We have identified a new mixed lineage leukemia (MLL) gene fusion partner in a patient with treatment-related acute myeloid leukemia (AML) presenting a t(2;11)(q37;q23) as the only cytogenetic abnormality. Fluorescence in situ hybridization demonstrated a rearrangement of the MLL gene and molecular genetic analyses identified a septin family gene, SEPT2, located on chromosome 2q37, as the fusion partner of MLL. RNA and DNA analyses showed the existence of an in-frame fusion of MLL exon 7 with SEPT2 exon 3, with the genomic breakpoints located in intron 7 and 2 of MLL and SEPT2, respectively. Search for DNA sequence motifs revealed the existence of two sequences with 94.4% homology with the topoisomerase II consensus cleavage site in MLL intron 7 and SEPT2 intron 2. SEPT2 is the fifth septin family gene fused with MLL, making this gene family the most frequently involved in MLL-related AML (about 10% of all known fusion partners). The protein encoded by SEPT2 is highly homologous to septins 1, 4 and 5 and is involved in the coordination of several key steps of mitosis. Further studies are warranted to understand why the septin protein family is particularly involved in the pathogenesis of MLL-associated leukemia.
The mixed lineage leukemia (MLL) gene codes for a multi-domain molecule that is a major regulator of class I homeobox (HOX) gene expression, directly interacting with HOX promoter regions (Milne et al., 2002; Nakamura et al., 2002). As HOX genes play a key role in the regulation of hematopoietic development, it seems plausible that deregulation of MLL activity might result in abnormal patterns of HOX gene expression in hematopoietic stem cells or progenitors (Daser and Rabbitts, 2005; Li et al., 2005; Slany, 2005). Normally, HOX expression is high in hematopoietic stem cells and becomes gradually extinguished during differentiation (Grier et al., 2005). A failure to downregulate HOX expression inhibits hematopoietic maturation and can lead to leukemia (Grier et al., 2005).
Abnormalities of 11q23 involving the MLL gene are found in several hematological malignancies, including acute lymphoblastic leukemia (ALL) and acute myeloid leukemia (AML) (Huret, 2005). The overall incidence of MLL-associated leukemia is around 3 and 8–10% for AML and ALL, respectively (Daser and Rabbitts, 2005). Rearrangements of MLL can also be found in a proportion of patients with therapy-related leukemia after treatment with topoisomerase II inhibitors, such as anthracyclines (e.g. doxorubicin and epirubicin) and epipodophyllotoxins (e.g. etoposide and tenoposide) (Pui and Relling, 2000). The presence of an MLL gene abnormality (MLL gene fusion or exon duplication) is associated with poor prognosis in ALL and is an intermediate prognostic factor in AML (Pui and Relling, 2000; Daser and Rabbitts, 2005; Li et al., 2005; Popovic and Zeleznik-Le, 2005; Slany, 2005).
One of the most notable features of MLL is the extraordinary diversity of the fusion partners. To date, 71 different chromosome bands have been described to be rearranged with 11q23 and about 50 fusion genes have been cloned (Huret, 2005). The most common MLL fusion partners are AF4 (4q21), AF6 (6q27), AF9 (9p23), AF10 (10p12), ELL (19p13.1) and ENL (19p13.3) (Nakamura et al., 2002; Daser and Rabbitts, 2005). The fusion genes encode chimeric proteins harboring the NH2-terminal amino acids of MLL and the COOH-terminal amino acids of the partner protein (Daser and Rabbitts, 2005; Li et al., 2005; Slany, 2005). The major contribution of the fusion partners investigated so far seems to be to convert the rearranged MLL protein to a potent transcriptional activator (Daser and Rabbitts, 2005). However, several MLL fusion partners affected by chromosomal translocations have not yet been identified (Huret, 2005). In the present study, we have identified the SEPT2 gene as a novel fusion partner of MLL in a patient with treatment-related AML presenting a t(2;11)(q37;q23) as the only cytogenetic abnormality.
A 54-year-old female was diagnosed with a breast adenocarcinoma in 1987 (T2N0M0; treated with radical mastectomy) and with a contralateral breast adenocarcinoma in 2001 (T1N0M0; treated with radical mastectomy, followed by six courses of chemotherapy with 5-fluorouracil, epirubicin and cyclophosphamide). In 2004, this patient developed pancytopenia and the diagnosis of therapy-related AML was established (AML-M4 according to the French–American–British classification). Blood count was hemoglobin 8.4 g/dl, platelets 34 × 109/l, and leukocytes 4.18 × 109/l with 2% circulating blasts. Bone marrow was infiltrated with 66.5% blasts. She was treated with two courses of chemotherapy (cytarabine, daunorubicin and cyclosporin), followed by an additional course of high-dose cytarabine and allogeneic bone marrow transplantation (BMT). The patient has no evidence of disease at the time of writing.
The bone marrow showed a t(2;11)(q37;q23) as the only cytogenetic abnormality (Figure 1a), which suggested the involvement of the MLL gene located in 11q23. Fluorescence in situ hybridization (FISH) analysis on bone marrow metaphases demonstrated the rearrangement of MLL, with the telomeric part of the gene being translocated to the der(2) (Figure 1b). Subsequent karyotype and FISH studies performed with 1–3 months intervals were normal, both before and after BMT.
The previous identification of four septin genes involved in rearrangements with the MLL gene (Megonigal et al., 1998; Osaka et al., 1999; Taki et al., 1999a; Ono et al., 2002; Kojima et al., 2004), combined with a GenBank search of putatively expressed genes on chromosomal band 2q37, prompted us to hypothesize that the MLL fusion partner on 2q37 was SEPT2. Reverse transcription–polymerase chain reaction (RT–PCR) with an antisense primer located on SEPT2 exon 9 and three MLL sense primers located on exons 5, 6 and 7 (Table 1) showed the presence of PCR fragments of 1233, 956, and 885 bp, respectively, suggestive of an MLL-SEPT2 rearrangement resulting from fusion of MLL exon 7 with SEPT2 exon 3 (Figure 2a). Additional RT–PCR analysis with sense primers located on MLL exons 7 and 8 and antisense primers located on SEPT2 exons 3 and 4 gave additional support to this hypothesis, as expected amplification fragments of 241 and 275 bp were observed with the MLL exon 7 primer and the SEPT2 antisense primers, but no amplification was detected with the MLL exon 8 primer (Figure 2b). Sequencing of the amplification products followed by a BLAST search confirmed that MLL exon 7 was fused in-frame with nucleotide 431 of the SEPT2 transcript (GenBank Accession no. NM_001008491) (Figure 2c). No mutation or deletion was detected in the MLL-SEPT2 breakpoint region. This fusion is expected to give rise to a chimeric fusion protein where the N-terminus of MLL is fused to almost the entire open-reading frame of SEPT2, except for the first three amino acids. The putative MLL-SEPT2 fusion protein of 1764 amino acids contains 1406 amino acids from the NH2-terminal part of MLL and 358 amino acids from the COOH-terminal part of SEPT2.
For the identification of the genomic MLL-SEPT2 fusion, the SEPT-Int2AS-2 antisense primer was used in combination with the MLL-Ex7S and MLL-Int7S sense primers, giving rise to amplification products of 1240 and 1047 bp, respectively (Figure 3a). No amplification products were observed when primers MLL-Ex7S and MLL-Int7S were used with the antisense primer SEPT2-Int2AS-1 (Figure 3a). The results suggested that the genomic DNA breakpoint was located 3′ of MLL-Int7S and 5′ of SEPT2-Int2AS-1. Partial sequencing of the amplification products showed that the breakpoints were located 252 bp downstream of MLL exon 7 and 447 bp downstream of SEPT2 exon 2 (Figure 3b and c).
As our patient had been treated with epirubicin, a known topoisomerase II inhibitor, we searched for topoisomerase II consensus cleavages sites (Abeysinghe et al., 2003) near the vicinity of the breakpoint region. Using SeqTools (Rasmussen, 2004), we found two sequences with 94.4% homology with the topoisomerase II consensus cleavage site (one mismatch): one located in MLL intron 7 (IndexTermATTAGCAGGTGGGTTTAG, nucleotide position 125–141 bp downstream of MLL exon 7) and the other in SEPT2 intron 2 (IndexTermGTCACCAGGCTGGAGTGC, nucleotide position 184–201 bp downstream of SEPT2 exon 2). We also searched the breakpoint junction (15 bp either side) for repetitive DNA sequence elements and motifs known to be associated with site-specific recombination, cleavage and gene rearrangement (Abeysinghe et al., 2003; Chuzhanova et al., 2003), but we could not find any evidence of their presence. Finally, using RepeatMasker (Smit et al., 2004), we also searched both MLL intron 7 and SEPT2 intron 2 for low complexity DNA sequences and interspersed repeats. We found a 296 bp Alu repeat in SEPT2 intron 2 located at nucleotides 148–309 downstream of SEPT2 exon 2.
Identical translocations to the one we present have previously been reported in three patients with leukemia (DeLozier-Blanchet et al., 1985; Winick et al., 1993; Fischer et al., 1997), but no molecular genetic investigation of this chromosomal rearrangement had so far been described. The MLL fusion partner we have identified, SEPT2, belongs to an evolutionarily conserved family of genes that encode a P loop-based GTP-binding domain flanked by a polybasic domain and (usually) a coiled-coil region (Hall and Russell, 2004; Russell and Hall, 2005). The SEPT2 protein possesses all the three domains and shares a very high homology with septins 1, 4 and 5 (Hall and Russell, 2004). Recently, it has been shown that there are at least 13 human septin genes (Russell and Hall, 2005). Four of them (SEPT5, SEPT6, SEPT9 and SEPT11) have already been cloned as MLL fusion partners (Megonigal et al., 1998; Osaka et al., 1999; Taki et al., 1999a; Ono et al., 2002; Kojima et al., 2004), with the N-terminal moiety of MLL fused to almost the entire open-reading frame of the partner septin gene (Russell and Hall, 2005). This is the fifth septin family gene fused with the MLL gene described so far, making the septins the protein family most frequently involved in rearrangements with the MLL gene. Septins constitute now nearly 10% of all fusion partners identified to date, suggesting that the involvement of this protein family in MLL-related leukemia is not a chance event. This hypothesis is supported by the fact that all the reported MLL-septin fusions are in frame and the breakpoints are found at the very 5′-end of known septin open-reading frames (Megonigal et al., 1998; Osaka et al., 1999; Taki et al., 1999a; Ono et al., 2002; Kojima et al., 2004).
We postulate that other septins may be involved in rearrangements with the MLL gene. For instance, several reports (Berger et al., 1987; Marosi et al., 1992; Harrison et al., 1998; Satake et al., 1999) have shown the existence of a molecular rearrangement of the MLL gene with a not yet identified fusion partner gene in 17q23, where the SEPT4 gene is located (Table 2). Furthermore, the existence of known MLL partner genes in chromosomal bands where septin genes are mapped (Table 2) does not exclude the possibility that they also may be rearranged with MLL. In fact, several MLL partner genes share the same chromosomal locations, like AF4 and SEPT11 (4q21) (Gu et al., 1992; Kojima et al., 2004), AF5q31 and GRAF (5q31) (Taki et al., 1999b; Borkhardt et al., 2000), FBP17 and AF9Q34 (9q34) (Fuchs et al., 2001; von Bergh et al., 2004), CBL and LARG (11q23) (Savage et al., 1991; Kourlas et al., 2000), AF15Q14 and MPFYVE (15q14) (Hayette et al., 2000; Chinwalla et al., 2003), LASP1 and AF17 (17q21) (Prasad et al., 1994; Strehl et al., 2003) and EEN and ENL (19p13.3) (Tkachuk et al., 1992; So et al., 1997). These data suggest that detailed molecular analysis is essential for the identification of other rearrangements involving the MLL gene.
Although MLL is a remarkably promiscuous leukemia-associated gene, current data suggest that these fusion partners fall into two distinct categories: those with a potent transactivation domain and nuclear localization and those that are located in the cytoplasm and possess potential oligomerization motifs (Nakamura et al., 2002; Daser and Rabbitts, 2005; Li et al., 2005). The septins do not possess an activation domain and there is no currently available evidence that they have other than a cytoplasmatic localization, but they are believed to oligomerize via their coiled-coil domain. Oligomerization of MLL-fused septins could then facilitate deregulated activity of MLL with recruitment of transcriptional activators. One exception is SEPT9, which lacks the coiled-coil domain present in other MLL-fused septins (Russell and Hall, 2005) and presumably has an alternative domain involved in the formation of oligomers. A recent report showed that oligomerization of MLL-SEPT6 is essential to immortalize hematopoietic progenitors in vitro and that the GTP-binding domain may have a role in the formation of dimers (Ono et al., 2005).
Although the presently available data suggest that the involvement of septins in MLL-related leukemia is only related to their capacity to oligomerize, the possibility that they have oncogenic activity of their own cannot be completely ruled out. Septins have roles in cytokinesis, vesicle traffic, polarity determination, microtubule and actin dynamics, and can form membrane diffusion barriers (Russell and Hall, 2005). For instance, SEPT2, the first human septin to be systematically studied, was shown to be required for cytokinesis and to bind actin and associate with focal adhesions (Kinoshita et al., 1997; Surka et al., 2002). Additionally, recent data support the idea that mammalian septins can form a novel scaffold at the midplane of the mitotic spindle that coordinates several key steps of mitosis, suggesting that SEPT2 can have a role in chromosome congression and segregation, and that altered expression of SEPT2 might lead to disordered chromosomal dynamics and underlie the development of aneuploidy (Spiliotis et al., 2005). However, the question of whether and how the normal function of SEPT2 is altered by its fusion to MLL remains to be elucidated.
Topoisomerase II inhibitor-related AML can be distinguished from other therapy-related leukemia by its genetic signature: balanced translocations involving the MLL gene (Pui and Relling, 2000). The identification of two sequences with 94.4% homology with the topoisomerase II consensus cleavage site in our patient, one located in MLL intron 7 and the other in SEPT2 intron 2, provides support to a link between topoisomerase II inhibitor therapy and the origin of the MLL-SEPT2 fusion gene in this particular case. Nevertheless, one must be cautious in interpreting this finding, as topoisomerase II consensus cleavages sites are either short, highly redundant or both, so their chance occurrence at breakpoint junctions is unlikely to be infrequent. Therefore, their presence at a given translocation breakpoint should not be automatically taken to imply that they are directly involved in the mechanisms of rearrangement (Abeysinghe et al., 2003). In addition, the presence of a 296 bp Alu repeat in SEPT2 intron 2 can, in theory, be related with the MLL-SEPT2 formation, as Alu sequences found in the vicinity of breakpoint regions can mediate the corresponding rearrangement by non-homologous recombination (Rudiger et al., 1995).
In summary, we have identified SEPT2 as the MLL fusion partner in therapy-related AML with a t(2;11)(q37;q23). This is the fifth septin that has been found fused with MLL in acute leukemia, but the precise role played by this family of genes in this disease remains incompletely known. A more detailed characterization of the functions of septins may contribute to a better understanding of MLL-mediated leukemogenesis.
Abeysinghe SS, Chuzhanova N, Krawczak M, Ball EV, Cooper DN . (2003). Hum Mutat 22: 229–244.
Berger R, Flandrin G, Bernheim A, Le Coniat M, Vecchione D, Pacot A et al. (1987). Cancer Genet Cytogenet 29: 9–21.
Borkhardt A, Bojesen S, Haas OA, Fuchs U, Bartelheimer D, Loncarevic IF et al. (2000). Proc Natl Acad Sci USA 97: 9168–9173.
Chinwalla V, Chien A, Odero M, Neilly MB, Zeleznik-Le NJ, Rowley JD . (2003). Oncogene 22: 1400–1410.
Chuzhanova N, Abeysinghe SS, Krawczak M, Cooper DN . (2003). Hum Mutat 22: 245–251.
Daser A, Rabbitts TH . (2005). Semin Cancer Biol 15: 175–188.
DeLozier-Blanchet CD, Cabrol C, Werner-Favre C, Beris P, Engel E . (1985). Cancer Genet Cytogenet 16: 95–102.
Fischer K, Frohling S, Scherer SW, McAllister Brown J, Scholl C, Stilgenbauer S et al. (1997). Blood 89: 2036–2041.
Fuchs U, Rehkamp G, Haas OA, Slany R, Konig M, Bojesen S et al. (2001). Proc Natl Acad Sci USA 98: 8756–8761.
Grier DG, Thompson A, Kwasniewska A, McGonigle GJ, Halliday HL, Lappin TR . (2005). J Pathol 205: 154–171.
Gu Y, Nakamura T, Alder H, Prasad R, Canaani O, Cimino G et al. (1992). Cell 71: 701–708.
Hall PA, Russell SE . (2004). J Pathol 204: 489–505.
Harrison CJ, Cuneo A, Clark R, Johansson B, Lafage-Pochitaloff M, Mugneret F et al. (1998). Leukemia 12: 811–822.
Hayette S, Tigaud I, Vanier A, Martel S, Corbo L, Charrin C et al. (2000). Oncogene 19: 4446–4450.
Huret JL . (2005). Atlas of Genetics and Cytogenetics in Oncology and Haematology http://www.infobiogen.fr/services/chromcancer/Genes/MLL.html.
Ida K, Kitabayashi I, Taki T, Taniwaki M, Noro K, Yamamoto M et al. (1997). Blood 90: 4699–4704.
ISCN (1995). An International System for Human Cytogenetic Nomenclature. Karger: Basel.
Kinoshita M, Kumar S, Mizoguchi A, Ide C, Kinoshita A, Haraguchi T et al. (1997). Genes Dev 11: 1535–1547.
Kojima K, Sakai I, Hasegawa A, Niiya H, Azuma T, Matsuo Y et al. (2004). Leukemia 18: 998–1005.
Kourlas PJ, Strout MP, Becknell B, Veronese ML, Croce CM, Theil KS et al. (2000). Proc Natl Acad Sci USA 97: 2145–2150.
Li ZY, Liu DP, Liang CC . (2005). Leukemia 19: 183–190.
Marosi C, Koller U, Koller-Weber E, Schwarzinger I, Schneider B, Jager U et al. (1992). Cancer Genet Cytogenet 61: 14–25.
Megonigal MD, Rappaport EF, Jones DH, Williams TM, Lovett BD, Kelly KM et al. (1998). Proc Natl Acad Sci USA 95: 6413–6418.
Milne TA, Briggs SD, Brock HW, Martin ME, Gibbs D, Allis CD et al. (2002). Mol Cell 10: 1107–1117.
Nakamura T, Mori T, Tada S, Krajewski W, Rozovskaia T, Wassell R et al. (2002). Mol Cell 10: 1119–1128.
Ono R, Nakajima H, Ozaki K, Kumagai H, Kawashima T, Taki T et al. (2005). J Clin Invest 115: 919–929.
Ono R, Taki T, Taketani T, Kawaguchi H, Taniwaki M, Okamura T et al. (2002). Cancer Res 62: 333–337.
Osaka M, Rowley JD, Zeleznik-Le NJ . (1999). Proc Natl Acad Sci USA 96: 6428–6433.
Poirel H, Rack K, Delabesse E, Radford-Weiss I, Troussard X, Debert C et al. (1996). Blood 87: 2496–2505.
Popovic R, Zeleznik-Le NJ . (2005). J Cell Biochem 95: 234–242.
Prasad R, Leshkowitz D, Gu Y, Alder H, Nakamura T, Saito H et al. (1994). Proc Natl Acad Sci USA 91: 8107–8111.
Pui CH, Relling MV . (2000). Br J Haematol 109: 13–23.
Rasmussen SW . (2004). SEQ Tools Version 8.2 – Build 101 http://www.seqtools.dk.
Rudiger NS, Gregersen N, Kielland-Brandt MC . (1995). Nucleic Acids Res 23: 256–260.
Russell SE, Hall PA . (2005). Br J Cancer 93: 499–503.
Satake N, Maseki N, Nishiyama M, Kobayashi H, Sakurai M, Inaba H et al. (1999). Leukemia 13: 1013–1017.
Savage PD, Shapiro M, Langdon WY, Geurts van Kessel AD, Seuanez HN, Akao Y et al. (1991). Cytogenet Cell Genet 56: 112–115.
Slany RK . (2005). Hematol Oncol 23: 1–9.
Smit AFA, Hubley R, Green P . (2004). RepeatMasker Open-3.0 http://www.repeatmasker.org.
So CW, Caldas C, Liu MM, Chen SJ, Huang QH, Gu LJ et al. (1997). Proc Natl Acad Sci USA 94: 2563–2568.
Sobulo OM, Borrow J, Tomek R, Reshmi S, Harden A, Schlegelberger B et al. (1997). Proc Natl Acad Sci USA 94: 8732–8737.
Spiliotis ET, Kinoshita M, Nelson WJ . (2005). Science 307: 1781–1785.
Strehl S, Borkhardt A, Slany R, Fuchs UE, Konig M, Haas OA . (2003). Oncogene 22: 157–160.
Surka MC, Tsang CW, Trimble WS . (2002). Mol Biol Cell 13: 353235–353245.
Taki T, Kano H, Taniwaki M, Sako M, Yanagisawa M, Hayashi Y . (1999b). Proc Natl Acad Sci USA 96: 14535–14540.
Taki T, Ohnishi H, Shinohara K, Sako M, Bessho F, Yanagisawa M et al. (1999a). Cancer Res 59: 4261–4265.
Taki T, Sako M, Tsuchida M, Hayashi Y . (1997). Blood 89: 3945–3950.
Tkachuk DC, Kohler S, Cleary ML . (1992). Cell 71: 691–700.
von Bergh AR, Wijers PM, Groot AJ, van Zelderen-Bhola S, Falkenburg JH, Kluin PM et al. (2004). Genes Chromosomes Cancer 39: 324–334.
Winick NJ, McKenna RW, Shuster JJ, Schneider NR, Borowitz MJ, Bowman WP et al. (1993). J Clin Oncol 11: 209–217.
Yamamoto K, Seto M, Iida S, Komatsu H, Kamada N, Kojima S et al. (1994). Blood 83: 2912–2921.
This research was supported by Liga Portuguesa Contra o Cancro – Núcleo Regional do Norte.
About this article
Cite this article
Cerveira, N., Correia, C., Bizarro, S. et al. SEPT2 is a new fusion partner of MLL in acute myeloid leukemia with t(2;11)(q37;q23). Oncogene 25, 6147–6152 (2006). https://doi.org/10.1038/sj.onc.1209626
- fusion oncogenes
- therapy-related AML
Septin 9_i2 is downregulated in tumors, impairs cancer cell migration and alters subnuclear actin filaments
Scientific Reports (2017)
BMC Cancer (2009)
A variant-type MLL/SEPT9 fusion transcript in adult de novo acute monocytic leukemia (M5b) with t(11;17)(q23;q25)
International Journal of Hematology (2008)
Mammalian Genome (2007)