Elsevier

DNA Repair

Volume 5, Issues 9–10, 8 September 2006, Pages 1109-1118
DNA Repair

Etoposide and illegitimate DNA double-strand break repair in the generation of MLL translocations: New insights and new questions

https://doi.org/10.1016/j.dnarep.2006.05.018Get rights and content

Abstract

Faithful repair of chromosomal double-strand breaks (DSBs) is central to genome integrity and the suppression of genome rearrangements including translocations that are a hallmark of leukemia, lymphoma, and soft-tissue sarcomas [B. Elliott, M. Jasin, Double-strand breaks and translocations in cancer, Cell. Mol. Life Sci. 59 (2002) 373–385; D.C. van Gent, J.H. Hoeijmakers, R. Kanaar, Chromosomal stability and the DNA double-stranded break connection, Nat. Rev. Genet. 2 (2001) 196–206]. Chemotherapy agents that target the essential cellular enzyme topoisomerase II (topo II) are known promoters of DSBs and are associated with therapy-related leukemias. There is a clear clinical association between previous exposure to etoposide and therapy-related acute myeloid leukemia (t-AML) characterized by chromosomal rearrangements involving the mixed lineage leukemia (MLL) gene on chromosome band 11q23 [C.A. Felix, Leukemias related to treatment with DNA topoisomerase II inhibitors, Med. Pediatr. Oncol. 36 (2001) 525–535]. Most MLL rearrangements initiate within a well-characterized 8.3 kb region that contains both putative topo II cleavage recognition sequences and repetitive elements leading to the logical hypothesis that MLL is particularly susceptible to aberrant cleavage and homology-mediated fusion to repetitive elements located on novel chromosome partners. In this review, we will discuss the findings and implications of recent attempts to confirm this hypothesis.

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.

References (94)

  • N. Osheroff et al.

    Mechanism of action of topoisomerase II-targeted antineoplastic drugs

    Adv. Pharmacol.

    (1994)
  • J. Pedersen-Bjergaard et al.

    Two different classes of therapy-related and de-novo acute myeloid leukemia?

    Cancer Genet. Cytogenet.

    (1991)
  • J. Pedersen-Bjergaard et al.

    The balanced and the unbalanced chromosome aberrations of acute myeloid leukemia may develop in different ways and may contribute differently to malignant transformation

    Blood

    (1994)
  • C.A. Felix

    Secondary leukemias induced by topoisomerase-targeted drugs

    Biochim. Biophys. Acta

    (1998)
  • M.J. Ratain et al.

    Therapy-related acute myeloid leukemia secondary to inhibitors of topoisomerase II: from the bedside to the target genes

    Ann. Oncol.

    (1992)
  • M.R. Nowrousian et al.

    Impact of chemotherapy regimen and hematopoietic growth factor on mobilization and collection of peripheral blood stem cells in cancer patients

    Ann. Oncol.

    (2003)
  • P.D. Aplan et al.

    Site-specific DNA cleavage within the MLL breakpoint cluster region induced by topoisomerase II inhibitors

    Blood

    (1996)
  • A. Ng et al.

    Genotoxicity of etoposide: greater susceptibility of MLL than other target genes

    Cancer Genet. Cytogenet.

    (2006)
  • J.E. Ploski et al.

    Characterization of DNA fragmentation events caused by genotoxic and non-genotoxic agents

    Mutat. Res.

    (2001)
  • J. Libura et al.

    Therapy-related acute myeloid leukemia-like MLL rearrangements are induced by etoposide in primary human CD34+ cells and remain stable after clonal expansion

    Blood

    (2005)
  • C. Richardson

    RAD51, genomic stability, and tumorigenesis

    Cancer Lett.

    (2005)
  • A. Slupianek et al.

    BCR/ABL regulates mammalian RecA homologs, resulting in drug resistance

    Mol. Cell

    (2001)
  • A.J. Pierce et al.

    NHEJ deficiency and disease

    Mol. Cell

    (2001)
  • S.J. Collis et al.

    Evasion of early cellular response mechanisms following low level radiation-induced DNA damage

    J. Biol. Chem.

    (2004)
  • K. Oguchi et al.

    Missense mutation and defective function of ATM in a childhood acute leukemia patient with MLL gene rearrangement

    Blood

    (2003)
  • B. Elliott et al.

    Double-strand breaks and translocations in cancer

    Cell. Mol. Life Sci.

    (2002)
  • D.C. van Gent et al.

    Chromosomal stability and the DNA double-stranded break connection

    Nat. Rev. Genet.

    (2001)
  • C.A. Felix

    Leukemias related to treatment with DNA topoisomerase II inhibitors

    Med. Pediatr. Oncol.

    (2001)
  • J.A. Ross

    Maternal diet and infant leukemia: a role for DNA topoisomerase II inhibitors?

    Int. J. Cancer Suppl.

    (1998)
  • J.A. Ross et al.

    Maternal exposure to potential inhibitors of DNA topoisomerase II and infant leukemia (United States): a report from the Children's Cancer Group

    Cancer Causes Control

    (1996)
  • L.G. Spector et al.

    Maternal diet and infant leukemia: the DNA topoisomerase II inhibitor hypothesis: a report from the children's oncology group

    Cancer Epidemiol. Biomarkers Prev.

    (2005)
  • F.E. Alexander et al.

    Transplacental chemical exposure and risk of infant leukemia with MLL gene fusion

    Cancer Res.

    (2001)
  • R. Strick et al.

    Dietary bioflavonoids induce cleavage in the MLL gene and may contribute to infant leukemia

    Proc. Natl. Acad. Sci. U.S.A.

    (2000)
  • Y. Gu et al.

    Sequence analysis of the breakpoint cluster region in the ALL-1 gene involved in acute leukemia

    Cancer Res.

    (1994)
  • O.A. Bernard et al.

    Molecular basis of 11q23 rearrangements in hematopoietic malignant proliferations

    Genes Chromosomes Cancer

    (1995)
  • P.H. Domer et al.

    Molecular analysis of 13 cases of MLL/11q23 secondary acute leukemia and identification of topoisomerase II consensus-binding sequences near the chromosomal breakpoint of a secondary leukemia with the t(4; 11)

    Leukemia

    (1995)
  • M. Negrini et al.

    Potential topoisomerase II DNA-binding sites at the breakpoints of a t(9; 11) chromosome translocation in acute myeloid leukemia

    Cancer Res.

    (1993)
  • S.A. Schichman et al.

    ALL-1 partial duplication in acute leukemia

    Proc. Natl. Acad. Sci. U.S.A.

    (1994)
  • S.A. Schichman et al.

    ALL-1 tandem duplication in acute myeloid leukemia with a normal karyotype involves homologous recombination between Alu elements

    Cancer Res.

    (1994)
  • A.O. Sperry et al.

    Dysfunction of chromosomal loop attachment sites: illegitimate recombination linked to matrix association regions and topoisomerase II

    Proc. Natl. Acad. Sci. U.S.A.

    (1989)
  • M. Stanulla et al.

    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

    Mol. Cell. Biol.

    (1997)
  • S. Dong et al.

    Breakpoint clusters of the PML gene in acute promyelocytic leukemia: primary structure of the reciprocal products of the PML-RARA gene in a patient with t(15; 17)

    Genes Chromosomes Cancer

    (1993)
  • A.R. Mistry et al.

    DNA topoisomerase II in therapy-related acute promyelocytic leukemia

    N. Engl. J. Med.

    (2005)
  • A.R. Jeffs et al.

    The BCR gene recombines preferentially with Alu elements in complex BCR-ABL translocations of chronic myeloid leukaemia

    Hum. Mol. Genet.

    (1998)
  • A.R. Jeffs et al.

    Nonrandom distribution of interspersed repeat elements in the BCR and ABL1 genes and its relation to breakpoint cluster regions

    Genes Chromosomes Cancer

    (2001)
  • J. Zucman-Rossi et al.

    Chromosome translocation based on illegitimate recombination in human tumors

    Proc. Natl. Acad. Sci. U.S.A.

    (1998)
  • C. Richardson et al.

    Frequent chromosomal translocations induced by DNA double-strand breaks

    Nature

    (2000)
  • Cited by (0)

    1

    Present address: Program in Molecular Biology, Sloan-Kettering Institute, Memorial Sloan Kettering Cancer Center, New York, NY 10021, United States.

    View full text