoma. METHODS: We included 41 patients with isocitrate dehydrogenase 1/2-wildtype glioblastoma, who received 12 or more cycles of temozolomide therapy between June 2006 and December 2019. We evaluated the outcome between 16 patients who continued temozolomide therapy beyond 12 cycles up to 24 cycles (≥13 cycles group) and 25 patients wherein temozolomide therapy was discontinued at 12 cycles (12 cycles group). RESULTS: The median progression-free survival and survival time after completing 12 cycles (residual progression-free survival and residual overall survival) did not differ between the 12 cycles group and ≥13 cycles group (residual progression-free survival: 11.3 vs. 9.2 months, P = 0.61, residual overall survival: 25.7 vs. 30.2 months, P = 0.76). Multivariate analysis including temozolomide therapy beyond 12 cycles, age at 12 cycles, Karnofsky performance status at 12 cycles, residual tumor at 12 cycles, maintenance therapy regimen and O-6-methylguanine deoxyribonucleic acid methyltransferase promoter methylation status revealed that extended temozolomide therapy beyond 12 cycles was not correlated with residual progression-free survival and residual overall survival (P = 0.80 and P = 0.41, respectively) but Karnofsky performance status at 12 cycles ≥80 was significantly associated with increased residual overall survival (P = 0.0012). CONCLUSIONS: Continuing temozolomide beyond 12 cycles confers no clinical benefit over the discontinuation of temozolomide at 12 cycles. Karnofsky performance status at 12 cycles ≥80 may serve as a novel predictive factor for long-term survival.

p53 m6A modulation sensitizes hepatocellular carcinoma to apatinib through apoptosis.

Hepatocellular carcinoma (HCC) is insidious and prone to metastasis and recurrence. Currently, no effective treatment is available for HCC. Furthermore, HCC does not respond to various radio- and chemotherapies, and the molecular mechanism of treatment resistance is unclear. Here, we found that p53 n6-methyladenosine (m6A) played a decisive role in regulating HCC sensitivity to chemotherapy via the p53 activator RG7112 and the vascular endothelial growth factor receptor inhibitor apatinib. Our results reveal that p53 activation plays a crucial role in chemotherapy-induced apoptosis and reducing cell viability. Moreover, decreasing m6A methyltransferase (e.g., methyltransferase-like 3, METTL3) expression through chemotherapeutic drug combinations reduced p53 mRNA m6A modification. p53 mRNA m6A modification blockage induced by S-adenosyl homocysteine or siRNA-mediated METTL3 inhibition enhanced HCC sensitivity to chemotherapy. Importantly, we observed that downregulation of METTL3 and upregulation of p53 expression by oral administration of chemotherapy drugs triggered apoptosis and xenograft tumor growth inhibition in nude mice. Based on these findings, we hypothesize that a METTL3-m6A-p53 axis could be a potential target in HCC therapy.

Looking for the skeleton in the closet-rare genetic diagnoses in patients with diabetes and skeletal manifestations.

AIMS: The precision medicine approach of tailoring treatment to the individual characteristics of each patient has been a great success in monogenic diabetes subtypes, highlighting the importance of accurate clinical and genetic diagnoses of the type of diabetes. We sought to describe three unique cases of childhood-onset diabetes in whom skeletal manifestations led to the revelation of a rare type of diabetes. METHODS : Case-scenarios and review of the literature. RESULTS: Case 1: A homozygous mutation in TRMT10A, a tRNA methyltransferase, was identified in a 15-year-old boy with new-onset diabetes, developmental delay, microcephaly, dysmorphism, short stature and central obesity. The progressive apoptosis of pancreatic beta cells required insulin replacement therapy, with increased demand due to an unfavorable body composition. Case 2: Congenital generalized lipodystrophy type 1 was suspected in an adolescent male with an acromegaloid facial appearance, muscular habitus, and diabetes who presented with a pathological fracture in a cystic bone lesion. A homozygous mutation in AGPAT2, an acyl transferase which mediates the formation of phospholipid precursors, was identified. Leptin replacement therapy initiation resulted in a remarkable improvement in clinical parameters. Case 3: A 12-year-old boy with progressive lower limb weakness and pain was diagnosed with diabetic ketoacidosis. Diffuse diaphyseal osteosclerosis compatible with the diagnosis of Camurati-Engelmann disease and a heterozygous mutation in TGFß1 were identified. Preservation of euglycemia by insulin replacement relieved pain, suggesting that the diabetic milieu may have augmented TGFß1 overexpression. CONCLUSION: Unraveling the precise genetic cause for the clinical manifestations led to the prediction of phenotypic manifestations, and enhanced the clinical outcomes.

Historical expectations with DNA methyltransferase inhibitor monotherapy in MDS: when is combination therapy truly "promising"?

DNA methyltransferase inhibitors (DNMTIs) for patients with higher risk myelodysplastic syndromes (HR-MDS) have low complete remission rates and are not curative. Early DNMTI combination clinical trials in HR-MDS are often termed "promising," but many randomized trials subsequently failed to show benefit. Clearer understanding of when a combination is likely to improve upon DNMTI monotherapy would inform randomized studies. We reviewed MDS azacitidine or decitabine monotherapy studies. We collected baseline demographics including International Prognostic Scoring System (IPSS) risk, DNMTI, disease characteristics; and response variables including survival and marrow and hematologic responses. Aggregate estimates across studies were calculated using meta-analyses techniques. Using a binomial design, we estimated the necessary operating characteristics to design a phase 2 study showing improved efficacy of a combination over monotherapy. Among 1908 patients, the overall response rate (ORR) was 24% (n = 464; 95% confidence interval [CI], 0.22-0.26): 267 complete response (CR, 14%), 68 partial response (4%), and 129 marrow complete remission (7%). Among 1604 patients for whom a hematologic response was reported, 476 (30%; 95% CI, 0.27-0.32) reported hematologic improvement (HI). More patients treated with azacitidine achieved HI (38%; 95% CI, 0.35-0.41) compared with decitabine (15%; 95% CI, 0.13-0.19), whereas the marrow ORR rate was higher with decitabine (29%; 95% CI, 0.26-0.33) compared with azacitidine (21%; 95% CI, 0.19-0.23). CR rates were similar between DNMTIs: 13% with azacitidine and 16% with decitabine. Variables that influence MDS response include the specific DNMTI backbone and the distribution of IPSS risk of patients enrolled on a trial. Considering these factors can help identify which early combination approaches are worth assessing in larger randomized trials.

NSUN6, an RNA methyltransferase of 5-mC controls glioblastoma response to temozolomide (TMZ) via NELFB and RPS6KB2 interaction.

Nop2/Sun RNA methyltransferase (NSUN6) is an RNA 5-methyl cytosine (5mC) transferase with little information known of its function in cancer and response to cancer therapy. Here, we show that NSUN6 methylates both large and small RNA in glioblastoma and controls glioblastoma response to temozolomide with or without influence of the MGMT promoter status, with high NSUN6 expression conferring survival benefit to glioblastoma patients and in other cancers. Mechanistically, our results show that NSUN6 controls response to TMZ therapy via 5mC-mediated regulation of NELFB and RPS6BK2. Taken together, we present evidence that show that NSUN6-mediated 5mC deposition regulates transcriptional pause by accumulation of NELFB and the general transcription factor complexes (POLR2A, TBP, TFIIA, and TFIIE) on the preinitiation complex at the TATA binding site to control translation machinery in glioblastoma response to alkylating agents. Our findings open a new frontier into controlling of transcriptional regulation by RNA methyltransferase and 5mC.