Genome-Wide Scanning of Epigenomic Alterations in Childhood Leukemia

To study how epigenetic changes contribute to childhood leukemia, and to identify new disease biomarkers, we combined an approach of pharmacologic inhibition of epigenetic modifications of chromatin with gene expression arrays (Affymetrix Exon 1.0; and miRNA array, UCSF-designed) to reveal precise epigenetic aberrations on a global scale in defined subsets of leukemia cell lines that recapitulate common genetic subtypes of leukemia. From these analyses, we identified >200 genes up-regulated by 5-aza-2′-deoxycytidine and trichostatin A treatment (including genes encoding both mRNA and miRNA). We validated 50 selected genes using real-time quantitative PCR. We also tested promoter regions for methylation in the cell lines and selected pediatric leukemia bone marrows using bisulfite-DNA sequencing. A panel of leukemia-specific methylation targets was discovered, which includes some well-known genes (such as HOXA5 and DAPK) and several novel candidates in childhood leukemia (eg., CD82 and KLF4). To extend these observations, we performed methylation microarrays on DNA samples isolated from the studied cell lines as well as bone marrows of 35 childhood leukemia patients with defined cytogenetics, using Illumina Methylation Cancer Panel I. This panel includes a total of 1,505 CpG loci from 807 genes. We also assayed corresponding archived newborn bloods (Guthrie cards) from 32 cases, and 19 control Guthrie cards of children matched to the cases based on gender and age, to guage the impact of congenital methylation events on childhood leukemia. Many targets genes, including known tumor suppressor genes and genes involved in DNA repair, cell cycle control and differentiation were found to be methylated in leukemia cell lines and the leukemic bone marrow samples, but not in Guthrie card controls. In addition, the methylation status of a core group of 67 CpG sites can differentiate the common subtypes of childhood leukemia – those with TEL-AML1, E2A-PBX1, high hyperdiploidy, and normal karyotypes. Several genes concordantly methylated in Guthrie cards and leukemic bone marrow may represent prenatal epimutations. With the integration of such assays, we have constructed a panel of potential candidate genes and miRNAs involved in development of childhood leukemia which have aberrant promoter methylation. Our studies lay a foundation for a comprehensive understanding of the full extent of epigenetic changes in development of childhood leukemia and how they may be exploited for therapeutic purposes as well as for early detection of this disease.