Epigenetic changes, including DNA methylation, are a common finding in human malignancies. Research inchronic lymphocytic leukemia (CLL) has focused on genetic deletions that promote impaired apoptosis, but todate very few genes have been identified. In preliminary studies we have utilized Restriction LandmarkGenomic Scanning (RLGS), a two-dimensional gel electrophoresis that allows detection of altered DNAmethylation patterns, to study methylation in ten CLL patient samples. Our results strongly indicate that DNAmethylation, via gene silencing, contributes significantly to the pathogenesis of CLL. Specifically, wedemonstrated marked variation in the amount of aberrant methylation in patient samples ranging from 2.5-8.1%as compared to normal B cells. To fully establish the role of methylation in CLL and to exploit these alterationsin the clinic, we propose more detailed studies outlined below. Our hypothesis for this Project is that DNAmethylation contributes significantly to the development and progression of CLL.
In Specific Aim 1, RLGS willbe used on 100 CLL patients of different genotypes. RLGS profiles of CLL patients samples will be comparedwith CD19+ selected normal B-lymphocytes to identify novel CLL methylation sequences specific to CLL. Inaddition, methylation patterns between favorable and un-favorable genotypes will be compared to identify themost promising gene targets to pursue for investigation in Specific Aim 2.
In Specific Aim 2, we plan to clone asmany as 200 novel methylated sequences identified from aim 1 using genomic libraries that will guide thesearch for new tumor suppressor genes in CLL. Furthermore we propose experiments to perform abiostatistical evaluation of DNA methylation patterns in CLL.
In aim 3 these genes will then be analyzed in alarge cohort of CLL patient samples obtained from the national cooperative oncology groups, and results will becorrelated with response to therapy, progression-free survival and overall survival.
In Specific Aim 4, novelcandidate cancer genes, DAPK1, ID4 and DERMO1, whose expression is altered by DNA methylation, will bestudied in more detail in order to determine potential importance to the pathogenesis and progression of CLL.
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