21 Translocation in Leukemogenesis

The 8;21 translocation in leukemogenesis

Luke F Peterson1 and Dong-Er Zhang*,1

1Department of Molecular and Experimental Medicine, The Scripps Research Institute, Mail Drop: MEM-L51, La Jolla, CA 92037, USA

A common chromosomal translocation in acute myeloid of 13 exons distributed over at least 87 kb (Figure 1) leukemia (AML) involves the AML1 (acute myeloid and also has alternative spliced forms (Wolford and leukemia 1, also called RUNX1, core binding factor Prochazka, 1998). The breakpoints within the AML1 protein (CBFa), and PEBP2aB) gene on chromosome 21 locus are clustered in the classically recognized intron 5, and the ETO (eight-twenty one, also called MTG8) gene with three breakpoint cluster regions (BCR) (Zhang on chromosome 8. This translocation generates an AML1- et al., 2002). On the other hand, the breakpoints are ETO fusion protein. t(8;21) is associated with 12% of de clustered in two introns of ETO (Tighe et al., 1993; novo AML cases and up to 40% in the AML subtype M2 Tighe and Calabi, 1995), with one BCR in intron 1a of the French–American-British classification. Further- (Tighe and Calabi, 1995) and three BCRs in intron 1b more, it is also reported in a small portion of M0, M1, and (Xiao et al., 2001; Zhang et al., 2002). These breakpoints M4 AML samples. Despite numerous studies on the create the same chimeric transcript of AML1-ETO due function of AML1-ETO, the precise mechanism by which to the lack of any splice acceptor site in exon 1b of ETO the fusion protein is involved in leukemia development (Kozu et al., 1993; de Greef et al., 1995; Xiao et al., is still not fully understood. In this review, we will discuss 2001). Interestingly, the clusters of AML1 and ETO structural aspects of the fusion protein and the accumu- breakpoints are associated with topoisomerase II DNA lated knowledge from in vitro analyses on AML1-ETO cleavage and DNase I hypersensitive sites (Zhang et al., functions, and outline putative mechanisms of its leuke- 2002). Such phenomenon is also present in translocation mogenic potential. breakpoints of other genes of leukemia-associated Oncogene (2004) 23, 4255–4262. doi:10.1038/sj.onc.1207727 translocations (Felix, 1998). In addition, complex translocations involving chromosomes 8 and 21 also Keywords: translocation; myeloid leukemia; RUNX; express the same AML1-ETO fusion gene, suggesting AML; ETO; MTG8 that the breakpoint regions are the same (Kozu et al., 1993; de Greef et al., 1995). Overall, no translocation has been identified between AML1 and ETO that occurs outside these intron boundaries that produce the AML1-ETO fusion protein. Structure of acute myeloid leukemia 1 (AML1)-eight- AML1 is a member of the RUNX (PEBP2a, twenty one (ETO) core binding factor protein (CBFa), AML) family, which includes AML1 (RUNX1, PEBP2aB, CBFa2), The 8;21 translocation with breaks at 8q22 and 21q22.3 AML2 (RUNX3, PEBP2aC, CBFa3), and AML3 was first reported by Dr Janet Rowley in 1973 during (RUNX2, PEBP2aA, CBFa1). All RUNX proteins the analysis of a leukemia patient sample (Rowley, contain a runt homology DNA binding domain, 1973). Two genes (AML1 and ETO) located at these which is highly homologous to the Drosophila Runt breakpoints were finally identified in the early 1990s by protein. Runt is involved in segmentation and sexual several groups (Gao et al., 1991; Miyoshi et al., 1991; determination (Kania et al., 1990; Duffy and Gergen, Erickson et al., 1992, 1994; Nisson et al., 1992; Shimizu 1991). Besides the DNA binding runt homology et al., 1992). As shown in Figure 1, the classical domain, AML1 also contains a transactivation domain recognized organization of the AML1 gene on chromo- (Meyers et al., 1995; Kurokawa et al., 1996), nuclear some 21 consists of nine exons and produces at least matrix attachment signal (NMTS) (Zeng et al., 1998), three proteins (Miyoshi et al., 1995). Further fine and two inhibitory domains (Imai et al., 1998; Kanno mapping revealed that AML1 gene locus spans 260 kb et al., 1998; Levanon et al., 1998). Furthermore, AML1 and there are several additional potential exons (Leva- forms a heterodimer with the CBFb facilitating efficient non et al., 2001). The expression of AML1 is regulated DNA binding (Ogawa et al., 1993). In the 8;21 by two independent promoters (Ghozi et al., 1996) and translocation, AML1 contributes the N-terminal region, alternative splicing (Miyoshi et al., 1995; Levanon et al., including runt homology domain, thus making AML1- 1996, 2001). The ETO gene on chromosome 8 consists ETO able to bind AML1 target gene promoters (Figure 2). The ETO protein belongs to the ETO family, which *Correspondence: D-E Zhang; E-mail: [email protected] includes ETO (MTG8), ETO2 (MTG16, MTG8-related The 8;21 translocation in leukemogenesis LF Peterson and D-E Zhang 4256

Figure 2 AML1-ETO-interacting proteins. AML1-ETO contains the N-terminal sequences of AML1, including the RHD. Sub- sequent a.a.’s are of ETO, containing the four NHR1–4. Indicated as well are the regions containing the NMTS of ETO (broken line arrows). Known RHD and ETO/AML1-ETO-interacting proteins (or family of proteins) are shown. The dimerization domain of AML1-ETO and ETO family members is located in NHR2 Figure 1 Genomic structure of t(8;21). Chromosome 8 containing the ETO gene is made up of 13 exons spanning approximately 87 kb, which can give four alternative splice forms and is regulated by two promoters. Chromosome 21 contains the AML1 gene with Protein–protein interactions nine exons that give various alternative splice forms and is regulated by two promoters and spans 260 kb. The breakpoint cluster areas are denoted by the crossing lines between ETO and Since AML1-ETO contains the N-terminal portion of AML1. Owing to the absence of a splice acceptor in exon 1b of AML1 including the intact runt homology domain, it ETO, the mRNA of the fusion transcripts does not include this may interact with other transcription regulators known exon. White boxes and black boxes indicate translated and to bind to this region of AML1 (Figure 2). The first and untranslated exon sequences, respectively. Underlined numbers in the best known AML1 binding protein is its heterodimer the AML1-ETO mRNA denote exons contributed by the ETO gene partner CBFb (also called PEBP2b) that promotes efficient DNA binding of AML1 and is essential for the full activity of AML1 transcriptional activation (Ogawa et al., 1993; Wang et al., 1993, 1996a, b; Kanno 2 (MTGR2)), and MTGR1 (Calabi and Cilli, 1998; et al., 1998). Most protein–protein interactions invol- Kitabayashi et al., 1998). All three members of this ving the runt homology domain (RHD) of AML1 were family have four evolutionarily conserved Nervy homo- identified due to the proximity of the AML1 binding site logy domains termed Nervy homology regions (NHR) to the DNA binding sites of these transcription factors, 1–4 based on their homology to the Drosophila including Ets-1, LEF-1, C/EBPa, PU.1, MEF, Pax5, protein Nervy, which is involved in neuronal develop- and GATA1 (Zhang et al., 1996; Petrovick et al., 1998; ment during segmentation of Drosophila embryos Kim et al., 1999; Libermann et al., 1999; Mao et al., (Feinstein et al., 1995). NHR1 has homology with the 1999; Goetz et al., 2000; Gu et al., 2000; Elagib et al., Drosophila TATA-box-associated factor 110 (TAF110) 2003; Li et al., 2004). For example, AML1 binding sites and other TAFs (Erickson et al., 1994). The NHR2 overlap with Ets-1 sites and are close to LEF-1 sites in domain contains a hydrophobic amino-acid (a.a.) enhancers of several T-cell receptors (Hsiang et al., 1993; heptad repeat and is required for homo- and hetero- Wotton et al., 1994; Giese et al., 1995; Mayall et al., dimerization between the ETO family members 1997). AML1 associates with these adjacent transcrip- (Gelmetti et al., 1998; Kitabayashi et al., 1998; Zhang tion factors and enhances their interaction to DNA (Sun et al., 2001). NHR3 contains a predicted coiled-coil et al., 1995). Furthermore, C/EBPa and PU.1 bind to structure (Minucci et al., 2000). NHR4 is a myeloid- the adjacent sites of AML1 consensus sequence in Nervy-DEAF1 homology domain with two predicted human M-CSF receptor promoter. They interact with zinc-finger motifs (CxxCCxxC and a CxxCHxxC) the RHD of AML1 with differing potentials in the (Erickson et al., 1994; Gross and McGinnis, 1996). synergistic activation of the M-CSF receptor (Zhang These zinc-finger motifs of ETO are involved in protein– et al., 1996; Petrovick et al., 1998). protein interaction, but not protein–DNA interaction. The first ETO-interacting protein identified was its AML1-ETO contains almost the entire ETO protein family member MTGR1 (Kitabayashi et al., 1998). (Figure 2). MTGR1 shares 61% identity with ETO and also

Oncogene The 8;21 translocation in leukemogenesis LF Peterson and D-E Zhang 4257 contains four Nervy homology domains. The NHR2 Accumulating data indicate that NMTS of various domain plays a critical role in the interaction between transcription factors may influence the specific localiza- ETO and MTGR1. Another ETO family member, tion and local concentration of transcription factors and ETO2, also interacts with ETO via NHR2 domain provide additional specificities for these factors in gene (Davis et al., 1999). Using ETO as bait or silencing expression (Davie, 1997). In addition, many of the mediator for retinoic acid receptor and thyroid hormone corepressor proteins that interact with AML1-ETO are receptor (SMRT) as bait in two independent yeast two- associated with such nuclear matrix foci (Davie et al., hybrid protein interaction studies and based on the 1999; Wood et al., 2000). AML1 contains an NMTS on educated guess, three groups reported almost simulta- the C-terminal side of runt homology domain, which is neously that nuclear receptor corepressor (NCoR) and not included in the AML1-ETO fusion protein. Inter- SMRT bind to the NHR4 zinc-finger domain of ETO estingly, ETO contains one nuclear localization signal (Gelmetti et al., 1998; Lutterbach et al., 1998b; Wang (a.a. 241–280) and two NMTS (a.a. 197–387 and a.a. et al., 1998). NCoR and SMRT are nuclear hormone 438–552) (Odaka et al., 2000; Barseguian et al., 2002). It corepressors, which are released from nuclear hormone has been reported that AML1 and ETO do not share receptors upon ligand binding, allowing transcriptional the same subnuclear locations and that AML1-ETO is activation (Shibata et al., 1997; Chen and Li, 1998). colocalized with ETO in specific nuclear foci (McNeil These corepressor proteins are known to associate with et al., 1999; Odaka et al., 2000). Such relocalization of other proteins involved in transcriptional repression, the AML1 DNA binding activity by AML1-ETO may such as mSin3a and the histone deacetylase (HDAC) therefore contribute to the deregulation of AML1 target family (Shibata et al., 1997; Chen and Li, 1998). genes. Therefore, via NCoR and SMRT, AML1-ETO can assemble a transcription repressor complex on AML1 target regulatory sequence and negatively regulate gene Multiple transcriptional functions of AML1-ETO expression. It has been demonstrated that the NHR4 region plays a critical role in such negative regulation A main functional characteristic of AML1-ETO is its of the activity of AML1 target promoters (Gelmetti ability to bind DNA containing AML1 binding sites T et al., 1998; Lutterbach et al., 1998a, b; Wang et al., (TG /CGGT) (Meyers et al., 1993). Many transient 1998). Further analysis revealed that NCoR, mSin3a, transfection reporter gene studies looking at the and different HDAC family members can interact with transcriptional aspect of AML1-ETO suggest that various regions of ETO and also negatively regulate AML1-ETO negatively regulate AML1 target genes. AML1 targets (Amann et al., 2001; Hildebrand et al., Such targets include MDR-1, GM-CSF receptor, IL-3, 2001) (Figure 2). In addition, the dimerization of ETO c-fos, TCRb enhancer, immunoglobulin a promoter, or AML1-ETO with AML1-ETO also plays an im- and most recently identified p14ARF (Table 1). The portant role in the regulation of AML1 target promoter negative regulatory effect of AML1-ETO was first activities (Minucci et al., 2000; Zhang et al., 2001). reported from the analysis of AML1 transactivation Atrophin-1 is a protein widely expressed and involved in of TCRb enhancer (Meyers et al., 1995). This inhibition the neurodegenerative disease dentate-rubral and pallido- was attributed to the ETO portion of the protein and luysian atrophy. A yeast two-hybrid study for identify- a very low amount of AML1-ETO was required to ing associated proteins of atrophin-1 showed that five inhibit AML1 transactivation. In this early report, of the seven positive clones were ETO and one clone AML1-ETO alone did not show any negative effect on was ETO2 (Wood et al., 2000). Furthermore, atrophin-1 TCRb enhancer activity. A similar negative regulation is colocalized with ETO in the nucleus, repressing of AML1-ETO on AML1 transactivation of the GM- gene expression (Wood et al., 2000). Using yeast two- CSF promoter was also reported (Frank et al., 1995). hybrid and biochemical copurification approaches, Here, the repressor activity of AML1-ETO in the Kozu’s group reported that the N-terminal region of absence of AML1 cotransfection was detected. Re- ETO interacts with chaperon heat-shock protein cently, the inhibition of the promoter activity of cell (HSP90) and the NHR3 domain of ETO bind to the cycle protein p14ARF by AML1-ETO was reported, in regulatory subunit of type II cyclic AMP-dependent which the inhibition was dependent on the RHD and protein kinase (PKA RIIa) (Komori et al., 1999; NHR2–4 region of AML1-ETO (Linggi et al., 2002). Fukuyama et al., 2001). Other proteins that interact Interestingly, a mutant of AML1-ETO, AML1-ETOD395 with ETO were found by investigating the possibility of without ETO NHR2-4 showed the block of c-fos interaction due to the presence of both proteins in promoter induction in an AML1 binding site-dependent hematopoietic cells, such as PLZF (Melnick et al., manner in reporter assays. Furthermore, the induction 2000b), Gfi-1 (McGhee et al., 2003), and Bcl-6 of endogenous c-fos mRNA expression in the response (Chevallier et al., 2004). All these interactions highlight of G-CSF stimulation was also blocked in 32Dcl3 a mechanism for ETO function as a corepressor in the granulocytic progenitor cell expressing AML1-ETOD395 repressive activity of transcription factors. Thus the (Hwang et al., 1999). dominant-negative effect of AML1-ETO on AML1 and Besides the negative effect of AML1-ETO on gene other transcriptional regulators, discussed below, expression, AML1-ETO was also reported as a positive could be exacerbated through such interactions and regulator, which is either dependent on or independent dimerizations. of AML1 protein and AML1 DNA binding sites. As

Oncogene The 8;21 translocation in leukemogenesis LF Peterson and D-E Zhang 4258 Table 1 Reported enhancers and promoters regulated by AML1 and/or AML1-ETO in the hematopoietic compartment Promoter/enhancer Expression References

Germ-line IgA1 Lymphoid Pardali et al. (2000), Xie et al. (1999) BLK Lymhoid Libermann et al. (1999) TCRa, b, g, d chains Lymphoid Giese et al. (1995), Hernandez-Munain and Krangel (1995), Hsiang et al. (1993) IL-3 Myeloid/lymphoid Uchida et al. (1997) Granzyme B Lymphoid Babichuk and Bleackley (1997), Wargnier et al. (1995) CD3 Lymphoid Hallberg et al. (1992) GM-CSF Lymphoid/myeloid Frank et al. (1995), Takahashi et al. (1995) Mona/gads Lymphoid Guyot and Mouchiroud (2003) M-CSF receptor Myeloid Rhoades et al. (1996), Zhang et al. (1994) MPO Myeloid Nuchprayoon et al. (1994) Complement receptor(1 Lymphoid/myeloid Kim et al. (1999a) NP-3 Myeloid Westendorf et al. (1998) Mast cell protease 6 Mast cells Morii et al. (1999) Neutrophil elastase Myeloid Nuchprayoon et al. (1994) CD11c Myeloid Drescher et al. (2003) CD11a Lymphoid Puig-Kroger et al. (2000) CD36 Myeloid/monocytic Armesilla et al. (1996) Bactericidal/permeability- Myeloid/neutrophil Lennartsson et al. (2003) increasing protein (BPI) MIP-1a Lymphoid Bristow and Shore (2003), Choi et al. (2003) Glycoprotein aIIb Myeloid/megakaryocyte Elagib et al. (2003) MDR1 Myeloid/lymphoid Lutterbach et al. (1998a) Art-1 Myeloid/erythroid Harada et al. (2001) c-fos Myeloid/lymphoid Hwang et al. (1999) Bcl-2 Myeloid/lymphoid Klampfer et al. (1996) p21WAF1 Myeloid/lymphoid Lutterbach et al. (2000) p14ARF Myeloid/lymphoid Linggi et al. (2002) PKCb Myeloid/lymphoid Hug et al. (2004) Cyclin D3 Myeloid/lymphoid Bernardin-Fried et al. (2004)

‘Expression’ speciﬁes where the gene is commonly expressed in hematopoietic cells. Targets regulated by AML1 in other tissues are not included

shown in Table 1, the proximal promoter of human on the C/EBP binding site in the upstream region of the M-CSF receptor (c-fms) contains an AML1 binding site G-CSF receptor gene. Further analysis demonstrate that (Zhang et al., 1994). AML1 expression signiﬁcantly C/EBPe expression is increased by AML1-ETO and enhances the promoter activity. Furthermore, AML1 C/EBPe activates the G-CSF receptor promoter via the can synergize or collaborate with C/EBPa and PU.1 in C/EBP binding site (Shimizu et al., 2000). The precise the activation of M-CSF receptor promoter (Zhang mechanism by which AML1-ETO induces C/EBPe et al., 1996; Petrovick et al., 1998). AML1-ETO does expression is still not understood. These observations induce the M-CSF receptor activity by itself to a lower suggest that AML1-ETO is not just a negative re- level than AML1. However, AML1-ETO enhances gulator of AML1 targets, but that it also functions as AML1 transactivation in a dosage-dependent manner a transactivator of speciﬁc genes that contain AML1 (Rhoades et al., 1996). Further, increased AML1-ETO binding sites. It is not clear whether ETO interacts with expression to the level higher than AML1 expression any positive regulators, such as coactivators p300 or reduces AML1 transactivation. Such effect of AML1- CBP, in the activation of gene expression. ETO requires minimally the N-terminal portion of ETO, On the other hand, AML1-ETO also binds to other including both NHR1 and NHR2 domains (Rhoades transcription regulators via its RHD or ETO portion et al., 1996). The promoter of the Bcl-2 gene can also be and can regulate gene expression independent of any positively regulated via a consensus AML1 binding AML1 binding site. The RHD interacts with C/EBPa, DNA sequence by AML1-ETO in transient transfection PU.1, and MEF. AML1-ETO has been reported to assays, while AML1 has no effect on the reporter regulate negatively the transcriptional activity of these activity (Klampfer et al., 1996). Both RHD and C- factors. AML1-ETO competes with c-Jun in binding terminal region of AML1-ETO, including NHR4 to PU.1 and blocks the coactivator activity of c-Jun domain, are critical for the transactivation. In addition, (Vangala et al., 2002). It can also repress C/EBPa- the G-CSF receptor promoter is activated by AML1- dependent transcription through an adjacent AML1 ETO. The C-terminal deletion studies reveal that the N- binding site (Westendorf et al., 1998) or blocks C/EBPa- terminal portion of AML1-ETO, including the NHR2 positive autoregulatory regulation of the C/EBPa own domain, is necessary for the transactivation and the promoter (Pabst et al., 2001). The association with MEF C-terminal 200 a.a.’s are dispensable for this effect promotes the inhibition of MEF-induced transcription, (Shimizu et al., 2000). Interestingly, such activation is independent of AML1-ETO binding to DNA (Mao independent of the AML1 binding site but is dependent et al., 1999). Furthermore, AML1-ETO interacts with

Oncogene The 8;21 translocation in leukemogenesis LF Peterson and D-E Zhang 4259 PLZF and Gfi proteins via its ETO portion. PLZF is cellular behavior using either hematopoietic cell lines itself involved in myeloid leukemia in t(11;17) and or primary human or mouse bone marrow cells. In functions as a repressive transcription factor blocking various stably transfected AML1-ETO-expressing hema- myeloid cell growth and differentiation and promoting topoietic cell lines, including 32D, L-G, K562, MEL, apoptosis (Shaknovich et al., 1998). The region of and U937 cells, AML1-ETO is shown to block myeloid AML1-ETO between NHR1 and NHR2 is required for and erythroid differentiation (Ahn et al., 1998; the interaction with PLZF (Melnick et al., 2000b). ETO Kitabayashi et al., 1998; Le et al., 1998; Hwang et al., behaves as a corepressor of PLZF in the inhibition of 1999; Kohzaki et al., 1999; Shimizu et al., 2000; Amann promoter activity of IL-3 receptor via a PLZF DNA et al., 2001; Burel et al., 2001; Harada et al., 2001). In binding site. The interaction of NCoR/SMRT with the 32D and L-G cells, AML1-ETO blocks their differentia- NHR4 zinc-finger domain plays a critical role in this tion and promotes G-CSF-dependent proliferation (Ahn corepressor activity (Melnick et al., 2000b). However, et al., 1998; Kitabayashi et al., 1998; Kohzaki et al., AML1-ETO inhibits the repressor activity of PLZF 1999), which is accompanied by the upregulation of the (Melnick et al., 2000a). AML1-ETO reduces PLZF G-CSF receptor expression by increased C/EBPe. DNA binding and also disrupts the nuclear matrix (Shimizu et al., 2000). In U937 cells, however, the block compartmentalization of PLZF. Such effects of AML1- of differentiation is accompanied by a decreased C/ ETO are also dependent on its zinc-finger NHR4 EBPa expression (Burel et al., 2001; Pabst et al., 2001). domain (Melnick et al., 2000a). These are very interest- Furthermore, using stably transfected inducible AML1- ing reports showing the opposite effect of AML1-ETO ETO-expressing cell lines, AML1-ETO showed a and ETO on the regulation of gene expression. Proto- negative effect on cell cycle and survival, including a oncogene products Gfi-1 and Gfi-1b are related zinc- G1 arrest associated with decreased CDK4 and c-Myc finger DNA binding transcription repressors and are expression (Amann et al., 2001; Burel et al., 2001). More highly expressed in hematopoietic cells (Gilks et al., recently, it was shown that treating inducible AML1- 1993; Tong et al., 1998). They are essential factors for ETO-expressing U937 cells with an inhibitor of c-Jun N- normal hematopoiesis (Karsunky et al., 2002; Saleque terminal kinase (JNK) or coexpression of the JNK et al., 2002; Hock et al., 2003). It has been recently inhibitor protein 1 significantly reduced AML1-ETO reported that ETO binds to Gfi-1 and Gfi-1b via its promoted apoptosis (Elsasser et al., 2003). These NHR1 and NHR2 domains and facilitates the recruit- observations demonstrate that AML1-ETO has an ment of the HDAC complex to the repressor activity of effect on the cellular programs related to proliferation, these Gfi-1 proteins (McGhee et al., 2003). Whether and differentiation, and apoptosis. how AML1-ETO may affect Gfi-1 function has not been In retrovirally transduced primary human CD34 þ clarified yet. However, as described in more detail in the cells, AML1-ETO promoted self-renewal following an next section, expressing AML1-ETO in K562 and MEL initial block in colony formation and proliferation in cells and in primary erythroid progenitors block methylcellulose and liquid cultures, respectively (Mulloy erythroid differentiation (Le et al., 1998; Harada et al., et al., 2002). A similar approach showed that differ- 2001; Tonks et al., 2002). The possible interaction entiation of CD34 þ /CD13À erythroid cells were abro- between Gfi-1 and AML1-ETO may contribute to the gated in the presence of AML1-ETO (Tonks et al., disruption of the normal function of Gfi-1. 2002). Erythroid progenitors with AML1-ETO expres- Taken together, these reports about AML1-ETO in sion produced fewer number and smaller erythroid the regulation of gene expression indicate that AML1- colonies in colony-forming assays. According to the ETO may function as a transcription repressor or authors (Tonks et al., 2002), erythropoiesis can be activator. Its function may depend on the AML1 distinguished into two stages. The early stage is DNA binding site in the regulatory element of its target independent of EPO and the late stage is dependent on gene or may be through its interaction with other EPO. AML1-ETO expression inhibits EPO-independent transcription regulators. Therefore, such effect may be proliferation of these cells and slows down the G1 to related to the presence of other factors in specific cells, S phase transition of the cell cycle. It also blocks the the specific differentiation stage, or particular cellular growth arrest at the boundary of EPO-independent to condition. Additional studies related to the molecular EPO-dependent stage of erythropoiesis and results in the mechanism of AML1-ETO function in the regulation of expansion of erythroid progenitors (Tonks et al., 2002). gene expression will provide further insight into the role It is difficult to separate the promotion of self-renewal of AML1-ETO during leukemogenesis. from the inhibition of differentiation. The disruption of cell differentiation will keep the proliferation potential of early progenitor cells and promote their expansion. AML1-ETO in cell proliferation, differentiation, and Conceivably, the additional mutations that enhance survival the cell proliferation will result in the uncontrolled expansion of early progenitors, which initiates leuke- The disruption of normal cell proliferation, differentia- mogenesis. A more recent report indicates that retro- tion, and survival contribute to the development of viral-mediated AML1-ETO-expressing human CD34 þ leukemia. To understand the mechanism of how t(8;21) cells, after a long-term in vitro culture, can still fusion protein is involved in leukemogenesis, many differentiate into myeloid and lymphoid lineages, and groups have studied the effect of AML1-ETO on that most importantly they do not promote leukemia in

Oncogene The 8;21 translocation in leukemogenesis LF Peterson and D-E Zhang 4260 NOD/SCID mice (Mulloy et al., 2003). A similar in the presence of IL-3 (Shimada et al., 2000). An effect of AML1-ETO on growth and differentiation interesting recent finding is that AML1-ETO in the is observed in the in vitro assays of primary mouse inducible U937 cell line disrupts the expression of some bone marrow progenitor cells (Okuda et al., 1998; genes involved in DNA repair, such as FEN1, OGG1, Hug et al., 2002). Furthermore, the loss of the POLD2, and POLD3 (Alcalay et al., 2003). The null NHR2 and NHR4 domains together eliminates such mutants of these factors have all been suggested to play effect of AML1-ETO fusion protein (Hug et al., 2002). a role in rapid tumor progression and/or cancer Taken together, these observations suggest that AML1- susceptibility. Their dysregulated expression could con- ETO could exert its oncogenic potential by mechanisms tribute to genetic instability at the onset of acquiring of blocking differentiation and promoting the self- AML1-ETO and cause the accumulation of DNA renewal of stem cells and early progenitors. The ETO mutations involved in the progression of AML-M2 portion is critical for such effects. Furthermore, the phenotype, which is determined by the combinatory acquisition of secondary events that disrupt the inhibi- effect of the mutations and the presence of AML1-ETO. tion of cell cycle by AML1-ETO is necessary for These observations are further in support of a ‘second- leukemia development. Such potential is well supported ary mutagenic event(s)’ model proposed for AML1- by in vivo studies using animal models, which go beyond ETO leukemogenesis (Yuan et al., 2001; Higuchi et al., the scope of this review (Yergeau et al., 1997; Okuda 2002; Schwieger et al., 2002; Grisolano et al., 2003). et al., 1998; Yuan et al., 2001; de Guzman et al., 2002; Thus, finding the crucial pathways required for AML1- Higuchi et al., 2002; Schwieger et al., 2002; Grisolano ETO to fully transform a myeloid cell to leukemia et al., 2003). remains the great challenge. Microarray analysis to study the effect of AML1- ETO expression on gene regulation is a powerful technology used to elucidate the molecular mechanism of AML1-ETO in leukemogenesis. One such approach Summary using murine L-G granulocytic progenitor cells showed that the presence of AML1-ETO promotes the differ- The t(8;21) fusion protein AML1-ETO is a multi- entiation of these cells to a promyelocyte stage based on functional protein exemplified by its role in regulating the induction of various genes involved in azurophil diverse cellular programs such as differentiation, pro- granule formation at this early stage of differentiation liferation, apoptosis, and self-renewal in various in vitro (Shimada et al., 2002). This finding corroborates the and in vivo models. It regulates the expression of both observed phenotype of t(8;21) patient samples that AML1 target and non-AML1 target genes via its display such granules in their cytoplasm. A more recent interaction with various transcription regulators. study using U937 cells with inducible AML1-ETO Furthermore, additional mutations besides t(8;21) are expression and microarray analysis reveal that the necessary for the development of AML. The challenging expression of various genes are disrupted or enhanced questions with regard to t(8;21) involved leukemogenesis upon the induction of AML1-ETO (Alcalay et al., remain as: what are the critical target genes regulated 2003). Examples of disrupted targets are C/EBPa and b, by AML1-ETO and what are the molecular pathways which are involved in hematopoiesis (Chen et al., 1997; that cooperate with AML1-ETO in promoting the Zhang et al., 1997). Some examples of induced genes are oncogenesis of hematopoietic cells? The massive amount LMO1 and BCL11A, which have been implicated in of new information about the human and mouse lymphoid/myeloid malignancies (Aplan et al., 1997; Liu genomes and new technology development will greatly et al., 2003). Furthermore, using a differential display enhance our ability to address these interesting approach, Shimada et al. (2000) has reported that questions. AML1-ETO activate the expression of TIS11b (also called ERF-1 or cMG1) gene. Over expression of Acknowledgements TIS11b in L-G cells increases cell proliferation and We thank Robert Hines for editing this manuscript. This work delays cell differentiation in response to G-CSF treat- was supported by National Institute of Health Grants ment, but shows a slight reduction in cell proliferation CA072009 and CA096735.

References

Ahn MY, Huang G, Bae SC, Wee HJ, Kim WY and Aplan PD, Jones CA, Chervinsky DS, Zhao X, Ellsworth M, Ito Y. (1998). Proc. Natl. Acad. Sci. USA, 95, Wu C, McGuire EA and Gross KW. (1997). EMBO J., 16, 1812–1817. 2408–2419. Alcalay M, MeaniN, GelmettiV, FantozziA, FagioliM, Barseguian K, Lutterbach B, Hiebert SW, Nickerson J, Orleth A, Riganelli D, Sebastiani C, Cappelli E, Casciari C, Lian JB, Stein JL, van Wijnen AJ and Stein GS. (2002). Sciurpi MT, Mariano AR, Minardi SP, Luzi L, Muller H, Proc. Natl. Acad. Sci. USA, 99, 15434–15439. Di Fiore PP, Frosina G and Pelicci PG. (2003). J. Clin. Burel SA, Harakawa N, Zhou L, Pabst T, Tenen DG and Invest., 112, 1751–1761. Zhang DE. (2001). Mol. Cell. Biol., 21, 5577–5590. Amann JM, Nip J, Strom DK, Lutterbach B, Harada H, CalabiF and CilliV. (1998). Genomics, 52, 332–341. Lenny N, Downing JR, Meyers S and Hiebert SW. (2001). Chen JD and LiH. (1998). Crit. Rev. Eukaryot. Gene Expr., 8, Mol. Cell. Biol., 21, 6470–6483. 169–190.

Oncogene The 8;21 translocation in leukemogenesis LF Peterson and D-E Zhang 4261 Chen X, Liu W, Ambrosino C, Ruocco MR, Poli V, Romani Hwang ES, Hong JH, Bae SC, Ito Y and Lee SK. (1999). L, Quinto I, Barbieri S, Holmes KL, Venuta S and Scala G. FEBS Lett., 446, 86–90. (1997). Blood, 90, 156–164. ImaiY, Kurokawa M, Tanaka K, FriedmanAD, Ogawa S, Chevallier N, Corcoran CM, Lennon C, Hyjek E, Chadburn Mitani K, Yazaki Y and Hirai H. (1998). Biochem. Biophys. A, Bardwell VJ, Licht JD and Melnick A. (2004). Blood, 103, Res. Commun., 252, 582–589. 1454–1463. Kania MA, Bonner AS, Duffy JB and Gergen JP. (1990). Davie JR. (1997). Mol. Biol. Rep., 24, 197–207. Genes Dev., 4, 1701–1713. Davie JR, Samuel SK, Spencer VA, Holth LT, Chadee DN, Kanno T, Kanno Y, Chen LF, Ogawa E, Kim WY and Ito Y. Peltier CP, Sun JM, Chen HY and Wright JA. (1999). (1998). Mol. Cell. Biol., 18, 2444–2454. Biochem. Cell Biol., 77, 265–275. Karsunky H, Mende I, Schmidt T and Moroy T. (2002). Davis JN, Williams BJ, Herron JT, Galiano FJ and Meyers S. Oncogene, 21, 1571–1579. (1999). Oncogene, 18, 1375–1383. Kim WY, Sieweke M, Ogawa E, Wee HJ, Englmeier U, Graf T de Greef GE, Hagemeijer A, Morgan R, Wijsman J, Hoefsloot and Ito Y. (1999). EMBO J., 18, 1609–1620. LH, Sandberg AA and SacchiN. (1995). Leukemia, 9, Kitabayashi I, Ida K, Morohoshi F, Yokoyama A, Mitsuhashi 282–287. N, Shimizu K, Nomura N, Hayashi Y and Ohki M. (1998). de Guzman CG, Warren AJ, Zhang Z, Gartland L, Erickson Mol. Cell. Biol., 18, 846–858. P, Drabkin H, Hiebert SW and Klug CA. (2002). Mol. Cell. Klampfer L, Zhang J, Zelenetz AO, Uchida H and Nimer SD. Biol., 22, 5506–5517. (1996). Proc. Natl. Acad. Sci. USA, 93, 14059–14064. Duffy JB and Gergen JP. (1991). Genes Dev., 5, 2176–2187. KohzakiH, Ito K, Huang G, Wee HJ, MurakamiY and Ito Y. Elagib KE, Racke FK, Mogass M, Khetawat R, Delehanty LL (1999). Oncogene, 18, 4055–4062. and Goldfarb AN. (2003). Blood, 101, 4333–4341. Komori A, Sueoka E, Fujiki H, Ishii M and Kozu T. (1999). Elsasser A, Franzen M, Kohlmann A, Weisser M, Schnittger Jpn. J. Cancer Res., 90, 60–68. S, Schoch C, Reddy VA, Burel S, Zhang DE, Uefﬁng M, Kozu T, Miyoshi H, Shimizu K, Maseki N, Kaneko Y, Asou H, Tenen DG, Hiddemann W and Behre G. (2003). Oncogene, Kamada N and OhkiM. (1993). Blood, 82, 1270–1276. 22, 5646–5657. Kurokawa M, Tanaka T, Tanaka K, Ogawa S, Mitani K, Erickson P, Gao J, Chang KS, Look T, Whisenant E, YazakiY and HiraiH.(1996). Oncogene, 12, 883–892. Raimondi S, Lasher R, Trujillo J, Rowley J and Drabkin H. Le XF, Claxton D, Kornblau S, Fan YH, Mu ZM and (1992). Blood, 80, 1825–1831. Chang KS. (1998). Eur. J. Haematol., 60, 217–225. Erickson PF, Robinson M, Owens G and Drabkin HA. (1994). Levanon D, Bernstein Y, Negreanu V, Ghozi MC, Bar-Am I, Cancer Res., 54, 1782–1786. Aloya R, Goldenberg D, Lotem J and Groner Y. (1996). Feinstein PG, Kornfeld K, Hogness DS and Mann RS. (1995). DNA Cell Biol., 15, 175–185. Genetics, 140, 573–586. Levanon D, Glusman G, Bangsow T, Ben Asher E, Male DA, Felix CA. (1998). Biochim. Biophys. Acta, 1400, 233–255. Avidan N, Bangsow C, Hattori M, Taylor TD, Taudien S, Frank R, Zhang J, Uchida H, Meyers S, Hiebert SW and Blechschmidt K, Shimizu N, Rosenthal A, Sakaki Y, Lancet Nimer SD. (1995). Oncogene, 11, 2667–2674. D and Groner Y. (2001). Gene, 262, 23–33. Fukuyama T, Sueoka E, Sugio Y, Otsuka T, Niho Y, Akagi K Levanon D, Goldstein RE, Bernstein Y, Tang H, Goldenberg and Kozu T. (2001). Oncogene, 20, 6225–6232. D, Stifani S, Paroush Z and Groner Y. (1998). Proc. Natl. Gao J, Erickson P, Gardiner K, Le Beau MM, Diaz MO, Acad. Sci. USA, 95, 11590–11595. Patterson D, Rowley JD and Drabkin HA. (1991). Proc. Li FQ, Person RE, Takemaru K, Williams K, Meade-White Natl. Acad. Sci. USA, 88, 4882–4886. K, Ozsahin AH, Gungor T, Moon RT and Horwitz M. Gelmetti V, Zhang J, Fanelli M, Minucci S, Pelicci PG and (2004). J. Biol. Chem., 279, 2873–2884. Lazar MA. (1998). Mol. Cell. Biol., 18, 7185–7191. Libermann TA, Pan Z, Akbarali Y, Hetherington CJ, GhoziMC, BernsteinY, Negreanu V, Levanon D and Groner Boltax J, Yergeau DA and Zhang DE. (1999). J. Biol. Y. (1996). Proc. Natl. Acad. Sci. USA, 93, 1935–1940. Chem., 274, 24671–24676. Giese K, Kingsley C, Kirshner JR and Grosschedl R. (1995). Linggi B, Muller-Tidow C, van de LL, Hu M, Nip J, Serve H, Genes Dev., 9, 995–1008. Berdel WE, van der RB, Quelle DE, Rowley JD, Cleveland Gilks CB, Bear SE, Grimes HL and Tsichlis PN. (1993). Mol. J, Jansen JH, Pandolﬁ PP and Hiebert SW. (2002). Nat. Cell. Biol., 13, 1759–1768. Med., 8, 743–750. Goetz TL, Gu TL, Speck NA and Graves BJ. (2000). Mol. Liu P, Keller JR, Ortiz M, Tessarollo L, Rachel RA, Cell. Biol., 20, 81–90. Nakamura T, Jenkins NA and Copeland NG. (2003). Nat. Grisolano JL, O’Neal J, Cain J and Tomasson MH. (2003). Immunol., 4, 525–532. Proc. Natl. Acad. Sci. USA, 100, 9506–9511. Lutterbach B, Sun D, Schuetz J and Hiebert SW. (1998a). Mol. Gross CT and McGinnis W. (1996). EMBO J., 15, 1961–1970. Cell. Biol., 18, 3604–3611. Gu TL, Goetz TL, Graves BJ and Speck NA. (2000). Mol. Lutterbach B, Westendorf JJ, Linggi B, Patten A, Moniwa M, Cell. Biol., 20, 91–103. Davie JR, Huynh KD, Bardwell VJ, Lavinsky RM, Harada Y, Harada H, Downing JR and Kimura A. (2001). Rosenfeld MG, Glass C, Seto E and Hiebert SW. (1998b). Biochem. Biophys. Res. Commun., 284, 714–722. Mol. Cell. Biol., 18, 7176–7184. Higuchi M, O’Brien D, Kumaravelu P, Lenny N, Yeoh EJ and Mao S, Frank RC, Zhang J, Miyazaki Y and Nimer SD. Downing JR. (2002). Cancer Cell, 1, 63–74. (1999). Mol. Cell. Biol., 19, 3635–3644. Hildebrand D, Tiefenbach J, Heinzel T, Grez M and Maurer AB. Mayall TP, Sheridan PL, Montminy MR and Jones KA. (2001). J. Biol. Chem., 276, 9889–9895. (1997). Genes Dev., 11, 887–899. Hock H, Hamblen MJ, Rooke HM, Traver D, Bronson RT, McGhee L, Bryan J, Elliott L, Grimes HL, Kazanjian A, Cameron S and Orkin SH. (2003). Immunity, 18, 109–120. Davis JN and Meyers S. (2003). J. Cell Biochem., 89, Hsiang YH, Spencer D, Wang S, Speck NA and Raulet DH. 1005–1018. (1993). J. Immunol., 150, 3905–3916. McNeil S, Zeng C, Harrington KS, Hiebert S, Lian JB, Stein Hug BA, Lee SYD, Kinsler EL, Zhang J and Lazar MA. JL, van Wijnen AJ and Stein GS. (1999). Proc. Natl. Acad. (2002). Cancer Res., 62, 2906–2912. Sci. USA, 96, 14882–14887.

Oncogene The 8;21 translocation in leukemogenesis LF Peterson and D-E Zhang 4262 Melnick A, Carlile GW, McConnell MJ, Polinger A, Hiebert Sun W, Graves BJ and Speck NA. (1995). J. Virol., 69, SW and Licht JD. (2000a). Blood, 96, 3939–3947. 4941–4949. Melnick AM, Westendorf JJ, Polinger A, Carlile GW, Arai S, Tighe JE and Calabi F. (1995). Clin. Sci. (Lond.), 89, Ball HJ, Lutterbach B, Hiebert SW and Licht JD. (2000b). 215–218. Mol. Cell. Biol., 20, 2075–2086. Tighe JE, Daga A and Calabi F. (1993). Blood, 81, 592–596. Meyers S, Downing JR and Hiebert SW. (1993). Mol. Cell. Tong B, Grimes HL, Yang TY, Bear SE, Qin Z, Du K, Biol., 13, 6336–6345. El Deiry WS and Tsichlis PN. (1998). Mol. Cell. Biol., 18, Meyers S, Lenny N and Hiebert SW. (1995). Mol. Cell. Biol., 2462–2473. 15, 1974–1982. Tonks A, Pearn L, Tonks AJ, Pearce L, Hoy T, Phillips S, Minucci S, Maccarana M, Cioce M, De Luca P, Gelmetti V, Fisher J, Downing JR, Burnett AK and Darley RL. (2002). Segalla S, DiCroce L, GiavaraS, MatteucciC, GobbiA, Blood, 101, 2002–2006. Bianchini A, Colombo E, Schiavoni I, Badaracco G, Hu X, Vangala RK, Heiss-Neumann MS, Rangatia JS, Singh SM, Lazar MA, Landsberger N, NerviC and PelicciPG. (2000). Schoch C, Tenen DG, Hiddemann W and Behre G. (2002). Mol. Cell, 5, 811–820. Blood, 101, 2002–2004. Miyoshi H, Ohira M, Shimizu K, Mitani K, Hirai H, Imai T, Wang J, Hoshino T, Redner RL, Kajigaya S and Liu JM. Yokoyama K, Soeda E and OhkiM. (1995). Nucleic Acids (1998). Proc. Natl. Acad. Sci. USA, 95, 10860–10865. Res., 23, 2762–2769. Wang Q, Stacy T, Binder M, Marin-Padilla M, Sharpe AH Miyoshi H, Shimizu K, Kozu T, Maseki N, Kaneko Y and Speck NA. (1996b). Proc. Natl. Acad. Sci. USA, 93, and OhkiM. (1991). Proc. Natl. Acad. Sci. USA, 88, 3444–3449. 10431–10434. Wang Q, Stacy T, Miller JD, Lewis AF, Gu TL, Huang X, Mulloy JC, Cammenga J, Berguido FJ, Wu K, Zhou P, Bushweller JH, Bories JC, Alt FW, Ryan G, Liu PP, Comenzo RL, Jhanwar S, Moore MA and Nimer SD. Wynshaw-Boris A, Binder M, Marin-Padilla M, Sharpe AH (2003). Blood, 102, 4369–4376. and Speck NA. (1996a). Cell, 87, 697–708. Mulloy JC, Cammenga J, MacKenzie KL, Berguido FJ, Wang S, Wang Q, Crute BE, Melnikova IN, Keller SR and Moore MA and Nimer SD. (2002). Blood, 99, 15–23. Speck NA. (1993). Mol. Cell. Biol., 13, 3324–3339. Nisson PE, Watkins PC and Sacchi N. (1992). Cancer Genet. Westendorf JJ, Yamamoto CM, Lenny N, Downing JR, Cytogenet., 63, 81–88. Selsted ME and Hiebert SW. (1998). Mol. Cell. Biol., 18, Odaka Y, Mally A, Elliott LT and Meyers S. (2000). 322–333. Oncogene, 19, 3584–3597. Wolford JK and Prochazka M. (1998). Gene, 212, 103–109. Ogawa E, Inuzuka M, Maruyama M, Satake M, Wood JD, Nucifora Jr FC, Duan K, Zhang C, Wang J, Kim Naito-Fujimoto M, Ito Y and Shigesada K. (1993). Y, Schilling G, Sacchi N, Liu JM and Ross CA. (2000). Virology, 194, 314–331. J. Cell Biol., 150, 939–948. Okuda T, CaiZ, Yang S, Lenny N, Lyu CJ, van Deursen JM, Wotton D, Ghysdael J, Wang S, Speck NA and Owen MJ. Harada H and Downing JR. (1998). Blood, 91, 3134–3143. (1994). Mol. Cell. Biol., 14, 840–850. Pabst T, Mueller BU, Harakawa N, Schoch C, Haferlach T, Xiao Z, Greaves MF, Bufﬂer P, Smith MT, Segal MR, Behre G, Hiddemann W, Zhang DE and Tenen DG. (2001). Dicks BM, Wiencke JK and Wiemels JL. (2001). Leukemia, Nat. Med., 7, 444–451. 15, 1906–1913. Petrovick MS, Hiebert SW, Friedman AD, Hetherington CJ, Yergeau DA, Hetherington CJ, Wang Q, Zhang P, Tenen DG and Zhang DE. (1998). Mol. Cell. Biol., 18, Sharpe AH, Binder M, Marin-Padilla M, Tenen DG, 3915–3925. Speck NA and Zhang DE. (1997). Nat. Genet., 15, 303–306. Rhoades KL, Hetherington CJ, Rowley JD, Hiebert SW, Yuan Y, Zhou L, Miyamoto T, Iwasaki H, Harakawa N, Nucifora G, Tenen DG and Zhang DE. (1996). Proc. Natl. Hetherington CJ, Burel SA, Lagasse E, Weissman IL, Acad. Sci. USA, 93, 11895–11900. AkashiK and Zhang DE. (2001). Proc. Natl. Acad. Sci. Rowley JD. (1973). Ann. Genet., 16, 109–112. USA, 98, 10398–10403. Saleque S, Cameron S and Orkin SH. (2002). Genes Dev., 16, Zeng C, McNeil S, Pockwinse S, Nickerson J, Shopland L, 301–306. Lawrence JB, Penman S, Hiebert S, Lian JB, van Wijnen AJ, Schwieger M, Lohler J, Friel J, Scheller M, Horak I and Stein JL and Stein GS. (1998). Proc. Natl. Acad. Sci. USA, Stocking C. (2002). J. Exp. Med., 196, 1227–1240. 95, 1585–1589. Shaknovich R, Yeyati PL, Ivins S, Melnick A, Lempert C, Zhang DE, Fujioka K, Hetherington CJ, Shapiro LH, Waxman S, Zelent A and Licht JD. (1998). Mol. Cell. Biol., Chen HM, Look AT and Tenen DG. (1994). Mol. Cell. 18, 5533–5545. Biol., 14, 8085–8095. Shibata H, Spencer TE, Onate SA, Jenster G, Tsai SY, Zhang DE, Hetherington CJ, Meyers S, Rhoades KL, TsaiMJ and O’Malley BW. (1997). Recent Prog. Horm. Larson CJ, Chen HM, Hiebert SW and Tenen DG. (1996). Res., 52, 141–164. Mol. Cell. Biol., 16, 1231–1240. Shimada H, Ichikawa H, Nakamura S, Katsu R, Iwasa M, Zhang DE, Zhang P, Wang ND, Hetherington CJ, Kitabayashi I and Ohki M. (2000). Blood, 96, 655–663. Darlington GJ and Tenen DG. (1997). Proc. Natl. Acad. Shimada H, Ichikawa H and Ohki M. (2002). Leukemia, 16, Sci. USA, 94, 569–574. 874–885. Zhang J, Hug BA, Huang EY, Chen CW, GelmettiV, Shimizu K, Kitabayashi I, Kamada N, Abe T, Maseki N, Maccarana M, Minucci S, Pelicci PG and Lazar MA. Suzukawa K and OhkiM. (2000). Blood, 96, 288–296. (2001). Mol. Cell. Biol., 21, 156–163. Shimizu K, Miyoshi H, Kozu T, Nagata J, Enomoto K, ZhangY,StrisselP,StrickR,ChenJ,NuciforaG,LeBeauMM, MasekiN, Kaneko Y and OhkiM. (1992). Cancer Res., 52, Larson RA and Rowley JD. (2002). Proc. Natl. Acad. Sci. 6945–6948. USA, 99, 3070–3075.

Oncogene