Myeloma is characterized by stage-specific alterations in DNA methylation that occur early during myelomagenesis.

Epigenetic changes play important roles in carcinogenesis and influence initial steps in neoplastic transformation by altering genome stability and regulating gene expression. To characterize epigenomic changes during the transformation of normal plasma cells to myeloma, we modified the HpaII tiny fragment enrichment by ligation-mediated PCR assay to work with small numbers of purified primary marrow plasma cells. The nano-HpaII tiny fragment enrichment by ligation-mediated PCR assay was used to analyze the methylome of CD138(+) cells from 56 subjects representing premalignant (monoclonal gammopathy of uncertain significance), early, and advanced stages of myeloma, as well as healthy controls. Plasma cells from premalignant and early stages of myeloma were characterized by striking, widespread hypomethylation. Gene-specific hypermethylation was seen to occur in the advanced stages, and cell lines representative of relapsed cases were found to be sensitive to decitabine. Aberrant demethylation in monoclonal gammopathy of uncertain significance occurred primarily in CpG islands, whereas differentially methylated loci in cases of myeloma occurred predominantly outside of CpG islands and affected distinct sets of gene pathways, demonstrating qualitative epigenetic differences between premalignant and malignant stages. Examination of the methylation machinery revealed that the methyltransferase, DNMT3A, was aberrantly hypermethylated and underexpressed, but not mutated in myeloma. DNMT3A underexpression was also associated with adverse overall survival in a large cohort of patients, providing insights into genesis of hypomethylation in myeloma. These results demonstrate widespread, stage-specific epigenetic changes during myelomagenesis and suggest that early demethylation can be a potential contributor to genome instability seen in myeloma. We also identify DNMT3A expression as a novel prognostic biomarker and suggest that relapsed cases can be therapeutically targeted by hypomethylating agents.

Genome-wide hydroxymethylation tested using the HELP-GT assay shows redistribution in cancer.

5-hydroxymethylcytosine (5-hmC) is a recently discovered epigenetic modification that is altered in cancers. Genome-wide assays for 5-hmC determination are needed as many of the techniques for 5-methylcytosine (5-mC) determination, including methyl-sensitive restriction digestion and bisulfite sequencing cannot distinguish between 5-mC and 5-hmC. Glycosylation of 5-hmC residues by beta-glucosyl transferase (ß-GT) can make CCGG residues insensitive to digestion by MspI. Restriction digestion by HpaII, MspI or MspI after ß-GT conversion, followed by adapter ligation, massive parallel sequencing and custom bioinformatic analysis allowed us determine distribution of 5-mC and 5-hmC at single base pair resolution at MspI restriction sites. The resulting HpaII tiny fragment Enrichment by Ligation-mediated PCR with ß-GT (HELP-GT) assay identified 5-hmC loci that were validated at global level by liquid chromatography-mass spectrometry (LC-MS) and the locus-specific level by quantitative reverse transcriptase polymerase chain reaction of 5-hmC pull-down DNA. Hydroxymethylation at both promoter and intragenic locations correlated positively with gene expression. Analysis of pancreatic cancer samples revealed striking redistribution of 5-hmC sites in cancer cells and demonstrated enrichment of this modification at many oncogenic promoters such as GATA6. The HELP-GT assay allowed global determination of 5-hmC and 5-mC from low amounts of DNA and with the use of modest sequencing resources. Redistribution of 5-hmC seen in cancer highlights the importance of determination of this modification in conjugation with conventional methylome analysis.

High resolution methylome analysis reveals widespread functional hypomethylation during adult human erythropoiesis.

Differentiation of hematopoietic stem cells to red cells requires coordinated expression of numerous erythroid genes and is characterized by nuclear condensation and extrusion during terminal development. To understand the regulatory mechanisms governing these widespread phenotypic changes, we conducted a high resolution methylomic and transcriptomic analysis of six major stages of human erythroid differentiation. We observed widespread epigenetic differences between early and late stages of erythropoiesis with progressive loss of methylation being the dominant change during differentiation. Gene bodies, intergenic regions, and CpG shores were preferentially demethylated during erythropoiesis. Epigenetic changes at transcription factor binding sites correlated significantly with changes in gene expression and were enriched for binding motifs for SCL, MYB, GATA, and other factors not previously implicated in erythropoiesis. Demethylation at gene promoters was associated with increased expression of genes, whereas epigenetic changes at gene bodies correlated inversely with gene expression. Important gene networks encoding erythrocyte membrane proteins, surface receptors, and heme synthesis proteins were found to be regulated by DNA methylation. Furthermore, integrative analysis enabled us to identify novel, potential regulatory areas of the genome as evident by epigenetic changes in a predicted PU.1 binding site in intron 1 of the GATA1 gene. This intronic site was found to be conserved across species and was validated to be a novel PU.1 binding site by quantitative ChIP in erythroid cells. Altogether, our study provides a comprehensive analysis of methylomic and transcriptomic changes during erythroid differentiation and demonstrates that human terminal erythropoiesis is surprisingly associated with hypomethylation of the genome.

High resolution integrative analysis reveals widespread genetic and epigenetic changes after chronic in-vitro acid and bile exposure in Barrett's epithelium cells.

Barrett's epithelium (BE) is a premalignant condition resulting from chronic gastroesophageal reflux that may progress to esophageal adenocarcinoma (EAC). Early intervention holds promise in preventing BE progression. However, identification of high-risk BE patients remains challenging due to inadequate biomarkers for early diagnosis. We investigated the effect of prolonged chronic acid and bile exposure on transcriptome, methylome, and mutatome of cells in an in-vitro BE carcinogenesis (BEC) model. Twenty weeks acid and bile exposed cells from the BEC model (BEC20w) were compared with their naïve predecessors HiSeq Illumina based RNA sequencing was performed on RNA from both the cells for gene expression and mutational analysis. HELP Tagging Assay was performed for DNA methylation analysis. Ingenuity pathway, Gene Ontology, and KEGG PATHWAY analyses were then performed on datasets. Widespread aberrant genetic and epigenetic changes were observed in the BEC20w cells. Combinatorial analyses revealed 433 from a total of 863 downregulated genes had accompanying hypermethylation of promoters. Simultaneously, 690 genes from a total of 1,492 were upregulated with accompanying promoter hypomethylation. In addition, 763 mutations were identified on 637 genes. Ingenuity pathway analysis, Gene Ontology, and KEGG PATHWAY analyses associated the genetic and epigenetic changes in BEC20w cells with cellular and biological functions. Integration of high resolution comparative analyses of naïve BAR-T and BEC20w cells revealed striking genetic and epigenetic changes induced by chronic acid and bile exposure that may disrupt normal cellular functions and promote carcinogenesis. This novel study reveals several potential targets for future biomarkers and therapeutic development.

Aberrant epigenetic and genetic marks are seen in myelodysplastic leukocytes and reveal Dock4 as a candidate pathogenic gene on chromosome 7q.

Myelodysplastic syndromes (MDS) are characterized by abnormal and dysplastic maturation of all blood lineages. Even though epigenetic alterations have been seen in MDS marrow progenitors, very little is known about the molecular alterations in dysplastic peripheral blood cells. We analyzed the methylome of MDS leukocytes by the HELP assay and determined that it was globally distinct from age-matched controls and was characterized by numerous novel, aberrant hypermethylated marks that were located mainly outside of CpG islands and preferentially affected GTPase regulators and other cancer-related pathways. Additionally, array comparative genomic hybridization revealed that novel as well as previously characterized deletions and amplifications could also be visualized in peripheral blood leukocytes, thus potentially reducing the need for bone marrow samples for future studies. Using integrative analysis, potentially pathogenic genes silenced by genetic deletions and aberrant hypermethylation in different patients were identified. DOCK4, a GTPase regulator located in the commonly deleted 7q31 region, was identified by this unbiased approach. Significant hypermethylation and reduced expression of DOCK4 in MDS bone marrow stem cells was observed in two large independent datasets, providing further validation of our findings. Finally, DOCK4 knockdown in primary marrow CD34(+) stem cells led to decreased erythroid colony formation and increased apoptosis, thus recapitulating the bone marrow failure seen in MDS. These findings reveal widespread novel epigenetic alterations in myelodysplastic leukocytes and implicate DOCK4 as a pathogenic gene located on the 7q chromosomal region.

Cytokine-Regulated Phosphorylation and Activation of TET2 by JAK2 in Hematopoiesis.

Even though the Ten-eleven translocation (TET) enzymes catalyze the generation of 5-hydroxymethylcytosines required for lineage commitment and subsequent differentiation of stem cells into erythroid cells, the mechanisms that link extracellular signals to TET activation and DNA hydroxymethylation are unknown. We demonstrate that hematopoietic cytokines phosphorylate TET2, leading to its activation in erythroid progenitors. Specifically, cytokine receptor-associated JAK2 phosphorylates TET2 at tyrosines 1939 and 1964. Phosphorylated TET2 interacts with the erythroid transcription factor KLF1, and this interaction with TET2 is increased upon exposure to erythropoietin. The activating JAK2V617F mutation seen in myeloproliferative disease patient samples and in mouse models is associated with increased TET activity and cytosine hydroxymethylation as well as genome-wide loss of cytosine methylation. These epigenetic and functional changes are also associated with increased expression of several oncogenic transcripts. Thus, we demonstrate that JAK2-mediated TET2 phosphorylation provides a mechanistic link between extracellular signals and epigenetic changes during hematopoiesis. SIGNIFICANCE: Identification of TET2 phosphorylation and activation by cytokine-stimulated JAK2 links extracellular signals to chromatin remodeling during hematopoietic differentiation. This provides potential avenues to regulate TET2 function in the context of myeloproliferative disorders and myelodysplastic syndromes associated with the JAK2V617F-activating mutation.This article is highlighted in the In This Issue feature, p. 681.

Methylome profiling reveals distinct alterations in phenotypic and mutational subgroups of myeloproliferative neoplasms.

Even though mutations in epigenetic regulators frequently occur in myeloproliferative neoplasms, their effects on the epigenome have not been well studied. Furthermore, even though primary myelofibrosis (PMF) has a markedly worse prognosis than essential thrombocytosis or polycythemia vera, the molecular distinctions between these subgroups are not well elucidated. We conducted the HELP (HpaII tiny fragment enriched by LM-PCR) assay to study genome-wide methylation in polycythemia vera, essential thrombocytosis, and PMF samples compared with healthy controls. We determined that polycythemia vera and essential thrombocytosis are characterized by aberrant promoter hypermethylation, whereas PMF is an epigenetically distinct subgroup characterized by both aberrant hyper- and hypomethylation. Aberrant hypomethylation in PMF was seen to occur in non-CpG island loci, showing further qualitative differences between the disease subgroups. The differentially methylated genes in polycythemia vera and essential thrombocytosis were involved predominantly in cell signaling pathways and were enriched for binding sites of GATA1 and other transcription factors. In contrast, aberrantly methylated genes in PMF were involved in inflammatory pathways and were enriched for NF1, LEF1, and other transcription factors. Within the PMF subgroup, cases with ASXL1 disruptions formed an epigenetically distinct subgroup with relatively increased methylation. Cases of myeloproliferative neoplasms (MPN) with TET2 mutations showed decreased levels of hydroxymethylation and distinct set of hypermethylated genes. In contrast, the JAK2V617F mutation did not drive epigenetic clustering within MPNs. Finally, the significance of aberrant methylation was shown by sensitivity of MPN-derived cell lines to decitabine. These results show epigenetic differences between PMF and polycythemia vera/essential thrombocytosis and reveal methylomic signatures of ASXL1 and TET2 mutations.

Mechanism of action of lenalidomide in hematological malignancies.

Immunomodulatory drugs lenalidomide and pomalidomide are synthetic compounds derived by modifying the chemical structure of thalidomide to improve its potency and reduce its side effects. Lenalidomide is a 4-amino-glutamyl analogue of thalidomide that lacks the neurologic side effects of sedation and neuropathy and has emerged as a drug with activity against various hematological and solid malignancies. It is approved by FDA for clinical use in myelodysplastic syndromes with deletion of chromosome 5q and multiple myeloma. Lenalidomide has been shown to be an immunomodulator, affecting both cellular and humoral limbs of the immune system. It has also been shown to have anti-angiogenic properties. Newer studies demonstrate its effects on signal transduction that can partly explain its selective efficacy in subsets of MDS. Even though the exact molecular targets of lenalidomide are not well known, its activity across a spectrum of neoplastic conditions highlights the possibility of multiple target sites of action.