The emergence of
therapy-related
acute myeloid leukemia (t-AML) has been associated with
DNA topoisomerase II (TOP2)-targeted
drug treatments and
chromosomal translocations frequently involving the MLL, or ALL-1, gene. Two distinct mechanisms have been implicated as potential triggers of t-AML translocations: TOP2-mediated DNA cleavage and apoptotic higher-order
chromatin fragmentation. Assessment of the role of TOP2 in this process has been hampered by a lack of techniques allowing in vivo mapping of TOP2-mediated DNA cleavage at
nucleotide resolution in single-copy genes. A novel method, extension
ligation-mediated polymerase chain reaction (ELMPCR), was used here for mapping topoisomerase-mediated
DNA strand breaks and apoptotic DNA cleavage across a translocation-prone region of MLL in human cells. We report the first genomic map integrating translocation breakpoints and
topoisomerase I, TOP2, and apoptotic DNA cleavage sites at
nucleotide resolution across an MLL region harboring a t-AML translocation hotspot. This hotspot is flanked by a TOP2 cleavage site and is localized at one extremity of a minor apoptotic cleavage region, where multiple single- and double-strand breaks were induced by
caspase-activated apoptotic nucleases. This cleavage pattern was in sharp contrast to that observed approximately 200 bp downstream in the exon 12 region, which displayed much stronger apoptotic cleavage but where no double-strand breaks were detected and no t-AML-associated breakpoints were reported. The localization and remarkable clustering of the t-AML breakpoints cannot be explained simply by the DNA cleavage patterns but might result from potential interactions between TOP2
poisoning, apoptotic DNA cleavage, and DNA repair attempts at specific sites of higher-order
chromatin structure in apoptosis-evading cells. ELMPCR provides a new tool for investigating the role of
DNA topoisomerases in fundamental genetic processes and translocations associated with
cancer treatments involving topoisomerase-targeted drugs.