DNA Cleavage within the MLL Breakpoint Cluster Region Is a Specific Event Which Occurs as Part of Higher-Order Chromatin Fragmentation during the Initial Stages of Apoptosis

Abstract

A distinct population of therapy-related acute myeloid leukemia (t-AML) is strongly associated with prior administration of topoisomerase II (topo II) inhibitors. These t-AMLs display distinct cytogenetic alterations, most often disrupting the MLL gene on chromosome 11q23 within a breakpoint cluster region (bcr) of 8.3 kb. We recently identified a unique site within the MLL bcr that is highly susceptible to DNA double-strand cleavage by classic topo II inhibitors (e.g., etoposide and doxorubicin). Here, we report that site-specific cleavage within the MLL bcr can be induced by either catalytic topo II inhibitors, genotoxic chemotherapeutic agents which do not target topo II, or nongenotoxic stimuli of apoptotic cell death, suggesting that this site-specific cleavage is part of a generalized cellular response to an apoptotic stimulus. We also show that site-specific cleavage within the MLL bcr can be linked to the higher-order chromatin fragmentation that occurs during the initial stages of apoptosis, possibly through cleavage of DNA loops at their anchorage sites to the nuclear matrix. In addition, we show that site-specific cleavage is conserved between species, as specific DNA cleavage can also be demonstrated within the murine MLL locus. Lastly, site-specific cleavage during apoptosis can also be identified at the AML1 locus, a locus which is also frequently involved in chromosomal rearrangements present in t-AML patients. In conclusion, these results suggest the potential involvement of higher-order chromatin fragmentation which occurs as a part of a generalized apoptotic response in a mechanism leading to chromosomal translocation of the MLL and AML1 genes and subsequent t-AML.Nonrandom chromosomal aberrations, particularly chromosomal translocations, are frequently found in association with a wide spectrum of malignancies, most prominently leukemias and lymphomas (9,14,47). The available evidence suggests that these nonrandom chromosomal translocations are often causal events leading to malignant transformation (2, 40). However, the molecular mechanisms which cause these translocations remain largely unknown.In many cases, a powerful argument can be made that these translocations are the result of mistakes in normal V(D)J recombinase activity (4, 60). These arguments are based on identification of features that resemble normal V(D)J recombinase activity, such as cryptic heptamer sequences, nontemplated N-region nucleotide addition, and exonucleolytic deletion of germ line nucleotides at the translocation breakpoints (4, 60). Other factors that have been implicated in the generation of nonrandom translocations include homologous recombination events between Alu elements (52) and exposure to DNA-damaging agents (35,36). It has been recognized for some time that DNA-damaging cancer chemotherapeutic agents, such as the topoisomerase II (topo II) inhibitor etoposide (36) and the alkylating agent melphalan (35), can cause chromosomal translocations. For instance, phytohemagglutinin-stimulated peripheral blood ly...

Alternative genetic pathways and cooperating genetic abnormalities in the pathogenesis of therapy-related myelodysplasia and acute myeloid leukemia

“…Cytostatic agents belonging to the topoisomerase II inhibitors interfere with rejoining of these double-strand breaks, and this activity is believed in some way to increase the risk of two simultaneous, illegitimate recombinations between the broken DNA strands of adjacent chromosomes. Studies of the genomic break-points of cases belonging to pathways III-VI now support this hypothesis, 39,[57][58][59][60][61] as the break points colocalize with topoisomerase II breakage sites.…”

A DNA damage repair mechanism is involved in the origin of chromosomal translocations t(4;11) in primary leukemic cells

“…Recent data further suggest the existence of a broad range of agents capable of activating signal pathways, that disrupt chromatin and lead to apoptosis if allowed to proceed to completion. When interrupted prematurely, these signal pathways may produce translocations and thus allow the cell to escape the apoptotic fate (Stanulla et al, 1997b). Conceivably, these agents cleave chromatin at preferred sites, which may provide another explanation for the observed tight clustering of the translocation breakpoints in both genes.…”

Cleavage of the MLL gene by activators of apoptosis is independent of topoisomerase II activity

“…It is clear that genomic MLL is a target of site-specific cleavage at the intron 11/exon 12 border in a variety of human cells. 7,8,10 As shown in Figures 2 and 3, both the entire 8.3 kbp MLL BCR and smaller fragments of 367 and 780 bp were subject to cleavage when placed as episomes in cells that were subsequently triggered to undergo apoptosis. Of note, all episomal inserts that supported cleavage contained both a strong topoisomerase II consensus binding site (at 6864-6847 as noted in Broeker et al 16 ) and the target for apoptotic cleavage at 6768 bp (see Figure 3).…”

“…In particular, therapy-related breakpoints appear clustered towards the telomeric region of the BCR, a region that colocalizes with a DNase I hypersensitivity site and is also a target for site-specific cleavage induced by apoptotic agents. [7][8][9] We have demonstrated before that cells exposed to triggers of the apoptotic program may initiate MLL cleavage and subsequent translocation, within cells that have the potential to divide. 10 In addition, direct analysis has shown that the cleavage event is linked to the release of high molecular weight (B50 kbp) DNA fragments, an early event in apoptosis execution.…”