Molecular basis of Acute Myelogenous Leukemia

As bases moleculares da leucemia mielóide aguda

Abstracts

Acute Myelogenous Leukemia (AML) is frequently associated with recurring chromosomal translocations, which lead to the fusion of two genes encoding transcription factors. As the moieties of these fusion proteins retain part of the functional domains of the wild-type proteins, they may interfere directly or indirectly with the transcriptional regulation of the leukemic cell, conferring survival advantage. The majority of the transcription factors commonly involved in recurring chromosomal translocations may be grouped in one of the following families: core binding factor (CBF), retinoic acid receptor alpha (RARalpha), homeobox (HOX) family, and mixed lineage leukemia (MLL). In vivo analysis of the molecular basis of leukemogenesis through the generation of transgenic mouse models revealed that a common theme is the recruitment of transcriptional co-activators and co-repressors by these fusion proteins. However, the expression of the fusion protein is not sufficient to induce full blown leukemia, as evidenced in part by the long latencies required for disease development in the transgenic models of leukemia, and therefore, second mutagenic events may contribute to AML pathogenesis.

Acute myelogenous leukemia; fusion genes; transgenic models; core binding factors (CBF); retinoic acid receptor alpha(RARalpha); homeobox (HOX); mixed lineage leukemia (MLL) genes

A leucemia mielóide aguda (LMA) está freqüentemente associada a translocações cromossômicas recorrentes. Em muitos casos, os genes presentes nos pontos de quebra cromossômica são conhecidos e, quase todos codificam para fatores de transcrição. O gene híbrido, resultante da justaposição de exons de genes distintos, codifica para proteínas de fusão. Como estas retêm a maior parte dos domínios funcionais das proteínas selvagens, elas interferem direta ou indiretamente com regulação da transcrição gênica, conferindo vantagem à sobrevivência das células leucêmicas. A maioria dos fatores de transcrição afetados pelas translocações cromossômicas associadas a LMA pode ser agrupada numa das seguintes famílias: dos core binding factors (CBF), do receptor a do ácido retinóico (RARalfa), do homeobox (HOX), ou do mixed lineage leukemia (MLL). Estudos in vivo, empregando modelos transgênicos, revelaram um mecanismo molecular comum a estas proteínas de fusão: a desregulação da transcrição gênica via recrutamento de fatores co-ativadores ou co-repressores. Embora necessária, a expressão das proteínas de fusão não é suficiente para o desenvolvimento da leucemia. A existência de uma longa fase pré leucêmica observada nos modelos transgênicos sugere que outros eventos mutagênicos devam ocorrer para o desenvolvimento das leucemias.

Leucemia mielóide aguda; genes de fusão; modelos transgênicos; core binding factors (CBF); receptor a do ácido retinóico (RARalfa); homeobox (HOX); mixed lineage leukemia (MLL)

Artigo Especial / Special Article

Molecular basis of Acute Myelogenous Leukemia

Eduardo M. Rego

Acute Myelogenous Leukemia (AML) is frequently associated with recurring chromosomal translocations, which lead to the fusion of two genes encoding transcription factors. As the moieties of these fusion proteins retain part of the functional domains of the wild-type proteins, they may interfere directly or indirectly with the transcriptional regulation of the leukemic cell, conferring survival advantage. The majority of the transcription factors commonly involved in recurring chromosomal translocations may be grouped in one of the following families: core binding factor (CBF), retinoic acid receptor alpha (RARa), homeobox (HOX) family, and mixed lineage leukemia (MLL). In vivo analysis of the molecular basis of leukemogenesis through the generation of transgenic mouse models revealed that a common theme is the recruitment of transcriptional co-activators and co-repressors by these fusion proteins. However, the expression of the fusion protein is not sufficient to induce full blown leukemia, as evidenced in part by the long latencies required for disease development in the transgenic models of leukemia, and therefore, second mutagenic events may contribute to AML pathogenesis.

Keywords: Acute myelogenous leukemia, fusion genes, transgenic models, core binding factors (CBF), retinoic acid receptor a(RARa), homeobox (HOX), mixed lineage leukemia (MLL) genes

Introduction

The vast majority of leukemias are sporadic, and a result of acquired mutations in hematopoietic progenitor cells. Much has been learned about the molecular genetic basis of acute myelogenous leukemias (AML) through the cloning and characterization of these acquired mutations (1). Table 1 shows the most frequent chromosomal abnormalities in myeloid malignancies. A common theme is the association with balanced reciprocal translocations, which lead to the fusion of two genes encoding transcription factors. These transcription factors are frequently conserved in evolution, and are important in embryonic development as well as in normal hematopoiesis (2). Transcription factors involved in AML include core binding factor (CBF), retinoic acid receptor alpha (RARa), homeobox (HOX) family, and mixed lineage leukemia (MLL).

Core Binding Factor Translocations

CBF is a heterodimeric transcription factor comprised of CBFa (also known as AML1) and CBFb subunits. The complex recognizes and binds to the promoter region of several genes important in normal myeloid differentiation, for instance, the interleukin-3 (IL-3) and granulocyte-macrofage colony stimulating factor (GM-CSF) genes (3). The t(8;21) is detected in 20-25% of the subtype M2 of the French-American-British classification of acute leukemias and, approximately 90% of t(8;21)-positive patients have AML M2 morphological features. As a consequence of the t(8;21), the AML1 gene is translocated from chromosome 21 and fused to the eight-twenty-one (ETO) gene in chromosome 8 (4). The fusion protein produced retains important functional domains from the parental proteins and, therefore may disrupt the function of the normal AML1 or ETO proteins. In fact, there is a large body of evidence that supports that AML1-ETO fusion protein acts as a dominant negative inhibitor of the AML1 gene (5). The AML1 gene function is essential to normal embryonic development, as demonstrated by the fact that its inactivation by homologus recombination (knock out) resulted in early embryonic death due to central nervous system (CNS) hemorrhage (3, 6). In transcription activation assays, cells transfected with a reporter construct containing the TCRb enhancer (a known target of the AML1 transcription factor) coupled to a reporter enzyme, chloramphenicol acetyl transferase (CAT), presented enhanced expression of CAT when co-transfected with the AML1 gene. The co-transfection of the fusion gene AML1-ETO did not only result in CAT expression, but also was capable of completely abrogating the induction of CAT expression induced by the normal AML1 gene. Thus, suggesting that the fusion protein is a dominant negative inhibitor of AML1.

Compelling evidence that the AML1-ETO fusion is a dominant negative inhibitor of the wild-type AML1 gene has been obtained from the analysis of knock-in animals. Through this technology the human fusion gene is inserted into the wild-type locus of the murine AML1 gene, therefore under the control of the same transcription factors as the normal gene during the development of the mouse embryo. The phenotype of the AML1-ETO knock-in mice is identical to that of the AML1 knock-out mouse, with embryonic death at day 11.5 post-coitus (d.p.c.) with the characteristic pattern of CNS hemorrhage and lack of definitive hematopoiesis (6, 7).

The fusion protein CBFb/MYH11 is the product of inv(16) and shares many properties with the AML1/ETO fusion protein. The CBFb/MYH11 is a dominant negative inhibitor of CBF both in transcaptivation assays and during development. In fact, the CBFb/MYH11 knock in model presents the same phenotype as the AML1/ETO knock in, and both are identical to the phenotype of the AML1 and CBFb knock outs (7).

Finally, it should be emphasized that the TEL/AML1 fusion protein associated with pediatric acute lymphoblastic leukemia is also a dominant negative inhibitor of AML1, therefore consistent with the hypothesis that each of the CBF translocations gives rise to fusion proteins that are dominant negative inhibitors of wild-type CBF (8).

Retinoic Acid Receptor a Fusion Proteins

Acute promyelocytic leukemia (APL) is a distinct subtype of acute myelogenous leukemia (the M3 and M3-Variant subtypes according to the French-American-British classification of the acute leukemias) characterized by unique biological features such as: i) the expansion of leukemic cells blocked at the promyelocytic stage of the myelopoiesis; ii) its invariable association with reciprocal chromosomal translocations involving the retinoic acid receptor a (RARa) gene on chromosome 17, and iii) its exquisite sensitivity to the differentiating action of all-trans retinoic acid (ATRA) (9). At the molecular level, the RARa gene on chromosome 17 is fused to the promyelocytic leukemia gene (PML), to the promyelocytic leukemia zinc finger (PLZF) gene, to the nucleophosmin (NPM) gene, to the nuclear mitotic apparatus (NuMA) gene, or to the signal transducer and activator of transcription 5B (STAT 5B) gene (for brevity hereafter referred to as X genes) located on chromosomes 15, 11, 5, 11, or 17, respectively (10). These chromosomal translocations are reciprocal, thus leading to the generation of X-RARa and RARa-X fusion genes and the co-expression of their chimeric products in the leukemic blasts (11).

Structurally, the X-RARa fusion proteins retain most of the RARa functional domains (domains B-F), including the DNA- and ligand-binding domains. These are linked C-terminally to five different moieties from the various X proteins. These various N-terminal regions, although structurally distinct, normally mediate self-association of the various X proteins and, in turn, heterodimerization between X and X- RARa proteins (10, 12). Unlike RARa, which can activate transcription at physiological doses of retinoic acid, the various X- RARa fusion proteins function as aberrant and dominant transcriptional repressors through heterodimerization with retinoid-X receptor (RXR), and at least in part, through their ability to form repressive complexes with nuclear receptor co-repressors, such as Sin3A or Sin3B and histone deacetylases (HDACs) (12-14). In addition, the various X- RARa fusion proteins may interfere with their respective X pathways, through physical interaction (15, 16).

Much has been learned about APL pathogenesis and the role of the X- RARa and RARa-X fusion proteins through the analysis of TM models (17). Transgenic mice in which the PML-RARa fusion gene is expressed under the control of the human cathepsin G (hCG) or MRP8 promoter develop AML. After a long latency (ranging between 6 months to 1 year), 10-15% of these TM develop a lethal form of leukemia that closely resembles human APL, the leukemic population consists mainly of myeloid blasts and promyelocytes (18).

As in human APL, PML- RARa TM leukemia responds to RA treatment that induces the terminal differentiation of the leukemic blasts. Human APL patients harboring t(11;17) (PLZF-RARa), however, unlike other APL patients, do not respond to ATRA (18). In contrast to leukemia in hCG-PML- RARa TM, leukemia in hCG-PLZF-RARa TM lacks the distinctive block of differentiation at the promyelocytic stage of myelopoiesis and is characterized by leukocytosis and infiltration of all organs by terminally differentiated myeloid cells (a picture that resembles human chronic myeloid leukemia). As in hCG-PML- RARa TM, this leukemia develops after a long latency of approximately six months. However, the leukemic phenotype is now completely penetrant. Moreover, hCG-PLF- RARa TM do not respond to RA treatment (16, 19).

All together, these results demonstrate that: i) X- RARa proteins are necessary but not sufficient to APL leukemogenesis, as supported by the fact that leukemia occurs, but only after a long latency and that hCG-PLZF- RARa TM do not develop a classic APL phenotype; ii) the differential response to RA observed between t(11;17) and t(15;17)APL is mediated by the X- RARa fusion proteins.

Translocations involving HOX family members

Members of the HOX family of transcription factors are involved in translocations associated with human leukemias. In AML the best known example is the NUP98/HOXA9 fusion protein, generated by the t(7;11) (20). Nevertheless, HOX function is also indirectly associated with leukemia, for instance, translocations involving the MLL or the PLZF gene products disrupt the function of the respective wild-type proteins, which are thought to exert regulatory function on HOX expression (21, 22). There is a tightly regulated pattern of expression of HOX genes during hematopoiesis, HOXA9 for example is expressed in early progenitors cells, but downregulated during hematopoietic differentiation and is undetectable in terminally differentiated cells. A plausible mechanism of action of NUP98/HOXA9 is that over-expression of HOXA9 confers a proliferative advantage and inhibition of differentiation to leukemic cells. The fact that the HOXA9 DNA binding domain is required to the transformation activity of NUP98/HOXA9 fusion protein in 3T3 cells, reinforces this hypothesis (20).

In addition, to the deregulation of HOX function NUP98 deregulation may play a role in leukemogenesis. NUP98 is a component of the nuclear pore, and is possible that the fusion protein might affect transport of RNA through the nuclear pores. Moreover, the NUP98 moiety in NUP98/HOXA9 fusion protein has transactivating activity. However, further studies are required in order to elucidate the leukemogenic activity of NUP98/HOXA9 fusion protein.

Translocations involving MLL gene

MLL gene is also known as ALL1, HRX or Htrx, and is homologue to trithorax gene of Drosophila. It is involved in several chromosomal translocations associated with acute leukemias, with a remarkably diverse group of fusion partners (1, 23). In common, all these fusion proteins retain a truncated MLL gene product (although retaining different domains), suggesting that the MLL moiety is critical to leukemogenesis. In fact, knock-in mice harboring the MLL/AF9 fusion under the control of the MLL promoter region develop AML, and the MLL/AF4 fusion is capable of transforming primary hematopoietic cells (23).

The wild-type MLL is critical both in embryonic development and in hematopoiesis. Mice in which the MLL gene has been inactivated have a very early embryonic lethal phenotype, and mice heterozygous for the inactivated gene MLL +/- have abnormalities in the development of the axial skeleton. Since MLL is known to regulate HOX genes expression, these developmental abnormalities have been attributed to the deregulation of HOX function (23).

MLL contains AT hooks that are thought to bind to the minor groove of DNA and facilitate the recruitment of transcription factors of enhancer and promoter elements, therefore exerting modulatory function on target genes (yet not completely known).

Conclusion

In conclusion, chromosomal translocations leading to the generation of hybrid genes encoding fusion proteins play a key role in the pathogenesis of AML. In the last decade, the cloning and characterization of recurring chromosomal translocation breakpoints provided extraordinary insights into the mechanisms by which these fusion proteins affect gene expression causing resistance to apoptosis, and cell cycle deregulation, thus conferring survival advantage. Moreover, based on the encouraging data emerging from APL therapy with agents that target the leukemogenic fusion protein, it is expected that a detailed understanding of the molecular genetic basis of leukemia will lead to improvements in therapy.

As bases moleculares da leucemia mielóide aguda

Resumo

A leucemia mielóide aguda (LMA) está freqüentemente associada a translocações cromossômicas recorrentes. Em muitos casos, os genes presentes nos pontos de quebra cromossômica são conhecidos e, quase todos codificam para fatores de transcrição. O gene híbrido, resultante da justaposição de exons de genes distintos, codifica para proteínas de fusão. Como estas retêm a maior parte dos domínios funcionais das proteínas selvagens, elas interferem direta ou indiretamente com regulação da transcrição gênica, conferindo vantagem à sobrevivência das células leucêmicas. A maioria dos fatores de transcrição afetados pelas translocações cromossômicas associadas a LMA pode ser agrupada numa das seguintes famílias: dos core binding factors (CBF), do receptor a do ácido retinóico (RARa), do homeobox (HOX), ou do mixed lineage leukemia (MLL). Estudos in vivo, empregando modelos transgênicos, revelaram um mecanismo molecular comum a estas proteínas de fusão: a desregulação da transcrição gênica via recrutamento de fatores co-ativadores ou co-repressores. Embora necessária, a expressão das proteínas de fusão não é suficiente para o desenvolvimento da leucemia. A existência de uma longa fase pré leucêmica observada nos modelos transgênicos sugere que outros eventos mutagênicos devam ocorrer para o desenvolvimento das leucemias.

Palavras-chave: Leucemia mielóide aguda, genes de fusão, modelos transgênicos, core binding factors (CBF), receptor a do ácido retinóico (RARa), homeobox (HOX), mixed lineage leukemia (MLL)

Recebido: 12/07/2002

Aceito: 22/07/2002

Professor Associado, Departamento de Clínica Médica, Faculdade de Medicina de Ribeirão Preto, Universidade de São Paulo

Correspondence to: [e-mail]

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Publication Dates

• Publication in this collection
  14 Jan 2003
• Date of issue
  2002