DNA methylation is commonly thought of as a "molecular lock" that leads to permanent gene silencing. To investigate this notion, we tested 24 different HDAC inhibitors (HDACi) on colon cancer cells that harbor a GFP locus stably integrated and silenced by a hypermethylated CMV promoter. We found that HDACi efficiently reactivated expression of GFP and many other endogenous genes silenced by DNA hypermethylation. After treatment, all promoters were marked with active chromatin, yet DNA hypermethylation did not change. Thus, DNA methylation could not prevent gene reactivation by drug-induced resetting of the chromatin state. In evaluating the relative contribution of DNA methylation and histone modifications to stable gene silencing, we followed expression levels of GFP and other genes silenced by DNA hypermethylation over time after treatment with HDACi or DNA demethylating drugs. Reactivation of methylated loci by HDACi was detectable for only 2 weeks, whereas DNA demethylating drugs induced permanent epigenetic reprogramming. Therefore, DNA methylation cannot be considered as a lock for gene expression, but rather as a memory signal for long-term maintenance of gene silencing. These findings define chromatin as an important druggable target for cancer epigenetic therapy and suggest that removal of DNA methylation signals is required to achieve long-term gene reactivation.

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TET2 enzymatically converts 5-methylcytosine to 5-hydroxymethylcytosine as well as other covalently-modified cytosines and its mutations are common in myeloid leukemia. However, the exact mechanism and the extent to which TET2 mutations affect DNA methylation remain in question. Here we report on DNA methylomes in TET2 wild type (TET2-WT) and mutant (TET2-MT) cases of chronic myelomonocytic leukemia (CMML). We analyzed 85,134 CpG sites (28,114 sites in CpG islands (CGIs) and 57,020 in non-CpG islands (NCGIs)). TET2 mutations do not explain genome-wide differences in DNA methylation in CMML, and we found few and inconsistent differences at CGIs between TET2-WT and TET2-MT cases. By contrast, we identified 409 (0.71%) TET2-specific differentially methylated CpGs (tet2-DMCs) in NCGIs, 86% of which were hypermethylated in TET2-MT cases, suggesting a strikingly different biology of the effects of TET2 mutations at CGIs and NCGIs. DNA methylation of tet2-DMCs at promoters and non-promoters repressed gene expression. Tet2-DMCs showed significant enrichment at hematopoietic-specific enhancers marked by H3K4me1, and at binding sites for the transcription factor p300. Tet2-DMCs showed significantly lower 5-hydroxymethylcytosine in TET2-MT cases. We conclude that leukemia-associated TET2 mutations affect DNA methylation at NCGI regions containing hematopoietic-specific enhancers and transcription factor binding sites.

Genetic changes are infrequent in acute myeloid leukemia (AML) compared to other malignancies and often involve epigenetic regulators, suggesting that an altered epigenome may underlie AML biology and outcomes. In 96 AML cases including 65 pilot samples selected for cured/not-cured, we found higher CpG island (CGI) promoter methylation in cured patients. Expanded genome-wide digital restriction enzyme analysis of methylation (DREAM) data revealed a CGI methylator phenotype independent of IDH1/2 mutations we term AML-CIMP (A-CIMP+). A-CIMP was associated with longer overall survival (OS) in this dataset (median OS, years: A-CIMP+ = Not reached, A-CIMP− =1.17; P=0.08). For validation we used 194 samples from The Cancer Genome Atlas interrogated with Illumina 450k methylation arrays where we confirmed longer OS in A-CIMP (median OS, years: A-CIMP+ =2.34, A-CIMP− =1.00; P=0.01). Hypermethylation in A-CIMP favored CGIs (OR: CGI/non-CGI=5.21), and while A-CIMP was enriched in CEBPA (P=0.002) and WT1 mutations (P=0.02), 70% of cases lacked either mutation. Hypermethylated genes in A-CIMP function in pluripotency maintenance, and a gene expression signature of A-CIMP was associated with outcomes in multiple datasets. We conclude that CIMP in AML cannot be explained solely by gene mutations (e.g. IDH1/2, TET2), and that curability in A-CIMP+ AML should be validated prospectively.