Etoposide and illegitimate DNA double-strand break repair in the generation of MLL translocations: New insights and new questions
Introduction
Chromosomal double-strands breaks (DSBs) pose a threat to genome integrity since their aberrant repair has the potential to result in genome rearrangements, including translocations that are a hallmark of leukemia, lymphoma, and soft-tissue sarcomas [1], [2]. Chemotherapy agents that target the essential cellular enzyme topoisomerase II (topo II) are known promoters of DSBs and are associated with therapy-related leukemias characterized by balanced chromosomal translocations. There is a clear association between exposure to etoposide, therapy-related acute myeloid leukemia (t-AML), and chromosomal rearrangements involving the mixed lineage leukemia (MLL) gene on chromosome band 11q23 [3]. Evidence also suggests an association between de novo infant leukemia and in utero exposure to topo II poisons [4], [5], [6], [7], [8], [9], [10], [11]. However, despite significant efforts by many groups, the molecular mechanism for this phenomenon is not fully understood. The clinical data might suggest that the MLL 11q23 locus is particularly susceptible to etoposide-mediated breakage and subsequent illegitimate repair events that anneal heterologous chromosomes. Alternatively, etoposide may promote breakage and illegitimate repair along all chromosomes with relatively equal frequency, but the specific oncogenic fusions with MLL lead to a growth advantage, and ultimately tumorigenesis, in a critical hematopoietic stem-cell subpopulation. The answer undoubtedly lies somewhere in between.
11q23 rearrangements associated with both therapy-related and infant de novo leukemia cluster within a well-characterized 8.3 kb breakpoint cluster region (bcr) that encompasses exons 5–11 of the MLL gene (also known as ALL-1, Htrx, HRX) [12]. MLL rearrangements identified in patient-derived leukemic samples include reciprocal balanced translocations, deletions, duplications, or amplifications [13]. Translocations fuse MLL with more than 60 partner genes, with AF4, AF9, and ELL as the most common [14]. The bcr of MLL is AT-rich, contains Alu, LINE, and MER repetitive sequences, putative topo II cleavage recognition sites, as well as a scaffold and matrix attachment region (SAR/MAR) (Fig. 1) [12], [15], [16], [17], [18], [19], [20], [21], [22], [23]. All of these elements have been proposed to play a direct or indirect role in promoting 11q23 rearrangements. Similar motifs have been identified in other bcrs and recurrent translocations, including the fusions that produce PML-RARα [24], [25], BCR-ABL [26], [27], [28], and Ewing's sarcoma [1], [29]. However, the initial molecular events and mechanisms by which each of these specific rearrangements occurs remain controversial.
Balanced chromosomal translocations can theoretically be generated through any of the three DSB repair pathways—homologous recombination associated with crossover, non-homologous end-joining (NHEJ), and single-strand annealing (SSA) (Fig. 2). Multiple experimental systems have used the yeast derived I-SceI endonuclease to induce specific cellular DSBs at predetermined sites in the genome, and analyze the products of repair at the molecular level. (Although some results from I-SceI based systems will be discussed here, for a full review, see Weinstock et al., this issue.) This approach demonstrated that a chromosomal DSB can be repaired with homologous sequence on a heterologous chromosome [30], [31], [32], [33]. While a single DSB is sufficient to promote homology-directed interchromosomal repair, two or more DSBs are needed to result in translocations analogous to those identified in leukemic patient-derived samples [30], [31], [32], [33]. The lack of translocations induced by a single DSB may not be surprising since it would require (1) invasion into a sufficient length of homology and generation of a Holliday junction to promote a crossover, or (2) break-induced replication to the end of the chromosome, and both mechanisms are normally suppressed in mitotic cells. By contrast, multiple DSBs create multiple ends and may simply increase the odds that broken ends will interact and anneal by NHEJ or SSA. In addition, multiple breaks may alter chromatin architecture and loosen chromosome domain boundaries sufficient for the interactions required. Although I-SceI-based systems have provided a significant amount of information about the potential of mammalian cells for interchromosomal repair, DNA damaging agents such as etoposide induce damage at multiple sites in the genome, and pose new questions about the possible interaction between multiply damaged sites in the context of chromatin structure or the potential competition between sites for repair proteins within the nuclear architecture.
Section snippets
Etoposide-promoted damage of the MLL 11q23 locus
Topo II is an essential cellular enzyme that catalyzes changes in DNA topology via its cleavage-religation equilibrium. Chemotherapeutic drugs termed “topo II poisons” convert topo II into a DNA-damaging enzyme by disrupting the cleavage-religation equilibrium, most often by decreasing the rate of religation in a dose-dependent manner. Disruption of the cleavage-religation reaction results in accumulation of DSBs, activation of DNA damage sensors, cell cycle arrest, and initiation of apoptosis
Illegitimate repair of the MLL locus
To determine if etoposide-mediated cleavage of the MLL locus promotes rearrangements, initial studies detected damage-induced alterations by Southern blotting using an MLL fragment as a probe but without identification of the reciprocal partner [22], [58], [63]. These studies complemented in vitro cleavage assays described above. However, sequence information about rearranged repair products was not possible until the development and use of strategic PCR methods including inverse PCR,
A model for repair pathway choice
The repair products identified in ex vivo systems between duplexes with breaks induced by I-SceI, etoposide, mitoxantrone, and apoptosis are roughly similar with any differences observed most likely attributable to the reporters in place or techniques of analysis [25], [30], [63], [67], [76] suggesting that the damaging agent to produce a DSB does not alter the repair process. However, although DSBs produced by bleomycin, calicheamicin, high and low linear energy ionizing radiation, restriction
Specificity
Whether etoposide specifically or preferentially promotes MLL 11q23 translocations over rearrangements within other chromosomal loci has yet to be directly studied. Following exposure of human lymphocytes to 85 μM etoposide for 1 h, chrs. 1, 11, and 17 are involved in translocations more frequently than expected for length, but without mapping specific sites of damage [76]. Preferential rearrangement of these same chromosomes is also observed after treatment with the alkylating agent melphalan
Conclusion
Less than a decade has passed since the direct demonstration that mammalian mitotically growing cells can search genome wide for homology sufficient to repair a DSB, even if that homology exists on a heterologous chromosome [92]. Since that time multiple experimental models have clearly provided strength to the hypothesis that the repetitive elements scattered throughout the mammalian genome are sufficient to promote the balanced chromosomal translocations that are a hallmark of leukemias,
Acknowledgements
C.R. is an American Cancer Society Research Scholar and supported by NCI. J.L. is supported by a Marie Currie International Fellowship of the 6th European Community Framework Programme.
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Present address: Program in Molecular Biology, Sloan-Kettering Institute, Memorial Sloan Kettering Cancer Center, New York, NY 10021, United States.