Distribution of translocation breakpoints within the PML and RARA loci in t-APL cases arising following epirubicin and mitoxantrone. PML exons are represented by red boxes, RARA exons in blue and introns are represented by black lines. Arrows indicate the location of PML and RARA translocation breakpoints identified by long-range PCR and sequence analysis in patients with t-APL arising following mitoxantrone (red arrows) or epirubicin (green arrows). In 12 patients mitoxantrone was used for treatment of multiple sclerosis (MS). In the remaining 5 patients with mitoxantrone-related APL and the 6 patients with t-APL following epirubicin, these agents were used for prior breast cancer. Significant breakpoint clustering was observed, with a “hotspot” identified in PML intron 6 (position 1482–9) in mitoxantrone-related APL (following use of the drug for MS or breast cancer) and separate clusters associated with APL arising following epirubicin exposure. Chromosomal breakpoints were confirmed to be preferential sites of drug-induced topoisomerase II cleavage in functional assays (see Figure 2). Adapted from Mays et al.42 with permission.

Model for formation of the t(15;17) in a case of mitoxantrone-related t-APL (case F8) following topoII induced cleavage in PML and RARA loci with 4-base 5′ overhangs and aberrant DNA repair. Native PML and RARA sequences are red and blue, respectively. The processing includes exonucleolytic nibbling to form two-base (der(15)) or single-base (der(17)) homologies and creation of both breakpoint junctions by error-prone nonhomologous end-joining (NHEJ). In formation of the der(15), positions 1487–1488 on the antisense strand of PML are lost by exonucleolytic nibbling (pink) before NHEJ joins the indicated bases. Positions 1485–1487 on the sense strand of PML are lost by exonucleolytic nibbling (pink) and the der(17) forms by NHEJ. Template-directed polymerization of the relevant single-stranded overhangs fills in any gaps (light blue). Each RARA overhang is preserved completely. Adapted from Mistry et al.33 with permission.

Demonstration of mitoxantrone-induced topoisomerase II dependent DNA cleavage at translocation breakpoints in therapy-related APL. A) In vitro DNA topoisomerase II (topoII) cleavage assay carried out for a PML substrate containing the breakpoints of 4 treatment-related APL (t-APL) cases (F-8,-24,-25,-27) within the 8-bp breakpoint “hotspot” (positions 1482–1489). Patients received combination chemotherapy including the topoII poison mitoxantrone for breast cancer. Control reactions were carried out in the absence of topoII (lanes 1–4), and in the presence of etoposide (VP16), etoposide catechol (VP16-OH), etoposide quinone (VP16-Q) and mitoxantrone (Mit). Dideoxy sequencing reactions of the substrate are shown in lanes 5–8. Cleavage reactions were carried out by exposure to human topoIIα in the absence of drug (lane 9), and in the presence of etoposide (lane 10), etoposide catechol (lane 11), etoposide quinone (lane 12) and mitoxantrone (lane 13). Additional cleavage reactions were carried out to evaluate the heat-stability of cleavage complexes formed by incubation at 75°C for 10 min (lanes 14–18). The nucleotide shown by the dash is the 5′ side of the cleavage site (-1 position), which corresponds to the der(15) and der(17) translocation breakpoints in 4 cases of mitoxantrone-related APL (far right). The cleavage site at position 1484 was observed in the absence of drug, and in the presence of etoposide, both etoposide metabolites and mitoxantrone (lanes 9–13). Cleavage at this position was the strongest site observed in the presence of mitoxantrone (lane 13). Furthermore, the complexes formed at this site were shown to be heat-stable in the presence of mitoxantrone (lane 18). Interestingly, a cleavage site at position 1502 is also observed, which corresponds to a breakpoint detected in a case of de novo APL.
B) TopoII cleavage assay of normal homologue of der(15) and der(17) RARA translocation breakpoints in APL of one of the mitoxantrone-related cases (F-8). The substrate spanning positions 2603 to 2871 of RARA intron 2 contained the translocation breakpoints. Dash at right shows (−1) position of cleavage site corresponding to der(15) and der(17) translocation breakpoints (arrow far right). Adapted from Mistry et al.33 with permission.

Model summarizing formation of reciprocal translocation breakpoint junctions in treatment related APL directly by generation of drug-stimulated topoisomerase II cleavage complexes and near-precise or precise NHEJ repair. Adapted from Felix et al.22 with permission.