AML pathogenesis is a complex multistep process that involves an interplay of genetic and epigenetic aberrations. The time from disease onset to its full-blown clinical picture requires detailed knowledge about the timing of disease-driving molecular mechanisms to successfully interfere with these processes by targeted therapy. Here, we addressed the question whether epigenetic aberrations already contribute to the early events and processes in AML pathogenesis by utilizing a murine AML progression model with a stable down-regulation of the hematopoietic transcription factor PU.1 . We characterized DNA methylation dynamics over three stages of disease development and demonstrated that distinct DNA methylation changes occur early and subsequently expand during leukemogenesis. The reliability and relevance of methylation assessment by our genome-wide, array-based approach was corroborated by independently confirming 34 of 40 selected genes/chromosomal locations using quantitative, high-resolution mass spectrometry.
PCA with the DNA methylation values of all CGI array probes distinguished late leukemic stage from preleukemic and early leukemic stage animals. Preleukemic animals with low or absent myeloblasts already exhibited a high number of hypermethylated sequences, indicating extensive involvement of epigenetic mechanisms at this stage. These sequences represented 762 genes or other genomic locations. Approximately one-fourth of the preleukemic hypermethylated sequences were consistently hypermethylated throughout all leukemogenic stages, underscoring the pathogenic relevance of the affected genes for disease initiation and progression. Compared to hypermethylation, hypomethylation was considerably less abundant, probably due to its preferential occurrence outside of CGIs. Thus, our findings highlight that CGI hypermethylation accompanies AML onset and, therefore, may contribute to AML development.
From the preleukemic to the early leukemic stage, the number of hypermethylated probes appeared largely stable. At the late leukemic stage, however, the number of hypermethylated probes strikingly increased approximately 20-fold, accompanied by genomic diversification of DNA methylation. Cluster analysis of quantitative methylation values clearly discriminated between PU.1-wt and PU.1-kd animals.
The increase of aberrant DNA methylation abundance in the late leukemic stage cannot simply be explained by the mere increase in blast counts, but might rather be the consequence of a vigorous 'epigenetic' clonal evolution or of severe disturbance of the epigenetic machinery. When we examined the methylation levels in diverse hematopoietic progenitors at the preleukemic stage, all cell types displayed hypermethylation, similar to that of the bulk of myelogenic cells at this stage. Accordingly, we could so far neither dissect the AML cell of origin nor attribute leukemic expansion to the expansion of a distinct hematopoietic lineage.
Our study supports a model of an epigenetic outburst targeting distinct regions early in disease progression. This could be a consequence of genetic alterations in enzymes regulating epigenetic patterns, such as gene mutations found in human myeloid malignancies, including DNMT3a, TET2, IDH1, IDH2, EZH2, or ASXL1. With respect to the dramatic outburst of aberrant DNA methylation in the late stage, our AML-like mouse model differs from a recently reported chronic lymphocytic leukemia-like mouse model, where early DNA methylation events are followed by a gradual increase of aberrantly hypermethylated genomic regions over time .
We identified a wealth of known and novel AML-associated genes, epigenetically altered already at the preleukemic disease stage, and provide a repository of 762 early hypermethylated and 504 hypomethylated genes, together constituting a valuable resource for investigating potential key pathogenic factors in AML. Since methylation of cytosine is a reversible epigenetic modification, and demethylating drugs are already used in the clinical setting for treatment of both MDS and AML patients [55, 56], the novel early candidates identified in this study may point towards druggable mechanisms and pathways for targeted therapy. In line with observations by others [36, 37], a prominent role at disease onset can be ascribed to the Wnt signaling pathway, since members of this pathway, Fzd5, Fzd8, Fzd10, and Wnt3 (Additional file 7), were overrepresented among the early aberrantly methylated targets. The link between Wnt signaling and the PU.1-kd-driven AML mouse model is corroborated by earlier observations that PU.1 is targeted by Wnt pathway members .
We detected a considerable overlap between early aberrantly methylated genes and genes involved in human myeloid malignancies (MDS and AML) , indicating the relevance of the observed epigenetic changes in the mouse model for human disease. Hypermethylated genes in the preleukemic stage such as Cebpa and Hic1 have already been described as being hypermethylated as well in AML [57, 58]. Moreover, normal karyotype AML and MDS patients (of different WHO subtypes) displayed hypermethylation of the Wnt pathway members FZD5 and FZD8, as observed in the mouse model. We confirmed three additional candidates, PRDM16, ROBO3 and CXCL14, to be hypermethylated in the AML patient cohort. So far, none of these five genes has been validated as being aberrantly methylated in AML by a quantitative high resolution method, albeit FZD5, FZD8, ROBO3 and PRDM16 have been found in other genome-wide methylation screens of MDS samples . The concordant presence of aberrant methylation in these candidate genes already in early stages of our mouse model as well as in both MDS and AML suggests a disease driving potential of these aberrations.
It has been shown previously that binding of transcription factors to target DNA sequences may prevent their methylation . In line with this, knockdown of transcription factor PU.1 was associated with preleukemic hypermethylation at a considerable number of PU.1 target sequences derived from publicly available ChIP-Seq data . Looking closer at four selected PU.1 target genes by quantitative methylation analysis, we confirmed hypermethylation in both PU.1-kd animals and human AML samples. However, a correlation between PU.1 mRNA expression and methylation levels of the selected target genes BCOR, HES6, ITPKA and TAL1 could not be demonstrated in AML patients, suggesting other mechanisms than mere PU.1 down-regulation to be required for hypermethylation of these genes in human AML.
Taken together, our results suggest that the PU.1-kd mouse is a valuable model to study epigenetic changes during AML progression. The newly identified early hypermethylated genes are potential determinants for aberrant DNA methylation patterns in the disease course and, consequently, may contribute to disease development in humans. Early epigenetic changes are suspected drivers of malignancies and, hence, may offer the chance to identify suitable drug targets for early therapeutic intervention. As shown here, epigenetic profiling of tumor progression models is a promising strategy to highlight the role of epigenetics in disease initiation and progression.