The applicant has optimized the Restriction Landmark Genomic Scanning (RLGS) method for the detection of genetic and epigenetic alterations in human cancers. RLGS is a two-dimensional gel electrophoresis technique, in which up to 2,000 end-labeled NotI restriction sites are displayed in a single gel. Rapid cloning of RLGS spots is accomplished with the help of an arrayed NotI-EcoRV library. The use of methylation sensitive restriction enzymes (RLGS-M) allows scanning for altered DNA methylation pattern. It is well established that inactivation of tumor suppressor genes can lead to neoplastic transformation. The mechanisms of inactivation are incompletely understood, but include DNA methylation of the gene's promoter sequence. This has important therapeutic implications, because certain drugs used successfully to treat cancer result in demethylation of DNA. Here, the applicant and his colleagues propose to utilize the unique ability of RLGS-M to scan the genomes for promoter methylation in acute myeloid leukemias (AML). They hypothesize that DNA methylation promotes the survival of AML cells that resist intensive treatment. In preliminary studies, they have used RLGS-M to identify WIT1, a gene methylated in relapsed AML but not methylated at diagnosis or in normal tissue. The applicant and his colleagues propose four specific aims: I) to screen additional cases of AML with normal karyotype for evidence of DNA methylation; II) to clone those loci which are methylated at high frequencies; III) to correlate the findings with clinical outcome by testing a larger set of AML with methylation-sensitive-PCR; and IV) to clone novel genes linked to the methylated sites. The applicant hopes that the work will form the basis for therapeutic trials to demethylate such genes in the hope of improving the rate of cure with intensive treatment.
