Next-Generation Bisulfite Sequencing of Aml Reveals Widespread Acquisition of Epigenetic Abnormalities in Leukemic Stem Cells That Are Stably Retained in More Mature Leukemic Cell Fractions

Acute myeloid leukemia (AML) is a disease of aberrant hematopoietic differentiation believed to mirror the hierarchical pattern of hematopoiesis with leukemia stem cells (LSC) serving as the originating cell population from which the tumor arises. Like hematopoietic stem cells (HSC), leukemia stem cells are believed to be largely quiescent and therefore impervious to conventional chemotherapeutics resulting in relapse of disease despite achievement of clinical remission. The DNA methylation profiles of bulk leukemia cells differ significantly from normal CD34+ cells; however, less is known about the potential differences between epigenetic profiles of purified LSC and normal HSC. Moreover, the stability of the methylome as the LSC differentiate into mature leukemia progenitor cells (LPC) has not been studied. In order to address this LSC, LPC, and HSC were sorted from the bone marrow of AML patients and normal controls based on CD34, CD38, CD45 and ALDH activity. LSC were defined as CD34⁺ CD38^- ALDH^mid; LPC as CD34⁺ CD38⁺; and HSC as CD34⁺ CD38^- ALDH^high. These isolated fractions were used for genome-wide DNA methylation analysis by the next-generation enhanced reduced representation bisulfite sequencing (ERRBS) assay, which allowed for the comparison of the methylation landscapes of LSC and HSC, as well as those of LSC and LPC. A total of thirteen AML samples were examined for the presence of LSC, six of which did not have an ALDH^mid population but had instead an ALDH^high population. Because of their phenotypic similarity to normal HSC, these samples were not included in the present comparison against HSC. The methylomes of six independent LSC samples were compared to methylomes of five HSC. Sequenced ERRBS libraries were aligned against the human genome (hg19) and differentially methylated regions (DMR) were identified using a beta-binomial model and selecting regions with absolute mean methylation difference of >25% and false discovery rate (FDR) < 10%. The methylation profiles of LSC showed widespread genome wide differences relative to HSC; 39,162 regions were found to be hypermethylated in LSC while 5,408 regions were hypomethylated. DMRs were enriched at CpG islands as well as intra- and intergenic enhancers. Functional annotation of the DMRs to gene sets in the MSigDb database revealed enrichment for genes marked by the Polycomb repressive mark H3K27me3 (FDR = 1.86×10^-49). In order to determine whether epigenetic abnormalities observed at the LSC level were distinct from the epigenetic profiles observed in the more mature LPC fraction, we compared paired LSC and LPC specimens from 6 AML patients. Notably, LPC did not significantly differ in their epigenetic profiles from LSC, indicating that epigenetic abnormalities acquired at the LSC stage are stably transmitted through leukemic expansion to the more mature LPC fraction. In summary, we have identified widespread epigenetic abnormalities acquired at the LSC stage, of greater magnitude than was previously recognized by performing comparisons of leukemic cells to unfractionated CD34+ controls. Genes targeted by aberrant methylation in LSC are significantly enriched in Polycomb target genes, suggesting a potential role for Polycomb proteins in leukemic transformation. By contrast, no significant epigenetic differences were observed between the LSC and LPC fractions in AML, indicating that epigenetic abnormalities acquired at the LSC level are static through multiple cell generations.