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Etoposide induces chimeric Mll gene fusions¹

JAVIER G. BLANCO^*, MATHEW J. EDICK^* and MARY V. RELLING^*,,2

^* Department of Pharmaceutical Sciences, St. Jude Children’s Research Hospital, and
University of Tennessee, Memphis, Tennessee, USA

²Correspondence: St. Jude Children’s Research Hospital, 332 North Lauderdale, Memphis, TN 38105, USA. E-mail: mary.relling{at}stjude.org

SPECIFIC AIM

Our aim was to determine if we could detect the formation of chimeric Mll gene fusions in intact mammalian cells following etoposide treatment, to characterize any such fusion partners, and to determine whether such fusions were detectable after chemotherapy whose mechanism does not include topoisomerase II inhibition.

PRINCIPAL FINDINGS

1. Etoposide causes formation of Mll fusions whereas vincristine does not
In humans, topoisomerase II inhibitor-induced therapy-related acute leukemia is characterized by chimeric MLL gene fusions, with the vast majority of fusion points occurring in an 8.3-kb breakpoint cluster region (BCR) that encompasses exons 5 through 11. However, MLL partners with over 40 different genes to create leukemogenic gene fusions. The strategy to detect possible etoposide-induced murine Mll chimeric fusions, without bias as to the partner gene, is presented in the overall schematic depicted in Fig. 1 . Amplification of the circularized Mll BCR germline fragment yields a single band of 10.5 kb. The sizes of the Mll fusion products amplified by long-distance inverse-PCR are dictated by the location of the fusion point and the distance between the NcoI site on the 3' partner and the Mll fusion point. Alternatively-sized bands (other than 10.5 kb) were purified and sequenced to identify the nature of the chimeric partner genes.

View larger version (26K): [in this window] [in a new window]   Figure 1. Etoposide stabilizes topoisomerase II-induced double strand (DS) DNA breaks, which tend to occur in the breakpoint cluster region (BCR) of MLL in humans, and facilitates the formation of fusion genes. We hypothesized that in murine cells, Mll fusion genes might be formed in the region homologous to the human BCR after treatment with etoposide, and the strategy for detecting such fusions is depicted. NcoI (long vertical bars represent NcoI sites) excises Mll exons 5–13; following intramolecular ligation, inverse primers define a 10.5 kb fragment for germline Mll. PCR amplifies a 5' portion of Mll along with any fusion gene that lies between the breakpoint and an NcoI site in the 3' partner gene. The 5' NcoI site is invariant within Mll; germline Mll contains a second NcoI site 3' of exon 13, whereas the placement of an NcoI site in any fusion partner determines the size of the non-germline Mll products after circularization of digested DNA and long-distance inverse PCR amplification. Examples are shown for fragment formation from germline Mll and three Mll chimeric fusions.

Etoposide (100 µM for 8 h) induced the formation of Mll fusion products after 8 h of incubation at a higher frequency of 16.0 x 10^–6 cell ^–1 compared with no-drug controls (1.0x10^–6 cell ^–1, P=0.0002). The possibility that a nongenotoxic anticancer drug could also trigger the formation of Mll fusions was investigated by treating with the microtubule inhibitor vincristine. Vincristine caused the expected mitotic arrest, whereas etoposide resulted in the expected G2/M arrest when compared with no-drug controls (Fig. 2 A). However, at comparable levels of cytotoxicity (49±5% viability after 10 µM vincristine for 48 h and 55±5% viability after 100 µM etoposide for 8 h), vincristine produced a lower level of Mll genomic fusions (1.0x10^–6 cell^–1) than etoposide (11.0x10^–6 cell^–1, P=0.0047), and was comparable to the no-drug controls (1 in 1x10⁶ cells, P=1.0) (Fig. 2B ).

View larger version (35K): [in this window] [in a new window]   Figure 2. Comparisons among control, vincristine-, and etoposide-treated embryonic stem cells. A) Cell cycle distribution (left panels), indicating a mitotic arrest following vincristine and a G2/M arrest following etoposide. The corresponding gel electrophoresis of Mll PCR products (right panels) indicates the increased presence of non-germline Mll fusion products (lower molecular weight bands) following etoposide. B) At comparable levels of cell viability (left y-axis, open bars), the frequency of Mll fusions (x10–6, right y-axis, filled bars) was higher (P=0.0047) following etoposide (100 µM for 8 h) compared with vincristine (10 µM for 48 h).

2. The murine Mll fusions formed after etoposide shared many characteristics of human MLL chimeric fusions in topoisomerase II inhibitor-induced leukemias
We isolated 49 Mll fusion products following etoposide treatment. Each was unique, but they shared some common characteristics. First, 82% of the Mll partners were known or probable murine genes. The 49 partners represented 18 of the 19 murine autosomes, and included a number of genes whose human orthologs have been implicated in cancer. Of the fusions involving known or suspected genes, we confirmed that 100% of the Mll breakpoints and 94% of the partner breakpoints were localized in introns. The breakpoints were distributed in a 1.5 kb region of Mll between exons 9 and 11 (Fig. 3 A). Microhomologies (1–5 bp) were present at the fusion points in 74% of the products. Topoisomerase II motifs with perfect-match or one-mismatch sites in proximity to the fusion points (17–368 bp) were present in 44% of the chimeric products, with one Mll fusion having a topoisomerase II motif (one mismatch) located directly at the fusion point.

View larger version (25K): [in this window] [in a new window]   Figure 3. Characteristics of Mll fusion partners. A) Short regions of homology between partners at the breakpoint were demonstrated in 74% of products, as indicated in the example fusion product (top panel). Location of fusion points in Mll indicated by arrows. B) Each triangle represents the location of an Mll fusion partner on an idealized murine banded karyotype, with the location of Mll in chromosome 9 indicated by a solid dot. The frequency of chromosomes involved as fusion partners is indicated (bottom).

One of the more intriguing fusion partners was Runx1. The Mll breakpoint was in intron 10 and the breakpoint in Runx1 occurred in intron 4, in the region homologous to the human BCR in the human homologue, AML1. AML1 is involved in more leukemic translocations than any other human gene.

CONCLUSIONS AND SIGNIFICANCE

Topoisomerase II inhibitors are commonly used anticancer drugs that have been key to curing many adult and pediatric cancers, but their use is hampered by induction of secondary leukemias. Establishing reliable laboratory models to compare leukemogenic effect with cytotoxicity is a critical step to developing less leukemogenic but still equally efficacious regimens of these agents. However, there are no laboratory models of topoisomerase II-induced leukemias. Why are some regimens including topoisomerase II inhibitors leukemogenic while others are not? To address this, a model of topoisomerase II inhibitor-induced relevant gene rearrangements is needed. Herein, we present such a model.

The fusions we generated recapitulated many of the most important features of human leukemogenic MLL translocations, including targeting of the analogous breakpoint cluster region, drug-specific susceptibility, promiscuous involvement of partner genes, targeting intronic regions in both partners, and fusion with murine homologs of human leukemogenic genes. This opens the possibility of using pre-clinical mouse models for drug-induced second cancers, as part of a strategy to avoid leukemogenesis in the future.

FOOTNOTES

¹ To read the full text of this article, go to http://www.fasebj.org/cgi/doi/1096/fj.03-0638fje

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