|Transcription factor fusions in acute leukemia: variations on a theme|
|Joseph M Scandura, Piernicola Boccuni, Jorg Cammenga and Stephen D Nimer|
Laboratory of Molecular Aspects of Hematopoiesis, Sloan-Kettering Institute Memorial Sloan-Kettering Cancer Center, 1275 York Avenue, New York, NY 10021, USA
Correspondence to: S D Nimer, Laboratory of Molecular Aspects of Hematopoiesis, Sloan-Kettering Institute Memorial Sloan-Kettering Cancer Center, 1275 York Avenue, New York, NY 10021, USA. E-mail: firstname.lastname@example.org
The leukemia-associated fusion proteins share several structural or functional similarities, suggesting that they may impart a leukemic phenotype through common modes of transcriptional dysregulation. The fusion proteins generated by these translocations usually contain a DNA-binding domain, domains responsible for homo- or hetero-dimerization, and domains that interact with proteins involved in chromatin remodeling (e.g., co-repressor molecules or co-activator molecules). It is these shared features that constitute the 'variations on the theme' that underling the aberrant growth and differentiation that is the hallmark of acute leukemia cells.
Oncogene (2002) 21, 3422-3444 DOI: 10.1038/sj/onc/1205315
leukemia; transcription; PML; RAR; TGF; NUP98; AML1/ETO; CBF
The hallmark of acute leukemia is the clonal, malignant proliferation of immature hematopoietic cells and the French-American-British, or FAB, classification has been the standard system used to classify the acute leukemias. Acute myelogenous leukemia (AML) is divided into eight major FAB subtypes (M0-M7), which are defined by the degree to which differentiation along one of the myeloid lineages is evident by morphology and immunophenotype (Bennett et al., 1985). With the exception of acute promyelocytic leukemia, APL (M3), which is uniquely sensitive to retinoic acid, the initial treatment (induction chemotherapy) of these leukemias is identical. However, the choice of subsequent therapy, to 'consolidate' a complete remission, often depends upon the specific cytogenetic abnormality found in the leukemic blasts, rather than their morphology or degree of differentiation. While remission rates for patients with de novo AML do not vary greatly across FAB subtypes, long-term survival can vary considerably. Several deficiencies in the FAB classification of acute leukemia have been addressed by a recent WHO classification scheme that ascribes more significance to the presence of specific cytogenetic abnormalities and to dysplasia (Harris et al., 1999).
The accurate diagnosis of the acute leukemias is critical for selecting their proper treatment and for defining the biological basis for these diseases. There are 'lumpers' and 'splitters' when it comes to defining and classifying diseases of man. In this article, we will make the argument that in many ways, AML is a pathophysiologically homogeneous disease, despite several biological, as well as clinically important variations.
Basic biology of AML
The fundamental biological features of the malignant cell in AML are (1) its ability to proliferate continuously and (2) its aberrant or arrested differentiation. Much is known about the signals that control cell survival and proliferation, whereas the signals required for differentiation are less well defined. Although growth factor receptor overexpression is commonly seen in solid tumors (e.g., HER2 or EGF receptor overexpression) (Robertson et al., 2000), it has not been found in the acute leukemias. Cytokine overexpression is rarely seen in leukemic cells, although leukemic blasts can express growth factors that act in an autocrine or paracrine fashion (Fraser et al., 1994; Majka et al., 2001). Colony-stimulating factor receptor abnormalities have been found only rarely in acute leukemia (e.g., G-CSF receptor abnormalities in AML that develops in patients with congenital neutropenia), but recent studies demonstrate that activating mutations in the FLT3 receptor and in c-Kit (the receptor for stem cell factor) are quite common (Beghini et al., 2000; Horiike et al., 1997; Nakao et al., 1996; Rombouts et al., 2000; Yamamoto et al., 2001). These mutations will be covered in another article in this journal. We will focus on leukemia-associated abnormalities in transcription factor structure and function.
Recurrent cytogenetic abnormalities in AML
More than half of newly diagnosed cases of AML display detectable and usually single cytogenetic abnormalities (Mrozek et al., 2001). Balanced chromosomal translocations are the most specific genetic lesions in AML and may represent critical, early events in the genesis of the leukemic clone. The most common translocations are listed in Table 1, which also identifies the genes located at the translocation breakpoints. Genes encoding transcription factors are nearly always found at one of the breakpoints, and the fusion proteins that are formed by the translocations generally interfere with the normal function of one or both of the rearranged genes. Abnormalities in the untranslocated, presumably normal allele, have not been examined in many instances, but loss of the normal TEL allele is nearly always found in t(12;21) positive (i.e., TEL/AML1 positive) ALL (Takeuchi et al., 1997). In some instances, the reciprocal translocation also encodes a chimeric fusion protein.
The most well-studied genetic lesions are t(8;21) (q22;q22), t(15;17)(q22;12), inv(16)(p13;q22), and t(9;11) (p22;q23). Other common translocations in AML are t(9;22)(q34;q11), t(3;3)(q21;q26), t(8;16) (p11; p13), t(6;9)(p23;q34), t(7;11)(p15;p15), t(6;11) (q27;q23), t(11;19)(q23;p13.1), t(11;19)(q23;p13.3), t(16;16) (p13; q22), t(16;21)(p11;q22) and t(1;22) (p13;q13) (Mitelman et al., 2001; and reviewed in Mrozek et al. (2001)). The breakpoints for these translocations have been cloned and the genes involved are under intensive investigation. Recurrent numerical and unbalanced cytogenetic aberrations are also observed in AML, the most common are del(5q) and del(7q) but deletions of 9q, 11q, 12p and 20q, and trisomy 8 are also seen frequently. The critical genes disrupted by these abnormalities have not been clearly identified, thus they will not be discussed in this review.
Secondary leukemias are those that are associated with prior chemotherapy or that arise from an antecedent hematologic disorder such as a myelodysplastic syndrome or a myeloproliferative disease (e.g., chronic myelogenous leukemia). They are predominantly AML (90%) and are associated with an extremely poor prognosis (see Dann and Rowe (2001) and references therein). Chemotherapy-related leukemias were first described in survivors of Hodgkin's Disease treated with nitrogen mustard (mechlorethamine), but were later found in patients treated with other alkylating agents (procarbazine, lomustine, chlorambucil), and in patients receiving epipodophyllotoxins (teniposide and etoposide) or other drugs that target topoisomerase II (e.g., anthracyclines). Eighty-five per cent of therapy-related leukemias will occur within 10 years of the chemotherapy, and the risk of developing them generally relates to the cumulative dose of the offending agent. The cytogenetic lesions associated with therapy-related leukemias segregate with the class of the antecedent chemotherapy. Secondary leukemias induced by alkylating agents are most frequently associated with loss of all or part of chromosomes 5 or 7, usually present first with myelodysplasia, and have a long period of latency (5-7 years). AML associated with topoisomerase inhibitors occurs with a shorter latency (~2 years), in a younger population, and most commonly involves the translocation of chromosome 11q23 (the mixed-lineage leukemia, MLL, gene locus) to chromosomes 4, 9, and 19, causing the t(4;11), t(9;11) and t(19;11) translocations, respectively. Other translocations which commonly occur in therapy-related AML include t(3;21) (AML1/EVI1) and t(8;16) (MOZ/CBP). Translocations that are common in de novo AML, such as t(8;21), t(15;17), and inv(16), can also be seen in patients that develop AML after therapy for a prior malignancy; whether these are truly 'therapy-related AML' or not cannot be determined.
Clinical syndromes: association with specific cytogenetic abnormalities
The karyotype is an independent predictor of remission rates and overall survival in AML (Bloomfield et al., 1998; Grimwade et al., 2001; Keating et al., 1988; Leith et al., 1997; Mrozek et al., 1997; Visani et al., 2001) and the common cytogenetic abnormalities are highly-associated with the morphology of the leukemic blasts and with particular clinical features of the disease. The t(8;21) translocation is found predominantly in the FAB M2 subtype of AML (Mrozek et al., 2001). These myeloblasts usually show 'aberrant' expression of CD19, have a predisposition to form granulocytic sarcomas, and are associated with eosinophilia (Andrieu et al., 1996; Jacobsen et al., 1984; Swirsky et al., 1984). t(8;21) positive AML has a good prognosis in adults, although the expression of CD56, a marker of natural killer cells, is sometimes seen and may portend a poorer outcome (Baer et al., 1997; Byrd and Weiss, 1994). Inv(16) and the much less common t(16;16), are highly associated with the FAB M4Eo subtype of AML, acute myelomonocytic leukemia with characteristic bone marrow and peripheral blood eosinophilia (Larson et al., 1986; Le Beau et al., 1983). These leukemias also have a better than average prognosis and are associated with extramedullary disease (Le Beau et al., 1983), such as granulocytic sarcomas, and rarely, CNS involvement (Marlton et al., 1995). The inv(16), t(16;16), and t(8;21) positive AML disrupt the 'core binding factor complex' and are sometimes referred to as CBF leukemias. Adult patients with these cytogenetic abnormalities have been shown to benefit from the inclusion of high-dose cytarabine chemotherapy in their consolidation treatment regimen (Bloomfield et al., 1998).
The FAB M3 subtype of AML, acute promyelocytic leukemia (APL) is almost uniformly (99%) associated with the t(15;17) translocation (Pandolfi, 2001). APL is often associated with a low white blood cell count at diagnosis and with disseminated intravascular coagulation, a complication that led to high mortality in the past. The most striking characteristic of t(15;17)-positive AML is the unique capacity of the affected promyelocytes (and blasts) to fully differentiate to neutrophils when treated with pharmacologic doses of all-trans retinoic acid (ATRA) (Castaigne et al., 1990; Huang et al., 1988; Warrell, 1996). ATRA therapy alone usually leads to a complete remission of APL and when this remission is consolidated with an anthracycline-containing regimen, the long-term survival of these patients is more than 75% (Soignet et al., 1997). Relapsed APL, but not other types of AML, is highly responsive to treatment with arsenic trioxide (As2O3), a drug recently approved by the FDA for this indication (Soignet et al., 2001).
Translocations involving 11q23 are seen in up to 80% of leukemias diagnosed in the very young (less than 24 months) (Raimondi et al., 1999; Kalwinsky, 1990; Satake et al., 1999). In these infants, 11q23 abnormalities are generally associated with hyperleukocytosis at diagnosis and poor prognosis (Gibbons et al., 1990; Kalwinsky et al., 1990; Thirman et al., 1993). Prenatal rearrangement of 11q23 has been detected in blood spots from fetuses that subsequently developed leukemia during infancy; hence, the rearrangement is an early event during leukemogenesis (Gale et al., 1997). It can also be found in non-affected twins suggesting that it is not sufficient for the disease (Gill Super et al., 1994). Maternal exposure to flavinoids (natural topoisomerase II inhibitors) may be an important predisposing factor in its pathogenesis (Ross et al., 1996; Strick et al., 2000). Disorders of 11q23 are also associated with exposure to topoisomerase II inhibitors in adults and often portend a poor prognosis (reviewed in Dann and Rowe (2001)). 11q23 translocations are usually found in AML of the FAB M4 or M5 subtype (Mrozek et al., 2001).
Classical models of oncogenesis
Classical models of oncogenesis involve proto-oncogene activation and/or the inactivation of tumor suppressor gene function. The normal immunoglobulin and T-cell receptor gene rearrangements that occur during B- and T-cell development predispose to translocations that can lead to aberrant oncogene expression and contribute to lymphoid cell transformation (reviewed in Rabbitts (1994)). Gene rearrangements do not normally occur during myeloid development; thus, activation of proto-oncogenes by overexpression is uncommon in myelogenous leukemia - one notable exception being the overexpression of the EVI-1 gene resulting from the t(3;3)(q21;q26). Similarly, the biallelic loss of tumor suppressor gene function by deletion and mutation has not been found in AML, although p53 mutations are associated with the loss of the short arm of chromosome (17p) (Lai et al., 1995). Thus classical tumor suppressor gene inactivation mechanisms, where one allele is deleted and the other mutated, may not be common in AML. Nonetheless, haploinsufficiency or point mutations in certain transcription factor genes involved in myeloid differentiation have been reported in AML. For instance, familial platelet disorder with predisposition to AML (FPD/AML) is a rare autosomal-dominant disease that manifests as quantitative and qualitative platelet defects and a predisposition to develop AML. It is caused by nonsense and missense mutations that disrupt the DNA-binding activity or transcriptional activity of AML1 (Song et al., 1999). Acquired dominant negative mutations of C/EBP have been reported, and may occur in up to 17% of M2 AML with a normal karyotype (Pabst et al., 2001b). Gene expression can also be extinguished by epigenetic mechanisms, such as promoter hypermethylation, which may be important in the progression of myeloid leukemias.
Leukemia-associated translocations: the paradigm of fusion proteins
The most common consequence of the myeloid leukemia-associated translocations is the generation of a chimeric gene that codes for a novel fusion protein. Characterization of the fusion proteins involved in leukemogenesis has proceeded rapidly, revealing their effects on hematopoietic progenitor cell behavior, and identifying transcriptionally-regulated target genes and interacting proteins. The familiar concepts of proto-oncogene activation and tumor suppressor inactivation can be blurred by these fusion proteins, because they may possess unique capabilities not shared by either of the fusion partners. Such capabilities can uniquely contribute to leukemogenesis although, in most cases, other abnormalities, or 'second hits', appear to be necessary for the development of leukemia.
Mechanism of leukemia-associated fusion proteins
Translocations that disrupt the function of a small number of genes account for a large percentage of the cytogenetic abnormalities in AML. The translocations t(8;21), inv(16) and t(16;16), which together account for ~25% of AML (Langabeer et al., 1997a,b), each affect the function of the heterodimeric transcription factor, core binding factor (CBF). CBF is composed of two subunits: AML1 (also known as RUNX1, CBF2, and PEBP2B), which is encoded by a gene located at 21q22, is directly involved in the translocations t(8;21), t(3;21), t(16;21) and t(12;21) (found in ALL); and, CBF, which is encoded by a gene at 16q22, and is fused to the MYH11 gene (Liu et al., 1993) in the inv(16) and the t(16;16). APL accounts for ~10% of AML (Grimwade et al., 2001) and is always associated with a translocation involving the RAR gene located at 17q12. In 99% of cases, the translocation is t(15;17)(q22;q12) which fuses RAR to PML, generating a PML/RAR fusion protein. In very rare cases, APL is associated with the t(11;17)(q23;q12), t(5;17)(q35;q12), t(17;17)(q11;q12) or t(11;17)(q13;q12) translocations, resulting in the fusion of RAR to promyelocytic leukemia zinc finger (PLZF), nucleophosmin (NPM), Stat5b or NuMA genes, respectively (Arnould et al., 1999; Chen et al., 1993; Redner et al., 1996; Wells et al., 1996).
Though less common than the CBF and RAR fusions, several other genes are recurrently involved in leukemia-associated translocations. Fusions involving the MLL gene result from 11q23 abnormalities and, though common in infant leukemias, occur in less than 3% of adult AML (Grimwade et al., 2001). NUP98, TEL and CBP/p300 gene rearrangements are even less frequent in AML (Grimwade et al., 2001; Mitelman et al., 2002; Mrozek et al., 2001). Nonetheless, the leukemia-associated fusion proteins share structural and functional similarities, suggesting that they may impart a leukemic phenotype through common modes of transcriptional dysregulation. The fusion proteins generated by these translocation usually contain a DNA-binding domain, domains responsible for homo- and hetero-dimerization, regions that target the proteins to distinct subnuclear locations and domains that interact with proteins involved in chromatin remodeling (e.g., corepressor molecules) or that themselves impact on chromatin structure (e.g. have histone acetyltransferase activity). It is these shared features that constitute the 'variations on the theme' of aberrant growth and differentiation of acute leukemia cells.
CBF leukemias (AML1 and CBF)
The AML1 (RUNX1) protein binds to the 'core' enhancer motif, TG(T/c)GGT, found in various viral and cellular enhancer and promoter regions, with CBF, its non-DNA-binding heterodimerization partner (Meyers et al., 1993). The normal functions of both AML1 and CBF are critical to hematopoietic development as mice lacking either gene die in utero and fail to develop definitive hematopoieis (Okuda et al., 1996; Wang et al., 1996a,b). Several isoforms of AML1 have been described but it appears that the 453 and 479 amino acid isoforms of AML1, AML1b and AML1B (or AML1c), are of greatest significance in hematopoiesis. AML1B contains a central runt homology domain (RHD) that is required for DNA binding and for the interaction of AML1 with CBF, ETS proteins (MEF, PU.1, ETS-1, ELF-1), Smads, and other transcription factors (such as C/EBP) (Jakubowiak et al., 2000; Kim et al., 1999a,b; Mao et al., 1999; Pardali et al., 2000; Petrovick et al., 1998; Uchida et al., 1997; Wotton et al., 1994; Xie et al., 1999; Zhang et al., 1996b; Zhang and Derynck, 2000). Other regions of AML1B have been shown to be important for its interactions with chromatin remodeling factors, such as the co-activator molecules CBP/p300 and ALY (Bruhn et al., 1997; Kitabayashi et al., 1998b), the co-repressors mSin3 and N-CoR (Amann et al., 2001; Lutterbach et al., 1998b; Wang et al., 1998a), and with other nuclear proteins such as YAP, LEF-1, Ear-2, HES-1 (Ahn et al., 1998; Chen et al., 1997; Giese et al., 1995; McLarren et al., 2000, 2001; Yagi et al., 1999) (see Figure 1). A C-terminal VWRPY motif, found in all members of the Runt-related (RUNX) family of transcription factors (Aronson et al., 1997), interacts with mammalian homologues of the Drosophila Groucho repressor proteins, including TLE1 and TLE2 (Imai et al., 1998). The N-terminal 40 amino acids in AML1B inhibit its in vitro binding to DNA, possibly via intramolecular interactions (Uchida et al., 1997).
The AML1 nuclear-localization signal (NLS) is located within the RHD and it serves to bring CBF into the nucleus with AML1 (Lu et al., 1995). AML1B has a C-terminal context-dependent transactivation domain and a C-terminal nuclear matrix targeting signal (Zeng et al., 1997, 1998). AML1B colocalizes within the nuclear matrix with a subset of hyperphosphorylated RNA polymerase II. This subnuclear location appears to be important for transactivation by AML1B (Zeng et al., 1998). In most cases, AML1 functions as a transcriptional activator, yet AML1 can down-regulate p21WAF/CIP1 promoter activity in NIH3T3 cells and it does interact with co-repressor molecules such as mSin3 (Lutterbach et al., 1998b, 2000). Given the large number of nuclear proteins that demonstrate functional and cooperative interactions with AML1, it is possible that AML1 acts as a central regulatory platform that coordinates the interactions between enhancer-binding transcription factors, components of the basic transcriptional apparatus and proteins involved in chromatin remodeling.
The t(8;21) translocation generates a chimeric protein that contains the N-terminal portion of AML1B (truncated just after the RHD) and almost the entire ETO protein (Erickson et al., 1992; Miyoshi et al., 1993). ETO (Eight Twenty One) is a member of a family of nuclear proteins that bear homology to the Drosophila protein, nervy (Feinstein et al., 1995). These proteins contain four regions of significant homology, referred to as 'nervy-homology regions' (NHR) 1, 2, 3 and 4 (see Figure 1). ETO does not bind DNA itself but appears to be involved in the regulation of transcription. The NHR1 bears some homology to the TATA-binding associated factor, TAF110 (Erickson et al., 1994), and it contains a region necessary for the nuclear import and subnuclear localization of ETO (Odaka et al., 2000); its function is otherwise unclear. NHR2 contains a HHR motif (i.e., hydrophobic heptad repeat) that mediates the formation of stable ETO : ETO dimers (Lutterbach et al., 1998a). This region is also involved in AML1/ETO homodimerization and its heterodimerization with ETO, and MTGR1 (and possibly MTG16) (Kitabayashi et al., 1998a; Lutterbach et al., 1998a). The precise partners of AML1/ETO in AML cells is not known but it is likely that the interactions of AML1/ETO with itself or with other ETO-like proteins helps regulate its function in the cell. The function of NHR3 is poorly defined whereas NHR4 contains a zinc-finger motif referred to as the MYND domain (Myeloid Nervy and Deaf-1) that together with the HHR motif is involved in the recruitment of transcriptional corepressor proteins. The MYND domain interacts with the nuclear corepressor (NCoR) and silencing mediator of retinoid and thyroid (SMRT) proteins (Gelmetti et al., 1998; Lutterbach et al., 1998a; Wang et al., 1998a). ETO also interacts directly and tightly with mSin3A and can directly interact with the class 1 histone deacetylases (HDACs), HDAC-1, HDAC-2 and HDAC-3 (Amann et al., 2001; Hildebrand et al., 2001). Little is known about the normal functions of ETO and ETO 'knock out' mice do not appear to have hematopoietic defects (Calebi et al., 2001). Nonetheless a ETO has been shown to interact with PLZF and to enhance the ability of PLZF to repress transcription (Melnick et al., 2000b).
The AML1/ETO fusion protein can dominantly inhibit the normal function of AML1B both in vitro and in vivo, and this effect is believed to result from the interaction of AML1/ETO with corepressor molecules, such as N-CoR, SMRT, and mSin3. The multiple domains of AML1/ETO that can interact with co-repressors may lead to tighter and perhaps 'constitutive' binding of these molecules to the fusion protein. Additionally, the truncated portion of AML1 in the AML1/ETO fusion protein, lacks the transcriptional activating domain and the p300/CBP interacting domain of AML1B and is therefore severely limited in its capacity to activate transcription. In vivo evidence of the dominant negative activity of AML1/ETO is revealed by the nearly identical phenotype of mice with AML1/ETO 'knocked-into' the AML1 locus and mice with both AML1 alleles 'knocked-out' (Okuda et al., 1996, 1998).
Expression of AML1/ETO has also been shown to up-regulate several genes. AML1/ETO can transactivate BCL-2 and AP-1 reporter constructs (Frank et al., 1999; Klampfer et al., 1996) and can up-regulate the ubiquitin-specific protease, UBP43 (Liu et al., 1999). AML1 and AML1/ETO interact synergistically to up-regulate the M-CSF receptor (Rhoades et al., 1996). The mechanism by which AML1/ETO brings about the transactivation of these genes has not been thoroughly explored and may be indirect; yet, the loss of C-terminal AML1B repressor 'domains' in the fusion protein implies that the mechanism could also be direct. Further, the AML1 portion of the fusion protein may contribute to gene upregulation by altering the activity of the ETO portion. In fact, although ETO augments transcriptional repression by PLZF, AML1/ETO impairs its repression (Melnick et al., 2000a,b).
In t(12;21)-positive ALL, almost the entire AML1B protein is fused to the N-terminus of TEL. TEL is a transcriptional repressor (Chakrabarti and Nucifora, 1999; Kwiatkowski et al., 1998) and it is required for hematopoiesis within the bone marrow compartment (Wang et al., 1997, 1998b). TEL binds DNA through an ETS domain, however, this portion of TEL is deleted in the fusion protein. Instead, TEL/AML1 binds DNA via the RHD contained in its AML1 portion, redirecting the repressor functions of TEL to AML1 targets. Similar to ETO, TEL provides TEL/AML1 with a dimerization motif that is required for repression of target genes (Hiebert et al., 1996). This region of TEL also directly binds corepressor molecules (Wang and Hiebert, 2001) and like AML1/ETO, the TEL/AML1 fusion protein has been shown to repress AML1B target genes (Fenrick et al., 1999; Hiebert et al., 1996; Uchida et al., 1999). The remaining TEL allele is nearly always deleted in t(12;21)-positive ALL (Golub et al., 1995; Sato et al., 1995) suggesting that complete loss of TEL function is more advantageous than its incomplete loss or that conditions favoring homodimerization of TEL/AML1 over its heterodimerization with TEL are more beneficial to the growth or development of leukemia (McLean et al., 1996). It is not clear whether such loss of heterozygosity is a general phenomenon in CBF leukemias as the status of the remaining alleles of other translocated genes (e.g., the untranslocated ETO allele in t(8;21)-positive AML) have not been thoroughly characterized. Fusion of TEL to AML1 localizes the TEL/AML1 fusion protein to TEL bodies, which are cell cycle specific nuclear speckles (Chakrabarti et al., 2000). This location is distinct from the location of wild-type AML1.
Inv(16) and t(16;16) fuse the CBF gene to the MYH11 gene which encodes the smooth muscle myosin heavy chain (SMMHC) (Liu et al., 1993), generating a CBF/SMMHC fusion protein. Although the SMMHC protein is not known to play a role in normal hematopoiesis, the CBF/SMMHC fusion protein resembles several of the other leukemia-associated fusion proteins. CBF/SMMHC binds AML1 and, through this interaction, may itself make contact with DNA (Liu et al., 1996b; Lutterbach et al., 1999). A C-terminal coiled-coil motif in SMMHC provides a motif for CBF/SMMHC to form dimers and perhaps higher order oligomers (Cao et al., 1998; Liu et al., 1993). Like the AML1 fusion proteins, CBF/SMMHC acts as a repressor of AML1 function. The SMMHC portion of CBF/SMMHC contains a cryptic repressor domain that can directly repress the activity of AML1 when bound to DNA at transcriptionally-active sites (Kanno et al., 1998; Lutterbach et al., 1999). As for AML1/ETO, TEL/AML1 and PML/RAR, deletion of the dimerization motif in CBF/SMMHC abrogates its repressor function (Cao et al., 1998). However, CBF/SMMHC may also function by sequestering AML1 from such transcriptionally-active sites (Adya et al., 1998; Kanno et al., 1998). Nonetheless, the effects of CBF/SMMHC on the available pool of AML1 protein and on its cellular localization are not resolved; how much of the fusion protein is nuclear vs cytoplasmic and other vital issues have not been fully defined.
APL-related fusion proteins
RAR is a nuclear hormone receptor that confers the transcriptional response to retinoic acid (reviewed in Chambon (1996)). RAR binds to specific DNA sequences referred to as retinoic acid responsive elements (RARE) [a PuGGTCA sequence spaced by five nucleotides (DR5)], in a heterodimeric complex with retinoid-X receptors (RXR). RARs have a common domain structure consisting of an N-terminal DNA-binding domain, a linker domain and a C-terminal ligand-binding (i.e., retinoic acid binding) domain (see Figure 1). In the absence of retinoic acid (RA), RAR/RXR complexes repress transcription through interactions with NCoR, SMRT, mSin3A and mSin3B and the recruitment of HDACs (reviewed in Grunstein (1997)). Normally, physiologic concentrations of RA induce a conformational change in the RARs causing release of these corepressor molecules and recruitment of transcriptional coactivator molecules, leading to activation of gene transcription.
Translocations involving the RAR gene on chromosome 17q12 are invariably associated with APL. The common t(15;17) translocation gives rise to the PML/RAR fusion protein and, in a significant subset of patients, the reciprocal RAR/PML transcript is also present (reviewed in Alcalay et al. (2001)). This contrasts with the CBF leukemias where reciprocal fusion proteins involving the AML1 and CBF gene are not found.
The PML/RAR fusion protein contains most of the RAR protein, including its DNA- and ligand-binding domains, fused to almost the entire PML protein. PML is normally concentrated in subnuclear 'PODs' or 'nuclear bodies' which are complex macromolecular structures that contain numerous other nuclear proteins (Dyck et al., 1994; Koken et al., 1994; Weis et al., 1994; Zhong et al., 2000b).
Both the ring finger and coiled-coil moieties of PML are required for its localization in these nuclear bodies and for its interaction with nuclear proteins within these structures such as p53, CBP, Daxx, pRb, sp100, sp140 and perhaps ETO (reviewed in Zhong et al. (2000b)). Like TEL (and Ran Gap1) (Chakrabarti et al., 2000), PML is post-translationally modified by SUMO-1, an ubiquitin-like molecule. Normally, the coiled-coil domain of PML mediates homodimer formation, but in the fusion protein, the presence of this domain allows for heterodimer formation with PML/RAR and for the homodimerization of PML/RAR (Lin and Evans, 2000; Minucci et al., 2000). Expression of PML/RAR causes delocalization of not only the normal PML protein, but of other nuclear body components as well (Zhong et al., 2000a). Similarly, the subnuclear localization of AML1 is redirected within the nuclear matrix, by its fusion to ETO in the AML1/ETO chimeric protein (McNeil et al., 1999; Minucci et al., 2000; Odaka et al., 2000). The expression of AML1/ETO also causes reorganization of PML nuclear bodies (McNeil et al., 2000) but the significance of this finding has not been thoroughly elucidated.
The PML/RAR fusion protein binds to RAREs (Lin and Evans, 2000), to RXR and to RA with the same affinity as the native RAR (reviewed in He et al. (1999); Melnick and Licht (1999)). Like RAR, PML/RAR binds corepressor molecules and, in the absence of RA, represses transcription of RA-responsive genes. However, unlike RAR, physiologic concentrations of RA are not sufficient to release corepressor molecules from PML/RAR, and repression of RA-responsive promoters is maintained (Grignani et al., 1998; He et al., 1998; Lin et al., 1998). Nonetheless, pharmacologic concentrations of RA, such as can be achieved with the administration of ATRA, can convert PML/RAR from a repressor of RA target genes to an activator (Grignani et al., 1998; He et al., 1998; Lin et al., 1998). This likely explains why APL is uniquely sensitive to treatment with retinoic acid (Soignet et al., 1997; Warrell, 1996). Whether the efficacy is wholly due to a restored RA response is not clear, as ATRA treatment also restores the normal subnuclear localization of PML, and other nuclear body proteins as well (Dyck et al., 1994; Faretta et al., 2001; Koken et al., 1994; Weis et al., 1994).
The ability of the PML coiled-coiled domain in the PML/RAR fusion protein to induce dimerization is critical to its ability to negatively regulate RAR-mediated transcription (Lin and Evans, 2000; Minucci et al., 2000). Loss of a normal PML allele increases the leukemia potential of PML/RAR in transgenic mice, as does loss of the normal RAR allele. This suggests that homodimerization of the fusion proteins may be important in the generation or maintenance of the leukemic phenotype, and also that the fusion protein functions as a more effective dominant negative on PML and RAR function when there are fewer normal copies of these genes.
The t(11;17) translocation generates a PLZF/RAR fusion protein, and a reciprocal RAR/PLZF protein (Chen et al., 1993); both fusion proteins can suppress RAR and PLZF signaling (He et al., 2000). Like TEL ( and to some extent ETO), PLZF is a transcriptional repressor that provides the fusion protein with a second binding domain for corepressor molecules (Grignani et al., 1998; He et al., 1998; Lin et al., 1998; Ruthardt et al., 1997). The interaction between the PLZF portion of the fusion protein and corepressor molecules, is unaffected by even pharmacologic doses of RA (Grignani et al., 1998; He et al., 1998; Lin et al., 1998). Thus, neither patients harboring the t(11;17) translocation, nor PLZF/RAR transgenic mice respond to treatment with ATRA (He et al., 1997; Licht et al., 1995).
The coiled-coil region of PML, the POZ domain of PLZF and the NHR2 region of ETO are responsible for the oligomerization and subnuclear localization of the PML/RAR, PLZF/RAR and AML1/ETO fusion proteins, respectively (Dong et al., 1996; Lin and Evans, 2000; Lutterbach et al., 1998a; Minucci et al., 2000). These domains also bring about delocalization of the native, untranslocated RAR, PML, PLZF and AML1 proteins (reviewed in Stein et al. (2000)). Furthermore, oligomerization is believed to enhance the recruitment of corepressor molecules, like NCoR, thereby augmenting the transcriptional repression mediated by these fusion proteins (Lin and Evans, 2000). Supporting this assertion is the demonstration that an heterologous dimerization domain can substitute for PML when fused to RAR (Minucci et al., 2000).
11q23 Leukemias (MLL)
The MLL gene is very large, spanning 90 kb and containing 36 exons. It codes for a 3969 amino acid protein (431 kDa) that is the human homolog of the Drosophila trithorax gene (TRX), a positive regulator of homeobox gene expression (Breen and Harte, 1991; Rasio et al., 1996). The N-terminal portion of MLL contains an 'AT hooks' region that is believed to bind to the minor groove of AT-rich DNA segments (see Figure 1). This DNA-binding motif is retained in each of the MLL fusion proteins identified and is similar to that found in the HMG-I(Y) family of high mobility group (HMG) proteins. Proteins containing AT-hooks help regulate transcription by inducing changes in DNA conformation that permit the association of transcription factors with regulatory regions of DNA (reviewed in Aravind and Landsman (1998) and Jones and Kadonaga (2000)). Unlike the sequence-specific DNA-binding domains found in other leukemia-associated fusion proteins, the AT-hooks in MLL appear to recognize specific DNA structures rather than sequence (Broeker et al., 1996; Zeleznik-Le et al., 1994). The N-terminal portion of MLL also contains a region homologous to the regulatory, but not catalytic, domain of a DNA methyltransferase (Ma et al., 1993) and is capable of repressing transcription from reporter constructs (Broeker et al., 1996; Prasad et al., 1995). MLL contains four centrally-located PHD (plant homeodomain) zinc-fingers that mediate its homodimerization and its interactions with other nuclear proteins (Fair et al., 2001). Between the third and fourth PHD-fingers is an atypical bromo domain of unknown function (Kouzarides, 2000) and following the PHD-fingers is a region that contains transcriptional activating activity (Broeker et al., 1996; Zeleznik-Le et al., 1994). The C-terminal portion of MLL, known as the SET domain, mediates homodimerization of the native MLL protein (Rozovskaia et al., 2000) and allows MLL to interact with components of the mammalian SWI/SNF complex (e.g., INI1/hSNF5) which are involved in ATP-dependent chromatin remodeling (Rozenblatt-Rosen et al., 1998).
The MLL gene is involved in more than 40 leukemia-associated translocations and over 20 of its fusion partners have been identified. The most common translocations are t(9;11)(p22;q23), t(4;11)(q21;q23) (associated with infant ALL), t(11;19)(q23;p13.3), t(11;19)(q23;p13.1), t(6;11)(q27;q23), t(11;16)(q23;p13), and t(11;22)(q23;q13) giving rise to the MLL/AF9, MLL/AF4, MLL/ENL (or MLL/EEN), MLL/ELL, MLL/AF6, MLL/CBP, and MLL/p300 fusion proteins, respectively.
All translocations involving the MLL gene result in fusion transcripts that code for the N-terminal AT-hooks and methyltransferase domain of MLL fused in frame to a C-terminal portion of its translocation partner. Thus, the DNA-binding region and repressor domain of MLL is transferred to the partner protein whereas the regions capable of activating transcription are lost. Both the PHD fingers and SET domain, which mediate MLL homodimerization, are also deleted from the fusion proteins. MLL is a nuclear protein that has been reported to partition to an aberrant subnuclear location when fused to a translocation partner protein (Joh et al., 1996; Rogaia et al., 1997; Yano et al., 1997). Thus, several of the structural themes evident in the other leukemia-associated fusion proteins re-emerge in the MLL fusions.
The C-terminal portion of the MLL fusion partners ENL, AF9, and AF4, contains a transcriptional activating domain retained in the MLL fusion protein (Prasad et al., 1995; Rubnitz et al., 1994). When fused to the MLL fragment, the transactivation domain of ENL is both necessary and sufficient for the ability of MLL/ENL to immortalize murine myeloid cells in vitro (Lavau et al., 1997; Slany et al., 1998). The presence of a transcriptional activation domain in several of the MLL fusion proteins suggests that this activity may be important to the leukemic phenotype associated with the fusion proteins, through direct or indirect mechanisms (Luo et al., 2001). Consistent with this theory are the fusions of MLL with p300 and CBP; the coactivator and histone acetyltransferase (HAT) activities of these MLL partners are retained in the fusion proteins. The role that dimer formation plays in the activity of the MLL fusion proteins is currently less clear than with other transcription factor fusion proteins.
Nucleoporin protein fusions
Macromolecules are transported in and out of the nucleus through nuclear pore complexes (NPCs) that contain nucleoporins, and at least three groups of soluble factors; karyopherins, Ran, and Ran-interacting proteins. Karyopherins recognize the nuclear localization or nuclear export sequences on macromolecules (such as transcription factor proteins). The karyopherin-TF complex moves through the NPC to its destination inside the nucleus and this movement requires the interaction of karyopherins with nucleoporins (Radu et al., 1995; Yaseen and Blobel, 1997), the GTPase activity of the Ran protein, and several Ran-interacting proteins (such as RanGAP or p10).
Nucleoporins (Nup) are the target of at least 12 chromosomal rearrangements in leukemia (mostly t-AML) and myelodysplastic syndrome (MDS) (Table 2). Ten of these rearrangements involve the nucleoporin, NUP98 (Borrow et al., 1996; Nakamura et al., 1996), and two involve NUP214 (also known as CAN) (Fornerod et al., 1995; Kraemer et al., 1994). In the majority of cases, the NUP98 gene (located on chromosome 11p15) is fused to a homeobox gene such as HOXA9 on chromosome 7p15, PMX1 on 1q24, or HOXD13 on 2q31 (Borrow et al., 1996; Nakamura et al., 1996, 1999; Nishiyama et al., 1999; Raza-Egilmez et al., 1998). Other NUP98 fusion partners include DDX10 (a putative RNA helicase), DNA topoisomerase I (TOP1), and the transcriptional coactivators p52 and p75-lens epithelium-derived growth factor (LEDGF) (Ahuja et al., 1999, 2000; Arai et al., 1997). In all of the NUP98 fusion proteins, the N-terminal portion of NUP98 is fused to the C-terminal portion of its partner (see Figure 1). The normal NUP98 gene generates an mRNA that encodes a precursor protein that is proteolytically cleaved to yield two nucleoporins, NUP98 (from its N-terminal portion) and NUP96 (from its C-terminal portion) (Fontoura et al., 1999). All of the fusion transcripts that involve NUP98 lack the portion encoding NUP96, but how this affects the expression and subcellular localization of NUP96, or the function of the NPC, is not known.
The nucleoporin containing fusion proteins all contain the nucleoporin FG repeats that act as docking sites for karyopherins during nuclear transport (Radu et al., 1995). This suggests that the nucleoporin fusions contribute to leukemogenesis because they alter nuclear transport. NUP98 is normally located at the nuclear side of the nuclear pore complex, whereas several of the chimeric nucleoporins, including NUP98/HOXA9, have been localized to the inside of the nucleus instead of the normal nucleoporin location at the nuclear rim (Fornerod et al., 1995; Kasper et al., 1994, 1999). Its presence in the nucleus appears to be required for its transforming ability, suggesting that the aberrant localization of NUP98 fusions can lead to their inappropriate interactions with key transcriptional regulatory proteins required for myeloid differentiation.
Hox gene dysregulation appears to be a recurrent theme in leukemia pathogenesis (Look, 1997; Thorsteinsdottir et al., 2001) and overexpression of Hoxa9 in murine bone marrow cells induces AML after a latency period (Kroon et al., 1998) and expression of NUP98/HOXA9, in hematopoietic stem cells has been shown to cause leukemia in mice (Kroon et al., 2001). The nucleoporin fusion proteins likely affect the subcellular localization and the regulatory activity of homeobox genes. Thus, the nucleoporin fusion proteins act as aberrant transcription factors and also bind to soluble transport factors. It is not yet known how NUP98/HOXA9 is imported into the nucleus and how it interferes with the nucleocytoplasmic distribution of transcription factors important for myeloid differentiation. There is evidence that the FG repeat region of NUP98, retained in the NUP98/HOXA9 fusion, interacts with the transcriptional co-activators CBP and p300 (Kasper et al., 1999). If NUP98/HOXA9 causes leukemic transformation by interfering with myeloid differentiation, its nuclear location suggests that this may occur at the transcriptional level.
Effects on proliferation
It is likely that the genesis of acute leukemia requires mutations that promote the proliferation, and prevent the normal apoptosis, of hematopoietic progenitor cells. Pathways associated with proliferation, cell death and differentiation vs self renewal are often affected in cancer (for review see Reya et al. (2001)); thus, it is not surprising that the transcription factors involved in leukemogenesis interact directly or indirectly with these pathways. Nonetheless, the effects of leukemia-associated fusion proteins on apoptosis and cell cycle have not been easily predictable, nor ones that would clearly promote leukemogenesis. Defining the cell type and context-specific effects of the leukemia-associated fusion proteins is key to understanding their relevant effects on the growth of hematopoietic cells.
Effects on apoptosis
Like true oncogenes, one striking feature of many of these proteins is that, although they are expressed in the leukemia cell, their expression is often poorly, or not, tolerated when expressed in other cell types. The U937 cell line has been used to study many of these fusion proteins, because it tolerates their presence, but why this cell line behaves differently that others is not clear. Thus, studies in vitro do not always agree with studies conducted using leukemic blasts obtained from patients with AML. For example, we reported that the Kasumi-1 and SKNO-1 cell lines, derived from patients with t(8;21) AML and expressing high levels of AML1/ETO, express high levels of the anti-apoptotic protein BCL2 (Klampfer et al., 1996). AML1/ETO increased BCL2 promoter activity in a cell line-dependent fashion (in U937 cells) (Klampfer et al., 1996) and decreased transcription from BAX (a pro-apoptotic protein) reporter constructs in several cell lines (unpublished data). Similarly, BCL2 levels were found to be up-regulated when AML1/ETO was expressed in the murine myeloid precursor cell line, 32Dcl3 (Kohzaki et al., 1999). However, when AML1/ETO expression was driven from a tetracycline-inducible promoter in an engineered U937 cell line, upregulation of AML1/ETO induced apoptosis and downregulation of BCL2 expression (Burel et al., 2001). Yet, others have found no effect of AML1/ETO on BCL-2 levels using an identical engineered cell line strategy (M Lubbert, personal communication), and we found a transient increase in BCL-2 levels using an independently generated tet-off U937 inducible system. When myeloblasts obtained from adult patients with t(8;21) AML were studied low levels of BCL2 were found (Banker et al., 1998; Shikami et al., 1999), whereas relatively high levels of BCL2 were seen in t(8;21) samples from children (Banker et al., 1998). This may correlate with the prognosis of t(8;21) AML, which is much better in adults than in children.
A consistent story has emerged from studies of the APL-associated fusion partners in transgenic mice (recently reviewed in Pandolfi (2001)). PML appears to play an important role in apoptosis, as bone marrow progenitors from PML-/- mice are refractory to multiple apoptotic signals such as Fas, TNF, and interferons (Wang et al., 1998d). Within the nuclear bodies, PML colocalizes and physically interacts with p53 and Daxx, both mediators of apoptotic stimuli. PML acts as a transcriptional co-activator with p53 and can act as a positive regulator of p53 dependent apoptosis (Guo et al., 2000). Similarly, FAS-mediated apoptosis is abrogated when the interaction between PML and Daxx is disrupted (Zhong et al., 2000c). PML/RAR disrupts nuclear body structure and is a dominant-negative inhibitor of PML function, thus it is not surprising that hematopoietic progenitors from PML/RAR transgenic mice are resistant to both p53-dependent and p53-independent apoptotic stimuli (Wang et al., 1998d). Interestingly, p53 mutations are rare in APL, suggesting that inactivation of this pathway by the PML/RAR fusion protein obviates the need for inactivation of p53 by mutation (Longo et al., 1993). Context-dependent effects of the expression of this fusion protein (reviewed in He et al. (1999)) are also found: whereas expression of PML/RAR in U937 cells makes them resistant to apoptotic stimuli (Grignani et al., 1993), the expression of PML/RAR in several other hematopoietic and non-hematopoietic cell lines induces apoptosis and arrest growth (Ferrucci et al., 1997).
PLZF, which is also found in the nuclear bodies, also appears to facilitate pro-apoptotic signals; the skeletal abnormalities found in PLZF-/- mice are believed to result from the loss of the pro-apoptotic and growth inhibitory effects of PLZF in the limb bud (Barna et al., 2000). Consistent with these observations, overexpression of PLZF in 32Dcl3 cells causes increased apoptosis and G1 cell cycle arrest (Shaknovich et al., 1998).
Though not as well characterized, MLL and its fusion proteins have been shown to interact with mediators of apoptosis. Overexpressed ELL binds to p53 and can disrupt its ability to regulate transcription, suggesting that the MLL/ELL fusion could have anti-apoptotic effects (Shinobu et al., 1999). Consistent with this, p53 mutations do not appear to be important for the development of 11q23-leukemias (Megonigal et al., 1998). MLL has been shown to interact with GADD34 (a protein up-regulated by DNA damage) and augment apoptosis associated its expression whereas, MLL/ENL, MLL/AF9 and MLL/ELL all inhibit GADD34-induced apoptosis. This suggests that the MLL fusion proteins can also interfere with p53-independent apoptosis (Adler et al., 1999).
Effects on cell cycle
Overexpression of AML1 in 32Dcl3 cells can promote cell-cycle progression (Strom et al., 2000) by shortening G1 and it can repress p21WAF/CIP1 promoter activity in NIH3T3 cells (Lutterbach et al., 2000). In contrast, AML1/ETO slows cell cycle progression through G1 in U937 cells (Burel et al., 2001) and up-regulates p21WAF/CIP1 mRNA levels in human CD34+ cells (our unpublished observations). It is by no means clear that the effects of the wild-type and chimeric AML1 proteins on the cell cycle are related to their regulation of p21WAF/CIP1 expression. For instance, expression of CBF/SMMHC in 32Dcl3 cells slows cell growth and increases the proportion of cells in G1 (Cao et al., 1997); yet, CBF/SMMHC has also been shown to enhance repression of the p21WAF/CIP1 promoter by AML1B (Lutterbach et al., 1999). Thus, increases in p21WAF/CIP1 activity may not be required for slowing of the cell cycle by the 'CBF'-fusion proteins. In an attempt to define the effects that an exogenous repression domain has when directed to core binding factor DNA binding sites, a tamoxifen-sensitive, KRAB-AML1-ER fusion protein was constructed and shown to slow cell growth and prolong the G1 phase of the cell cycle upon activation of the KRAB repression domain (Lou et al., 2000). Using representational differential analysis (RDA), the authors found that activation of the repression domain was associated with down-regulated CDK4. Similarly, CDK4 and c-myc are down-regulated by expression of AML1/ETO, yet enforced expression of these proteins cannot prevent AML1/ETO-induced cell cycle arrest (Burel et al., 2001).
APL-associated fusion partners also affect the cell cycle machinery. PML/RAR up-regulates cyclin-A1 expression (Muller et al., 2000); a finding consistent with the high levels of cyclin-A1 that have been found in samples from patients with APL (Yang et al., 1999). PML appears to be a negative regulator of cell growth as PML-/- mouse embryo fibroblasts (mefs) proliferate faster than wild-type cells and are able to form colonies in soft agar (Wang et al., 1998c). Conversely, overexpression of PML in a variety of cells leads to growth suppression and G1 arrest (Le et al., 1998; Mu et al., 1997). Interestingly, PML seems to be crucial for the growth-inhibitory activity of RA, and its absence abrogates the RA-dependent transactivation of p21WAF/CIP1 (Liu et al., 1996a).
It is likely that through alterations in cell cycle regulation the self-renewal and differentiation of hematopoietic progenitors is perturbed. Clearly, AML1/ETO expression alters the behavior of murine and human hematopoietic progenitor cells. Hematopoietic stem cells derived from AML1/ETO knock-in mice demonstrate increased self-renewal and readily give rise to immortalized cell lines in vitro (Okuda et al., 1998). Similarly, the expression of AML1/ETO in hematopoietic cells obtained from a tet-inducible transgenic mouse, causes a dramatic increase in their efficiency of serial replating in methylcellulose cultures (Rhoades et al., 2000). Differentiation also appears to be intimately related to cell cycle regulation as C/EBP, a lineage-restricted transcription factor that is down-regulated by AML1/ETO (Burel et al., 2001; Pabst et al., 2001a; Westendorf et al., 1998), has recently been shown to repress E2F activity and promote cell cycle arrest (Johansen et al., 2001; Porse et al., 2001; Slomiany et al., 2000). Notably, it was shown that a C/EBP mutation similar to that found in some AML patients (Pabst et al., 2001b) does not repress E2F and mice, homozygous for these mutated C/EBP alleles that have defects in granulocytic differentiation (Porse et al., 2001).
A hallmark of acute myelogenous leukemia is the aberrant differentiation with the accumulation of blasts resembling a specific level of granulocytic maturation (the myeloblast). CBF/MYH11 expressing cells from chimeric mice show a block in granulocytic differentiation and, after a long latency and with low penetrance, developed myelomonocytic leukemia resembling the human disease (Castilla et al., 1999). Expression of AML1/ETO in chimeric mice or by retroviral transduction of murine bone marrow, led to the establishment of immortal cell lines with a multi-lineage potential (Okuda et al., 1998; Yergeau et al., 1997), and though slight phenotypic differences were observed in liver-derived compared to bone marrow-derived progenitors in the AML1/ETO chimeric mice, no block in terminal differentiation was observed (Okuda et al., 1998). Cell lines have been used to show a negative effect of AML1/ETO on differentiation. U937 cells differentiate towards mature granulocytes following treatment with vitamin D3 and TGF and this is blocked by the expression of AML1/ETO (Gelmetti et al., 1998). We have recently shown that AML1/ETO interferes with the TGF signaling pathway, which may explain this effect (Jakubowiak et al., 2000). TGF signaling is also affected by the AML1/EVII fusion protein, arguing that the disruption of this pathway could be a common mechanism in leukemia (Kurokawa et al., 1998a). Other groups have demonstrated that the differentiation of 32Dcl3 cells (Kohzaki et al., 1999), and of the L-G cell line (Kitabayashi et al., 1998b) are blocked by expression of AML1/ETO. Recently, a transgenic mouse model was generated that expresses AML1/ETO from the human MRP8 promoter (Yuan et al., 2001). These mice develop AML after treatment with DNA damaging agents and the leukemic cells resemble immature granulocytes suggesting their arrested differentiation. Additionally, retrovirally transduced human progenitor cells expressing PML/RAR demonstrate an arrest in maturation with an accumulation of promyelocytes resembling the phenotype observed in human APL (Grignani et al., 2000). When murine hematopoietic progenitors are transduced with NUP98/HOXA9 and transplanted into syngeneic mice they induce a myeloproliferative disease and eventually an AML-like disease with a myelomonocytic or monocytic phenotype (Kroon et al., 2001).
Insensitivity to antiproliferative signals
In addition to their effects on the expression of cell cycle regulatory proteins, or proteins that regulate apoptosis, there are now several indications that the leukemia-associated fusion proteins frequently disrupt normal anti-growth signals received by the cell. Dysregulated TGF signal transduction is an important, and common, property of malignant cells (Chen et al., 1998; Massague et al., 2000). Whereas primary cultures of normal cells are highly sensitive to growth-inhibition by TGF, most malignant cells are resistant to this effect (Fortunel et al., 2000; Fynan and Reiss, 1993). Disruption of TGF signaling in solid tumors is not complete: pro-oncogenic effects of TGF, such as the stimulation of invasion and angiogenesis, may be preserved despite the disruption of growth inhibition (Engel et al., 1998). Smad proteins, the intracellular mediators of TGF signaling, function as tumor suppressors and inactivating mutations in Smads are found in many solid tumors (Massague et al., 2000; Xu and Attisano, 2000). Recently inactivating mutations have been identified in rare patients with AML (Imai et al., 2001).
TGF is known to maintain early hematopoietic progenitor cells in a quiescent, undifferentiated state (Batard et al., 2000; Pierelli et al., 2000; Van Ranst et al., 1996). This effect is at least partially due to its induction of the CDK inhibitors p21WAF/CIP1 (Ducos et al., 2000) and p27KIP1 (Mahmud et al., 1999) and possibly due to decreased expression of cytokine receptors on the cell surface (Dubois et al., 1994; Jacobsen et al., 1991, 1993). Early hematopoietic progenitors normally produce TGF (Majka et al., 2001) that can act in an autocrine fashion (Batard et al., 2000), and neutralization of TGF by monoclonal antibodies has been reported to recruit early progenitors into the cell cycle and induce them to differentiate (Batard et al., 2000; Dao et al., 1998; Ducos et al., 2000; Pierelli et al., 2000; Weekx et al., 1999). Lineage-restricted progenitor cells are generally less responsive to growth inhibition by TGF (Cashman et al., 1999, Sargiacomo et al., 1991), although some effects on apoptosis and differentiation have been observed (Cashman et al., 1999; Dybedal et al., 1997).
TGF signaling pathways intersect with signaling pathways activated by nuclear hormone receptors (Testa et al., 1993; Yanagisawa et al., 1999). Retinoic acid has been shown to augment TGF-mediated growth arrest of both leukemic cell lines (Turley et al., 1996) and normal, human CD34+ hematopoietic progenitor cells (Lardon et al., 1996; Turley et al., 1996). Conversely, TGF may sensitize cells to RA. The c-ski proto-oncogene family of proteins are normally expressed in hematopoietic progenitors (Pearson-White et al., 1995) and represent one point of convergence of these signaling pathways. Ski proteins negatively regulate Smad-mediated signal transduction (Luo et al., 1999; Sun et al., 1999a) and by binding corepressors (N-CoR/SMRT, mSin3A) and HDACs (Akiyoshi et al., 1999; Luo et al., 1999; Nomura et al., 1999; Tokitou et al., 1999) C-ski is involved in the repression of RA-responsive promoter constructs (Dahl et al., 1998). TGF stimulation leads to rapid, ubiquitin-mediated down-regulation of c-ski (Stroschein et al., 1999; Sun et al., 1999b), potentially relieving repression of RA target genes. High concentrations of retinoids can block transformation of chicken erythroblasts by v-ski and reverse its inhibition of RAR-mediated transcription (Dahl et al., 1998). Smad 3 and vitamin D receptors directly interact (Yanagisawa et al., 1999) and the expression of AML1/ETO, PML/RAR or PLZF/RAR in U937 cells, has been shown to disrupt the differentiation of these cells induced by the combination of TGF and vitamin D3 (Gelmetti et al., 1998; Ruthardt et al., 1997; Testa et al., 1993).
Smad proteins interact with a variety of transcription factors that impart DNA sequence specificity to the Smad nuclear complex. We, and others, have studied the interaction of Smads with AML1 and the AML1-containing fusion proteins. AML1B physically and functionally interacts with receptor activated Smads (Smad2 and Smad3) in the TGF-dependent activation of the germ-line immunoglobulin heavy chain promoter (Hanai et al., 1999; Jakubowiak et al., 2000; Pardali et al., 2000; Zhang and Derynck, 2000). TGF has been shown to stimulate the expression of proteins that are also regulated by AML1, which suggests that other interactions between Smads and AML1 may occur. Certainly there is a precedent, as interactions between Runx2 and Smad family members are critical for normal bone and cartilage formation (Leboy et al., 2001; Rodan and Harada, 1997).
The AML1/ETO fusion protein can interfere with some TGF-mediated signals. Expression of AML1/ETO in COS cells blocks TGF-induced promoter activity and this requires both the RHD and the C-terminal 174 amino acids of the fusion protein (Jakubowiak et al., 2000). The RHD binds Smad proteins whereas the C-terminus of ETO does not, rather it interacts with co-repressor molecules and HDACs. Interestingly, the C-terminus of AML1B (which is lost in the AML1/ETO fusion) can also interact with Smads (Jakubowiak et al., 2000; Pardali et al., 2000).
AML1/EVI1, the product of the t(3;21)(q26;q21) translocation, has also been shown to disrupt TGF-dependent promoter activation and block TGF-mediated growth inhibition 32Dcl3 cells (Kurokawa et al., 1998a,b; Sood et al., 1999). Smad3 makes two contacts with the EVI1 portion of AML1/EVI1 (Kurokawa et al., 1998a) and can interact with EVI1 itself (Kurokawa et al., 1998b; Sood et al., 1999). There are conflicting reports concerning the consequences of the interaction of EVI1 with Smad3. EVI1 has been shown to both enhance (Sood et al., 1999) and interfere with (Kurokawa et al., 1998a,b) TGF-mediated growth suppression and reporter activity in 32Dcl3 cells.
Other mechanisms for leukemia-associated mutations to disrupt TGF signal transduction have been demonstrated. For instance, oncogenic Ras mutations, which are fairly common in MDS and AML (Longo et al., 1993; Padua et al., 1998; Shen et al., 1987), can render cells unresponsive to the anti-proliferative effects of TGF (Kretzschmar et al., 1999). It is likely that only a subset of TGF-modulated genes will be affected by leukemia-associated mutations. However, disruption of the TGF program in the earliest progenitor cells could dysregulate their entry into the cell cycle at the same time that it disturbs their orderly differentiation.
Target genes and interacting proteins
Identification of critical target genes affected by the leukemia-associated fusion proteins is in its infancy but RA- and TGF-inducible genes, and those under the control of AML1, C/EBP, homeobox proteins (such as HOXA9) and AP-1 remain attractive targets by virtue of their frequent involvement, and the potential consequences of their aberrancy, in leukemia. The identification, by visual inspection, of consensus DNA binding sites within putative promoter sequences has led to transient transfection studies that have revealed a variety of cell type specific effects of the fusion proteins on gene transcription. However, the advent of microarray technologies, and techniques such as differential display, serial analysis of gene expression (SAGE), and representational difference analysis (RDA) now allow for a more unbiased opportunity to identify the true target genes of the leukemia-associated fusion proteins. Inducible gene expression systems and engineered cell lines will also aid in the identification of target genes and their role in leukemogenesis. As we define more of these cellular targets, it appears that the AML-associated fusion proteins may impair the cellular transcriptional machinery via several common pathways.
Many of the leukemia associated fusion proteins repress transcription of genes important for the normal differentiation of hematopoietic progenitor cells or affect genes that regulate cell proliferation and survival. AML1 is involved in the regulation of several genes important to hematopoiesis including granulocyte-macrophage colony-stimulating factor (GM-CSF) (Frank et al., 1995; Takahashi et al., 1995), interleukin-3 (IL-3) (Uchida et al., 1999), macrophage colony-stimulating factor (M-CSF) (Zhang et al., 1996a), myeloperoxidase (Britos-Bray and Friedman, 1997), neutrophil elastase (Nuchprayoon et al., 1994), granzyme B (Babichuk and Bleackley, 1997), the defensin NP-3 (Westendorf et al., 1998) and T-cell receptor //-chains (Hsiang et al., 1993; Prosser et al., 1992; Redondo et al., 1992). AML1/ETO inhibits the expression of several AML1-induced genes such as GM-CSF (Frank et al., 1995), IL-3 (Mao et al., 1999), and NP-3 (Westendorf et al., 1998) and similarly, TEL/AML1 interferes with AML1-dependent transcriptional activation of the TCR IL-3 and M-CSF receptor promoters (Fears et al., 1997; Hiebert et al., 1996; Uchida et al., 1999). The expression of AML1/ETO can also antagonize other effects of AML1B. For instance, whereas AML1B down-regulates p21WAF/CIP1 (Lutterbach et al., 2000), AML1/ETO is able to up-regulate its expression (unpublished observations).
It is also clear that AML1/ETO, and the other CBF leukemia-associated fusion proteins, have 'gain-of-function' activities not shared by constituent proteins. Although AML1/ETO can up-regulate expression of UBP43 (Klampfer et al., 1996; Liu et al., 1999) and can up- or down-regulate expression of the Bcl-2 or multidrug resistance gene, MDR1 promoters, these genes are not clearly regulated by the normal AML1B protein (Lutterbach et al., 1998a). AML1/ETO can also affect the expression of genes regulated by transcription factors with which it interacts. An AML1/ETO mutant that does not bind DNA, but that retains its ability to interact with MEF, can repress MEF-dependent activation of the IL-3 promoter (Mao et al., 1999). Inhibition by AML1/ETO C/EBP-induced activation of the NP-3 promoter (Westendorf et al., 1998) and of Smad-mediated activation of the Ig class switch promoter (Jakubowiak et al., 2000) has also been shown. Defining the functional and physical interactions between these fusion proteins and other regulatory proteins will be invaluable in furthering our understanding of leukemogenesis.
Other AML1/ETO target genes have been identified using differential display following expression of the fusion protein in the L-G cell line. One gene differentially expressed was TIS11b (ERF-1, cMG1) and, although overexpression of this protein lead to myeloid proliferation in response to granulocyte colony-stimulating factor (Shimada et al., 2000), the importance of TIS11b in t(8;21) AML is unclear.
Recent evidence suggests that C/EBP plays an important role in myelopoiesis. Abnormalities in C/EBP function have been identified in a variety of AML subtypes, but most commonly in those of the FAB M2 subtype that lack the t(8;21). C/EBP is involved in hematopoietic differentiation, and disruption of its activity alters myeloid development; C/EBP-/- mice lack mature granulocytes and accumulate myeloblasts (Zhang et al., 1997). Conversely, overexpression of C/EBP in the bipotential cell line U937 induces their granulocytic differentiation (Radomska et al., 1998). The heterozygous mutations in the C/EBP gene that have been identified in 15% of AML FAB M2 leukemia with a normal karyotype (Pabst et al., 2001b) commonly generate a truncated 30 kD protein missing the N-terminal DNA-binding domain. The remaining allele of C/EBP is unaffected, which suggests that the mutant behaves as a dominant negative, or that C/EBP gene dosage is important during hematopoietic differentiation. C/EBP activity may be perturbed in AML via several mechanisms. AML1/ETO down-regulates transcription from the C/EBP promoter and low level expression of C/EBP protein and mRNA were found in samples from patients with t(8;21)-positive AML, compared to other AML subtypes (Pabst et al., 2001a). However, the absence of an animal model for t(8;21) AML, makes difficult the complete evaluation of any target genes.
The APL-associated translocations involve RAR and because these fusion proteins impair RA-responsive gene promoters in vitro it is assumed that these fusion proteins lead to the inhibition of RA-responsive target genes. The identification of potential RA target genes altered by PML/RAR is not complete, but it includes C/EBP (Lian et al., 2001), a protein involved in terminal myeloid differentiation. PLZF/RAR can repress transcription from RA-responsive promoters and ETO appears able to enhance this effect (Melnick et al., 2000b). Effects of AML1/ETO on RA-targets were reported by P Pelicci last year (personal communication).
Members of the AP-1 family of TFs may also be important targets of the leukemia-associated fusion proteins. These bZIP proteins bind DNA as dimers (e.g., Jun/Fos heterodimers) and both stimulatory and inhibitory members of the family have been identified. AML1/ETO up-regulates AP-1 activity in hematopoietic and non-hematopoietic cells, and using an NIH3T3 transformation model, we demonstrated that the transforming activity of wild-type and mutant AML1/ETO proteins correlated with their ability to up-regulate AP-1 (Frank et al., 1999). The expression of AML1/ETO also led to increased amounts of phospho-Jun and phospho-ATZ, suggesting increased activity of the MAPK pathway (or decreased activity of a MAPK phosphatase) may be important. We evaluated the effects of other CBF-fusions on NIH3T3 cells. Like AML1/ETO, CBF/SMMHC can transform NIH3T3 cells (Hajra et al., 1995), although AML1/EVI-1 did not. We found only AML1/ETO activated c-Jun and AP-1 activity (unpublished data). In contrast, Hirai and colleagues have previously shown that AML1/EVI-1 increases AP-1 activity, due to its EVI-1 moiety (Tanaka et al., 1994).
PML/RAR has also been shown to enhance AP-1 activity, similar to AML1/ETO. The precise role of altered AP-1 activity in AML is not clear, but AP-1 does play a vital role in the response of cells to mitogens such as phorbol esters, and both v-jun and v-fos are powerful oncogenes. The absence of Jun B, which negatively regulates AP-1 activity, has been shown to lead to myeloproliferative disorder in mice (Jochum et al., 1999; Passegue et al., 2001). Thus, increased AP-1 activity may provide a proliferative signal to the leukemia cell.
Models of leukemia
Animal models have been used to identify the hematopoietic phenotypes induced by the leukemia-associated fusion proteins and to define the biological pathways that they influence. Both knock-in and transgenic mouse (TG) approaches have been widely used to study the AML1 and RAR-related fusion proteins. Limitations of the transgenic approach include: (i) the need to find the correct tissue specific promoter for each fusion gene; (ii) differences in expression levels in different founders that result from dissimilar integration sites and gene copy number; and (iii) the existence of both endogenous wild-type copies of the genes involved in the translocation (causing a potentially confounding gene dosage effect). Knock-in mice express the fusion protein under its natural promoter, which means expression in the right tissue at the normal developmental stage and at the appropriate level. The knock-in system somewhat more closely mimics the gene dosage effects found in the human cancer due to the loss of one copy of one of the two genes involved in the chromosomal rearrangement. However, knock-in approaches may exhibit dominant lethality if genes dysregulated by the fusion gene are indispensable for embryonic development. Although transgenic or knock-in mice generally contain only one translocation product, crossing of transgenic mice, as has been done for PML/RAR and RAR/PML, can help define the effects of the reciprocal fusion transcript as well.
The Lox-P/Cre-recombinase system has been exploited for the de novo creation of chromosomal translocations during mouse development. Lox-P sites are introduced on two different chromosomes and chromosomal translocation is brought about by the transient expression of Cre-recombinase in embryonic stem cells (Smith et al., 1995; Van Deursen et al., 1995). This approach has been used to produce chromosomal rearrangements identical to those found in human AML, such as the t(8;21) and t(4;11) (Collins et al., 2000).
AML1/ETO and AML1
Mice lacking AML1 (or CBF) demonstrate the vital role of AML1 activity in the development of definitive hematopoiesis. AML1 homozygous null embryos show normal morphogenesis and yolk-sac derived erythropoiesis, but lack fetal liver hematopoiesis and die at embryonic day 11.5-12.5 (E11.5-12.5) with a particular pattern of central nervous system hemorrhage (Okuda et al., 1996; Wang et al., 1996a). AML1-/- ES cells retain their capacity to differentiate into primitive erythroid cells in vitro; however, no myeloid or erythroid progenitors of definitive hematopoietic origin were detected in either the yolk sac or fetal livers of mutant embryos. This hematopoietic defect is intrinsic to the stem cells, as AML1-/- ES cells fail to contribute to hematopoiesis in chimeric animals. Thus, AML1-regulated target genes appear to be essential for definitive hematopoiesis (Okuda et al., 1996). AML1+/- mice, which have disruption of one copy of the AML1 gene, are born with normal frequency and exhibit no significant phenotype. However, these heterozygous mice appear to have significantly reduced numbers of erythroid and myeloid progenitors (Wang et al., 1996a).
AML1/ETO knock-in mice are not a model for AML because these mice also die in mid-gestation from central nervous system hemorrhage and they exhibit a block in fetal liver hematopoiesis (Okuda et al., 1998); this phenotype is very similar to that seen in AML1 or CBF null mice (Okuda et al., 1996; Wang et al., 1996a,b), clearly indicating that AML1/ETO blocks normal AML1 function. However, yolk sac cells from AML1/ETO+ mice differentiate into macrophages in hematopoietic colony forming unit assays, unlike AML1-/- or CBF-/- cells, which form no colonies in vitro (Yergeau et al., 1997). Additionally, AML1/ETO positive fetal liver cells contains dysplastic multilineage hematopoietic progenitors that display an abnormally high self-renewal capacity in vitro (Okuda et al., 1998). These data indicate that AML1/ETO has unique in vivo gains-of-function that may be critical to its role in leukemogenesis (Yergeau et al., 1997).
To circumvent the lethal effects of AML1/ETO on embryonic development, transgenic mice were generated with AML1/ETO expressed from the human MRP8 promoter, which is active in myeloid cells (Yuan et al., 2001). These mice do not develop leukemia during their lifespan, and their hematopoiesis appears normal; however, it is unclear in which cellular compartment(s) the expression of AML1/ETO is sustained. D Zhang and colleagues generated transgenic mice whose expression of AML1/ETO is regulated by tetracycline (Rhoades et al., 2000). In the presence of tetracycline, expression of AML1/ETO is undetectable and mouse development proceeds normally. After birth, tetracycline is withdrawn, allowing for the continual expression of AML1/ETO in bone marrow cells. These mice do not develop leukemia during the normal murine lifespan of 24 months. However, expression of AML1/ETO was variable or undetectable in different bone marrow compartments, which may be problematic. Nonetheless, abnormal stem cell proliferation was seen in vitro, suggesting increased self-renewal in the AML1/ETO expressing progenitors. This and several other experimental models suggests that AML1/ETO needs additional genetic changes to give rise to AML (Rhoades et al., 2000; Yuan et al., 2001). To determine whether additional mutations that cooperate with AML1/ETO are necessary, newborn offspring from the breeding of transgenic heterozygous mice and wild-type mice, were injected with a very large dose of the mutagen ENU (mice received 300 mg/kg ENU). Following this treatment, five out of nine (55%) of the transgenic mice developed AML; all of the other transgenic mice and all of the wild-type littermates developed acute T-lymphoblastic leukemia. Recent studies, using a conditional knock-in approach to express AML1-ETO in adult mouse hematopoietic cells, showed that AML1-ETO expression was not sufficient for leukomogenesis. Treatment with ENU lead to granulocytic sarcomas, that were trasnplantable (Higuchi et al., 2002) in 31% of mice.
One issue that we have evaluated is whether the expression of AML1/ETO in human cells gives rise to a phenotype different from that seen when it is expressed in murine cells. To address this question, we used retroviral gene transfer to express AML1/ETO in human CD34+ cells. We found that expression of AML1/ETO inhibited colony formation by committed progenitor cells (CFU) but it enhanced the growth of the more immature stem cells (cobblestone area-forming cells), resulting in their long term survival. These AML1/ETO-expressing stem cells retained their ability to generate progenitor cells and they continued to express CD34 after 5-6 weeks in long-term culture. Thus, AML1/ETO appears to enhance the growth, survival and possibly the self renewal of pluripotent stem cells when expressed in human cells (Mulloy et al., 2002).
CBF/SMMHC mouse models
CBF/SMMHC knock-in mice were generated by inserting the relevant part of the human MYH11 cDNA into the mouse CBF gene through homologous recombination. These CBF/SMMHC knock-in mice lack definitive hematopoiesis and develop fatal CNS hemorrhage around embryonic day 12.5, similar to that seen in CBF or AML1 null mice (Okuda et al., 1996; Wang et al., 1996a,b). This is consistent with a dominant negative function of CBF/SMMHC (Castilla et al., 1996). ES cells with the knocked-in CBF/MYH11 gene contribute to the erythroid lineage but not the myeloid lineage in chimeric mice. When CBF/SMMHC chimeric mice were injected with a single dose (100 mg/kg) of N-ethyl-N-nitrosourea (ENU), a potent DNA alkylating mutagen, none of the wild-type mice developed leukemia, however 84% (21/25) of the treated CBF/SMMHC chimeras developed a myelomonocytic type of acute leukemia after 2-6 months. This leukemia was transplantable into isogenic recipients. Overall, these data suggest that CBF/SMMHC may dictate the disease specificity but that additional 'hits' are required for leukemic transformation (Castilla et al., 1999).
The hMRP8 promoter element has also been used to generate CBF/SMMHC transgenic mice that express the fusion protein in myeloid cells. CBF/SMMHC transgenic mice have impaired neutrophil maturation. Although the mice had normal numbers of circulating neutrophils, the bone marrow contained increased numbers of immature myeloid cells with dysplastic features (Kogan et al., 1998). These mice did not develop leukemia, suggesting that expression of the CBF/SMMHC fusion protein from the hMRP8 promoter may occur 'too late' to affect leukemogenesis.
To circumvent the potential embryonic lethality of the AML1/MDS1/EVI1 (AME) fusion protein, (Cuenco et al., 2000) expressed AME in mouse bone marrow cells via retroviral transduction, and used these cells for bone marrow transplant experiments. Mice transplanted with AME transduced bone marrow cells develop AML 5-13 months after transplantation and this leukemia is transplantable into secondary recipients with a shorter latency (Cuenco et al., 2000). EVI-1 is itself overexpressed in AML with t(3;3) or inv(3) abnormalities, and it shares many characteristics with AML1/EVI-1. Whether the aberrant expression of EVI-1 (via the fusion protein) contributes to the development of leukemia in this model is not known.
Numerous APL TG mouse models have been developed, and in general they demonstrate the importance of both the RAR fusion protein and the reciprocal fusion protein in leukemogenesis. It is clear from these studies that the compartment in which the fusion protein is expressed is an important determinant of the phenotype. Expression of PML/RAR in transgenic mice under control of the CD11b promoter did not result in leukemia, and in fact total white blood cell and granulocyte counts did not differ between PML/RAR transgenic and control mice. However, marked reduction of myeloid progenitors in the peripheral blood and impaired myelopoiesis was revealed by treating mice with sub-lethal irradiation (Early et al., 1996). In contrast, expression of PML/RAR from the promyelocyte-specific human cathepsin G (hCG) promoter, which is active in early myeloid cells, resulted in increased numbers of immature and mature myeloid cells in the peripheral blood, bone marrow and spleen. Approximately 30% of these TG mice developed acute myeloid leukemia after a long latency period (Grisolano et al., 1997). Pandolfi and colleagues have reported abnormal hematopoiesis resembling a myeloproliferative disorder in a similarly-constructed hCG-PML/RAR TG mouse and over a period of 12-14 months, 10% of these TG mice progressed to an acute leukemia that closely mimicked human APL (He et al., 1997). TG mice constructed with the expression of PML/RAR driven from the hMRP8 promoter also demonstrated abnormal granulocytic differentiation and developed APL with a low penetrance, though with a somewhat shorter latency (3-9 months) than that seen with the hCG-transgenics (Brown et al., 1997). Similar to its effects on the human disease, ATRA has been shown to induce the differentiation of leukemic cells derived from the TG mice in vitro, and can induce a remission of both the preleukemic state and of APL, in vivo (Brown et al., 1997; Grisolano et al., 1997; He et al., 1997).
The t(15;17) translocation causes haploinsufficiency of both the normal PML and RAR genes, thus a more faithful mouse model of APL was generated by crossing hCG PML/RAR mice with PML (-/-) animals. The reduced dose of PML resulted in a dramatic increase in the incidence of leukemia, and accelerated leukemia development, compared to PML/RAR TG mice (Rego et al., 2001). These results suggest that: PML functions as a tumor suppressor in vivo and that PML haploinsufficiency, and the functional impairment of PML by PML/RAR, are critical cooperating events in APL pathogenesis (Rego et al., 2001).
PLZF/RAR and NPM/RAR transgenic mice have also been generated. PLZF/RAR TG mice develop a chronic myeloid leukemia-like disorder within 3 months of birth (Cheng et al., 1999). NPM/RAR TG mice show a spectrum of phenotypes from typical APL to CML occurring relatively late in life (from 12 to 15 months). Bone marrow cells from NPM/RAR TG mice can be induced to differentiate by ATRA whereas bone marrow cells from PLZF/RAR transgenic mice cannot (Cheng et al., 1999; Rego et al., 2000). Although PLZF/RAR TG mice develop a leukemia that does not recapitulate the promyelocytic features of APL, PLZF/RAR-RAR/PLZF double TG mice develop leukemia with classic APL features. RAR/PLZF TG mice do not develop leukemia, demonstrating that RAR/PLZF (which interferes with PLZF transcriptional repression) is a critical cooperating event in APL pathogenesis (He et al., 2000).
Mice transplanted with bone marrow cells expressing the NUP98/HOXA9 fusion protein (through retroviral transduction) develop a myeloproliferative disease that progresses to AML (Kroon et al., 2001). The NUP98 portion of the fusion protein appears to be responsible for transforming a clinically silent pre-leukemic phase, observed when HOXA9 is overexpressed, into a chronic MPD. Co-expression of NUP98/HOXA9 and Meis1 accelerates the transformation of this MPD to AML, identifying a genetic interaction similar to that reported for HoxA9 and Meis1 (Kroon et al., 2001).
These animal models, and the ongoing thorough analysis of AML patient samples, are proving vitally important in the search for 'second hits'. ENU mutagenesis shows the need for second hits in the CBF leukemias, whereas the two fusion products generated in APL (PML/RAR and RAR/PML) may constitute the first and second hit in that disease. A variety of retroviral (insertional) mutagenesis schemes are being employed to more rapidly identify the necessary cooperating mutations.
The acquisition of an AML1/ETO fusion gene may be an early event in leukemogenesis but it appears insufficient to induce a leukemic phenotype. AML1/ETO transcripts are found in bone marrow cells from patients with long-standing complete remissions and they are also detectable in early progenitors that can give rise to B cells and CFU-GEMM's (Miyamoto et al., 2000). The recent findings of FLT3 ITD and activating point mutations in c-KIT (and FLT3) in a significant fraction of AML patients, suggests that proliferative signals may be important as the second signal. Rare cases where activated kinases (such as BCR/ABL or TEL/PDGFR) are found with CBF leukemias also point to the importance of such proliferative signals.
Implications for future therapy
The t(9;22) and t(15;17) were the first chromosomal translocations to be identified in leukemia, and the resulting fusion proteins have been the subject of intensive laboratory investigation. Now, 40 years after the original description of the Philadelphia chromosome, both APL and CML can be treated with highly specific therapies that are less toxic and more effective than the treatments that preceded them. ATRA, which binds to the heterodimerized RAR portion of the PML/RAR fusion protein has become the 'poster child' for differentiation therapy and, though not curative by itself, ATRA based induction therapy for APL has become the standard worldwide and has helped make this disease the most curable form of adult acute leukemia.
Earlier this year, the first tyrosine kinase inhibitor designed to specifically treat a molecular lesion found only in malignant cells, was approved for the treatment of CML in the United States. Gleevec (formerly, STI-571), an inhibitor of the BCR/ABL tyrosine-kinase (which also inhibits c-kit, PDGFR and ABL), appears to be highly effective in treating chronic phase CML, with complete cytogenetic and molecular remissions being observed in 40 and 10% of patients, respectively. Though it is too early to know whether this will translate into a survival benefit, Gleevec has provided relief for patients unable to tolerate interferon- or whose disease does not adequately respond to interferon-. Given the highly recurrent genetic abnormalities involved in the pathogenesis of AML, and their common mechanisms of action (e.g., dimerization and recruitment of co-repressor molecules), it is tempting to speculate that similar, tailored therapies are on the horizon.
We thank Ms Ellie Park for her assistance in preparing this manuscript for publication. This work was supported in part by NIH RO1 grant DK 52208 (SD Nimer), and by a Leukemia and Lymphoma Society SCOR grant (SD Nimer), a Clinical Scholars Award CA09512 from the NCI and the Charles A. Dana Foundation (JM Scandura), and by the Renny Saltzman Leukemia Research Fund (SD Nimer).
Adler HT, Chinery R, Wu DY, Kussick SJ, Payne JM, Fornace JrAJ, Tkachuk DC. (1999). Mol. Cell. Biol., 19: 7050-7060. MEDLINE
Adya N, Stacy T, Speck NA, Liu PP. (1998). Mol. Cell. Biol., 18: 7432-7443. MEDLINE
Ahn MY, Huang G, Bae SC, Wee HJ, Kim WY, Ito Y. (1998). Proc. Natl. Acad. Sci. USA, 95: 1812-1817. MEDLINE
Ahuja HG, Felix CA, Aplan PD. (1999). Blood, 94: 3258-3261. MEDLINE
Ahuja HG, Hong J, Aplan PD, Tcheurekdjian L, Forman SJ, Slovak ML. (2000). Cancer Res., 60: 6227-6229. MEDLINE
Akiyoshi S, Inoue H, Hanai J, Kusanagi K, Nemoto N, Miyazono K, Kawabata M. (1999). J. Biol. Chem., 274: 35269-35277. Article MEDLINE
Alcalay M, Orleth A, Sebastiani C, Meani N, Chiaradonna F, Casciari C, Sciurpi MT, Gelmetti V, Riganelli D, Minucci S, Fagioli M, Pelicci PG. (2001). Oncogene, 20: 5680-5694. MEDLINE
Amann JM, Nip J, Strom D, Lutterbach B, Harada H, Lenny N, Downing JR, Meyers S, Hiebert SW. (2001). Mol. Cell. Biol., 21: 6470-6483. MEDLINE
Andrieu V, Radford-Weiss I, Troussard X, Chane C, Valensi F, Guesnu M, Haddad E, Viguier F, Dreyfus F, Varet B, Flandrin G, Macintyre E. (1996). Br. J. Haematol., 92: 855-865. MEDLINE
Arai Y, Hosoda F, Kobayashi H, Arai K, Hayashi Y, Kamada N, Kaneko Y, Ohki M. (1997). Blood, 89: 3936-3944. MEDLINE
Aravind L, Landsman D. (1998). Nucleic Acids Res., 26: 4413-4421. Article MEDLINE
Arnould C, Philippe C, Bourdon V, Gr goire MJ, Berger R, Jonveaux P. (1999). Hum. Mol. Genet., 8: 1741-1749. MEDLINE
Aronson BD, Fisher AL, Blechman K, Caudy M, Gergen JP. (1997). Mol. Cell. Biol., 17: 5581-5587. MEDLINE
Babichuk CK, Bleackley RC. (1997). J. Biol. Chem., 272: 18564-18571. MEDLINE
Baer MR, Stewart CC, Lawrence D, Arthur DC, Byrd JC, Davey FR, Schiffer CA, Bloomfield CD. (1997). Blood, 90: 1643-1648. MEDLINE
Banker DE, Radich J, Becker A, Kerkof K, Norwood T, Willman C, Appelbaum FR. (1998). Clin, Cancer Res., 4: 3051-3062.
Barna M, Hawe N, Niswander L, Pandolfi PP. (2000). Nat. Genet., 25: 166-172. Article MEDLINE
Batard P, Monier MN, Fortunel N, Ducos K, Sansilvestri-Morel P, Phan T, Hatzfeld A, Hatzfeld JA. (2000). J. Cell Sci., 113: 383-390. MEDLINE
Beghini A, Peterlongo P, Ripamonti CB, Larizza L, Cairoli R, Morra E, Mecucci C. (2000). Blood, 95: 726-727. MEDLINE
Bennett JM, Catovsky D, Daniel MT, Flandrin G, Galton DA, Gralnick HR, Sultan C. (1985). Ann. Intern. Med., 102: 620-625.
Bloomfield CD, Lawrence D, Byrd JC, Carroll A, Pettenati MJ, Tantravahi R, Patil SR, Davey FR, Berg DT, Schiffer CA, Arthur DC, Mayer RJ. (1998). Cancer Res., 58: 4173-4179. MEDLINE
Borrow J, Shearman AM, Stanton JrVP, Becher R, Collins T, Williams AJ, Dube I, Katz F, Kwong YL, Morris C, Ohyashiki K, Toyama K, Rowley J, Housman DE. (1996). Nat. Genet., 12: 159-167. MEDLINE
Breen TR, Harte PJ. (1991). Mech. Dev., 35: 113-127. MEDLINE
Britos-Bray M, Friedman AD. (1997). Mol. Cell. Biol., 17: 5127-5135. MEDLINE
Broeker PL, Harden A, Rowley JD, Zeleznik-Le N. (1996). Curr. Top. Microbiol. Immunol., 211: 259-268. MEDLINE
Brown D, Kogan S, Lagasse E, Weissman I, Alcalay M, Pelicci PG, Atwater S, Bishop JM. (1997). Proc. Natl. Acad. Sci. USA, 94: 2551-2556. Article MEDLINE
Bruhn L, Munnerlyn A, Grosschedl R. (1997). Genes Dev., 11: 640-653. MEDLINE
Burel SA, Harakawa N, Zhou L, Pabst T, Tenen DG, Zhang DE. (2001). Mol. Cell. Biol., 21: 5577-5590. MEDLINE
Byrd JC, Weiss RB. (1994). Cancer, 73: 2107-2112. MEDLINE
Calebi F, Pannell R, Pavloska G. (2001). Mol. Cell. Biol., 21: 5658-5866. MEDLINE
Cao W, Adya N, Britos-Bray M, Liu PP, Friedman AD. (1998). J. Biol. Chem., 273: 31534-31540. MEDLINE
Cao W, Britos-Bray M, Claxton DF, Kelley CA, Speck NA, Liu PP, Friedman AD. (1997). Oncogene, 15: 1315-1327. MEDLINE
Cashman JD, Clark-Lewis I, Eaves AC, Eaves CJ. (1999). Blood, 94: 3722-3729. MEDLINE
Castaigne S, Chomienne C, Daniel MT, Ballerini P, Berger R, Fenaux P, Degos L. (1990). Blood, 76: 1704-1709. MEDLINE
Castilla LH, Garrett L, Adya N, Orlic D, Dutra A, Anderson S, Owens J, Eckhaus M, Bodine D, Liu PP. (1999). Nat. Genet., 23: 144-146. Article MEDLINE
Castilla LH, Wijmenga C, Wang Q, Stacy T, Speck NA, Eckhaus M, Marin-Padilla M, Collins FS, Wynshaw-Boris A, Liu PP. (1996). Cell, 87: 687-696. MEDLINE
Chakrabarti SR, Nucifora G. (1999). Biochem. Biophys. Res. Commun., 264: 871-877. Article MEDLINE
Chakrabarti SR, Sood R, Nandi S, Nucifora G. (2000). Proc. Natl. Acad. Sci. USA, 97: 13281-13285. Article MEDLINE
Chambon P. (1996). FASEB J., 10: 940-954. MEDLINE
Chen HI, Einbond A, Kwak SJ, Linn H, Koepf E, Peterson S, Kelly JW, Sudol M. (1997). J. Biol. Chem., 272: 17070-17077. Article MEDLINE
Chen YG, Hata A, Lo RS, Wotton D, Shi Y, Pavletich N, Massague J. (1998). Genes Dev., 12: 2144-2152. MEDLINE
Chen Z, Brand NJ, Chen A, Chen SJ, Tong JH, Wang ZY, Waxman S, Zelent A. (1993). EMBO J., 12: 1161-1167. MEDLINE
Cheng GX, Zhu XH, Men XQ, Wang L, Huang QH, Jin XL, Xiong SM, Zhu J, Guo WM, Chen JQ, Xu SF, So E, Chan LC, Waxman S, Zelent A, Chen GQ, Dong S, Liu JX, Chen SJ. (1999). Proc. Natl. Acad. Sci. USA, 96: 6318-6323. Article MEDLINE
Collins EC, Pannell R, Simpson EM, Forster A, Rabbitts TH. (2000). EMBO Rep., 1: 127-132. MEDLINE
Cuenco GM, Nucifora G, Ren R. (2000). Proc. Natl. Acad. Sci. USA, 97: 1760-1765. MEDLINE
Dahl R, Kieslinger M, Beug H, Hayman MJ. (1998). Proc. Natl. Acad. Sci. USA, 95: 11187-11192. MEDLINE
Dann EJ, Rowe JM. (2001). Best Pract. Res. Clin. Haematol., 14: 119-137. MEDLINE
Dao MA, Taylor N, Nolta JA. (1998). Proc. Natl. Acad. Sci. USA, 95: 13006-13011. Article MEDLINE
Dong S, Zhu J, Reid A, Strutt P, Guidez F, Zhong HJ, Wang ZY, Licht J, Waxman S, Chomienne C, Chomienne C, Chen E, Zelent A, Chen SJ. (1996). Proc. Natl. Acad. Sci. USA, 93: 3624-3629. Article MEDLINE
Dubois CM, Ruscetti FW, Stankova J, Keller JR. (1994). Blood, 83: 3138-3145. MEDLINE
Ducos K, Panterne B, Fortunel N, Monier MN, Hatzfeld J. (2000). J. Cell. Physiol., 184: 80-85. Article MEDLINE
Dybedal I, Guan F, Borge OJ, Veiby OP, Ramsfjell V, Nagata S, Jacobsen SE. (1997). Blood, 90: 3395-3403. MEDLINE
Dyck JA, Maul GG, Miller JrWH, Chen JD, Kakizuka A, Evans RM. (1994). Cell, 76: 333-343. MEDLINE
Early E, Moore MA, Kakizuka A, Nason-Burchenal K, Martin P, Evans RM, Dmitrovsky E. (1996). Proc. Natl. Acad. Sci. USA, 93: 7900-7904. Article MEDLINE
Engel ME, Datta PK, Moses HL. (1998). J. Cell Biochem. Suppl., 31: 111-122.
Erickson P, Gao J, Chang KS, Look T, Whisenant E, Raimondi S, Lasher R, Trujillo J, Rowley J, Drabkin H. (1992). Blood, 80: 1825-1831. MEDLINE
Erickson PF, Robinson M, Owens G, Drabkin HA. (1994). Cancer Res., 54: 1782-1786. MEDLINE
Fair K, Anderson M, Bulanova E, Mi H, Tropschug M, Diaz MO. (2001). Mol. Cell. Biol., 21: 3589-3597. MEDLINE
Faretta M, Di Croce L, Pelicci PG. (2001). Semin. Hematol., 38: 42-53. MEDLINE
Fears S, Gavin M, Zhang DE, Hetherington C, Ben-David Y, Rowley JD, Nucifora G. (1997). Proc. Natl. Acad. Sci. USA, 94: 1949-1954. MEDLINE
Feinstein PG, Kornfeld K, Hogness DS, Mann RS. (1995). Genetics, 140: 573-586. MEDLINE
Fenrick R, Amann JM, Lutterbach B, Wang L, Westendorf JJ, Downing JR, Hiebert SW. (1999). Mol. Cell. Biol., 19: 6566-6574. MEDLINE
Ferrucci PF, Grignani F, Pearson M, Fagioli M, Nicoletti I, Pelicci PG. (1997). Proc. Natl. Acad. Sci. USA, 94: 10901-10906. Article MEDLINE
Fontoura BM, Blobel G, Matunis MJ. (1999). J. Cell Biol., 144: 1097-1112. MEDLINE
Fornerod M, Boer J, van Baal S, Jaegle M, von Lindern M, Murti KG, Davis D, Bonten J, Buijs A, Grosveld G. (1995). Oncogene, 10: 1739-1748. MEDLINE
Fortunel NO, Hatzfeld A, Hatzfeld JA. (2000). Blood, 96: 2022-2036. MEDLINE
Frank R, Zhang J, Uchida H, Meyers S, Hiebert SW, Nimer SD. (1995). Oncogene, 11: 2667-2674. MEDLINE
Frank RC, Sun X, Berguido FJ, Jakubowiak A, Nimer SD. (1999). Oncogene, 18: 1701-1710. MEDLINE
Fraser JK, Guerra JJ, Nguyen CY, Indes JE, Gasson JC, Nimer SD. (1994). Mol. Cell. Biol., 14: 2213-2221. MEDLINE
Fynan TM, Reiss M. (1993). Crit. Rev. Oncog., 4: 493-540. MEDLINE
Gale KB, Ford AM, Repp R, Borkhardt A, Keller C, Eden OB, Greaves MF. (1997). Proc. Natl. Acad. Sci. USA, 94: 13950-13954. Article MEDLINE
Gelmetti V, Zhang J, Fanelli M, Minucci S, Pelicci PG, Lazar MA. (1998). Mol. Cell. Biol., 18: 7185-7191. MEDLINE
Gibbons B, Katz FE, Ganly P, Chessells JM. (1990). Br. J. Haematol., 74: 264-269. MEDLINE
Giese K, Kingsley C, Kirshner JR, Grosschedl R. (1995). Genes Dev., 9: 995-1008. MEDLINE
Gill Super HJ, Rothberg PG, Kobayashi H, Freeman AI, Diaz MO, Rowley JD. (1994). Blood, 83: 641-644. MEDLINE
Golub TR, Barker GF, Bohlander SK, Hiebert SW, Ward DC, Bray-Ward P, Morgan E, Raimondi SC, Rowley JD, Gilliland DG. (1995). Proc. Natl. Acad. Sci. USA, 92: 4917-4921. MEDLINE
Grignani F, De Matteis S, Nervi C, Tomassoni L, Gelmetti V, Cioce M, Fanelli M, Ruthardt M, Ferrara FF, Zamir I, Seiser C, Lazar MA, Minucci S, Pelicci PG. (1998). Nature, 391: 815-818. Article MEDLINE
Grignani F, Ferrucci PF, Testa U, Talamo G, Fagioli M, Alcalay M, Mencarelli A, Peschle C, Nicoletti I. (1993). Cell, 74: 423-431. MEDLINE
Grignani F, Valtieri M, Gabbianelli M, Gelmetti V, Botta R, Luchetti L, Masella B, Morsilli O, Pelosi E, Samoggia P, Pelicci PG, Peschle C. (2000). Blood, 96: 1531-1537. MEDLINE
Grimwade D, Walker H, Harrison G, Oliver F, Chatters S, Harrison CJ, Wheatley K, Burnett AK, Goldstone AH. (2001). Blood, 98: 1312-1320. Article MEDLINE
Grisolano JL, Wesselschmidt RL, Pelicci PG, Ley TJ. (1997). Blood, 89: 376-387. MEDLINE
Grunstein M. (1997). Nature, 389: 349-352. Article MEDLINE
Guo A, Salomoni P, Luo J, Shih A, Zhong S, Gu W, Paolo Pandolfi P. (2000). Nat. Cell. Biol., 2: 730-736. Article MEDLINE
Hajra A, Liu PP, Wang Q, Kelley CA, Stacy T, Adelstein RS, Speck NA, Collins FS. (1995). Proc. Natl. Acad. Sci. USA, 92: 1926-1930. MEDLINE
Hanai J, Chen LF, Kanno T, Ohtani-Fujita N, Kim WY, Guo WH, Imamura T, Ishidou Y, Fukuchi M, Shi MJ, Stavnezer J, Kawabata M, Miyazono K, Ito Y. (1999). J. Biol. Chem., 274: 31577-31582. Article MEDLINE
Harris NL, Jaffe ES, Diebold J, Flandrin G, Muller-Hermelink HK, Vardiman J, Lister TA, Bloomfield CD. (1999). J. Clin. Oncol., 17: 3835-3849. MEDLINE
He LZ, Bhaumik M, Tribioli C, Rego EM, Ivins S, Zelent A, Pandolfi PP. (2000). Mol. Cell, 6: 1131-1141. MEDLINE
He LZ, Guidez F, Tribioli C, Peruzzi D, Ruthardt M, Zelent A, Pandolfi PP. (1998). Nat. Genet., 18: 126-135. MEDLINE
He LZ, Merghoub T, Pandolfi PP. (1999). Oncogene, 18: 5278-5292. MEDLINE
He LZ, Tribioli C, Rivi R, Peruzzi D, Pelicci PG, Soares V, Cattoretti G, Pandolfi PP. (1997). Proc. Natl. Acad. Sci. USA, 94: 5302-5307. Article MEDLINE
Hiebert SW, Sun W, Davis JN, Golub T, Shurtleff S, Buijs A, Downing JR, Grosveld G, Roussell MF, Gilliland DG, Lenny N, Meyers S. (1996). Mol. Cell. Biol., 16: 1349-1355. MEDLINE
Hildebrand D, Tiefenbach J, Heinzel T, Grez M, Maurer AB. (2001). J. Biol. Chem., 276: 9889-9895. MEDLINE
Horiike S, Yokota S, Nakao M, Iwai T, Sasai Y, Kaneko H, Taniwaki M, Kashima K, Fujii H, Abe T, Misawa S. (1997). Leukemia, 11: 1442-1446. MEDLINE
Hsiang YH, Spencer D, Wang S, Speck NA, Raulet D. (1993). J. Immunol., 150: 3905-3916. MEDLINE
Huang ME, Ye YC, Chen SR, Chai JR, Lu JX, Zhoa L, Gu LJ, Wang ZY. (1988). Blood, 72: 567-572. MEDLINE
Imai Y, Kurokawa M, Izutsu K, Hangaishi A, Maki K, Ogawa S, Chiba S, Mitani K, Hirai H. (2001). Oncogene, 20: 88-96. MEDLINE
Imai Y, Kurokawa M, Tanaka K, Friedman AD, Ogawa S, Mitani K, Yazaki Y, Hirai H. (1998). Biochem. Biophys. Res. Commun., 252: 582-589. Article MEDLINE
Jacobsen RJ, Temple MJ, Sacher RA. (1984). Br. J. Haematol., 57: 539-540. MEDLINE
Jacobsen SE, Ruscetti FW, Dubois CM, Lee J, Boone TC, Keller JR. (1991). Blood, 77: 1706-1716. MEDLINE
Jacobsen SE, Ruscetti FW, Roberts AB, Keller JR. (1993). J. Immunol., 151: 4534-4544. MEDLINE
Jakubowiak A, Pouponnot C, Berguido F, Frank R, Mao S, Massague J, Nimer SD. (2000). J. Biol. Chem., 275: 40282-40287. MEDLINE
Jochum W, Passegue E, Wagner EF. (1999). Verh. Dtsch. Ges. Pathol., 83: 216-217. MEDLINE
Joh T, Kagami Y, Yamamoto K, Segawa T, Takizawa J, Takahashi T, Ueda R, Seto M. (1996). Oncogene, 13: 1945-1953. MEDLINE
Johansen LM, Iwama A, Lodie TA, Sasaki K, Felsher DW, Golub TR, Tenen DG. (2001). Mol. Cell. Biol., 21: 3789-3806. MEDLINE
Jones KA, Kadonaga JT. (2000). Genes Dev., 14: 1992-1996. MEDLINE
Kalwinsky DK, Raimondi SC, Schell MJ, Mirro JrJ, Santana VM, Behm F, Dahl GV, Williams D. (1990). J. Clin. Oncol., 8: 75-83. MEDLINE
Kanno Y, Kanno T, Sakakura C, Bae SC, Ito Y. (1998). Mol. Cell. Biol., 18: 4252-4261. MEDLINE
Kasper LH, Brindle PK, Schnabel CA, Pritchard CE, Cleary ML, van Deursen JM. (1999). Mol. Cell. Biol., 19: 764-776. MEDLINE
Keating MJ, Smith TL, Kantarjian H, Cork A, Walters R, Trujillo JM, McCredie KB, Gehan EA, Freireich EJ. (1988). Leukemia, 2: 403-412. MEDLINE
Kim JH, Lee S, Rho JK, Choe SY. (1999a). Int. J. Biochem. Cell. Biol., 31: 933-940.
Kim WY, Sieweke M, Ogawa E, Wee HJ, Englmeier U, Graf T, Ito Y. (1999b). EMBO J., 18: 1609-1620. Article MEDLINE
Kitabayashi I, Ida K, Morohoshi F, Yokoyama A, Mitsuhashi N, Shimizu K, Nomura N, Hayashi Y, Ohki M. (1998a). Mol. Cell. Biol., 18: 846-858. MEDLINE
Kitabayashi I, Yokoyama A, Shimizu K, Ohki M. (1998b). EMBO J., 17: 2994-3004. MEDLINE
Klampfer L, Zhang J, Zelenetz A, Uchida H, Nimer SD. (1996). Proc. Natl. Acad. Sci. USA, 93: 14059-14064. Article MEDLINE
Kogan SC, Lagasse E, Atwater S, Bae SC, Weissman I, Ito Y, Bishop JM. (1998). Proc. Natl. Acad. Sci. USA, 95: 11863-11868. MEDLINE
Kohzaki H, Ito K, Huang G, Wee HJ, Murakami Y, Ito Y. (1999). Oncogene, 18: 4055-4062. MEDLINE
Koken MH, Puvion-Dutilleul F, Guillemin MC, Viron A, Linares-Cruz G, Stuurman N, de Jong L, Szostecki C, Calvo F, Chomienne C. (1994). EMBO J., 13: 1073-1083. MEDLINE
Kouzarides T. (2000). EMBO J., 19: 1176-1179. MEDLINE
Kraemer D, Wozniak RW, Blobel G, Radu S. (1994). Proc. Natl. Acad. Sci. USA, 91: 1519-1523. MEDLINE
Kretzschmar M, Doody J, Timokhina I, Massague J. (1999). Genes Dev., 13: 804-816. MEDLINE
Kroon E, Krosl J, Thorsteinsdottir U, Baban S, Buchberg AM, Sauvageau G. (1998). EMBO J., 17: 3714-3725. Article MEDLINE
Kroon E, Thorsteinsdottir U, Mayotte N, Nakamura T, Sauvageau G. (2001). EMBO J., 20: 350-361. MEDLINE
Kurokawa M, Mitani K, Imai Y, Ogawa S, Yazaki Y, Hirai H. (1998a). Blood, 92: 4003-4012. MEDLINE
Kurokawa M, Mitani K, Irie K, Matsuyama T, Takahashi T, Chiba S, Yazaki Y, Matsumoto K, Hirai H. (1998b). Nature, 394: 92-96. Article MEDLINE
Kwiatkowski BA, Bastian LS, Bauer JrTR, Tsai S, Zielinska-Kwiatkowska AG, Hickstein DD. (1998). J. Biol. Chem., 273: 17525-17530. MEDLINE
Lai JL, Preudhomme C, Zandecki M, Flactif M, Vanrumbeke M, Lepelley P, Wattel E, Fenaux P. (1995). Leukemia, 9: 370-381. MEDLINE
Langabeer SE, Walker H, Gale RE, Wheatley K, Burnett AK, Goldstone AH, Linch DC. (1997a). Br. J. Haematol., 96: 736-739. MEDLINE
Langabeer SE, Walker H, Rogers JR, Burnett AK, Wheatley K, Swirsky D, Goldstone AH, Linch DC. (1997b). Br. J. Haematol., 99: 925-928. MEDLINE
Lardon F, Snoeck HW, Haenen L, Lenjou M, Nijs G, Weekx SF, Van Ranst PC, Berneman ZN, Van Bockstaele DR. (1996). Leukemia, 10: 1937-1943. MEDLINE
Larson RA, Williams SF, Le Beau MM, Bitter MA, Vardiman JW, Rowley JD. (1986). Blood, 68: 1242-1249. MEDLINE
Lavau C, Szilvassy SJ, Slany R, Cleary ML. (1997). EMBO J., 16: 4226-4237. MEDLINE
Le Beau MM, Larson RA, Bitter MA, Vardiman JW, Golomb HM, Rowley JD. (1983). N. Engl. J. Med., 309: 630-636. MEDLINE
Le XF, Vallian S, Mu ZM, Hung MC, Chang KS. (1998). Oncogene, 16: 1839-1849. MEDLINE
Leboy P, Grasso-Knight G, D'Angelo M, Volk SW, Lian JV, Drissi H, Stein GS, Adams SL. (2001). J. Bone Joint Surg. Am., 83-A: S15-S22. MEDLINE
Leith CP, Kopecky KJ, Godwin J, McConnell T, Slovak ML, Chen IM, Head DR, Appelbaum FR, Willman CL. (1997). Blood, 89: 3323-3329. MEDLINE
Lian Z, Wang L, Yamaga S, Bonds W, Beazer-Barklay Y, Kluger Y, Gerstein M, Newberger PE, Berliner N, Weissman SM. (2001). Blood, 98: 513-524. Article MEDLINE
Licht JD, Chomienne C, Goy A, Chen A, Scott AA, Head DR, Michaux JL, Wu Y, DeBlasio A, Miller JrWH. (1995). Blood, 85: 1083-1094. MEDLINE
Lin RJ, Evans RM. (2000). Mol. Cell, 5: 821-830. MEDLINE
Lin RJ, Nagy L, Inoue S, Shao W, Miller JrWH, Evans RM. (1998). Nature, 391: 811-814. Article MEDLINE
Liu LQ, Ilaria JrR, Kingsley PD, Iwama A, van Etten RA, Palis J, Zhang DE. (1999). Mol. Cell. Biol., 19: 3029-3038. MEDLINE
Liu M, Iavarone A, Freedman LP. (1996a). J. Biol. Chem., 271: 31723-31728. MEDLINE
Liu P, Tarle SA, Hajra A, Claxton DF, Marlton P, Freedman M, Siciliano MJ, Collins FS. (1993). Science, 261: 1041-1044. MEDLINE
Liu PP, Wijmenga C, Hajra A, Blake TB, Kelley CA, Adelstein RS, Bagg A, Rector J, Cotelingam J, Willman CL, Collins FS. (1996b). Genes Chromosomes Cancer, 16: 77-87. MEDLINE
Longo L, Trecca D, Biondi A, Lo Coco F, Grignani F, Maiolo AT, Pelicci PG, Neri A. (1993). Leuk. Lymphoma, 11: 405-410. MEDLINE
Look AT. (1997). Science, 278: 1059-1064. MEDLINE
Lou J, Cao W, Bernardin F, Ayyanathan K, Rauscher IF, Friedman AD. (2000). Oncogene, 19: 2695-2703. MEDLINE
Lu J, Maruyama M, Satake M, Bae SC, Ogawa E, Kagoshima H, Shigesada K, Ito Y. (1995). Mol. Cell. Biol., 15: 1651-1661. MEDLINE
Luo K, Stroschein SL, Wang W, Chen D, Martens E, Zhou S, Zhou Q. (1999). Genes Dev., 13: 2196-2206. Article MEDLINE
Luo RT, Lavau C, Du C, Simone F, Polak PE, Kawamata S, Thirman MJ. (2001). Mol. Cell. Biol., 21: 5678-5687. MEDLINE
Lutterbach B, Hou Y, Durst KL, Hiebert SW. (1999). Proc. Natl. Acad. Sci. USA, 96: 12822-12827. Article MEDLINE
Lutterbach B, Sun D, Schuetz J, Hiebert SW. (1998a). Mol. Cell. Biol., 18: 3604-3611. MEDLINE
Lutterbach B, Westendorf JJ, Linggi B, Isaac S, Seto E, Hiebert SW. (2000). J. Biol. Chem., 275: 651-656. MEDLINE
Lutterbach B, Westendorf JJ, Linggi B, Patten A, Moniwa M, Davie JR, Huynh KD, Bardwell VJ, Lavinsky RM, Rosenfeld MG, Glass C, Seto E, Hiebert SW. (1998b). Mol. Cell. Biol., 18: 7176-7184. MEDLINE
Ma Q, Alder H, Nelson KK, Chatterjee D, Gu Y, Nakamura T, Canaani E, Croce CM, Siracusa LD, Buchberg AM. (1993). Proc. Natl. Acad. Sci. USA, 90: 6350-6354. MEDLINE
Mahmud N, Katayama N, Nishii K, Sugawara T, Komada Y, Mitani H, Araki H, Ohishi K, Watanabe M, Masuya M, Nishikawa M, Minami N, Ohashi H, Shiku H. (1999). Br. J. Haematol., 105: 470-477. MEDLINE
Majka M, Janowska-Wieczorek A, Ratajczak J, Ehrenman K, Pietrzkowski Z, Kowalska MA, Gewirtz AM, Emerson SG, Ratajczak MZ. (2001). Blood, 97: 3075-3085. Article MEDLINE
Mao S, Frank RC, Zhang J, Miyazaki Y, Nimer SD. (1999). Mol. Cell. Biol., 19: 3635-3644. MEDLINE
Marlton P, Keating M, Kantarjian H, Pierce S, O'Brien S, Freireich EJ, Estey E. (1995). Leukemia, 9: 965-971. MEDLINE
Massague J, Blain SW, Lo RS. (2000). Cell, 103: 295-309. MEDLINE
McLarren KW, Lo R, Grbavec D, Thirunavukkarasu K, Karsenty G, Stifani S. (2000). J. Biol. Chem., 275: 530-538. MEDLINE
McLarren KW, Theriault FM, Stifani S. (2001). J. Biol. Chem., 276: 1578-1584. MEDLINE
McLean TW, Ringold S, Neuberg D, Stegmaier K, Tantravahi R, Ritz J, Koeffler HP, Takeuchi S, Janssen JW, Seriu T, Bartram CR, Sallan SE, Gilliland DG, Golub TR. (1996). Blood, 88: 4252-4258. MEDLINE
McNeil S, Javed A, Harrington KS, Lian JB, Stein JL, van Wijnen AJ, Stein GS. (2000). J. Cell Biochem., 79: 103-112. Article MEDLINE
McNeil S, Zeng C, Harrington KS, Hiebert S, Lian JB, Stein JL, van Wijnen AJ, Stein GS. (1999). Proc. Natl. Acad. Sci. USA, 96: 14882-14887. MEDLINE
Megonigal MD, Rappaport EF, Nowell PC, Lange BJ, Felix CA. (1998). Oncogene, 16: 1351-1356. MEDLINE
Melnick A, Licht JD. (1999). Blood, 93: 3167-3215. MEDLINE
Melnick A, Carlile GW, McConnell MJ, Polinger A, Hiebert SW, Licht JD. (2000a). Blood, 96: 3939-3947. MEDLINE
Melnick AM, Westendorf JJ, Polinger A, Carlile GW, Arai S, Ball HJ, Lutterbach B, Hiebert SW, Licht JD. (2000b). Mol. Cell. Biol., 20: 2075-2086. MEDLINE
Meyers S, Downing JR, Hiebert SW. (1993). Mol. Cell. Biol., 13: 6336-6345. MEDLINE
Minucci S, Maccarana M, Cioce M, De Luca P, Gelmetti V, Segalla S, Di Croce L, Giavara S, Matteucci C, Gobbi A, Bianchini A, Colombo E, Schiavoni I, Badaracco G, Hu X, Lazar MA, Landsberger N, Nervi C, Pelicci PG. (2000). Mol. Cell, 5: 811-820. Article MEDLINE
Mitelman F, Johansson B, Mertens F. (2002). http://cgap.nci.nih.gov/chromosomes/mitelman.
Miyamoto T, Weissman IL, Akashi K. (2000). Proc. Natl. Acad. Sci. USA, 97: 7521-7526. Article MEDLINE
Miyoshi H, Kozu T, Shimizu K, Enomoto K, Maseki N, Kaneko Y, Kamada N, Ohki M. (1993). EMBO J., 12: 2715-2721. MEDLINE
Mrozek K, Heinonen K, Bloomfield CD. (2001). Baillieres Best Pract. Res. Clin. Haematol., 14: 19-47.
Mrozek K, Heinonen K, de la Chapelle A, Bloomfield CD. (1997). Semin. Oncol., 24: 17-31. MEDLINE
Mu ZM, Le XF, Vallian S, Glassman AB, Chang KS. (1997). Carcinogenesis, 18: 2063-2069. Article MEDLINE
Muller C, Yang R, Park D, Serve H, Berdel WE, Koeffler HP. (2000). Blood, 96: 3894-3899. MEDLINE
Mulloy JC, Cammerga J, Mackinzie KL, Berguido FJ, Moore MA, Nimier SD. (2002). Blood, 99: 15-23. Article MEDLINE
Nakamura T, Largaespada DA, Lee MP, Johnson LA, Ohyashiki K, Toyama K, Chen SJ, Willman CL, Chen IM, Feinberg AP, Jenkins NA, Copeland NG, Shaughnessy JrJD. (1996). Nat. Genet., 12: 154-158. MEDLINE
Nakamura T, Yamazaki Y, Hatano Y, Miura I. (1999). Blood, 94: 741-747. MEDLINE
Nakao M, Yokota S, Iwai T, Kaneko H, Horiike S, Kashima K, Sonoda Y, Fujimoto T, Misawa S. (1996). Leukemia, 10: 1911-1918. MEDLINE
Nishiyama M, Arai Y, Tsunematsu Y, Kobayashi H, Asami K, Yabe M, Kato S, Oda M, Eguchi H, Ohki M, Kaneko Y. (1999). Genes Chromosomes Cancer, 26: 215-220. MEDLINE
Nomura T, Khan MM, Kaul SC, Dong HD, Wadhwa R, Colmenares C, Kohno I, Ishii S. (1999). Genes Dev., 13: 412-423. MEDLINE
Nuchprayoon I, Meyers S, Scott LM, Suzow J, Hiebert S, Friedman AD. (1994). Mol. Cell. Biol., 14: 5558-5568. MEDLINE
Odaka Y, Mally A, Elliott LT, Meyers S. (2000). Oncogene, 19: 3584-3597. MEDLINE
Okuda T, Cai Z, Yang S, Lenny N, Lyu CJ, van Deursen JM, Harada H, Downing JR. (1998). Blood, 91: 3134-3143. MEDLINE
Okuda T, van Deursen J, Hiebert SW, Grosveld G, Downing JR. (1996). Cell, 84: 321-330. MEDLINE
Pabst T, Mueller BU, Harakawa N, Schoch C, Haferlach T, Behre G, Hiddemann W, Zhang DE, Tenen DG. (2001a). Nat. Med., 7: 444-451. Article MEDLINE
Pabst T, Mueller BU, Zhang P, Radomska HS, Narravula S, Schnittger S, Behre G, Hiddemann W, Tenen DG. (2001b). Nat. Genet., 27: 263-270. Article MEDLINE
Padua RA, Guinn BA, Al-Sabah AI, Smith M, Taylor C, Pettersson T, Ridge S, Carter G, White D, Oscier D, Chevret S, West R. (1998). Leukemia, 12: 887-892. MEDLINE
Pandolfi PP. (2001). Hum. Mol. Genet., 10: 769-775. Article MEDLINE
Pardali E, Xie XQ, Tsapogas P, Itoh S, Arvanitidis K, Heldin CH, ten Dijke P, Grundstrom T, Sideras P. (2000). J. Biol. Chem., 275: 3552-3560. Article MEDLINE
Passegue E, Jochum W, Schorpp-Kistner M, Mohle-Steinlein U, Wagner EF. (2001). Cell, 104: 21-32. MEDLINE
Pearson-White S, Deacon D, Crittenden R, Brady G, Iscove N, Quesenberry PJ. (1995). Blood, 86: 2146-2155. MEDLINE
Petrovick MS, Hiebert SW, Friedman AD, Hetherington CJ, Tenen DG, Zhang DE. (1998). Mol. Cell. Biol., 18: 3915-3925. MEDLINE
Pierelli L, Marone M, Bonanno G, Mozzetti S, Rutella S, Morosetti R, Rumi C, Mancuso S, Leone G, Scambia G. (2000). Blood, 95: 3001-3009. MEDLINE
Porse BT, Pedersen TA, Xu X, Lindberg B, Wewer UM, Friis-Hansen L, Nerlov C. (2001). Cell, 107: 247-258. MEDLINE
Prasad R, Yano T, Sorio C, Nakamura T, Rallapalli R, Gu Y, Leshkowitz D, Croce CM, Canaani E. (1995). Proc. Natl. Acad. Sci. USA, 92: 12160-12164. MEDLINE
Prosser HM, Wotton D, Gegonne A, Ghysdael J, Wang S, Speck NA, Owen MJ. (1992). Proc. Natl. Acad. Sci. USA, 89: 9934-9938. MEDLINE
Rabbitts TH. (1994). Nature, 372: 143-149. MEDLINE
Radomska HS, Huettner CS, Zhang P, Cheng T, Scadden DT, Tenen DG. (1998). Mol. Cell. Biol., 18: 4301-4314. MEDLINE
Radu A, Moore MS, Blobel G. (1995). Cell, 81: 215-222. MEDLINE
Raimondi SC, Chang MN, Ravindranath Y, Behm FG, Gresik MV, Steuber CP, Weinstein HJ, Carroll AJ. (1999). Blood, 94: 3707-3716. MEDLINE
Rasio D, Schichman SA, Negrini M, Canaani E, Croce CM. (1996). Cancer Res., 56: 1766-1769. MEDLINE
Raza-Egilmez SZ, Jani-Sait SN, Grossi M, Higgins MJ, Shows TB, Aplan PD. (1998). Cancer Res., 58: 4269-4273. MEDLINE
Redner RL, Rush EA, Faas S, Rudert WA, Corey SJ. (1996). Blood, 87: 882-886. MEDLINE
Redondo JM, Pfohl JL, Hernandez-Munain C, Wang S, Speck NA, Krangel MS. (1992). Mol. Cell. Biol., 12: 4817-4823. MEDLINE
Rego EM, He LZ, Warrell JrRP, Wang ZG, Pandolfi PP. (2000). Proc. Natl. Acad. Sci. USA, 97: 10173-10178. MEDLINE
Rego EM, Wang ZG, Peruzzi D, He LZ, Cordon-Cardo C, Pandolfi PP. (2001). J. Exp. Med., 193: 521-529. MEDLINE
Reya T, Morrison SJ, Clarke MF, Weissman IL. (2001). Nature, 414: 105-111. Article MEDLINE
Rhoades KL, Hetherington CJ, Harakawa N, Yergeau DA, Zhou L, Liu LQ, Little MT, Tenen DG, Zhang DE. (2000). Blood, 96: 2108-2115. MEDLINE
Rhoades KL, Hetherington CJ, Rowley JD, Hiebert SW, Nucifora G, Tenen DG, Zhang DE. (1996). Proc. Natl. Acad. Sci. USA, 93: 11895-11900. Article MEDLINE
Robertson SC, Tynan J, Donoghue DJ. (2000). Trends Genet., 16: 368-. MEDLINE
Rodan GA, Harada S. (1997). Cell, 89: 677-680. MEDLINE
Rogaia D, Grignani F, Carbone R, Riganelli D, LoCoco F, Nakamura T, Croce CM, Di Fiore PP, Pelicci PG. (1997). Cancer Res., 57: 799-802. MEDLINE
Rombouts WJ, Blokland I, Lowenberg B, Ploemacher RE. (2000). Leukemia, 14: 675-683. MEDLINE
Ross JA, Potter JD, Reaman GH, Pendergrass TW, Robison LL. (1996). Cancer Causes Control, 7: 581-590. MEDLINE
Rozenblatt-Rosen O, Rozovskaia T, Burakov D, Sedkov Y, Tillib S, Blechman J, Nakamura T, Croce CM, Mazo A, Canaani E. (1998). Proc. Natl. Acad. Sci. USA, 95: 4152-4157. Article MEDLINE
Rozovskaia T, Rozenblatt-Rosen O, Sedkov Y, Burakov D, Yano T, Nakamura T, Petruck S, Ben-Simchon L, Croce CM, Mazo A, Canaani E. (2000). Oncogene, 19: 351-357. MEDLINE
Rubnitz JE, Morrissey J, Savage PA, Cleary ML. (1994). Blood, 84: 1747-1752. MEDLINE
Ruthardt M, Testa U, Nervi C, Ferrucci PF, Grignani F, Puccetti E, Peschle C, Pelicci PG. (1997). Mol. Cell. Biol., 17: 4859-4869. MEDLINE
Sargiacomo M, Valtieri M, Gabbianelli M, Pelosi E, Testa U, Camagna A, Peschle C. (1991). Ann. NY Acad. Sci, 628: 84-91. MEDLINE
Satake N, Maseki N, Nishiyama M, Kobayashi H, Sakurai M, Inaba H, Katano N, Horikoshi Y, Eguchi H, Miyake M, Seto M, Kaneko Y. (1999). Leukemia, 13: 1013-1017. MEDLINE
Sato Y, Suto Y, Pietenpol J, Golub TR, Gilliland DG, Davis EM, Le Beau MM, Roberts JM, Vogelstein B, Rowley JD. (1995). Blood, 86: 1525-1533. MEDLINE
Shaknovich R, Yeyati PL, Ivins S, Melnick A, Lempert C, Waxman S, Zelent A, Licht JD. (1998). Mol. Cell. Biol., 18: 5533-5545. MEDLINE
Shen WP, Aldrich TH, Venta-Perez G, Franza JrBR, Furth ME. (1987). Oncogene, 1: 157-165. MEDLINE
Shikami M, Miwa H, Nishii K, Takahashi T, Sekine T, Mahmud N, Nishikawa M, Shiku H, Kamada N, Kita K. (1999). Leukemia, 13: 358-368. MEDLINE
Shimada H, Ichikawa H, Nakamura S, Katsu R, Iwasa M, Kitabayashi I, Ohki M. (2000). Blood, 96: 655-663. MEDLINE
Shinobu N, Maeda T, Aso T, Ito T, Kondo T, Koike K, Hatakeyama M. (1999). J. Biol. Chem., 274: 17003-17010. MEDLINE
Slany RK, Lavau C, Cleary ML. (1998). Mol. Cell. Biol., 18: 122-129. MEDLINE
Slomiany BA, D'Arigo KL, Kelly MM, Kurtz DT. (2000). Mol. Cell. Biol., 20: 5986-5997. MEDLINE
Smith AJ, De Sousa MA, Kwabi-Addo B, Heppell-Parton A, Impey H, Rabbitts P. (1995). Nat. Genet., 9: 376-385. MEDLINE
Soignet S, Fleischauer A, Polyak T, Heller G, Warrell JrRP. (1997). Cancer Chemother. Pharmacol., 40: ((Suppl)) S25-S29. Article MEDLINE
Soignet SL, Frankel SR, Douer D, Tallman MS, Kantarjian H, Calleja E, Stone RM, Kalaycio M, Scheinberg DA, Steinherz P, Sievers EL, Coutre S, Dahlberg S, Ellison R, Warrell JrRP. (2001). J. Clin. Oncol., 19: 3852-3860. MEDLINE
Song WJ, Sullivan MG, Legare RD, Hutchings S, Tan X, Kufrin D, Ratajczak J, Resende IC, Haworth C, Hock R, Loh M, Felix C, Roy DC, Busque L, Kurnit D, Willman C, Gewirtz AM, Speck NA, Bushweller JH, Li FP, Gardiner K, Poncz M, Maris JM, Gilliland DG. (1999). Nat. Genet., 23: 166-175. Article MEDLINE
Sood R, Talwar-Trikha A, Chakrabarti SR, Nucifora G. (1999). Leukemia, 13: 348-357. MEDLINE
Stein GS, van Wijnen AJ, Stein JL, Lian JB, Montecino M, Zaidi K, Javed A. (2000). J. Cell Biochem., 35: ((Suppl)) 84-92.
Strick R, Strissel PL, Borgers S, Smith SL, Rowley JD. (2000). Proc. Natl. Acad. Sci. USA, 97: 4790-4795. MEDLINE
Strom D, Nip J, Westendorf JJ, Linggi B, Lutterbach B, Downing JR, Lenny N, Hiebert SW. (2000). J. Biol. Chem., 275: 3438-3445. MEDLINE
Stroschein SL, Wang W, Zhou S, Zhou Q, Luo K. (1999). Science, 286: 771-774. Article MEDLINE
Sun Y, Liu X, Eaton EN, Lane WS, Lodish HF, Weinberg RA. (1999a). Mol. Cell, 4: 449-509.
Sun Y, Liu X, Ng-Eaton E, Lodish HF, Weinberg RA. (1999b). Proc. Natl. Acad. Sci. USA, 96: 12442-12447. Article MEDLINE
Swirsky DM, Li YS, Matthews JG, Flemans RJ, Rees JK, Hayhoe FG. (1984). Br. J. Haematol., 56: 199-213. MEDLINE
Takahashi A, Satake M, Yamaguchi-Iwai Y, Bae SC, Lu J, Maruyama M, Zhang YW, Oka H, Arai N, Arai K. (1995). Blood, 86: 607-616. MEDLINE
Takeuchi S, Seriu T, Bartram CR, Golub TR, Reiter A, Miyoshi I, Gilliland DG, Koeffler HP. (1997). Leukemia, 11: 1220-1223. MEDLINE
Tanaka T, Nishida J, Mitani K, Ogawa S, Yazaki Y, Hirai H. (1994). J. Biol. Chem., 269: 24020-24026. MEDLINE
Testa U, Masciulli R, Tritarelli E, Pustorino R, Mariani G, Martucci R, Barberi T, Camagna A, Valtieri M, Peschle C. (1993). J. Immunol., 150: 2418-2430. MEDLINE
Thirman MJ, Gill HJ, Burnett RC, Mbangkollo D, McCabe NR, Kobayashi H, Ziemin-van der Poel S, Kaneko Y, Morgan R, Sandberg AA, Chaganti RSK, Larson RA, LeBeau MM, Diaz MO, Rowley JD. (1993). N. Engl. J. Med., 329: 909-914. MEDLINE
Thorsteinsdottir U, Kroon E, Jerome L, Blasi F, Sauvageau G. (2001). Mol. Cell. Biol., 21: 224-234. MEDLINE
Tokitou F, Nomura T, Khan MM, Kaul SC, Wadhwa R, Yasukawa T, Kohno I, Ishii S. (1999). J. Biol. Chem., 274: 4485-4488. MEDLINE
Turley JM, Falk LA, Ruscetti FW, Kasper JJ, Francomano T, Fu T, Bang OS, Birchenall-Roberts MC. (1996). Cell Growth Differ., 7: 1535-1544. MEDLINE
Uchida H, Downing JR, Miyazaki Y, Frank R, Zhang J, Nimer SD. (1999). Oncogene, 18: 1015-1022. MEDLINE
Uchida H, Zhang J, Nimer SD. (1997). J. Immunol., 158: 2251-2258. MEDLINE
Van Deursen J, Fornerod M, Van Rees B, Grosveld G. (1995). Proc. Natl. Acad. Sci. USA, 92: 7376-7380. MEDLINE
Van Ranst PC, Snoeck HW, Lardon F, Lenjou M, Nijs G, Weekx SF, Rodrigus I, Berneman ZN, Van Bockstaele DR. (1996). Exp. Hematol., 24: 1509-1515. MEDLINE
Visani G, Bernasconi P, Boni M, Castoldi GL, Ciolli S, Clavio M, Cox MC, Cuneo A, Del Poeta G, Dini D, Falzetti D, Fanin R, Gobbi M, Isidori A, Leoni F, Liso V, Malagola M, Martinelli G, Mecucci C, Piccaluga PP, Petti MC, Rondelli R, Russo D, Sessarego M, Specchia G, Testoni N, Torelli G, Mandelli F, Tura S. (2001). Leukemia, 15: 903-909. MEDLINE
Wang L, Hiebert SW. (2001). Oncogene, 20: 3716-3725. MEDLINE
Wang J, Hoshino T, Redner RL, Kajigaya S, Liu JM. (1998a). Proc. Natl. Acad. Sci. USA, 95: 10860-10865. Article MEDLINE
Wang LC, Kuo F, Fujiwara Y, Gilliland DG, Golub TR, Orkin SH. (1997). EMBO J., 16: 4374-4383. Article MEDLINE
Wang LC, Swat W, Fujiwara Y, Davidson L, Visvader J, Kuo F, Alt FW, Gilliland DG, Golub TR, Orkin SH. (1998b). Genes Dev., 12: 2392-2402. MEDLINE
Wang Q, Stacy T, Binder M, Marin-Padilla M, Sharpe AH, Speck NA. (1996a). Proc. Natl. Acad. Sci. USA, 93: 3444-3449. Article MEDLINE
Wang Q, Stacy T, Miller JD, Lewis AF, Gu TL, Huang X, Bushweller JH, Bories JC, Alt FW, Ryan G, Liu PP, Wynshaw-Boris A, Binder M, Marin-Padilla M, Sharpe AH, Speck NA. (1996b). Cell, 87: 697-708. MEDLINE
Wang ZG, Delva L, Gaboli M, Rivi R, Giorgio M, Cordon-Cardo C, Grosveld F, Pandolfi PP. (1998c). Science, 279: 1547-1551. Article MEDLINE
Wang ZG, Ruggero D, Ronchetti S, Zhong S, Gaboli M, Rivi R, Pandolfi PP. (1998d). Nat. Genet., 20: 266-272. Article MEDLINE
Warrell JrRP. (1996). Annu. Rev. Med., 47: 555-565. MEDLINE
Weekx SF, Plum J, Van Cauwelaert P, Lenjou M, Nijs G, De Smedt M, Vanhove M, Muylaert P, Van Bockstaele DR, Berneman ZN, Snoeck HW. (1999). Leukemia, 13: 1266-1272. MEDLINE
Weis K, Rambaud S, Lavau C, Jansen J, Carvalho T, Carmo-Fonseca M, Lamond A, Dejean A. (1994). Cell, 76: 345-356. MEDLINE
Wells RA, Hummel JL, De Koven A, Zipursky A, Kirby M, Dube I, Kamel-Reid S. (1996). Leukemia, 10: 735-740. MEDLINE
Westendorf JJ, Yamamoto CM, Lenny N, Downing JR, Selsted ME, Hiebert SW. (1998). Mol. Cell. Biol., 18: 322-333. MEDLINE
Wotton D, Ghysdael J, Wang S, Speck NA, Owen MJ. (1994). Mol. Cell. Biol., 14: 840-850. MEDLINE
Xie XQ, Pardali E, Holm M, Sideras P, Grundstrom T. (1999). Eur. J. Immunol., 29: 488-498. Article MEDLINE
Xu J, Attisano L. (2000). Proc. Natl. Acad. Sci. USA, 97: 4820-4825. Article MEDLINE
Yagi R, Chen LF, Shigesada K, Murakami Y, Ito Y. (1999). EMBO J., 18: 2551-2562. MEDLINE
Yamamoto Y, Kiyoi H, Nakano Y, Suzuki R, Kodera Y, Miyawaki S, Asou N, Kuriyama K, Yagasaki F, Shimazaki C, Akiyama H, Saito K, Nishimura M, Motoji T, Shinagawa K, Takeshita A, Saito H, Ueda R, Ohno R, Naoe T. (2001). Blood, 97: 2434-2439. Article MEDLINE
Yanagisawa J, Yanagi Y, Masuhiro Y, Suzawa M, Watanabe M, Kashiwagi K, Toriyabe T, Kawabata M, Miyazono K, Kato S. (1999). Science, 283: 1317-1321. Article MEDLINE
Yang R, Nakamaki T, Lubbert M, Said J, Sakashita A, Freyaldenhoven BS, Spira S, Huynh V, Muller C, Koeffler HP. (1999). Blood, 93: 2067-2074. MEDLINE
Yano T, Nakamura T, Blechman J, Sorio C, Dang CV, Geiger B, Canaani E. (1997). Proc. Natl. Acad. Sci. USA, 94: 7286-7291. MEDLINE
Yaseen NR, Blobel G. (1997). Proc. Natl. Acad. Sci. USA, 94: 4451-4456. MEDLINE
Yergeau DA, Hetherington CJ, Wang Q, Zhang P, Sharpe AH, Binder M, Marin-Padilla M, Tenen DG, Speck NA, Zhang DE. (1997). Nat. Genet., 15: 303-306. MEDLINE
Yuan Y, Zhou L, Miyamoto T, Iwasaki H, Harakawa N, Hetherington CJ, Burel SA, Lagasse E, Weissman IL, Akashi K, Zhang DE. (2001). Proc. Natl. Acad. Sci. USA, 98: 10398-10403. MEDLINE
Zeleznik-Le NJ, Harden AM, Rowley JD. (1994). Proc. Natl. Acad. Sci. USA, 91: 10610-10614. MEDLINE
Zeng C, McNeil S, Pockwinse S, Nickerson J, Shopland L, Lawrence JB, Penman S, Hiebert S, Lian JB, van Wijnen AJ, Stein JL, Stein GS. (1998). Proc. Natl. Acad. Sci. USA, 95: 1585-1589. MEDLINE
Zeng C, van Wijnen AJ, Stein JL, Meyers S, Sun W, Shopland L, Lawrence JB, Penman S, Lian JB, Stein GS, Hiebert SW. (1997). Proc. Natl. Acad. Sci. USA, 94: 6746-6751. MEDLINE
Zhang DE, Hetherington CJ, Meyers S, Rhoades KL, Larson CJ, Chen HM, Hiebert SW, Tenen DG. (1996a). Mol. Cell. Biol., 16: 1231-1240. MEDLINE
Zhang DE, Hohaus S, Voso MT, Chen HM, Smith LT, Hetherington CJ, Tenen DG. (1996b). Curr. Top Microbiol. Immunol., 211: 137-147. MEDLINE
Zhang DE, Zhang P, Wang ND, Hetherington CJ, Darlington GJ, Tenen DG. (1997). Proc. Natl. Acad. Sci. USA, 94: 569-574. Article MEDLINE
Zhang Y, Derynck R. (2000). J. Biol. Chem., 275: 16979-16985. Article MEDLINE
Zhong S, Muller S, Ronchetti S, Freemont PS, Dejean A, Pandolfi PP. (2000a). Blood, 95: 2748-2752. MEDLINE
Zhong S, Salomoni P, Pandolfi PP. (2000b). Nat. Cell Biol., 2: E85-E90. Article MEDLINE
Zhong S, Salomoni P, Ronchetti S, Guo A, Ruggero D, Pandolfi PP. (2000c). J. Exp. Med., 191: 631-640. MEDLINE
Figure 1 Structural domains within the leukemia-associated fusion proteins. Illustrated are structural motifs found in the leukemia-associated fusion proteins. Selected CBF leukemia-associated fusion proteins are shown in (a). DNA-binding regions are denoted by their proximity to a double-helix and are shaded blue. Activator and repression domains, and the co-activators or co-repressors with which they interact are shown in green and red, respectively. Dimerization motifs are presented with teeth. Nuclear and subnuclear localization signals are flagged. (b) Depicts the common APL-associated fusions. The scheme is essentially the same as for the CBF fusions although an additional ligand-binding domain is labeled. (c) Presents four of the more common MLL fusions and the relevant structural motifs present in its fusion partners. (d) Diagrams the NUP98/HOXA9 fusion protein, which is representative of the NUP98/Homeobox fusion proteins
Table 1 Common translocations found in AML. Listed are the more common cytogenic abnormalities found in AML. They are grouped as translocations involving Core Binding Factor (CBF), APL-associated lesions, translocations involving MLL, and those involving NUP98. Within each group, they are ordered roughly according to their prevalence. (For excellent reviews see Mrozek et al., 2001 and Mitelman et al., 2002)
Table 2 Functional features of the leukemia-associated fusion proteins. Listed are the more common AML-associated fusion proteins resulting from balanced translocations. As in Table , they are grouped as translocations involving Core Binding Factor (CBF), APL-associated lesions, translocations involving MLL, and those involving NUP98. IUP codes for variable bases are used as follows: Y, representing pyrimidines T or C; R, representing purines G or A; and M, representing A or C
|13 May 2002, Volume 21, Number 21, Pages 3422-3444|
|Table of contents Previous Article Next [PDF]