Near-precise interchromosomal recombination and functional DNA topoisomerase II cleavage sites at MLL and AF-4 genomic breakpoints in treatment-related acute lymphoblastic leukemia with t(4;11) translocation

We analyzed the der(11) and der(4) genomic breakpoint junctions of a t(4;11 ) in the leukemia of a patient previously administered etoposide and dactin omycin by molecular and biochemical approaches to gain insights about the t ranslocation mechanism and the relevant drug exposure. The genomic breakpoi nt junctions were amplified by PCR. Cleavage of DNA substrates containing t he normal homologues of the MLL and AF-4 translocation breakpoints was exam ined in vitro upon incubation with human DNA topoisomerase II alpha and eto poside, etoposide catechol, etoposide quinone, or dactinomycin. The der(11) and der(4) genomic breakpoint junctions both involved MLL intron 6 and AF- 4 intron 3. Recombination was precise at the sequence level except for the overall gain of a single templated nucleotide. The translocation breakpoint s in MLL and AF-4 were DNA topoisomerase 11 cleavage sites. Etoposide and i ts metabolites, but not dactinomycin, enhanced cleavage at these sites. Ass uming that DNA topoisomerase 11 was the mediator of the breakage, processin g of the staggered nicks induced by DNA topoisomerase 11, including exonucl eolytic deletion and template-directed polymerization, would have been requ ired before ligation of the ends to generate the observed genomic breakpoin t junctions. These data are inconsistent with a translocation mechanism inv olving interchromosomal recombination by simple exchange of DNA topoisomera se 11 subunits and DNA-strand transfer; however, consistent with reciprocal DNA topoisomerase 11 cleavage events in MLL and AF-4 in which both breaks became stable, the DNA ends were processed and underwent ligation. Etoposid e and/or its metabolites, but not dactinomycin, likely were the relevant ex posures in this patient.