Nikolopoulos Theodoros, Bochalis Eleftherios, Chatzilygeroudi Theodora, Chondrou Vasiliki, Dereki Irene, Athanasopoulou Katerina, Zafeiropoulos John, Bourikas Kyriakos, Patrinos George P, Symeonidis Argiris, Sgourou Argyro
Biology Laboratory, School of Science and Technology, Hellenic Open University, Patras, Greece.
School of Health Sciences, Faculty of Medicine, Hematology Division, University of Patras, Patras, Greece.
Mol Med. 2025 Feb 14;31(1):59. doi: 10.1186/s10020-025-01123-7.
Patients with higher-risk (HR) myelodysplastic syndrome (MDS), ineligible for allogeneic hematopoietic stem cell transplantation (alloHSCT), require prompt therapeutic interventions, such as treatment with hypomethylating agents (HMAs) to restore normal DNA methylation patterns, mainly of oncosuppressor genes, and consequently to delay disease progression and increase overall survival (OS). However, response assessment to HMA treatment relies on conventional methods with limited capacity to uncover a wide spectrum of underlying molecular events.
We implemented liquid chromatography-tandem mass spectrometry (LC-MS/MS) to assess 5' methyl-2' deoxycytidine (5mdC), 5' hydroxy-methyl-2'-deoxycytidine (5hmdC) levels and global adenosine/thymidine ([dA]/[T]) ratio in bone marrow aspirates from twenty-one HR MDS patients, pre- and post-HMA treatment. Additionally, targeted methylation analysis was performed by interpretation of NGS-methylation (MeD-seq) data obtained from the same patient cohort.
LC/MS-MS analysis revealed a significant hypomethylation status in responders (Rs), already established at baseline and a trend for further DNA methylation reduction post-HMA treatment. Non-responders (NRs) reached statistical significance for DNA hypomethylation only post-HMA treatment. The 5hmdC epigenetic mark was approximately detected at 37.5-40% among NRs and Rs, implying the impairment of the natural active demethylation pathway, mediated by the ten-eleven (TET) 5mdC dioxygenases. R and NR subgroups displayed a [dA]/[T] ratio < 1 (0.727 - 0.633), supporting high frequences of 5mdC transition to thymidine. Response to treatment, according to whole genome MeD-seq data analysis, was associated with specific, scattered hypomethylated DMRs, rather than presenting a global effect across genome. MeD-seq analysis identified divergent epigenetic effects along chromosomes 7, 9, 12, 16, 18, 21, 22, X and Y. Within statistically significant selected chromosomal bins, genes encoding for proteins and non-coding RNAs with reversed methylation profiles between Rs and NRs, were highlighted.
Implementation of powerful analytical tools to identify the dynamic DNA methylation changes in HR MDS patients undergoing HMA therapy demonstrated that LC-MS/MS exerts high efficiency as a broad-based but rapid and cost-effective methodology (compared to MeD-seq) to decode different perspectives of the epigenetic background of HR MDS patients and possess discriminative efficacy of the response phenotype to HMA treatment.
高危（HR）骨髓增生异常综合征（MDS）患者若不符合异基因造血干细胞移植（alloHSCT）条件，则需要迅速进行治疗干预，如使用去甲基化药物（HMA）进行治疗，以恢复正常的DNA甲基化模式，主要是抑癌基因的甲基化模式，从而延缓疾病进展并提高总生存期（OS）。然而，对HMA治疗的反应评估依赖于传统方法，其发现广泛潜在分子事件的能力有限。
我们采用液相色谱-串联质谱法（LC-MS/MS）评估21例HR MDS患者在接受HMA治疗前后骨髓穿刺液中5'-甲基-2'-脱氧胞苷（5mdC）、5'-羟基甲基-2'-脱氧胞苷（5hmdC）水平以及全局腺苷/胸腺嘧啶（[dA]/[T]）比值。此外，通过解读从同一患者队列获得的NGS甲基化（MeD-seq）数据进行靶向甲基化分析。
LC/MS-MS分析显示，反应者（Rs）在基线时就已存在显著的低甲基化状态，且在HMA治疗后有进一步降低DNA甲基化的趋势。无反应者（NRs）仅在HMA治疗后DNA低甲基化达到统计学意义。在NRs和Rs中，5hmdC表观遗传标记约为37.5 - 40%，这意味着由十一-易位（TET）5mdC双加氧酶介导的天然活性去甲基化途径受损。R和NR亚组的[dA]/[T]比值<1（0.727 - 0.633），支持5mdC向胸腺嘧啶转变的高频率。根据全基因组MeD-seq数据分析，治疗反应与特定的、分散的低甲基化差异甲基化区域（DMRs）相关，而非呈现全基因组的整体效应。MeD-seq分析确定了沿染色体7、9、12、16、18、21、22、X和Y的不同表观遗传效应。在具有统计学意义的选定染色体区域内，突出显示了Rs和NRs之间甲基化谱相反的蛋白质和非编码RNA编码基因。
采用强大的分析工具来识别接受HMA治疗的HR MDS患者动态DNA甲基化变化表明，LC-MS/MS作为一种基础广泛但快速且经济高效的方法（与MeD-seq相比），在解码HR MDS患者表观遗传背景的不同方面具有很高的效率，并且对HMA治疗的反应表型具有鉴别效力。
