Background: Epigenetic regulators as well as epigenetic marks play an essential role in regulating normal hematopoiesis. Epigenetic alterations are one of the early events reflecting changes in cell identify during the transition of a pre-leukemic condition into a malignant phenotype. Capturing epigenetic alterations at early stages of pre-leukemic conditions could benefit the diagnosis and understanding the disease progression during hematopoietic malignant transformation. DNA 5-hydroxymethylation (5hmC) is the one of the major oxidation products of 5-methycytosine (5mC) mediated by the TET protein family dioxygenase. 5hmC has been reported to be involve in the regulation of chromatin accessibility and gene transcription. The dynamic changes of 5hmC have been reported as a hallmark in myeloid malignancies. In our study, we applied an improved 5hmC profiling method (sCMSIP) developed in our lab to systematically profile 5hmC and evaluate the 5hmC dynamics in patients with myeloid malignancies.
Methods: In order to profile 5hmC using low amount of input DNA isolated from patient bone-marrow aspirates, we improved our previously developed anti-CMSIP method to capture 5hmC enriched regions using ultra-low amount of genomic DNA (10-20 ng gDNA). Cytosine-5-methylenesulfonatem (CMS) is the 5hmC derivatives upon sodium bisulfite treatment. A home-made anti-CMS antibody was developed to specifically recognize CMS genome-wide after sodium bisulfite treatment. The traditional anti-CMSIP has been widely used to study the genome-wide 5hmC distribution in various biological systems, including mESC, HSC and immune cells. We further optimized the immunoprecipitation procedure to profile genome-wide 5hmC with ultra-low amount of genomic DNA (sensitive CMSIP, sCMSIP). Using this improved CMS-IP method, we performed genome-wide 5hmC analysis in ~100 individuals including healthy donors (n = 10), patients with MDS (n = 62), AML (n = 4) and CMML (n = 13). In parallel, we performed RRBS (DNA methylation) and RNA-seq (transcriptome) analysis on matched samples. We then performed integrative bioinformatics analyses to unveil the potential diagnostic and prognostic values of 5hmC in myeloid neoplasms.
Results and Conclusions: Based on our analysis, we found distinct epigenetic landscapes and transcriptomes between healthy donor and patients with myeloid malignancies. Interestingly, we could further separate patients with MDS into three clusters by comparing their 5hmC landscapes with normal, AML and CMML individuals, suggesting that 5hmC might be a sensitive epigenetic mark to reflect the disease status of MDS. Furthermore, we identified a positive correlation between 5hmC distribution and the blast ratio in paired patients before and after hypomethylating agent (HMA) treatment. These findings suggest that 5hmC might be used as a prognostic marker for HMA treatment. Detailed analyses further elucidate the impaired 5hmC enrichment in various key transcription factors that are essential for HSC function and myeloid lineage specification.
Garcia-Manero:Amphivena: Consultancy, Research Funding; Helsinn: Research Funding; Novartis: Research Funding; AbbVie: Research Funding; Celgene: Consultancy, Research Funding; Astex: Consultancy, Research Funding; Onconova: Research Funding; H3 Biomedicine: Research Funding; Merck: Research Funding.
Asterisk with author names denotes non-ASH members.