DNA Topoisomerase II Poisons and the Etiology of Acute Leukemia in Infants.
Authors: Kolaris Christos P. , Slater Diana J. , Perl Anna Sechser , Rappaport Eric F. , Osheroff Neil , Felix Carolyn A. 
Published: April 2020

Background: Chromosomal translocations leading to MLL chimeric oncoproteins are the primary aberrations in infant acute leukemias, but how these translocations are created has not been established. A Children’s Oncology Group population epidemiology study recently validated that maternal consumption of dietary items containing topoisomerase II interacting compounds during pregnancy increases the risk of infant AML with MLL translocations. An inactivating polymorphism in the gene encoding NQO1, which detoxifies the topoisomerase II poison p-benzoquinone, also is associated with an increased risk of MLL-rearranged acute leukemias in infants, with the greatest risk for cases with t(4;11). Although there is heterogeneity, it has been suggested that infant leukemias share the same biased MLL translocation breakpoint distribution 3′ in the breakpoint cluster region (bcr) as secondary leukemias arising following chemotherapeutic topoisomerase II poisons. These observations led us to investigate the relationship between topoisomerase II cleavage complexes induced by dietary substances or p-benzoquinone and translocation breakpoints 3′ in the bcr.

Methods: Southern blot analysis was used to identify the MLL rearrangement in the AML of an infant male harboring the inactivating NQO1 polymorphism, and the MLL-AF9 genomic breakpoint junction was characterized by panhandle variant PCR. DNA substrates containing the normal homologues of the breakpoint sequences were utilized in topoisomerase II in vitro cleavage assays in the presence of p-benzoquinone or dietary substances to determine whether topoisomerase II cleavage sites were stimulated by these agents at the observed translocation breakpoints.

Results: There was a single MLL bcr rearrangement in the infant AML, and the cloning demonstrated a breakpoint junction fusing MLL bcr position 6774, 6775, 6776, 6777, 6778, 6779, 6780 or 6781 in intron 8 to position 35940, 35941, 35942, 35943, 35944, 35945, 35946 or 35947 relative to the start of AF9 intron 5. Identical 5′-GTTCTTA-3′ sequences suggesting the DNA double strand break repair mechanism of NHEJ were present at both breakpoints. The normal homologues of the breakpoints in MLL and AF9 were reciprocally cleaved by topoisomerase II in the presence of p-benzoquinone, and the cleavage sites were resolved to form the breakpoint junction. The MLL substrate contained another topoisomerase II cleavage site at position 6760 that not only was the strongest cleavage site in this substrate with the enzyme alone, but also was enhanced by p-benzoquinone, genistein, genistin, quercitin, and catechin at levels comparable to or greater than the anticancer drug etoposide. Further analysis of 3′ MLL bcr substrates revealed cleavage stimulation by natural topoisomerase II poisons near additional translocation breakpoints.

Conclusions: These results establish the relationship between topoisomerase II mediated damage from p-benzoquinone and dietary exposures and MLL translocation breakpoints. MLL translocation breakpoints in infant leukemias can be explained by topoisomerase II cleavage. The combined evidence from population epidemiology studies on maternal-fetal exposures, the NQO1-leukemia association implicating p-benzoquinone, genomic breakpoint junction cloning and topoisomerase II cleavage assays corroborate that topoisomerase II poisons damage MLL in acute leukemia in infants.