Mechanisms in Leukemia-Associated Chromosomal Translocations: Novel, High-Throughput Sequencing Method to Detect Topoisomerase II (TOP2) Cleavage Complexes Genome-Wide with Exact Base Resolution,

Abstract 3445

Associations of chemotherapeutic TOP2 poisons with secondary leukemia have implicated TOP2-mediated DNA damage in balanced chromosomal translocations underlying many common forms of leukemia. “TOP2 poisons” convert native TOP2 into a cellular toxin by disrupting the cleavage/re-ligation equilibrium, either by decreasing the reverse rate of re-ligation or increasing the forward rate of cleavage, thus increasing cleavage complexes and causing DNA strand breaks that can promote recombination or initiate apoptosis. Besides anticancer chemotherapy, several dietary substances and the benzene metabolite p-benzoquinone are TOP2 poisons. Population and molecular epidemiology, translocation breakpoint junction sequences, temporal origins of translocations, and correlations of TOP2 in vitro cleavage sites with translocation breakpoints have pointed to a model in which TOP2 is the DNA damage mediator and resolution of TOP2 cleavage complexes, whether induced by chemotherapy, dietary substances, environmental toxins or ROS mediated damage, forms translocation breakpoint junctions. Still, the DNA damage mechanism(s) remain controversial. Investigation of cleavage complexes at the DNA sequence level in a human hematopoietic progenitor cell model that approximates target cells for translocations is the critical next step in testing this model. We invented a high-throughput sequencing-based method (Provisional Patent Filed) to address cause-and-effect relationships between TOP2 cleavage complexes and translocation breakpoints in the context of DNA topological structure in the chromatin of human hematopoietic cells.

TOP2 relaxes supercoiled DNA by transiently cleaving and re-ligating both strands of the double helix. Each subunit of the TOP2 homodimer forms a phosphodiester bond with the base 3' to the cleavage. This creates a fleeting covalent TOP2-DNA intermediate called the cleavage complex with 4-base staggered DNA ends tethered by the enzyme. We implemented, refined, optimized and validated a novel assay system for immunodepletion of the alpha isoform of TOP2 (TOP2A) including DNA-bound TOP2A, with concomitant isolation and purification of the DNA from cleavage complexes in human hematopoietic cells. By taking advantage of the covalent phosphodiester bonds between TOP2A and DNA, the activity of calf intestinal phosphatase (CIP) (i.e. hydrolysis of phosphodiester bonds via removal of 5' phosphates) was used for a purpose never used before: to release DNA from cleavage complexes at exact sites of cleavage. These steps were performed in CEM cells and, to better mimic target cells for translocations, fresh cord blood mononuclear cells (MNCs) from three newborn infants. Western blot and Q-PCR analyses proved that we achieved: 1) isolation and immunodepletion of TOP2A and TOP2A-bound DNA, 2) CIP release of TOP2A-bound DNA from the cleavage complexes, and 3) quantitative enrichment of DNA amplicons near known MLL translocation breakpoint hotspots over that obtained using a negative control antibody for immunodepletion. By morphology and immunophenotype, the cord blood MNCs contained lymphocyte, monocyte, and minor CMP and GMP populations, and they mainly were in G0/G1 by cell cycle analysis. Even though TOP2A cleavage complex enrichment was evident, on Western blot analysis the TOP2A in the cord blood MNCs appeared predominantly degraded, which is consistent with known TOP2A cell cycle dependence and downregulation in quiescent cells.

This validation forms the basis for the next steps in the assay to localize cleavage complexes at single base resolution genome-wide through high-throughput sequencing of DNA ends created by TOP2 and mapping them to the genome. This strategy comprises an entirely novel application of high-throughput sequencing with many possible future uses to define TOP2 cleavage complexes, as well as other adaptations to identify covalent DNA modifications with exact base precision. Secondary leukemias are a growing problem, and the incidence of infant leukemia where TOP2 poisons also have been implicated is increasing. Solving how DNA breaks arise and form chromosomal translocations would have profound implications for anticancer treatment and leukemia prevention.