Certain pediatric leukemias share a common underlying cause with treatment–related secondary leukemias. Both diseases involve translocations in the KMT2A gene, in which a portion of this gene is swapped out with DNA from a “partner” gene on a separate chromosome. The resulting recombination causes an abnormal genetic rearrangement called a translocation that leads to leukemia, which is cancer of the blood cells. Patients with these types of leukemias tend to have poor outcomes.

As a step toward better understanding these forms of cancer, a joint effort by University of Pennsylvania and Children’s Hospital of Philadelphia researchers has applied an innovative new genome sequencing technique to catalog the sites of DNA cleavage by the enzyme topoisomerase II, called TOP2.

The work was led by Brian D. Gregory, an associate professor in Penn’s Department of Biology in the School of Arts & Sciences; Xiang Yu, a postdoctoral researcher in Gregory’s lab; Carolyn A. Felix, the Joshua Kahan Endowed Chair in Pediatric Leukemia Research and an attending physician at CHOP and a professor of pediatrics in Penn’s Perelman School of Medicine, and James W. Davenport, a research associate in the Felix lab.

They reported their findings in the journal Genome Research.

“This tool opens new possibilities to better understand and eventually be able to manipulate TOP2 cutting to prevent the rearrangements that give rise to leukemias,” said Felix, study co–leader.

The translocations that lead to infant leukemias and treatment–related secondary leukemias involve the action of the enzyme TOP2. This is because TOP2 plays a helping hand during DNA transcription and replication by cleaving the two DNA strands and easing the tangles and torsion that occurs during these processes, thus allowing them to untwist or pass through one another, then repairing the break. Translocations arise when the “repairs” result in mismatching and joining DNA from two locations in the genome.

Certain chemotherapeutic agents are TOP2 poisons and, while they can effectively kill cancer cells, they sometimes lead to abnormal DNA rejoining and cause translocations, which are the hallmark of the treatment–related secondary leukemias. Felix and colleagues have also previously reported that babies exposed in utero to TOP2 poisons, found in some foods in maternal diet, are at an increased risk of developing infant leukemia.

Felix’s lab has identified several specific leukemia–causing translocations between the KMT2A gene and partner genes, but wanted a more efficient way to identify all of the points in the genome that are cleaved by TOP2 to be able to confirm the role of TOP2 cleavage in the DNA damage that is repaired incorrectly to create translocations.

That’s where Gregory’s group came in. Over several years they had developed a technique to perform genome–wide sequencing of locations where an enzyme makes a covalent bond to DNA, which is what TOP2 does while snipping strands of DNA.

“We designed a way to pull down the DNA bound to TOP2, then break that bond so only the DNA covalently attached to TOP2 is free to be sequenced to single base–pair precision,” Gregory said. “This enabled us to map, for the first time, topoisomerase II cleavage on a genome–wide scale.”

The team performed the analysis on a human leukemia cell line derived from a patient with leukemia, obtaining all of the cleavage sites, then repeated the technique on the same cells treated with either chemotherapy drugs or other TOP2 poisons found in food or the environment.