Development of assays for genome wide analysis of cytosine methylation and their applications in tumor models

1. Epigenomic studies in Barrett’s esophagus and Esophageal cancer

Barretts esophagus is a premalignant condition that affects millions of Americans and can progress to esophageal adenocarcinoma. There is a need to identify pathogenic insights into the process of malignant transformation so that cases of BE with the highest risk of transformation can be identified and then be targeted by aggressive therapeutic interventions. We have developed genome wide methylation assays (nano-HELP assay, in collaboration with the Greally lab) that can be used for studies with small numbers of primary cells. This platform was used to study the epigenome of Barrett’s esophagus and esophageal adenocarcinoma (In collaboration with Anirban Maitra (MD Anderson) and Stephen Meltzer (Johns Hopkins).

Figure 4
Although a combination of genomic and epigenetic alterations are implicated in the multistep transformation of normal squamous esophageal epithelium to Barrett esophagus, dysplasia and adenocarcinoma, the combinatorial effect of these changes was unknown and had not been studied in a genome wide manner. By integrating genome-wide DNA methylation, copy number and transcriptome datasets obtained from endoscopic biopsies of neoplastic progression within the same individual, we found that the previously reported global hypomethylation phenomenon in cancer has its origins at the earliest stages of epithelial carcinogenesis. Promoter hypomethylation appeared to synergize with gene amplification leading to upregulation of a chr4q21 chemokine cluster and other transcripts during Barrett neoplasia. In contrast, gene-specific hypermethylation observed at a restricted number of loci, and in combination with hemi-allelic deletions, appeared to lead to downregulation of selected transcripts during multistep progression. We also observed that epigenetic regulation during epithelial carcinogenesis was not restricted to traditionally defined "CpG islands", but also occurred through a mechanism of differential methylation outside of these regions. Finally, we determined that chemokine ligands CXCL1 and CXCL3 are upregulated due to loss of promoter methylation and can serve as potential serum biomarkers in Barrett’s esophagus and adenocarcinoma. Studies are now underway in collaboration with Dr Stephen Meltzer (Johns Hopkins) and Anirban Maitra (MD Anderson) to evaluate the role of non coding alterations in this model.

Alvarez H, Opalinska J, Zhou L, Sohal D, Fazzari M, Yu Y, Montagna C, Montgomery E, Canto M, Dunbar K, Wang J, Roa J, Mo Y, Bhagat T, Ramesh K, Cannizzaro, Mollenhauer, Thompson, Suzuki M, Meltzer S, Melnick A, Greally JM, Maitra A, Verma A Widespread hypomethylation occurs early and synergizes with gene amplification during esophageal carcinogenesis. PLOS Genetics 2011 Mar;7(3) PMID: 21483804

Wu W, Bhagat T, Yang X, Song J, Cheng Y, Agarwal R, Abraham J, Ibrahim S, Bartenstein M, Hussain Z, Suzuki M, Yu Y, Eng C, Greally J, Verma A*, Meltzer SJ* Co-Corresponding Hypomethylation of Noncoding DNA Regions and Overexpression of the Long Noncoding RNA, AFAP1-AS1, in Barrett’s Esophagus and Esophageal Adenocarcinoma
Gastroenterology (In Press) 2013 PMID: 23333711

Yang X, Song JH, Cheng Y, Wu W, Bhagat T, Yu Y, Abraham JM, Ibrahim S, Ravich Roland W, Khashab M, Singh VK, Shin EJ, Yang X, Verma A, Meltzer SJ, Mori Y. Long Noncoding RNA HNF1A-AS1 Regulates Proliferation and Migration in Esophageal Adenocarcinoma Cells Gut 2013 (In Press)

Bajpai M, Kessel R, Bhagat T, Nischal S, Yu Y, Verma A*, Das K *Co-Corresponding
High resolution integrative analysis reveals widespread genetic and epigenetic changes after chronic in-vitro acid and bile exposure in Barrett's epithelium cells Genes Chromosome Cancer 2013 (In Press)


2. Development of HELP-GT assay for 5hmC determination:

Figure 5
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 (β-GT) can make CCGG residues insensitive to digestion by MspI. Restriction digestion by HpaII, MspI or MspI after β-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 β-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.

Bhattacharyya S, Yu Y, Suzuki M, Campbell N, Mazdo J, Vasanthakumar A, Bhagat TD, Nischal S, Christopeit M, Parekh S, Steidl U, Godley L, Maitra A, Greally JM, Verma A.

Genome wide hydroxymethylation tested using the HELP-GT assay shows redistribution in cancer. Nuc. Acid. Res. 2013;In Press.

3. Development of methylation based prognostic signatures in AML: (in collaboration with the Steidl Lab.)

Acute myeloid leukemia (AML) is characterized by a differentiation block of hematopoietic stem and progenitor cells. Frequently, it is associated with mutations in epigenetic modifiers. We hypothesized that analysis of DNA methylation changes during normal hematopoietic differentiation can yield epigenetic signatures that can identify novel, developmentally distinct, prognostic subgroups of AML. Genome-wide cytosine methylation analysis of highly purified human long-term hematopoietic stem cells (HSC), short-term HSC, common myeloid progenitors, and megakaryocyte-erythrocyte progenitors was performed by the nano-HELP assay. We observed that the most striking epigenetic changes occurred during commitment from short-term HSC to common myeloid progenitors and were predominantly characterized by loss of methylation. An epigenetic signature of HSC commitment was developed that proved to be highly prognostic in other independent large AML patient cohorts, regardless of treatment and epigenetic mutations. The epigenetic signature was superior to commitment-based gene expression signatures, and a stem-like methylome was independently predictive of poorer overall survival in AML.

Bartholdy B, Christopeit M, Will B, Mo Y, Barreyro L, Yu Y, Bhagat T, Okoye-Okafor U, Todorova T, Greally J, Levine RL, Melnick A, Verma A*, Steidl U. *Co-Corresponding

A human hematopoietic stem cell-commitment related DNA cytosine methylation signature is prognostic for overall survival in acute myeloid leukemia Journal of Clinical Investigation 2014 (In Press) 

4. DNA methylation alterations in Myeloproliferative syndromes :

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.

Nischal S, Bhattacharyya S, Christopeit N, Yu Y, Zhou L, Bhagat T, Sohal S, Will B, Mo Y, Suzuki M, Pardanani A, McDevitt M, Maciejewski J, Melnick A, Greally J, Steidl U, Moliterno A, Verma A

Methylome profiling reveals distinct alterations in phenotypic and mutational subgroups of myeloproliferative neoplasms Cancer Res, 2012 Dec 27. PMID: 23066032  

5. Genome wide alterations in DNA methylation in Myeloma

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.

Heuck C, Mehta J, Bhagat T, Gundabolu K, Yu Y, Khan S, Chrysofakis G, Schinke C, Tariman J, Vickrey E, Pulliam N, Nischal S, Zhou L, Bhattacharya S, Meagher R, Hu C, Maqbool S, , Suzuki M, Parekh S, Reu F, Steidl U, Greally J, Verma A*, Singhal S* Co-Corresponding 

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

Journal of Immunology (In Press) 2013

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