Department of Information Engineering, University of Florence, Florence, Italy.
Institute for Biomedical Technologies, National Research Council, Segrate, Milano, Italy.
Commun Biol. 2023 Apr 8;6(1):382. doi: 10.1038/s42003-023-04756-8.
Aberrant DNA methylation at CpG dinucleotides is a cancer hallmark that is associated with the emergence of resistance to anti cancer treatment, though molecular mechanisms and biological significance remain elusive. Genome scale methylation maps by currently used methods are based on chemical modification of DNA and are best suited for analyses of methylation at CpG rich regions (CpG islands). We report the first high coverage whole-genome map in cancer using the long read nanopore technology, which allows simultaneous DNA-sequence and -methylation analyses on native DNA. We analyzed clonal epigenomic/genomic evolution in Acute Myeloid Leukemias (AMLs) at diagnosis and relapse, after chemotherapy. Long read sequencing coupled to a novel computational method allowed definition of differential methylation at unprecedented resolution, and showed that the relapse methylome is characterized by hypermethylation at both CpG islands and sparse CpGs regions. Most differentially methylated genes, however, were not differentially expressed nor enriched for chemoresistance genes. A small fraction of under-expressed and hyper-methylated genes at sparse CpGs, in the gene body, was significantly enriched in transcription factors (TFs). Remarkably, these few TFs supported large gene-regulatory networks including 50% of all differentially expressed genes in the relapsed AMLs and highly-enriched in chemoresistance genes. Notably, hypermethylated regions at sparse CpGs were poorly conserved in the relapsed AMLs, under-represented at their genomic positions and showed higher methylation entropy, as compared to CpG islands. Analyses of available datasets confirmed TF binding to their target genes and conservation of the same gene-regulatory networks in large patient cohorts. Relapsed AMLs carried few patient specific structural variants and DNA mutations, apparently not involved in drug resistance. Thus, drug resistance in AMLs can be mainly ascribed to the selection of random epigenetic alterations at sparse CpGs of a few transcription factors, which then induce reprogramming of the relapsing phenotype, independently of clonal genomic evolution.
在 CpG 二核苷酸处发生的异常 DNA 甲基化是癌症的一个标志，与对癌症治疗产生耐药性有关，尽管分子机制和生物学意义仍不明确。目前使用的方法进行的全基因组甲基化图谱基于 DNA 的化学修饰，最适合分析富含 CpG 的区域（CpG 岛）的甲基化。我们报告了使用长读长纳米孔技术在癌症中进行的首次高覆盖率全基因组图谱，该技术允许在天然 DNA 上同时进行 DNA 序列和 - 甲基化分析。我们分析了急性髓系白血病 (AML) 在诊断和化疗后复发时的克隆表观基因组/基因组进化。长读测序与一种新的计算方法相结合，允许以前所未有的分辨率定义差异甲基化，并表明复发的甲基组学以 CpG 岛和稀疏 CpG 区域的过度甲基化为特征。然而，大多数差异甲基化基因既没有差异表达，也没有富集化疗耐药基因。稀疏 CpG 基因体中表达下调和高度甲基化的少数基因显著富集转录因子 (TF)。值得注意的是，这些少数 TF 支持包括复发 AML 中 50%的所有差异表达基因的大型基因调控网络，并且高度富集于化疗耐药基因。值得注意的是，稀疏 CpG 处的过度甲基化区域在复发 AML 中保存不佳，在其基因组位置的代表性不足，并且与 CpG 岛相比，显示出更高的甲基化熵。对可用数据集的分析证实了 TF 与其靶基因的结合以及在大型患者队列中相同基因调控网络的保守性。复发的 AML 仅携带少数患者特异性结构变体和 DNA 突变，显然与耐药性无关。因此，AML 中的耐药性主要归因于少数转录因子稀疏 CpG 处随机表观遗传改变的选择，这随后诱导复发表型的重新编程，而与克隆基因组进化无关。
