Molecular analysis of t(15;17) genomic breakpoints in secondary acute promyelocytic leukemia arising after treatment of multiple sclerosis

Abstract

Therapy-related acute promyelocytic leukemia (t-APL) with t(15;17) translocation is a well-recognized complication of cancer treatment with agents targeting topoisomerase II. However, cases are emerging after mitoxantrone therapy for multiple sclerosis (MS). Analysis of 12 cases of mitoxantrone-related t-APL in MS patients revealed an altered distribution of chromosome 15 breakpoints versus de novo APL, biased toward disruption within PML intron 6 (11 of 12, 92% vs 622 of 1022, 61%: P = .035). Despite this intron spanning approximately 1 kb, breakpoints in 5 mitoxantrone-treated patients fell within an 8-bp region (1482-9) corresponding to the "hotspot" previously reported in t-APL, complicating mitoxantrone-containing breast cancer therapy. Another shared breakpoint was identified within the approximately 17-kb RARA intron 2 involving 2 t-APL cases arising after mitoxantrone treatment for MS and breast cancer, respectively. Analysis of PML and RARA genomic breakpoints in functional assays in 4 cases, including the shared RARA intron 2 breakpoint at 14 446-49, confirmed each to be preferential sites of topoisomerase IIalpha-mediated DNA cleavage in the presence of mitoxantrone. This study further supports the presence of preferential sites of DNA damage induced by mitoxantrone in PML and RARA genes that may underlie the propensity to develop this subtype of leukemia after exposure to this agent.

Figure 1

Characterization of t(15;17) breakpoints within the PML and RARA loci. The location of breakpoints indicated by ▾ in the 14 patients (numbers correspond with UPNs in Tables 1 and 2) within the PML gene on chromosome 15 (A; bcr3 region and bcr1/2 region) and intron 2 of RARA on chromosome 17 (B) are shown. Breakpoint locations are numbered according to the following GenBank accession numbers: PML intron 6 (bcr 1), S57791; PML intron 3 (bcr 3), S51489; and RARA intron 2, AJ297538.

Figure 2

Investigation of t(15;17) translocation mechanism in UPN 2 by in vitro topoisomerase IIα DNA cleavage assay. Chromosomal breakpoint junctions were examined in an in vitro topoisomerase IIα cleavage assay using substrates containing PML (A) and RARA (B) translocation breakpoints in the APL case of UPN 2. Reactions in lane 1 were performed without DNA topoisomerase IIα and lanes 2 to 5 show dideoxy sequencing reactions. DNA cleavage reactions were performed in the presence of 147 nM of human DNA topoisomerase II alpha and in the absence (lanes 6 and 8) or presence of 20 μM mitoxantrone (lanes 7 and 9). Reactions in lanes 8 and 9 were incubated at 75°C to assess the heat stability of the cleavage products seen in lanes 6 and 7. In each case, the location of the relevant heat stable cleavage site is indicated by an arrow on the far right. (C) Native PML and RARA sequences are shown in red and blue, respectively. In the creation of the PML-RARA genomic fusion, processing includes exonucleolytic deletion to form a 2-base homologous overhang that facilitates repair via the error prone NHEJ pathway. In the creation of the reciprocal RARA-PML genomic fusion, 2-base homologies facilitate NHEJ repair, whereas in both instances polymerization of the relevant overhangs fills in any remaining gaps (shown black font).

Figure 3

Investigation of t(15;17) translocation mechanism in UPN 14 by in vitro topoisomerase IIα DNA cleavage assay. DNA cleavage assays are shown for PML (A) and RARA (B) genomic breakpoint regions. For the PML assay, the reverse complement of the substrate containing the “hotspot” region between 1482 and 1489 described by Mistry et al was used. Lanes 1 to 9 of each cleavage assay are described in the legend to Figure 2. (C) Native PML and RARA sequences are shown in red and blue, respectively. In the creation of PML-RARA, processing includes exonucleolytic deletion and repair via the NHEJ pathway. In the creation of RARA-PML, 2-base homologies facilitate repair via the NHEJ pathway, whereas in both instances polymerization of the relevant overhangs fills in any remaining gaps (shown in black font).