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10 September 2001, Volume 20, Number 40, Pages 5680-5694
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Common themes in the pathogenesis of acute myeloid leukemia
Myriam Alcalay1, Annette Orleth2, Carla Sebastiani2, Natalia Meani1, Ferdinando Chiaradonna1, Cristina Casciari2, Maria Teresa Sciurpi2, Vania Gelmetti2, Daniela Riganelli2, Saverio Minucci1, Marta Fagioli2 and Pier Giuseppe Pelicci1

1Department of Experimental Oncology, European Institute of Oncology, 20141 Milan, Italy

2Università degli Studi di Perugia, Policlinico Monteluce, 06100 Perugia, Italy

Correspondence to: M Alcalay, Department of Experimental Oncology, European Institute of Oncology, Via Ripamonti 435, 20141 Milan, Italy. E-mail: malcalay@ieo.it

Abstract

The pathogenesis of acute myeloid leukemia is associated with the appearance of oncogenic fusion proteins generated as a consequence of specific chromosome translocations. Of the two components of each fusion protein, one is generally a transcription factor, whereas the other partner is more variable in function, but often involved in the control of cell survival and apoptosis. As a consequence, AML-associated fusion proteins function as aberrant transcriptional regulators that interfere with the process of myeloid differentiation, determine a stage-specific arrest of maturation and enhance cell survival in a cell-type specific manner. The abnormal regulation of transcriptional networks occurs through common mechanisms that include recruitment of aberrant co-repressor complexes, alterations in chromatin remodeling, and disruption of specific subnuclear compartments. The identification and analysis of common and specific target genes regulated by AML fusion proteins will be of fundamental importance for the full understanding of acute myeloid leukemogenesis and for the implementation of disease-specific drug design. Oncogene (2001) 20, 5680-5694.

Keywords

acute myeloid leukemia; fusion proteins; transcription factors; differentiation; cell survival

The study of specific chromosome abnormalities in acute myeloid leukemia (AML) has provided much information of diagnostic and prognostic relevance, and has greatly increased our knowledge of the mechanisms underlying the pathogenesis of these diseases. Analysis of the karyotypes of leukemic patients has led to the finding that non-random, somatically acquired translocations and inversions are present in the vast majority of acute leukemias, and to the association of specific aberrations with specific subtypes of AML (Look, 1997, and references therein).

Chromosome translocations in AML give rise to gene fusions at the site of the chromosomal breaks. The coding exons of the two genes involved become juxtaposed, and form a single fusion gene, which gives origin to a novel hybrid protein with unique features. The most frequent targets of such events in AML are genes encoding transcriptional regulators, which, after recombination, give origin to functional hybrid genes encoding for fusion proteins with aberrant functions. These abnormal transcription factors, activated in particular subtypes of AML, are selective for defined stages of myeloid differentiation, suggesting that they interfere with transcriptional and functional networks that are essential at various stages of hematopoiesis.

A large number of diverse translocations has been described in AML (Mitelman, 1994). The most frequent are the t(8;21), t(15;17), inv(16) and t(9;11) which, together with their variants, account for approximately 40% of all AMLs (Look, 1997). These translocations encode for the AML1/ETO, PML/RARalpha, the CBFbeta/SMMHC, and MLL/AF9 fusion proteins, respectively. The biological properties of these fusion proteins have been widely investigated in recent years, and there is a large amount of evidence supporting their relevance in the pathogenesis of the corresponding leukemias. Many other chromosome translocations have been described in AML, including t(3;5), t(6,9), t(16;21), t(7;11), which are present, however, in less than 10% of cases. Not all of them have been studied in sufficient detail as to connect their presence in leukemic blasts directly with leukemogenesis. Random chromosome aberrations have been described in 30% of AML cases, whereas 20% of cases displays a normal karyotype (Look, 1997). This review will focus on the discussion of the biological activities of the four most frequent categories of AML-associated fusions, in an attempt to identify common features in the diverse leukemogenic pathways.

The PML/RARalpha fusion gene is formed as a consequence of the t(15,17), present in over 95% of cases of the M3 subtype of AML (acute promyelocytic leukemia, or APL) (Grignani et al., 1994). RARalpha, (retinoic acid receptor alpha), is a member of the steroid-thyroid receptor superfamily of nuclear hormone receptors (Mangelsdorf and Evans, 1995), preferentially expressed in myeloid cells (de The et al., 1989). PML (promyelocytic leukemia) is a nuclear protein that localizes in distinct matrix-associated structures known as nuclear bodies, which regulates senescence and apoptosis and functions as a growth suppressor (Zhong et al., 2000).

The rare cases of APL that do not display t(15;17) always carry RARalpha recombinations with partners located at other chromosomal sites. The t(11;17) generates a fusion between the RARalpha gene and the PLZF (promyelocytic zinc finger) gene, which encodes for a member of the POZ/zinc-finger family of transcription factors (Chen et al., 1993b). The t(5;17) fuses the NPM (nucleophosmin) gene to RARalpha (Redner et al., 1996). Although this is a rare translocation in APL, NPM represents an interesting target in that it is involved in other two chromosomal translocations found in hematopoietic malignancies: the t(3;5) of myelodysplastic syndrome and AMLs of diverse subtypes, which fuses NPM to MLF1 (myelodysplasia/myeloid leukemia factor 1) (Yoneda-Kato et al., 1996), and the t(2;5) of anaplastic large cell lymphoma, where NPM is fused to the tyrosine kinase ALK (anaplastic lymphoma kinase) (Morris et al., 1994). Other rare APL-associated translocations are another t(11;17), which fuses the nuclear mitotic apparatus protein NuMa to RARalpha (Wells et al., 1997), and the t(17;17), where RARalpha is fused to the signal transducer and activator of transcription STAT5b (Arnould et al., 1999). It is interesting to note that, at least in the case of the 15;17 and 11;17 translocations, the reciprocal fusion genes RARalpha/PML and RARalpha/PLZF are also transcribed and encode for expressed fusion proteins (Alcalay et al., 1992; Chen et al., 1993a). Although the functional properties of the latter remain largely unknown, transgenic model systems suggest they may have a role in promoting the leukemic phenotype (He et al., 2000; Zimonjic et al., 2000).

Characteristic of APL is the marked sensitivity of promyelocytic blasts to differentiation induced by retinoic acid (RA) both in vitro and in vivo (reviewed by Lin et al., 1999). This property resulted in the currently routine use of all-trans retinoic acid (ATRA) in APL therapy, and represents the first successful attempt of differentiation therapy that specifically targets the aberrant protein underlying disease onset (Castaigne et al., 1990; Huang et al., 1988). However, the use of ATRA as a single therapeutic agent is not sufficient: in the absence of additional chemotherapy, patients rapidly relapse, and become resistant to further ATRA treatment for reasons that remain to date unknown (Degos, 1992). RA sensitivity is directly connected to the type of translocation that causes APL. Patients bearing the t(11;17) do not respond to ATRA therapy, just as myeloid progenitors expressing the PLZF/RARalpha fusion do not differentiate upon RA treatment in vitro (Guidez et al., 1994; Ruthardt et al., 1997).

AML1 and CBFbeta represent two subunits of a transcriptional regulator known as core binding factor (CBF) or polyoma enhancer binding protein (PEBP2), which is fundamental for normal hematopoiesis (reviewed by Friedman, 1999). The AML1 gene recombines with the ETO (eight-twenty-one or MTG8) gene in the t(8;21), characteristic of the AML M2 subtype, but is also involved in other gene fusions with the EVI-1, MDS1 or EAP genes on chromosome 3, or the TEL gene on chromosome 12 (Friedman, 1999). The resulting AML1/EVI-1, AML1/MDS1, AML1/EAP fusion products are associated with the onset of acute myeloid leukemias of different subtypes, while the TEL/AML1 fusion is characteristic of childhood acute lymphoid leukemia. The diversity of the phenotypes resulting from the expression of different AML1 fusion proteins suggests that each AML1 partner contributes with functions that give rise to a specific leukemogenic process. The CBFbeta gene, instead, selectively recombines with the smooth muscle myosin heavy chain (SMMHC or MHY11) gene as a consequence of an inversion within chromosome 16, or of the more rare t(16;16), and is found predominantly in the M4eo subtype of AML (Liu et al., 1993).

The MLL/AF9 fusion, formed as a consequence of the t(9;11), is the most frequent of the multiple MLL fusions associated with AML (DiMartino and Cleary, 1999). It fuses the MLL (mixed lineage leukemia, or myeloid/lymphoid leukemia) gene on chromosome 11, which encodes for a human homologue of the Drosophila Trithorax transcriptional regulator, to the AF9 gene encoding for a putative transcription factor on chromosome 9 (Nakamura et al., 1993). Another translocation involving MLL frequently associated with AML is the t(11;19) (DiMartino and Cleary, 1999), which represents, in reality, a group of translocations, and three different genes mapping to chromosome 19(q13) have to date been identified in fusions with MLL. The ENL gene encodes for a putative transcriptional regulator, which, interestingly, presents a very high degree of overall homology with the AF9 gene product, suggesting common functional properties (Nakamura et al., 1993; Rubnitz et al., 1994). The ELL gene encodes for an RNA polymerase II elongation factor (Thirman et al., 1994), and the third partner of MLL on chromosome 19, EEN, is the human homologue of a member of a recently described murine SH3 domain-containing protein family (So et al., 1997).

The great heterogeneity of MLL translocation partners and of the resulting leukemic phenotypes suggests that MLL is a specific target for chromosomal breaks, whereas the recombination of the broken MLL gene is not restricted to specific sequences. The existence of leukemias with internal duplications of MLL and no recombination with other loci further supports this view, and raises the question of the functional relevance of MLL fusion partners in leukemogenesis (Caligiuri et al., 1994; Schichman et al., 1994). Breakpoints in the MLL locus, which spans 100 kb, cluster within an 8.5 kb region comprising exons 5-11, (Gu et al., 1992; Tkachuk et al., 1992), which contains 11 partial consensus sequences for Topoisomerase II, and one perfect consensus in exon 9 (Strissel et al., 1998). This finding could partly explain the high rate of MLL rearrangements found in leukemias that arise as a consequence of treatment with Topoisomerase II inhibitors. The MLL breakpoint cluster region also contains a number of Alu repeats, which may lead to homologous recombination events, and may be responsible for the partial duplications (Strout et al., 1998).

Regardless of subtype, AML is characterized by a defect in the normal process of maturation that converts a myeloid precursor cell into a mature white blood cell. The block of differentiation is variably associated with abnormal proliferation, enhanced cell survival and diminished response to apoptotic stimuli. The complex biological phenomena that participate in generating a leukemic phenotype remain largely unknown, but the analysis of the functional properties of several of the leukemia-associated fusion proteins has helped to elucidate fundamental steps in leukemogenesis. Although leukemias are heterogeneous in terms of phenotype, disease progression, prognosis and response to therapy, there are general mechanisms underlying leukemic transformation, which might prove to be of great importance in our understanding and management of these diseases.

AML-associated fusion proteins directly interfere with the myeloid differentiation program

Hematopoiesis is a stepwise process, characterized by the alternate expression of specific transcriptional regulators, growth factors and growth factor receptors, whose combination determines lineage commitment and maturation (reviewed by Tenen et al., 1997). AML-associated fusion proteins, which function as aberrantly activated transcriptional regulators, have been shown to affect hematopoietic differentiation in a variety of experimental models, and the specific stage of myeloid maturation arrest appears to be directly dependent on the nature of the fusion protein expressed.

Cell lines sensitive to differentiation agents (such as U937, 32D, K562) transfected with specific AML fusions, or cell lines carrying specific AML-associated chromosome alterations (NB4, Kasumi) have been widely used to analyse this property. Expression of PML/RARalpha, PLZF/RARalpha, NPM/RARalpha or AML1/ETO in U937 cells abolishes maturation upon treatment with diverse stimuli, such as vitamin D3 + TGFbeta (Gelmetti et al., 1998; Grignani et al., 1993; Ruthardt et al., 1997; Ferrucci and Pelicci, 2001, unpublished results). AML1/ETO expression in 32D cells also reduces differentiation after treatment with G-CSF (Kohzaki et al., 1999), whereas PML/RARalpha inhibits the response to low doses of RA in myeloid cell lines (Grignani et al., 1993). In these systems, the presence of a functional fusion protein is necessary to maintain the undifferentiated phenotype. Direct dependence of the block of differentiation on the fusion protein is clearly demonstrated by the use of inducible systems in U937 cells (Gelmetti et al., 1998; Grignani et al., 1993; Ruthardt et al., 1997). In cell lines stably expressing the AML1/ETO fusion protein or derived from AML patient blasts carrying the t(8;21), introduction of antisense oligonucleotides directed against the fusion junction abolishes the block of differentiation (Sakakura et al., 1994). Although these results were obtained in immortalized or transformed cell lines, recent reports describe an analogous differentiation block for APL fusion proteins by retroviral-mediated transduction of primary hematopoietic progenitors (murine lineage cells, and human CD34+ cells) (Grignani et al., 2000; Minucci et al., 2000).

Even more striking are the effects on hematopoietic differentiation after in vivo expression of AML fusion proteins. Mice transgenic for AML-associated fusion proteins have been generated with various approaches and although the resulting phenotypes largely depend on the promoters driving transcription of the transgenes, alterations in hematopoietic maturation have recurrently been observed. The defects of myeloid maturation depend on the specific fusion protein analysed, and correlate with the leukemic subtype the fusions were isolated from.

There are diverse mouse models for APL (reviewed by Rego and Pandolfi, 2001). Mice transgenic for PML/RARalpha display a slight impairment in neutrophil maturation, with accumulation of promyelocytic precursors in the bone marrow before the onset of a frank leukemia with common features to the human counterpart (Brown et al., 1997; Grisolano et al., 1997; He et al., 1997). Mice transgenic for other RARalpha fusions display more complex phenotypes in terms of resulting diseases. NPM/RARalpha transgenic mice develop various types of leukemia, of which the majority are associated with impaired myeloid differentiation (Cheng et al., 1999). PLZF/RARalpha transgenic mice develop a form of chronic myeloid leukemia, but the bone marrow blasts still retain the potential to differentiate terminally (Cheng et al., 1999; He et al., 1998). In the t(15;17) and t(11;17), the reciprocal transcripts may play a role in leukemogenesis, as suggested by animal models. The co-expression of PML/RARalpha and RARalpha/PML in transgenic mice increases the frequency of APL development (Zimonjic et al., 2000). Transgenic mice expressing the RARalpha/PLZF fusion present defective myelopoiesis, which is not followed by leukemia. However, as opposed to transgenic mice expressing the single fusion proteins, PLZF/RARalpha-RARalphaPLZF double transgenics develop leukemia with classic APL features (He et al., 2000). In the case of the t(11;17), the contribution of RARalpha/PLZF to leukemogenesis may be due to the presence of the DNA binding domain of PLZF in the context of this fusion protein, which may thus disturb the normal regulatory function of PLZF (Guidez et al., 1998).

The phenotypes of animals transgenic for fusions involving core binding factor subunits are even more dramatic. Mice heterozygous for either AML1/ETO or CBFbeta/SMMHC knock-in fail to develop definitive hematopoiesis, and die at day 13.5 or 12.5 of embryogenesis, respectively (Castilla et al., 1996; Okuda et al., 1998; Yergeau et al., 1997). Mice that inducibly express AML1-ETO under the control of a tetracycline-responsive element display a partial block of myeloid differentiation and an increased replating efficiency of progenitor cells in serial replating colony assays (Rhoades et al., 2000). Chimeric mice generated with heterozygous CBFbeta/SMMHC ES cells display defects in myeloid and lymphoid differentiation, and after treatment with an alkylating mutagen develop myelomonocytic leukemia (Castilla et al., 1999). Other transgenic mice in which the expression of CBFbeta/SMMHC is driven by the hMRP8 myeloid specific promoter do not develop spontaneous leukemia, but display an increase of immature neutrophilic cells in the bone marrow and impaired neutrophil differentiation of bone marrow cells in vitro (Kogan et al., 1998).

MLL fusion proteins also arrest differentiation of myeloid precursors. Although in vitro studies performed on cell lines do not demonstrate a clear-cut effect of MLL-fusions on myeloid maturation, in vivo expression of different MLL-AF9 or MLL-ELL transgenes in mice results in acute myeloid leukemias, which are preceded by a pre-leukemic phase characterized by an arrest of myeloid differentiation (Corral et al., 1996; Dobson et al., 1999; Lavau et al., 2000a). Also, MLL/ENL or MLL/ELL expression in purified murine hematopoietic precursor cells results in immortalization of immature myeloid progenitors (Lavau et al., 1997, 2000b).

A peculiarity of RARalpha fusions is the dose-dependent, dual response to RA. Cell lines expressing RARalpha fusions or blasts derived from APL patients do not respond to low doses of RA. Response to therapeutic doses of RA is, instead, diversified among the different RARalpha fusions, and recapitulates the sensitivity to ATRA treatment of APL patients bearing the different translocations. PML/RARalpha expression, in fact, confers an enhanced sensitivity to cell lines or APL blasts treated with RA in vitro (Chomienne et al., 1990; Grignani et al., 1993), whereas PLZF/RARalpha expression does not (Guidez et al., 1994; Ruthardt et al., 1997). The RA response displayed by in vitro models is confirmed by a similar therapeutic response to RA treatment in transgenic mouse models (Brown et al., 1997; Cheng et al., 1999; Grisolano et al., 1997; He et al., 1997). Less is known about the response to ATRA of patients bearing rare translocations; however, for the NuMa/RARalpha and NPM/RARalpha fusion proteins, RA sensitivity is predictable from the response of blasts in culture and of transgenic animals (Cheng et al., 1999; Redner et al., 1997; Wells et al., 1996).

Although the available evidence suggests that each fusion protein targets a specific stage of maturation and may require specific conditions to exert its leukemogenic potential, interference with normal myelopoiesis appears to be a common feature among all AML-associated fusion proteins. Furthermore, as exemplified by the RARalpha fusions of APL, response to specific differentiative stimuli is modulated by the fusion proteins.

AML fusion proteins regulate cell survival

Interference with terminal differentiation accounts for part of the leukemic phenotype, but other biological properties of AML-associated fusion proteins are clearly linked with clonal expansion. Indeed, another common functional characteristic of AML fusion proteins is their capacity to interfere with the regulation of cell survival. In the majority of cases, these proteins do not directly impair cell cycle regulation, but rather they enhance survival in conditions where a normal cell would undergo programmed cell death, such as growth factor deprivation. PML/RARalpha expression in U937 cells, for example, permits long term survival in low serum conditions (Grignani et al., 1993), and two other RARalpha fusions, PLZF/RARalpha and NPM/RARalpha present an analogous behavior in this cell system (Colombo and Pelicci, 2001, unpublished results). PML/RARalpha also abolishes TNFalpha induced apoptosis in U937 cells (Testa et al., 1998) and notably reduces apoptosis after growth factor deprivation of the GM-CSF dependent cell line TF-1 (Rogaia et al., 1995). More strikingly, PML/RARalpha expression protects purified human hematopoietic progenitor cells from apoptosis after growth factor deprivation (Grignani et al., 2000). In agreement with these findings, targeting of the PML/RARalpha transcript in the NB4 cell line with hammerhead rybozyme, which diminishes PML/RARalpha mRNA, induces apoptosis (Nason-Burchenal et al., 1998).

CBF-derived fusion proteins also affect cell survival. Expression of AML1/ETO in murine adult bone marrow hematopoietic progenitors results in immortalization of derived cell lines (Okuda et al., 1998; Yergeau et al., 1997) and myeloid precursors expressing AML1/ETO display an increased efficiency in serial replating colony assays (Rhoades et al., 2000). AML1/ETO was reported to activate the anti-apoptotic protein bcl-2 in cell lines bearing the t(8;21), but these results were not confirmed by analysis of bcl-2 levels in blasts form leukemic patients expressing the AML1/ETO fusion protein (Banker et al., 1998; Klampfer et al., 1996). Degradation of AML1/ETO in Kasumi cells through hammerhead rybozyme cleavage induces apoptotic cell death (Matsushita et al., 1999). The expression of CBFbeta/SMMHC fusion protein in Ba/F3 cells inhibits apoptosis after exposure to ionizing radiation or etoposide but not to growth factor withdrawal. This effect is connected to attenuation of p53 induction and is independent of p21 activity (Britos-Bray et al., 1998).

A number of direct and indirect evidences suggest that MLL fusion proteins also play a role in the control of cell survival. Cell lines carrying the t(4;11) exhibit prolonged cell survival after serum starvation (Kersey et al., 1998), and retrovirus mediated introduction of t(11,19) fusion products MLL/ELL or MLL/ENL immortalizes murine hematopoietic precursors (Lin- cells) in vitro (Lavau et al., 1997, 2000b). Although the mechanisms of immortalization remain largely unknown, there is some evidence that MLL fusions interfere with apoptosis: MLL/AF9, MLL/ELL and MLL/ENL fusion proteins can physically interact with the apoptosis enhancer GADD34 and abrogate GADD34-mediated apoptosis (Adler et al., 1999). Furthermore, the MLL/ELL fusion protein binds to p53 through its ELL moiety and suppresses p53-dependent transactivation in luciferase assays (Maki et al., 1999; Shinobu et al., 1999). In line with the findings described above, antisense oligonucleotides directed against the MLL/ENL junction inhibit cell growth and induce apoptosis of the KOCL33 cell line carrying the t(11:19) (Akao et al., 1998).

AML-associated fusion proteins appear capable of interfering with the cellular programs that regulate apoptosis, and it is plausible to hypothesize that cell survival independent from growth factor and cytokine stimulation plays an important role in the leukemogenic process. Paradoxically, expression of AML fusion proteins causes cell growth arrest and/or apoptosis in non-hematopoietic cell lines, and they are also capable of inducing apoptosis in non-permissive hematopoietic cell lines (for example, 32D cells for PML-RAR) (Ferrucci et al., 1997; Minucci and Pelicci, 2001, unpublished results). Although the mechanism(s) mediating anti-apoptotic properties in a defined cellular context, and triggering apoptosis in other cells are unknown, AML fusion proteins apparently select specific target cells where they exert a pro-survival function.

Transcriptional regulators involved in AML-associated fusions control myeloid differentiation

In the vast majority of cases, one of the two genes targeted by the chromosomal breaks in AML encodes for a protein that functions as a transcriptional regulator. The other partner can be variable, just as variable are its functional properties, and this may be associated with disease phenotype. Some genes are, in fact, involved in different chromosome translocations which give rise to different diseases, like the TEL/AML fusion of childhood acute lymphoblastic leukemia (ALL), or several MLL fusions also involved in ALL, and this suggests that the fusion with specific partners confers unique functional properties.

All RARalpha fusion proteins, which are the hallmark of APL, contain the same portion of the RARalpha protein at the C-terminus, fused to variable N-terminal portions of partner genes. The native RARalpha protein is a member of the family of retinoic acid receptors, which are structurally divided into six functional domains, including a zinc-finger domain with DNA binding capacity, a ligand binding domain, and two different domains involved in transcriptional regulation (reviewed by Mangelsdorf and Evans, 1995). RARs are ligand-dependent transcription factors, which act by binding as heterodimers with RXR to specific responsive elements (RAREs) localized in the promoter regions of regulated genes (Mangelsdorf and Evans, 1995). In the absence of ligand, RARs repress transcription by recruiting histone deacetylase through direct binding to co-repressors N-CoR and SMRT. RA releases this interaction, favoring recruitment of co-activators, and consequently transcription. (Mangelsdorf and Evans, 1995; Minucci and Ozato, 1996).

Disruptions of the various RAR loci in mice do not lead to defects in hematopoiesis (reviewed by Kastner et al., 1995). In particular, RARalpha-null mice have a normal granulocyte population. This observation would lead to discard the role of RARalpha as a major regulator of myeloid maturation, but it may also reflect the ability of other RA receptors to compensate for its function, as suggested by the impaired response to retinoids of granulocyte progenitors derived from RARalpha/RARgamma double mutants (Kastner et al., 2001). The analysis of myeloid progenitors derived from RARalpha-null mice revealed that RARalpha regulates granulopoiesis in a ligand-dependant manner. In the absence of RA (or when bound to an antagonist), RARalpha behaves as an inhibitor of differentiation, probably through a mechanism that involves transcriptional repression. In the presence of RA, RARalpha expression results in enhanced granulopoiesis (Kastner et al., 2001).

The identification of RARalpha regulated genes could help to gain insight about its role in hematopoiesis. The few direct targets known to date appear to be transcription factors, including the retinoic acid receptors themselves (Gudas, 1994). Homeobox genes, such as hoxb1 and CRAB-PII, whose coordinated expression is essential for hematopoiesis, are no longer induced by retinoic acid treatment of embryonic carcinoma cells with a homozygous disruption of the RARalpha gene, identifying them as true direct targets (Boylan et al., 1995; Gudas, 1994). Another recently identified target of RARalpha is the transcription factor C/EBPepsilon, a member of the C/EBP family preferentially expressed in myeloid cells. Forced expression of C/EBPepsilon in U937 cells mimics terminal granulocytic differentiation, including morphologic changes, increased CD11b/CD66b expression, and induction of secondary granule protein expression (Park et al., 1999). The cdk-inhibitor p21 is also a direct target of RARalpha, and its transcription is strongly induced during differentiation of U937 cells treated with RA (Liu et al., 1996a). Overexpression of p21 in U937 cells results in the cell-surface expression of monocyte/macrophage-specific markers even in the absence of differentiation agents, suggesting that ligand-modulated induction of the p21 gene facilitates myeloid differentiation (Liu et al., 1996b). All these data point towards an involvement of RARalpha-dependent regulation in the control of myeloid differentiation.

There is no ambiguity on the relevance of CBF transcription factors in hematopoiesis. CBFs are heterodimers including one of three DNA binding alpha subunits (known as AML1 or CBFalpha2, AML2 or CBFalpha3 and AML3 or CBFalpha1) and one beta subunit, which does not directly bind DNA but increases DNA-binding affinity of CBFalpha subunits (Levanon et al., 1994; Ogawa et al., 1993; Wang et al., 1993). The alpha subunits contain a Runt domain homologous to that of Drosophila transcriptional regulators involved in the control of segmentation and development. The Runt-homology domain (RHD) confers the ability of these proteins to bind a specific consensus DNA sequence and to interact with the CBFbeta subunit (Kagoshima et al., 1993; Meyers et al., 1993). CBFalpha subunits have other functionally relevant domains which are: a proline-serine-threonine rich region (PST), a nuclear matrix targeting signal (NMTS), which in conjunction to the PST domain forms a transactivation domain, a second transactivation domain, and a C-terminal VWRPY domain that binds the Groucho and TLE co-repressors (reviewed by Downing, 1999; Friedman, 1999). CBFbeta presents homology to Drosophila Runt-interacting proteins Brother and Big-brother, which have been shown to modulate the Runt-DNA interaction (Golling et al., 1996). Interaction with the CBFbeta subunit has been shown to protect the AML1 protein from rapid ubiquitin/proteasome-mediated degradation, and represents therefore an essential step in the generation of a functional CBF complex (Huang et al., 2001).

Expression of CBF subunits is not completely overlapping, suggesting cell-type specific functions. The CBFbeta subunit is widely expressed (Golling et al., 1996; Ogawa et al., 1993; Wang et al., 1993), whereas AML1 expression during development is limited to hematopoietic stem cells, endothelial cells of the aorta-gonad-mesonephros, condrogenic centers, olfactory and gustatory mucosa and neural ganglion cells; while after organogenesis it is confined to hematopoietic cells (Cai et al., 2000; Corsetti and Calabi, 1997; Simeone et al., 1995).

The CBF complex functions as a transcriptional activator. This function is consequential to the direct interaction of AML1 with p300, and subsequent recruitment of co-activator complexes that possess histone acetyltransferase activity to the target promoters (Kitabayashi et al., 1998). Many genes which are reported targets of CBF activation have relevant roles in hematopoietic maturation, such as myeloperoxidase, colony-stimulating factor 1 receptor, T-cell receptor subunits, neutrophil elastase, interleukin-3, GM-CSF, CD11a, and c-fos (Downing, 1999; Hwang et al., 1999; Puig-Kroger et al., 2000). CBF can also repress transcription from target genes. AML1 interacts with co-repressors, such as Groucho and Sin3, and negatively modulates the expression of direct target genes like the cdk-inhibitor p21 (Levanon et al., 1998; Lutterbach et al., 2000). Binding of co-activators to CBFalpha subunits antagonises the binding of co-repressors, and the concerted activities of these proteins may serve to modulate CBF function (McLarren et al., 2000).

The ALM1/CBPbeta complex cooperates with other lineage-restricted transcription factors such as c-Myb, C/EBPalpha, Ets family members PU.1 and MEF, HES-1, Pax5 and Smad, which bind adjacent sites on DNA (Downing, 1999; Jakubowiak et al., 2000; Kim et al., 1999; Libermann et al., 1999; Mao et al., 1999; McLarren et al., 2000). In most cases this functional synergy requires direct physical interaction of the transcription factors. Indirect interactions are instead involved in cooperation with other transcription factors, such as LEF1.

Perhaps the clearest evidence of CBF involvement in hematopoiesis is the analysis of the effects of a homozygous disruption of either the AML1 or the CBFbeta genes in mouse models, which results in identical phenotypes. Mice deficient for either AML1 or CBFbeta lack definitive hematopoiesis in the fetal liver, and die during mid-embryonic development due to hemorrages in the nervous system (Okuda et al., 1996; Sasaki et al., 1996; Wang et al., 1996a,b). Differentiation along the myeloid lineage appears to be more severely impaired, since AML1-/- embryonic stem cells retain the capacity to differentiate into primitive erythroid cells in vitro (Okuda et al., 1996). 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, and AML1-/- ES cells do not contribute to hematopoiesis in chimeric animals (Okuda et al., 1996). Recent studies suggest that AML1 is involved in the emergence of hematopoietic cells from endothelial cells, and that this process represents a major pathway of definitive hematopoiesis (Yokomizo et al., 2001). Consistent with these results, the Drosophila transcription factor Lozenge, which resembles AML1, is necessary for the development of Drosophila hematic crystal cells during embryonic and larval hematopoiesis (Lebestky et al., 2000).

The MLL native protein is the human homologue of Drosophila Trithorax, a regulator of homeobox gene expression (Djabali et al., 1992; Tkachuk et al., 1992). Trithorax binds to transcriptionally active loci and ensures that this active state is inherited from the progenitor to the daughter cells after mitosis. It is therefore not per se a transcriptional activator, but rather provides the cell with a memory of transcriptional state (Paro, 1993).

MLL is a large protein with several functional domains (DiMartino and Cleary, 1999). There are three regions of homology with Trithorax: two central PHD zinc-finger domains, not involved in DNA binding, but in protein-protein interactions, and a highly conserved C-terminal region known as SET domain, characteristic of a group of multifunctional chromatin regulators with activities in both eu- and heterochromatin (Jenuwein et al., 1998). In support of an MLL function in chromatin structure is the finding that MLL interacts, though the SET domain, with the INI/SNR1 protein, which is a component of the SWI/SNF chromatin remodeling complex (Rozenblatt-Rosen et al., 1998). In the N-terminal region there are AT hook motifs homologous to those of the high mobility group proteins HMGI-C and HMGI(Y), which bind to the minor groove of DNA and also play a role in regulating chromatin structure. A 47 AA region with homology to the non-catalytic domain of DNA-methyltransferase is present between the AT-hooks and the PHD zinc-finger domains. There are two regions of the MLL protein which can regulate transcription of a reporter gene when fused to the GAL4 DNA-binding domain: the methyltransferase homology region functions as a repressor, whereas a region between the PHD zinc-fingers and the SET domain acts as an activator (Zeleznik-Le et al., 1994).

MLL is expressed at high levels in differentiated myeloid cells and macrophages, at low levels in earlier hematopoietic progenitors and in lymphocytes, and is not expressed in erythroid cells (Corral et al., 1996; Hess et al., 1997). This pattern of expression suggests that MLL favors myelomonocytic differentiation. Mouse embryos deficient for MLL show a marked reduction in the number of myeloid and macrophage colonies in yolk sac cultures, although terminal differentiation does not appear to be impaired (Hess et al., 1997).

There are very few known targets of MLL. Considering Trithorax function, it is to be expected that MLL regulates homeobox gene expression. Homozygous deletion of MLL is lethal, but mice with a heterozygous deletion of MLL display growth retardation and hematopoietic abnormalities. The expression of two homeobox genes, hoxa7 and hoxc9 was absent in cells derived from the yolk sac of MLL-null mice, and severely altered in heterozygotes (Yu et al., 1995). Another homeobox gene, ARP1, was isolated as a consequence of its differential expression in MLL-/- versus MLL+/+ embryonic stem cells (Arakawa et al., 1998). Regulation of homeobox genes by MLL is of particular importance for the leukemogenic potential of MLL fusions, considering the relevance of this group of genes in regulation of hematopoiesis (Tenen et al., 1997).

The evidence discussed above suggests that AML-associated fusion proteins constantly involve transcriptional regulators that function in controlling myeloid lineage maturation. Less is known about the function of the respective fusion partners, in particular from the perspective of leukemogenesis. Yet, there are several evidences indicating that AML-associated fusions cannot simply be considered as dominant negative mutants of the damaged transcription factors, and likewise, some of the known features of the translocation partners point to an active role in the acquisition of transforming potential.

The PML protein is the most frequent partner of RARalpha in generating the fusions that underlie promyelocytic leukemogenesis. PML is a member of a family of proteins which contain the RBCC (RING B-box coiled-coil) motif which mediates protein-protein interactions (Zhong et al., 2000). PML overexpression results in growth suppression and cell death in a variety of cellular models (Fagioli et al., 1998; Mu et al., 1994; Quignon et al., 1998). Coherent with these results is the resistance of PML deficient mice to apoptotic stimuli such as ionizing radiations or anti-Fas antibody treatment (Wang et al., 1998b). PML binds to p53 within the nuclear bodies (Pearson et al., 2000), and induces cell senescence as a direct consequence of p53 activation through acetylation, in a process that involves multimerization with the CBP acetyltransferase within PML nuclear bodies (Pearson et al., 2000). Another RARalpha partner, PLZF, a transcriptional repressor belonging to the POZ/domain and Kruppel zinc finger (POK) family, also regulates cell survival. Its expression, in fact, has a dramatic growth suppressive effect accompanied by an increased rate of apoptosis in the myeloid cell line 32D (Shaknovich et al., 1998). PLZF has been shown to bind to and repress cyclin A2 promoter, a finding that could explain its function as a negative cell-cycle regulator (Yeyati et al., 1999). Disruption of the PLZF locus in mice results in skeletal defects and homeotic alterations of limb bud formation, compatible with its growth-inhibitory and pro-apoptotic function (Barna et al., 2000).

It is more difficult to envisage common functions for the RARalpha fusion partners if we consider the rare translocations. NPM is a major nuclear matrix protein whose expression is associated with an increased resistance to UV-induced cell death (Higuchi et al., 1998). Overexpression of NPM has been described in several leukemic cell lines and NPM is capable of inducing transformation of NIH3T3 cells (Kondo et al., 1997). NuMa is a nuclear mitotic apparatus protein (Ferhat et al., 1998), and Stat5b is a cytoplasmic signal transducer belonging to the JAK/Stat signaling pathway shown to promote cytokine-dependent survival and proliferation of differentiating myeloid progenitors (Kieslinger et al., 2000). Further knowledge of the possible mechanisms of interference with transcriptional regulation of each fusion partner is necessary to explain the pathogenesis of an identical disease in vivo. Recent reports indicate that protein-protein interaction domains of RARalpha partners are relevant for the functional properties of the fusions, suggesting that their main contribution to leukemogenesis may lie in the capacity to form complexes that alter the regulatory characteristics of the native transcription factors (Lin and Evans, 2000; Minucci et al., 2000).

The AML1 partner ETO has several characteristics that point towards an active role in conferring transforming potential to the fusion protein. ETO, the mammalian homologue of Drosophila Nervy is a nuclear phosphoprotein expressed in brain and CD34+ hematopoietic progenitors (Era et al., 1995). It functions as a co-repressor and binds directly to N-CoR and Sin3A, recruiting HDAC, thus forming complexes that alter chromatin structure and mediate transcriptional repression. (Gelmetti et al., 1998; Lutterbach et al., 1998b; Wang et al., 1998a). Oligomerization of ETO is obligatory for co-repressor interaction, and as for RARalpha partners, is of particular importance in the context of the AML1/ETO fusion protein (Minucci et al., 2000; Zhang et al., 2001). Interestingly, ETO functions as a co-repressor for PLZF in an HDAC-dependent manner (Melnick et al., 2000b). ETO transforms NIH3T3 cells and injection of ETO expressing cells into irradiated and splenectomized nude mice induces tumor formation (Wang et al., 1997).

It is, of course, much more difficult to find common functions among the various MLL partners. Then again, specific MLL fusions are clearly associated with specific subtypes of leukemia, and although lineage specificity may be determined by the particular stage of hematopoiesis struck by the translocation, the functional properties of the translocation partner may well play an important role. Of the partner genes more frequently associated with myeloid leukemogenesis, AF-9 and ENL share common structural motifs and have 56% sequence identity overall. The presence of nuclear localization signals and serine/proline rich regions within these proteins suggests that they may function as transcriptional activators (Nakamura et al., 1993; Rubnitz et al., 1994; Tkachuk et al., 1992). Other transcriptional regulators, such as CBP and p300, are also involved in translocations with MLL (DiMartino and Cleary, 1999). ELL, a fusion partner of MLL involved in t(11;19), is an RNA polymerase II elongation factor that increases the rate of transcription by suppressing transient pausing. Inducible expression of ELL in the human embryonic kidney cell 293 promotes arrest of cell growth followed by apoptosis through an unknown mechanism (Johnstone et al., 2001). Contrarily, other studies demonstrate that expression of ELL in Rat1 cells results in anchorage-independent growth and decreased growth factor requirement (Kanda et al., 1998). ELL binds to p53 and blocks its transactivating properties, and overexpression of ELL in BaF3 cells results in protection from p53-mediated apoptosis induced by genotoxic stress (Maki et al., 1999; Shinobu et al., 1999). As for RARalpha partners, it has also been suggested that the presence of protein-protein interaction domains in MLL partner proteins may be of primary relevance in the context of the fusion proteins (Dobson et al., 2000).

In the t(X;11) and t(6;11), MLL is fused with the forkhead transcription factors AFX and FKHRL1, respectively (Borkhardt et al., 1997; Hillion et al., 1997). The AFX and FKHRL1 proteins are human orthologues of the C. elegance DAF-16 protein, which is involved in life-span regulation (Lin et al., 1997; Ogg et al., 1997). Overexpression of AFX or FKHRL1 induces a block of cell-cycle progression at G1 phase, through a mechanism that involves transcriptional activation of the cell-cycle inhibitor p27, (Medema et al., 2000) or apoptosis through activation of fas-ligand expression (Brunet et al., 1999). Although these translocations represent rare events, they once again underline the relevance of the deregulation of senescence and apoptotic pathways in leukemogenesis.

In summary, it appears that AML-associated fusion proteins preferentially alter the function of specific transcription factors involved in the control of myeloid differentiation. This is not surprising, considering the relevance of coordinated transcriptional regulation in the process of myeloid maturation. The precise functions of the RARalpha, AML1, CBFbeta and MLL partners are less clear. No evidence links the fusion partners directly with myeloid differentiation. Although it is difficult to find a common theme in this very heterogeneous and scarcely characterized group of proteins, several of them seem to influence cell growth and survival, just as several of them bear protein-protein interaction domains that are retained in the context of the fusion proteins. It is possible, at least for some AML-associated fusions, to hypothesize that the native proteins disrupted in AML are involved on the one hand in controlling myeloid differentiation, and on the other in regulating growth and survival of myeloid precursor cells.

AML-associated fusion proteins disrupt common signaling pathways

Due to the variability of RARalpha, AML1 and MLL fusion partners, interpretations of their respective oncogenic mechanisms initially focused on their possible interference with the function of the native transcriptional regulatory functions. Recent findings clearly demonstrate that RARalpha and AML1 partners have a key role in determining leukemogenesis, and their respective fusion proteins must be considered as bi-functional proteins. For example, both PML and RAR components are required for the block of differentiation induced by PML/RARalpha expression (Grignani et al., 1996). A simple way to explain this observation is that the PML moiety juxtaposes a protein-protein interaction interface to RARalpha functional domains, favoring oligomerization. The function of PML in the context of the fusion is, therefore, to activate RARalpha (Minucci et al., 2000). Although there is no direct evidence yet, it is plausible to hypothesize that PML may also contribute an independent signaling function to the fusion protein. PML activates p53 and induces cell senescence and apoptosis, whereas PML/RARalpha increases cell survival by interfering with p53 activity through its PML moiety (Pearson and Pelicci, 2001, manuscript in preparation). It is possible, therefore, that each of the two components of the fusion protein determines a biological effect: enhanced cell survival by interference with PML function, and arrest of myeloid differentiation through abnormal RARalpha function.

There are, instead, controversial reports concerning the relevance of MLL partners for the transforming potential of MLL fusion proteins. A mere truncation of MLL at the breaksite has no influence in hematopoietic differentiation or tumor propensity (Corral et al., 1996), whereas a fusion of the same MLL portion with a bacterial lacZ gene, functionally equivalent to an MLL truncation, causes ES cell derived acute leukemias, albeit with long latency (Dobson et al., 1999). Furthermore, mutagenesis of the MLL/ENL fusion protein demonstrates that both MLL and ENL components are required to preserve transforming potential (Slany et al., 1998). It seems, therefore, that the C-terminal portion of the MLL-fusions also has a role in the leukemogenic mechanism, perhaps by contributing to abnormal protein-protein interactions.

Several studies in recent years highlight the importance in leukemogenesis of interference with the transcriptional status of the myeloid progenitor cells, and partially elucidate the molecular mechanisms that result in an abnormal transcriptional activity. Regulation of promoter activity requires chromatin remodeling, which involves the recruitment of at least two types of functions to the target promoters: the ATP-dependent SWI/SNF-like complex, and histone-modifying enzymes, which include histone acetyltransferases (HATs) and histone deacetylases (HDACs) (reviewed by Fry and Peterson, 2001). HATs are found in many promoter-bound complexes and favor transcriptional activation by acetylating histones. Conversely, HDACs function as transcriptional co-repressors and many of them are present in multimeric promoter-bound complexes (reviewed by Cress and Seto, 2000). Concerted interactions of transcription factors with co-activators and co-repressors via direct or indirect interactions ultimately regulate the transcriptional status of cells.

Chromatin remodeling and interaction of AML-fusions with co-repressor/HDAC complexes plays a key role in the pathogenesis of AMLs (Minucci and Pelicci, 1999). In APL, the recruitment of co-repressor/HDAC complexes to target promoters is responsible for both transcriptional repression of target genes and sensitivity to RA. PML/RARalpha and PLZF/RARalpha, in the absence of RA, recruit the nuclear co-repressor (N-CoR)-HDAC complex through the RARalpha CoR box, bind to target promoters, and repress transcription (Grignani et al., 1998; He et al., 1998; Lin et al., 1998; Wong and Privalsky, 1998). PLZF/RARalpha also contains a second CoR box in the PLZF amino-terminal region whose binding to the co-repressor complex is not modulated by RA. The different sensitivity to RA treatment in vivo and in vitro is a consequence of the fact that high doses of RA release HDAC activity from PML/RARalpha, thus permitting transcription of target genes, but is not sufficient to release repression from PLZF/RARalpha-bound promoters. The simultaneous treatment with RA and HDAC inhibitors can, instead, overcome the block of differentiation induced by PLZF/RARalpha.

AML1/ETO also binds co-repressor/HDAC complexes through the ETO moiety (Gelmetti et al., 1998; Lutterbach et al., 1998b; Wang et al., 1998a). For both RARalpha fusions and AML1/ETO, interaction with co-repressors is necessary for the differentiation block induced by the fusion proteins (Gelmetti et al., 1998; Grignani et al., 1998; He et al., 1998; Wang et al., 1999). Furthermore, both PML/RARalpha and AML1/ETO are part of high molecular weight nuclear complexes through the PML or ETO coiled-coil regions, and oligomerization is responsible for the abnormal recruitment of N-CoR, transcriptional repression, and impaired differentiation of primary hematopoietic precursors (Lin and Evans, 2000; Minucci et al., 2000).

Aberrant recruitment of co-repressor complexes to target promoters by AML-fusion proteins switches the transcriptional status from an active to an inactive state. In fact, many genes, such as GM-CSF, T cell receptor subunits, or the multidrug resistance gene (MDR), which are target of activation by CBF, are repressed by expression of AML1/ETO (Frank et al., 1995; Lutterbach et al., 1998a; Meyers et al., 1995). Repression of genes that are necessary for myeloid maturation may be the main mechanism underlying the differentiation block induced by AML-associated fusion proteins.

It is implicit that there will be genes whose transcription is activated by the fusion proteins through indirect mechanisms. However, there are also direct targets of aberrant transcriptional activation by AML-fusion proteins, such as bcl-2, whose mRNA levels increases after AML1/ETO or PML/RARalpha expression and in leukemic blasts of several subtypes of AML (Bradbury et al., 1997; Klampfer et al., 1996; Ferrucci and Pelicci, 2001, unpublished results). Cyclin A expression also increases after PML/RARalpha or PLZF/RARalpha expression through direct activation of Cyclin A promoter (Muller et al., 2000; Yeyati et al., 1999). It is interesting to note that both bcl-2 and Cyclin A overexpression are connected to increased cell survival (Funakoshi et al., 1997; Konopleva et al., 1999).

MLL-fusion proteins may also interfere with chromatin remodeling. MLL binds to the INI1 and SNR1 proteins that are homologous to members of the SWI/SNF complex through its SET domain (Rozenblatt-Rosen et al., 1998), which is lost in all MLL fusions proteins (Zeleznik-Le et al., 1994). This observation suggests a loss of the ability to recruit the SWI/SNF complex, which may result in an altered MLL function. However, this is probably a simplistic model, considering the variety of MLL partners, of which some are themselves associated with chromatin remodeling activity. AF9 and ENL, for example, are associated with the SWI/SNF complex (Cairns et al., 1996; Nakamura et al., 1993). CBP and p300, partners of MLL in rare translocations, retain the protein-protein interaction and the HAT activity domains in the context of the fusions, which might lead to abnormal chromatin remodeling either through constitutive activation of HAT activity or through recruitment of other acetyltransferase components of the co-activator complex (DiMartino and Cleary, 1999).

The importance of HDAC-dependent transcriptional repression as a mechanism of leukemogenesis is further suggested by the finding that blasts derived from AML patients of M2 and M4 subtypes become sensitive to RA after treatment with HDAC inhibitors (Ferrara et al., 2001). Tricostatin A, a potent HDAC inhibitor, is not toxic in adult mice and does not perturb mouse embryogenesis (Nervi et al., 2001). HDAC inhibitors may, therefore, become fundamental adjuvants in differentiation therapy of AML.

The formation of multimeric protein complexes including AML fusion proteins may also have other effects than the abnormal recruitment of transcriptional regulators to target promoters. The CBFbeta/SMMHC fusion protein, for example, forms multimers though the alpha helical region of the SMMHC moiety (Cao et al., 1997; Liu et al., 1996c). As a result, CBFbeta/SMMHC interferes with CBF activity by sequestering CBFalpha subunits in large complexes, which localize prevalently in the cytoplasm in adherent cells (Adya et al., 1998; Kanno et al., 1998) and in the nucleus in hematopoietic cells (Cao et al., 1997, 1998). Considering that CBFalpha subunits and CBFbeta/SMMHC form complexes that retain the capacity to bind DNA, CBFbeta/SMMHC may also interfere with CBFalpha transactivation through local effects on transcriptional complexes. The relevance of the dimerization interface in the context of the fusion is supported by the observation that deletion of the C-terminal portion of the SMMHC moiety abolishes interference with AML1 driven transactivation of a reporter gene (Cao et al., 1998). A cryptic repression domain has been described in the SMMHC domain, suggesting endogenous repressive potential of the fusion protein (Lutterbach et al., 1999). The CBFbeta/SMMHC fusion protein appears, therefore, to act as a dominant inhibitor of AML-1-dependent transactivation.

Disorganization of specific nuclear compartments is another common property of AML-fusion proteins, which may well play a part in altering the transcriptional machinery of the cell (reviewed by Faretta et al., 2001). Fundamental nuclear functions occur within specific nuclear compartments, and de-localization of their components, as well as disruption of the architecture of the compartments themselves, may result in deregulation of DNA transcription, replication, RNA processing, or post-translational modification of proteins.

PML localizes within specific nuclear structures called nuclear bodies (PML-NBs), ND10s, or PODs. PML-NBs are dynamic structures that contain proteins involved in diverse cellular functions, such as Sp100, SUMO-1, HP1, CBP, p53, p27, Daxx and BAX, whose integrity might directly or indirectly affect the transcriptional regulatory activity of its components (Faretta et al., 2001). PML/RARalpha expression causes a disruption of PML-NBs, which is reversed by RA treatment. Likewise, PLZF localizes within defined compartments, known as PLZF nuclear bodies (PLZF-NBs), which partially overlap with PML-NBs, and PLZF-NBs are disrupted by PLZF/RARalpha expression (Ruthardt et al., 1998). The finding that PML/RARalpha or NPM/RARalpha expression also disrupt PLZF-NBs points to common mechanisms in the pathogenesis of APL (Hummel et al., 1999; Koken et al., 1997).

De-localization is not peculiar to APL. MLL also localizes to specific subnuclear domains, which are disrupted by the expression of MLL-fusions (Rogaia et al., 1997). Native AML1 and ETO proteins are normally found in different nuclear compartments, and localization of the AML1/ETO fusion protein is determined by the ETO moiety (McNeil et al., 1999). This implies that, compared to wild type transcription factors, the fusion proteins act on target genes within a different regulatory context. Interestingly, expression of AML1/ETO causes reorganization of PML-NBs (McNeil et al., 2000; Stein et al., 2000) and influences PLZF localization by excluding it from the nuclear matrix and reducing its ability to bind to its cognate DNA-binding site through direct interaction (Melnick et al., 2000a).

In summary, AML fusion proteins de-regulate transcription through mechanisms that involve both the formation of aberrant complexes with other transcriptional regulators and the disruption of subnuclear compartmentalization. Furthermore, there appears to be functional interplay between specific AML-associated fusion proteins and the native molecules involved in other fusions, creating a link in the transcriptional pathways of different subtypes of AML.

Perspectives

Each AML-associated chromosome translocation almost invariably disrupts a gene that encodes for a transcription factor involved in the regulation of myeloid differentiation. Every step of hematopoietic maturation is characterized by the orderly and concerted expression of a specific set of genes, which requires tight transcriptional regulation. AML-associated fusion proteins interfere with the function of their native counterparts and determine a stage-specific arrest in the process of myeloid differentiation. In most cases, they de-regulate transcription through a mechanism that involves aberrant recruitment of co-repressor/HDAC complexes and interferes with chromatin remodeling (Figure 1). This observation has identified a new biological target for the treatment of these diseases. To date the only AML amenable to differentiation therapy is APL, and ATRA treatment has dramatically changed the prognosis of this disease in the past decade. Preliminary studies on the effect of simultaneous treatment of AML blasts with HDAC inhibitors and differentiation agents have given encouraging results, and open the possibility of introducing transcription-differentiation therapy in other subtypes of AML.

The partner genes involved in AML translocations encode for proteins that are much more heterogeneous in terms of function. Their contribution to the leukemogenic potential of the fusion proteins is less clear, however their presence appears to be fundamental for at least some of the biological effects. Many of them have physiological functions connected with the regulation cell survival and apoptosis, and their disruption may be involved in another fundamental property of AML-associated fusion proteins, which is that of enhancing cell survival in a cell-type specific manner. Although the mechanisms underlying this function are not yet clear, they may include an increased rate of transcription of anti-apoptotic genes and disorganization of specific subnuclear compartments like the nuclear bodies, which are of pivotal importance in the regulation of cell senescence and apoptosis (Figure 1). The pathways that regulate cell survival and apoptosis in myeloid cells represent other potential targets for the development of therapeutic agents that antagonize specific functions of the leukemogenic molecules.

Clearly, the alteration of regulated transcriptional networks in leukemic blasts will ultimately result in gene expression profiles that are specific for each subtype of AML. Analysis of global gene expression in AML cells with high-throughput methods, such as cDNA or oligonucleotide microarrays, will certainly represent a step ahead in identifying genes targeted by AML fusion proteins. One practical implication is the possibility of classifying diverse types of leukemia based uniquely on the analysis of gene expression patterns, as suggested by a pilot study performed with DNA microarrays (Golub et al., 1999). Recent investigations have identified sets of target genes regulated by the expression of AML-associated fusion proteins, and represent the first important step towards the understanding of the pathways that lead to a leukemic phenotype (Liu et al., 2000). Considering the existence of common mechanisms underlying leukemogenesis, a subset of targets that is common to more than one fusion protein is predictable, along with targets specific for each AML subtype. In a simplistic model, coherent with the evidence discussed in this review, we would expect to find that AML fusion proteins repress genes involved in myeloid differentiation or apoptosis, and activate genes that promote cell survival. PML/RARalpha and AML1/ETO regulate a set of common target genes whose transcriptional behavior reflects this prediction (Alcalay and Pelicci, 2001, unpublished results).

A careful, concerted analysis of the rapidly growing amount of information emerging from expression studies will almost certainly lead to a great leap ahead in our knowledge of the mechanisms underlying leukemogenesis, and help in the design of new therapeutic strategies.

Acknowledgements

The authors thank Dr Lucilla Luzi for the graphics included in this review.

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Figures

Figure 1 Schematic representation of proposed leukemogenic mechanisms. The top panel represents the nucleus of a normal myeloid progenitor, where both the native transcription factor (TF) and its partner protein (P) perform their physiological functions. The compass symbolizes the tight balance existing among the various destinies the cell can face: self-renewal, terminal differentiation, or programmed cell death. A transcription factor (TF) is shown as part of a co-activator complex including HAT, bound to DNA, and activating the transcription of a gene that promotes myeloid differentiation. Nuclear compartments, which include either native protein (TF or P), are organized into distinct subdomains. As a consequence of regulated transcriptional activity, normal myeloid progenitors can undergo self-renewal until they are committed to terminal differentiation, then their life-span is determined by mechanisms that regulate cell senescence and apoptosis. The bottom panel represents the nucleus of a leukemic blast. The fusion protein containing part of both the TF and the P proteins is shown as part of a co-repressor complex, which includes HDAC and is bound to DNA. Myeloid differentiation is blocked at a specific stage as a consequence of the expression of a specific fusion protein. Nuclear architecture is disrupted. The de-regulation of mechanisms that control cell senescence and apoptosis results in an increase of cell survival. The compass is broken

10 September 2001, Volume 20, Number 40, Pages 5680-5694
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