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Canonical Wnt signaling is mediated by a molecular "switch" that regulates the transcriptional properties of the T-cell factor (TCF) family of DNA-binding proteins. Members of the myeloid translocation gene (MTG) family of transcriptional corepressors are frequently disrupted by chromosomal translocations in acute myeloid leukemia, whereas MTG16 may be inactivated in up to 40% of breast cancer and MTG8 is a candidate cancer gene in colorectal carcinoma. Genetic studies imply that this corepressor family may function in stem cells. Given that mice lacking Myeloid Translocation Gene Related-1 (Mtgr1) fail to maintain the secretory lineage in the small intestine, we surveyed transcription factors that might recruit Mtgr1 in intestinal stem cells or progenitor cells and found that MTG family members associate specifically with TCF4. Coexpression of beta-catenin disrupted the association between these corepressors and TCF4. Furthermore, when expressed in Xenopus embryos, MTG family members inhibited axis formation and impaired the ability of beta-catenin and XLef-1 to induce axis duplication, indicating that MTG family members act downstream of beta-catenin. Moreover, we found that c-Myc, a transcriptional target of the Wnt pathway, was overexpressed in the small intestines of mice lacking Mtgr1, thus linking inactivation of Mtgr1 to the activation of a potent oncogene.
The t(8;21) chromosomal translocation that generates the fusion oncoprotein RUNX1-ETO predominates in leukemia patients of the French-American-British (FAB) class M2 subtype. The oncoprotein has the capacity to promote expansion of hematopoietic stem/progenitor cells and induces leukemia in association with other genetic alterations. Here, we show that RUNX1-ETO undergoes degradation in response to treatment with histone deacetylase inhibitors, one of which, depsipeptide (DEP), is currently undergoing phase II clinical testing in a variety of malignancies. These compounds induce turnover of RUNX1-ETO without affecting the stability of RUNX1-ETO partner proteins. In addition, RUNX1-ETO physically interacts with heat shock protein 90 (HSP90). DEP treatment interrupts the association of RUNX1-ETO with HSP90 and induces proteasomal degradation of RUNX1-ETO. DEP and the HSP90 antagonist 17-allylamino-geldanamycin (17-AAG) both triggered RUNX1-ETO degradation, but without any additive or cooperative effects. These findings may stimulate the development of more rational and effective approaches for treating t(8;21) patients using histone deacetylase inhibitors or HSP90 inhibitors.
Von Recklinghausen's disease is a relatively common familial genetic disorder characterized by inactivating mutations of the Neurofibromatosis-1 (NF1) gene that predisposes these patients to malignancies, including an increased risk for juvenile myelomonocytic leukemia. However, NF1 mutations are not common in acute myeloid leukemia (AML). Given that the RUNX1 transcription factor is the most common target for chromosomal translocations in acute leukemia, we asked if NF1 might be regulated by RUNX1. In reporter assays, RUNX1 activated the NF1 promoter and cooperated with C/EBPalpha and ETS2 to activate the NF1 promoter over 80-fold. Moreover, the t(8;21) fusion protein RUNX1-MTG8 (R/M), which represses RUNX1-regulated genes, actively repressed the NF1 promoter. R/M associated with the NF1 promoter in vivo and repressed endogenous NF1 gene expression. In addition, similar to loss of NF1, R/M expression enhanced the sensitivity of primary myeloid progenitor cells to granulocyte-macrophage colony-stimulating factor. Our results indicate that the NF1 tumor suppressor gene is a direct transcriptional target of RUNX1 and the t(8;21) fusion protein, suggesting that suppression of NF1 expression contributes to the molecular pathogenesis of AML.
Leukemic disease can be linked to aberrant gene expression. This often is the result of molecular alterations in transcription factors that lead to their misrouting within the nucleus. The acute myelogenous leukemia-related fusion protein AML1ETO is a striking example. It originates from a gene rearrangement t(8;21) that fuses the N-terminal part of the key hematopoietic regulatory factor AML1 (RUNX1) to the ETO (MTG8) repressor protein. AML1ETO lacks the intranuclear targeting signal of the wild-type AML1 and is directed by the ETO component to alternate nuclear matrix-associated sites. To understand this aberrant subnuclear trafficking of AML1ETO, we created a series of mutations in the ETO protein. These were characterized biochemically by immunoblotting and in situ by immunofluorescence microscopy. We identified two independent subnuclear targeting signals in the N- and C-terminal regions of ETO that together direct ETO to the same binding sites occupied by AML1ETO. However, each segment alone is targeted to a different intranuclear location. The N-terminal segment contains a nuclear localization signal and the conserved hydrophobic heptad repeat domain responsible for protein dimerization and interaction with the mSin3A transcriptional repressor. The C-terminal segment spans the nervy domain and the zinc finger region, which together support interactions with the corepressors N-CoR and HDACs. Our findings provide a molecular basis for aberrant subnuclear targeting of the AML1ETO protein, which is a principal defect in t(8;21)-related acute myelogenous leukemia.
The t(8;21)(q22;q22) translocation, which fuses the ETO gene on human chromosome 8 with the AML1 gene on chromosome 21 (AML1-ETO), is one of the most frequent cytogenetic abnormalities associated with acute myelogenous leukemia (AML). It is seen in approximately 12 to 15% of AML cases and is present in about 40% of AML cases with a French-American-British classified M2 phenotype. We have generated a murine model of the t(8;21) translocation by retroviral expression of AML1-ETO in purified hematopoietic stem cells (HSC). Animals reconstituted with AML1-ETO-expressing cells recapitulate the hematopoietic developmental abnormalities seen in the bone marrow of human patients with the t(8;21) translocation. Primitive myeloblasts were increased to approximately 10% of bone marrow by 10 months posttransplant. Consistent with this observation was a 50-fold increase in myeloid colony-forming cells in vitro. Accumulation of late-stage metamyelocytes was also observed in bone marrow along with an increase in immature eosinophilic myelocytes that showed abnormal basophilic granulation. HSC numbers in the bone marrow of 10-month-posttransplant animals were 29-fold greater than in transplant-matched control mice, suggesting that AML1-ETO expression overrides the normal genetic control of HSC pool size. In summary, AMLI-ETO-expressing animals recapitulate many (and perhaps all) of the developmental abnormalities seen in human patients with the t(8;21) translocation, although the animals do not develop leukemia or disseminated disease in peripheral tissues like the liver or spleen. This suggests that the principal contribution of AML1-ETO to acute myeloid leukemia is the inhibition of multiple developmental pathways.
The t(8;21) is one of the most frequent chromosomal translocations associated with acute leukemia. This translocation creates a fusion protein consisting of the acute myeloid leukemia-1 transcription factor and the eight-twenty-one corepressor (AML1 ETO), which represses transcription through AML1 (RUNX1) DNA binding sites and immortalizes hematopoietic progenitor cells. We have identified the p14(ARF) tumor suppressor, a mediator of the p53 oncogene checkpoint, as a direct transcriptional target of AML1 ETO. AML1 ETO repressed the p14(ARF) promoter and reduced endogenous levels of p14(ARF) expression in multiple cell types. In contrast, AML1 stimulated p14(ARF) expression and induced phenotypes consistent with cellular senescence. Chromatin immunoprecipitation assays demonstrated that AML1 ETO was specifically bound to the p14(ARF) promoter. In acute myeloid leukemia samples containing the t(8;21), levels of p14(ARF) mRNA were markedly lower when compared with other acute myeloid leukemias lacking this translocation. Repression of p14(ARF) may explain why p53 is not mutated in t(8;21)-containing leukemias and suggests that p14(ARF) is an important tumor suppressor in a large number of human leukemias.
The t(8;21), t(16;21), inv(16), and t(12;21) are some of the most frequent chromosomal translocations found in acute myeloid and acute lymphoblastic leukemia. The fusion proteins created by these chromosomal translocations are transcriptional repressors. A full understanding of the types of proteins that these fusion proteins recruit to repress transcription will not only clarify understanding of the molecular mechanism of action of these fusion proteins but also provide further targets for therapeutic intervention.
t(8;21) and t(16;21) create two fusion proteins, AML-1-ETO and AML-1-MTG16, respectively, which fuse the AML-1 DNA binding domain to putative transcriptional corepressors, ETO and MTG16. Here, we show that distinct domains of ETO contact the mSin3A and N-CoR corepressors and define two binding sites within ETO for each of these corepressors. In addition, of eight histone deacetylases (HDACs) tested, only the class I HDACs HDAC-1, HDAC-2, and HDAC-3 bind ETO. However, these HDACs bind ETO through different domains. We also show that the murine homologue of MTG16, ETO-2, is also a transcriptional corepressor that works through a similar but distinct mechanism. Like ETO, ETO-2 interacts with N-CoR, but ETO-2 fails to bind mSin3A. Furthermore, ETO-2 binds HDAC-1, HDAC-2, and HDAC-3 but also interacts with HDAC-6 and HDAC-8. In addition, we show that expression of AML-1-ETO causes disruption of the cell cycle in the G(1) phase. Disruption of the cell cycle required the ability of AML-1-ETO to repress transcription because a mutant of AML-1-ETO, Delta469, which removes the majority of the corepressor binding sites, had no phenotype. Moreover, treatment of AML-1-ETO-expressing cells with trichostatin A, an HDAC inhibitor, restored cell cycle control. Thus, AML-1-ETO makes distinct contacts with multiple HDACs and an HDAC inhibitor biologically inactivates this fusion protein.
The AML-1/ETO fusion protein, created by the (8;21) translocation in M2-type acute myelogenous leukemia (AML), is a dominant repressive form of AML-1. This effect is due to the ability of the ETO portion of the protein to recruit co-repressors to promoters of AML-1 target genes. The t(11;17)(q21;q23)-associated acute promyelocytic leukemia creates the promyelocytic leukemia zinc finger PLZFt/RAR alpha fusion protein and, in a similar manner, inhibits RAR alpha target gene expression and myeloid differentiation. PLZF is expressed in hematopoietic progenitors and functions as a growth suppressor by repressing cyclin A2 and other targets. ETO is a corepressor for PLZF and potentiates transcriptional repression by linking PLZF to a histone deacetylase-containing complex. In transiently transfected cells and in a cell line derived from a patient with t(8;21) leukemia, PLZF and AML-1/ETO formed a tight complex. In transient assays, AML-1/ETO blocked transcriptional repression by PLZF, even at substoichiometric levels relative to PLZF. This effect was dependent on the presence of the ETO zinc finger domain, which recruits corepressors, and could not be rescued by overexpression of co-repressors that normally enhance PLZF repression. AML-1/ETO also excluded PLZF from the nuclear matrix and reduced its ability to bind to its cognate DNA-binding site. Finally, ETO interacted with PLZF/RAR alpha and enhanced its ability to repress through the RARE. These data show a link in the transcriptional pathways of M2 and M3 leukemia. (Blood. 2000;96:3939-3947)
The ETO protein was originally identified by its fusion to the AML-1 transcription factor in translocation (8;21) associated with the M2 form of acute myeloid leukemia (AML). The resulting AML-1-ETO fusion is an aberrant transcriptional regulator due to the ability of ETO, which does not bind DNA itself, to recruit the transcriptional corepressors N-CoR, SMRT, and Sin3A and histone deacetylases. The promyelocytic leukemia zinc finger (PLZF) protein is a sequence-specific DNA-binding transcriptional factor fused to retinoic acid receptor alpha in acute promyelocytic leukemia associated with the (11;17)(q23;q21) translocation. PLZF also mediates transcriptional repression through the actions of corepressors and histone deacetylases. We found that ETO is one of the corepressors recruited by PLZF. The PLZF and ETO proteins associate in vivo and in vitro, and ETO can potentiate transcriptional repression by PLZF. The N-terminal portion of ETO forms complexes with PLZF, while the C-terminal region, which was shown to bind to N-CoR and SMRT, is required for the ability of ETO to augment transcriptional repression by PLZF. The second repression domain (RD2) of PLZF, not the POZ/BTB domain, is necessary to bind to ETO. Corepression by ETO was completely abrogated by histone deacetylase inhibitors. This identifies ETO as a cofactor for a sequence-specific transcription factor and indicates that, like other corepressors, it functions through the action of histone deactylase.