N-methyladenine DNA Modification in Glioblastoma

Abstract

SUMMARY Genetic drivers of cancer can be dysregulated through epigenetic modifications of DNA. While the critical role of DNA 5-methylcytosine (5mC) in the regulation of transcription is recognized, the functions of other non-canonical DNA modifications remain obsure. Here, we report the identification of novel N(6)-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 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.

No evidence for DNA N 6 -methyladenine in mammals

“…2A), clearly indicating misidentification of 6mdA. Next, we extended this analysis to 16 distinct 6mdA DIP-seq samples across four independent studies (6,7,9,19), revealing that most of the enriched peaks in all studies were located in repetitive elements overrepresented in both low complexity regions and simple repeats compared to other genomic fractions (52.7-and 27.7-fold, respectively) ( fig. S2, A and B), consistent with off-target binding (20).…”

The stress specific impact of ALKBH1 on tRNA cleavage and tiRNA generation

“…Interestingly, Alkbh1 overexpression led to translational repression of protein synthesis 17 , while its deletion impaired mitochondrial function 16 . In addition to these somewhat conflicting results on the role of Alkbh1 in cell growth and energy regulation, Alkbh1 did not exhibit its demethylase activity on m 1 A in patient derived glioma stem cells (GSCs) 18 . Notwithstanding, its knockdown in GSCs inhibited cell growth via its DNA N 6 -methyladenine demethylation activity 18 , in contrast to the lower levels of Alkbh1 observed in larger and more advanced gastric cancers indicating an opposing functional roles in both cancer types 19 .…”

“…In addition to these somewhat conflicting results on the role of Alkbh1 in cell growth and energy regulation, Alkbh1 did not exhibit its demethylase activity on m 1 A in patient derived glioma stem cells (GSCs) 18 . Notwithstanding, its knockdown in GSCs inhibited cell growth via its DNA N 6 -methyladenine demethylation activity 18 , in contrast to the lower levels of Alkbh1 observed in larger and more advanced gastric cancers indicating an opposing functional roles in both cancer types 19 . Alkbh1 was further shown to localize to mitochondrial RNA granules, exerting an important regulatory function on mitochondrial dynamics 20 .…”

ATAC-seq identifies thousands of extrachromosomal circular DNA in cancers and cell lines

“…Inverse PCR on exonuclease resistant extrachromosmal DNA (highly enriched in circular DNA) and FISH on metaphase spreads confirmed the presence of the identified somatically mosaic eccDNA. To provide additional evidence of the success of ATAC-seq in identifying eccDNA, we analyzed an ATAC-seq library generated from patient derived GBM cell lines (Xie et al, 2018) and identified the eccDNA harboring EGFR gene, which is known to be amplified through the formation of eccDNA in GBM. Finally, we analyzed ATAC-seq data from GBM and LGG generated by the TCGA consortium to identify hundreds of eccDNAs even before their amplification in the tumor was apparent as a copy number variation by hybridization to SNP arrays.…”

“…Recent studies have provided further evidence that this oncogenic amplification occurs on eccDNA (deCarvalho et al, 2018;Turner et al, 2017;Xu et al, 2019). To check if we can detect the eccDNA in ATAC-seq data generated from GBM cell lines we turned to six ATAC-seq libraries generated from GBM cell lines developed from a single glioblastoma patient (Xie et al, 2018). We ran the Circle_finder pipeline combining all the six libraries (GSM3318539, GSM3318540, GSM3318541, GSM3318542, GSM3318543 and GSM3318544) and found 58 eccDNAs varying in size from few hundred bases to few megabases.…”