Although epigenetic components play a major role in driving tumor progression in many human cancers, the methylation landscape in cancer epigenomes is still largely unexplored. Systematic sequence-based methylation analyses are notably absent and as a result, the potential clinical value of specific methylation differences and their biological impacts in cancers remain largely untapped. By identifying the aberrant methylation """"""""hot spots"""""""" in the cancer epigenome, we can target these genes for therapeutic intervention and develop them into DNA methylation biomarkers for early detection, diagnosis, prognosis, and monitoring the response to therapy. However, to fully understand the interactions between methylation and clinical behaviors, new methods are needed to determine single-base-level specific methylation patterns across the genome. As an important clinical model for our work, we will examine subsets of chronic lymphocytic leukemia (CLL) to discover DNA methylation alterations that distinguish the sub-types of CLL and suggest underlying mechanisms for differential clinical behaviors and tumor progression. Successful completion of this study will substantially influence the clinical management of CLL patients and allow """"""""up-front"""""""" administration of epigenetic therapies. To accomplish this, we will develop a high-throughput, large-scale, sequencing-based approach to provide efficient methods for deeply exploring the CLL methylome. In our preliminary study, we demonstrated that bisulfite sequencing can be carried out using an innovative massively parallel sequencing system (454-sequencing) that is capable of analyzing millions of DNA bases in a single run. This new generation of bisulfite sequencing will provide highly quantitative single methyl-cytosine resolution for specific methylation mapping in multiple CpG islands (CGIs). In this R33 application, we propose to optimize and develop a prototype high-throughput bisulfite sequencing method for ultra-deep analyses of DNA methylation patterns in primary CLL samples from CD38+ and CD38- CLL B-cells and test the hypothesis that the clinical behavior of subclasses of CLL can be defined in part by their distinct DNA methylation profiles that in turn affect multiple genes and signaling pathways. Specifically, we will: (1) develop a multiplexed amplicon preparation method for high-throughput, ultra-deep bisulfite sequencing;(2) develop a genome-scale approach for bisulfite sequencing of methylation-enriched genomic DNA libraries;(3) apply the innovative high-throughput bisulfite sequencing method to investigation of the CLL methylome. We believe that the technology developed will revolutionize the current analytical methods of DNA methylation, provide digital profiles of aberrant DNA methylation for individual human diseases and offer a deep-sequencing, robust method for epigenetic classification of disease subtypes.
Epigenetic processes control the packaging and function of the human genome and contribute to normal and pathologic states, including human cancer. Recent discoveries also have established that epigenetic alterations may play a major role in driving tumor initiation and progression. These are compelling reasons for applying advanced technologies to deciphering the cancer epigenome in its entirety. We propose to develop a high-throughput bisulfite genomic sequencing method for genome-wide quantitative analysis of DNA methylation in leukemic CLL cells and the associated bioinformatic tools for the analysis and visualization of the massive bisulfite sequencing data. This proposal will provide, for the first time, a large-scale digital definition of the DNA methylation landscape across the CLL methylome. We believe that the technology developed will revolutionize the current analytical methods of DNA methylation. With this highly informative method, we may identify genes critical in tumor progression that are influenced by aberrant DNA methylation changes. One of the clinical goals of this application is to discover epigenetic markers in the CD38+ and CD38- CLL cells that can define differential clinical behaviors in subclasses of CLL. Such a definition of the CLL methylome would substantially change the clinical management of these CLLs and allow up-front administration of therapies in more aggressive forms of CLL and also newer epigenetic or demethylating therapies. The considerable potential of the proposed method for deciphering complex epigenetic regulation and identifying novel epigenetic targets is an exciting opportunity for realizing advances in our understanding of CLL based on exquisitely specific mapping of cytosine methylation in the CLL genome.
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