N-methyladenine DNA Modification in Glioblastoma
Authors: Zhi Huang
Ryan C Gimple
Pengcheng Wang
Yinsheng Wang
Leo J Y Kim
Andrew E Sloan
Maike Sander
Tao P Wu
Chun Zeng
Jeremy N Rich
David U Gorkin
Zheng Li
Li Jiang
Amber E Kerstetter-Fogle
Yang Yu
Tyler E Miller
Qiulian Wu
Kui Zhai
Alexis V Dowiak
Cheng Zhang
Christine Lee-Poturalski
Andrew Z Xiao
Stephen C Mack
Yinghui Sui
Xiaoguang Fang
Briana C Prager
Shideng Bao
Kaixuan Lin
Qi Xie
Journal: Cell (2018)
Discipline: MOLECULAR BIOLOGY & GENETICS
Keywords: DNA methylation; H3K9me3; N(6)-methyladenine; brain tumor; cancer stem cell
Affiliations: Department of Medicine, Division of Regenerative Medicine, University of California, San Diego, La Jolla, CA 92037, USA., Department of Genetics and Yale Stem Cell Center, Yale School of Medicine, New Haven, CT 06520, USA., Department of Medicine, Division of Regenerative Medicine, University of California, San Diego, La Jolla, CA 92037, USA; Department of Pathology, Case Western Reserve University, Cleveland, OH 44120, USA., Department of Medicine, Division of Regenerative Medicine, University of California, San Diego, La Jolla, CA 92037, USA; Department of Pathology, Case Western Reserve University, Cleveland, OH 44120, USA; Cleveland Clinic Lerner College of Medicine, Case Western Reserve University, Cleveland, OH 44195, USA., Department of Chemistry, University of California, Riverside, Riverside, CA 92521, USA., Center for Epigenomics, Department of Cellular and Molecular Medicine, University of California, San Diego, La Jolla, CA 92037, USA., Departments of Pediatrics and Cellular and Molecular Medicine, Pediatric Diabetes Research Center, University of California, San Diego, La Jolla, CA, 92093, USA., Department of Pathology, Case Western Reserve University, Cleveland, OH 44120, USA., Department of Pharmacology, Case Western Reserve University, Cleveland, OH 44195, USA., Department of Stem Cell Biology and Regenerative Medicine, Lerner Research Institute, Cleveland Clinic, Cleveland, OH 44195, USA., Department of Pediatrics, Division of Hematology and Oncology, Baylor College of Medicine, Texas Children's Hospital, Houston, TX 77030, USA., Case Comprehensive Cancer Center, Case Western Reserve University School of Medicine, Cleveland, OH 44106, USA; Department of Neurological Surgery, University Hospitals - Cleveland Medical Center, Cleveland, OH 44106, USA., Department of Genetics and Yale Stem Cell Center, Yale School of Medicine, New Haven, CT 06520, USA. Electronic address: ., Department of Medicine, Division of Regenerative Medicine, University of California, San Diego, La Jolla, CA 92037, USA; Department of Neurosciences, University of California, San Diego, School of Medicine, La Jolla, CA 92037, USA. Electronic address: .

Abstract:  Genetic drivers of cancer can be dysregulated through epigenetic modifications of DNA. Although the critical role of DNA 5-methylcytosine (5mC) in the regulation of transcription is recognized, the functions of other non-canonical DNA modifications remain obscure. Here, we report the identification of novel N6-methyladenine (N6-mA) DNA modifications in human tissues and implicate this epigenetic mark in human disease, specifically the highly malignant brain cancer glioblastoma. Glioblastoma markedly upregulated N6-mA levels, which co-localized with heterochromatic histone modifications, predominantly H3K9me3. N6-mA levels were dynamically regulated by the DNA demethylase ALKBH1, depletion of which led to transcriptional silencing of oncogenic pathways through decreasing chromatin accessibility. Targeting the N6-mA regulator ALKBH1 in patient-derived human glioblastoma models inhibited tumor cell proliferation and extended the survival of tumor-bearing mice, supporting this novel DNA modification as a potential therapeutic target for glioblastoma. Collectively, our results uncover a novel epigenetic node in cancer through the DNA modification N6-mA. 

Retracted: No

PubPeer comments:
 I am wondering whether the high levels of 6mA reported in glioblastoma cells and primary tumors ( 0.1% 6mA dA) could be due to mycoplasma contamination. Indeed, it was previously found that 50% glioblastoma samples are infected with mycoplasma - a potential source of artifactual 6mA detection in mammalian samples: https: www.ncbi.nlm.nih.gov pmc articles PMC3573938 https: www.ncbi.nlm.nih.gov pmc articles PMC4723534 It should also be noted that Liu et al 2016 tested Alkbh1 activity on 6mA methylated DNA and found no evidence of DNA 6mA demethylase activity. , 
 I wonder if the Kaplan-Meier (K-M) survival curve that is shown in Fig. 7C, with patients split into in ALKBH1 high and low expressors, is a bit of cherry picking. It seems only this particular dataset (TCGA-GBM, Agilent array) gives a significant p-value for better survival of ALKBH1 low patients, all other GBM or glioma datasets (including TCGA-GBM U133 and TCGA-GBM RNA-Seq; shown below) on http: gliovis.bioinfo.cnio.es give no significant result. I think that weak point in the data should have been mentioned discussed in the paper. ![file] , 
 Authors should check another recent well executed study showing that previous reports of 6mA in higher organisms are artifactual: No evidence for DNA N6-methyladenine in mammals https: advances.sciencemag.org content 6 12 eaay3335 , 
 #1 The sequencing data which shows no contamination. The alkbh1 activities has been confirmed by several biochemistry and structure studies published recently. , 
 Recently there is a paper reported that there are Intratumor bacteria found in many tumor types including GBM and mostly these bacteria are intracellular in both cancer cells and immune cells. https: science.sciencemag.org content 368 6494 973 So how can we exclude the possibility that the detected 6mA in GBM is from the intracellular bacteria? Thanks. , 
 Readers should become aware of a recent paper Critical assessment of DNA adenine methylation in eukaryotes using quantitative deconvolution , that could not reproduce the finding that 6mA modification is enriched in glioblastoma stem cells and primary glioblastoma. From the text: We also collected and examined two glioblastoma brain tissue samples because glioblastoma stem cells and primary glioblastoma were reported to have a 6mA A level of ~1000 ppm by dot blotting and mass spectrometry (11). We obtained 570,283, 247,700, and 280,763 SMRT CCS reads from the PBMC sample and the two glioblastoma brain tissues, respectively, for single-molecule 6mA analysis. Of these, 99.53%, 99.88%, and 99.86% of CCS reads were mapped to the human reference genome, indicating highly pure samples. The 6mA A levels estimated by 6mASCOPE in glioblastoma samples were ~10u20136, with 3 ppm for glioblastoma-1 (CI, 1 to 16 ppm) and 2 ppm for glioblastoma-2 (CI, 1 to 13 ppm) (Fig. 5A) (31). This level is comparable to the negative controls with extremely low 6mA A levels: HEKWGA (1 ppm; CI, 1 to 6 ppm) and native HEK293 (1 ppm; CI, 1 to 6 ppm), when the confidence intervals are taken into consideration. In the PBMC sample, the 6mA A level estimation of 17 ppm (CI, 4 to 63 ppm) by 6mASCOPE is consistent with the measurements of UHPLCMS MS (Fig. 5A). These data suggested either that the abundance of 6mA, if present in glioblastoma and PBMCs, was much lower than the reported levels in the recent studies (glioblastoma, ~1000 ppm; lymphocytes, ~510 ppm) or that 6mA A levels may be highly heterogeneous or variable between different samples of the same cell type, the same tissue, or a specific disease. Motif enrichment analysis did not support a reliable motif in these samples (fig. S14) ,