The high incidence of balanced translocations in leukemia is a nonrandom event. Different types of leukemia are associated with specific balanced translocations (26
). In acute myeloid leukemias, the two most frequent balanced translocations are t(8;21) and t(15;17). About 20% of newly diagnosed acute myeloid leukemia patients show evidence of the presence of one of these translocations. In the current study, we provide strong evidence that AML1-ETO, PML-RARα. and the related PLZF-RARα share important target genes. Importantly, the findings in the cell lines translated directly to differential gene expression in primary AML patient samples at the time of diagnosis. Our further analyses deciphered TCF and LEF signaling as an oncogenic pathway that was specifically induced by several AML fusion proteins.
Identification of relevant target genes in the microarray data set was performed with a two-step procedure. In a first step, we identified 1,945 genes as potential targets for the fusion proteins. The large number of genes demanded further selection. Therefore, only genes which showed consistent regulation by all three fusion proteins were considered significant. This algorithm limited the number of identified genes to 52. Currently, no established single method that allows easy identification of all truly regulated genes from microarray analyses is available (6
). The combination of different algorithms as used in our study is very likely to increase the percentage of biologically relevant genes.
The number of repressed genes (n = 38) in our experiments was more than twice the number of induced genes (n = 14). Since AML1-ETO, PML-RARα, and PLZF-RARα act primarily as transcriptional repressors on most of their target gene promoters, the larger number of repressed genes could be expected.
So far, most studies have focused on identification of target genes of a single aberration. For example, AML1-ETO specifically represses the p14ARF
tumor suppressor (25
). Therefore, it is not unexpected that most of the genes identified in our study have not previously been regarded as target genes for AML fusion proteins.
Among the genes that were repressed by the fusion proteins, 29% were induced at least twofold by TSA in the fusion protein-expressing U937 cell lines and more than 30% could be induced by TSA in AML1-ETO-containing Kasumi leukemia cells. Some of these genes act as differentiation-associated transcription factors (e.g., Id2 and MEF) (7
). The functional relevance of these genes needs to be studied further.
Several of the induced genes regulate effects downstream of Wnt signaling (Table ). Regulation of the protein levels of catenins is central to the activation of this pathway. While β-catenin levels are predominantly regulated by the protein degradation machinery, this mechanism is less effective for plakoglobin (γ-catenin) (49
). This is in accordance with our data that plakoglobin was induced by all three fusion proteins on the mRNA as well as on the protein level. Importantly, plakoglobin was also expressed at significantly higher levels in fusion protein-positive AML patient samples compared to AML patient samples that did not express a fusion protein. Thus, our a priori hypothesis, derived from microarray experiments in a cell culture system, was independently confirmed with primary AML patient samples in vivo.
The protein level of β-catenin was also increased in cells expressing one of the fusion proteins. However, β-catenin was not regulated on the mRNA level (data not shown). While we cannot exclude that the fusion proteins have direct effects on β-catenin stability, the increased levels of β-catenin protein may well be a direct consequence of the transcriptional regulation of plakoglobin. Other groups have shown several mechanisms that mediate induction of β-catenin by plakoglobin. Overexpression of plakoglobin results in the accumulation of endogenous β-catenin in the nucleus (31
). This localization within the nucleus could reduce the accessibility of plakoglobin to the cytosolic degradation machinery. In addition, plakoglobin interacts with the APC protein and thereby possibly elevates the level of β-catenin by interfering with its degradation (31
Taken together, our data indicate that, in addition to the direct influence of plakoglobin on TCF- and LEF-dependent promoters, plakoglobin-induced stabilization of the β-catenin protein may contribute to the observed induction of Wnt target genes in hematopoietic cells.
Further analyses deciphered the mechanisms leading to plakoglobin induction. AML fusion proteins activate the plakoglobin promoter through an indirect mechanism which is dependent on the corepressor and histone deacetylase recruiting mechanisms. The direct effector protein is not known yet. Site-directed mutagenesis of a CCAAT site in the plakoglobin promoter did not alter promoter activation by AML1-ETO (data not shown).
Plakoglobin is a structural protein in the organization of cell-cell contacts, but it also plays a role in the Wnt signaling pathway (60
). In this context, plakoglobin functions as a coactivator of the TCF and LEF transcription factors. The Wnt signaling pathway plays a crucial role in cell fate and differentiation decisions in a variety of organs (8
). In addition, downstream signaling through the TCF and LEF transcription factors induces proliferation and cellular transformation (43
). Wnt signaling pathway alterations are a major factor in the pathogenesis of several types of cancer, with colon cancer being the best-studied example.
The importance of this pathway in the pathogenesis of leukemia was largely unknown. Wnt proteins are able to determine the cell fate of hematopoietic progenitors, and expression of several members of the Wnt family led to an expansion of multilineage progenitor cells (2
). While this article was under consideration, it was reported that Wnt3A plays an important role in self-renewal of hematopoietic stem cells (46
). This is an intriguing observation, given that AML fusion proteins are presumed to function partially by enhancing self-renewal (39
). It is possible that the self-renewal capacity conferred by AML fusion proteins is mediated by Wnt signaling pathway activation.
This is the first report to demonstrate that plakoglobin and TCF and LEF factors have a role in leukemia. Interestingly, several additional recent findings provide hints that hematopoiesis and leukemia might be more closely associated with the Wnt signaling pathway than was previously anticipated. For example, Wnt signaling prevents adipocyte differentiation by inhibiting C/EBPα (47
). C/EBPα is a major inducer of granulopoiesis and is frequently mutated in the FAB M2 subtype of AML (42
). Also, ATRA was shown to inhibit β-catenin-TCF and β-catenin-LEF signaling, and β-catenin interacted with PML (9
In the current study, we provide evidence that AML1-ETO and PML-RARα induce TCF and LEF transcription factor-dependent activity. In Western blots, plakoglobin appeared as a double band, possibly due to posttranslational modification. Plakoglobin induction was associated with the formation of plakoglobin/LEF-1 and β-catenin/LEF-1 complexes in vivo. Subsequently, we demonstrated plakoglobin binding to the c-myc
promoter activity, and increased c-myc
expression. Plakoglobin preferentially induces c-myc
compared to other Wnt target genes (22
). For the first time, we demonstrated plakoglobin induction by AML fusion proteins in hematopoietic cells. Plakoglobin in turn activates Wnt signaling and leads to expression of functionally relevant target genes. While expression of c-myc
and cyclin D1 was clearly induced by the fusion proteins, these genes were not identified in the microarray analyses, which is likely to be based on the strict selection criteria in the microarray analyses. This strict regulation decreased the number of false-positives but also implied that several genuine target genes were not detected. With regard to c-myc
, the mechanisms that lead to its induction by AML fusion proteins are indirect. This provides a possible explanation for why Myc was not found to be induced in the microarray analyses. The c-Myc oncoprotein appears to be regulated by AML1-ETO in several ways. In addition to our findings, it has been described that c-myc
is induced by AML1-ETO via C/EBPα inhibition (19
The functional relevance of Wnt signaling induced by AML fusion proteins via plakoglobin induction was verified in vitro and in vivo. In primary hematopoietic cells, retroviral transduction of plakoglobin retained the immature phenotype of the progenitor cells. The 32D cell line showed increased colony formation potential and proliferative activity upon plakoglobin transfection. Interestingly, plakoglobin enhanced the leukemogenic potential of 32D cells in syngeneic mice. Injection of plakoglobin-expressing 32D cells caused rapid death in almost all mice, and their organs were diffusely infiltrated by blast-like hematopoietic cells. The latency period of leukemia development in the 32D-plakoglobin-injected mice was comparable to the time it took for transforming FLT3 internal tandem duplication mutations in our previous experiments to lead to leukemia-related death (34
). The bone marrow-infiltrating cells grew in culture like to the initial 32D-plakoglobin cells and still depended on interleukin-3 (data not shown). Thus, no secondary mutations appeared to be necessary for leukemia-like growth of 32D-plakoglobin cells in vivo. The leukemias induced by AML1-ETO in a mouse model also grew factor dependently in vitro (17
). Thus, plakoglobin effects in 32D cells are in accordance with AML1-ETO effects in the mouse model. While some of the mice injected with the 32D control cells also died, their deaths occurred much later and the organs were less heavily infiltrated.
Taken together, our analyses identified shared targets of fusion proteins derived from the most frequent translocations in AML. PML-RARα, PLZF-RARα, and AML1-ETO induced plakoglobin expression in cell lines as well as in primary patient samples. Induction of plakoglobin expression resulted in transcriptional activation of the TCF and LEF transcription factors. The TCF and LEF target genes, cyclin D1 and c-myc, which are well known oncogenes, are induced by the fusion proteins, and typical functional consequences of oncogenic Wnt signaling were observed. These data provide evidence that AML-associated fusion proteins contribute to leukemogenesis by specifically targeting the Wnt signaling pathway.