Leukemic HRX Fusion Proteins Inhibit GADD34-Induced Apoptosis and Associate with the GADD34 and hSNF5/INI1 Proteins

Abstract

One of the most common chromosomal abnormalities in acute leukemia is a reciprocal translocation involving the HRX gene (also called MLL, ALL-1, or HTRX) at chromosomal locus 11q23, resulting in the formation of HRX fusion proteins. Using the yeast two-hybrid system and human cell culture coimmunoprecipitation experiments, we show here that HRX proteins interact directly with the GADD34 protein. We have found that transfected cells overexpressing GADD34 display a significant increase in apoptosis after treatment with ionizing radiation, indicating that GADD34 expression not only correlates with apoptosis but also can enhance apoptosis. The amino-terminal third of the GADD34 protein was necessary for this observed increase in apoptosis. Furthermore, coexpression of three different HRX fusion proteins (HRX-ENL, HRX-AF9, and HRX-ELL) had an anti-apoptotic effect, abrogating GADD34-induced apoptosis. In contrast, expression of wild-type HRX gave rise to an increase in apoptosis. The difference observed here between wild-type HRX and the leukemic HRX fusion proteins suggests that inhibition of GADD34-mediated apoptosis may be important to leukemogenesis. We also show here that GADD34 binds the human SNF5/INI1 protein, a member of the SNF/SWI complex that can remodel chromatin and activate transcription. These studies demonstrate, for the first time, a gain of function for leukemic HRX fusion proteins compared to wild-type protein. We propose that the role of HRX fusion proteins as negative regulators of post-DNA-damage-induced apoptosis is important to leukemia progression.The disruption of the human homologue of the Drosophila Trithorax (trx) gene, HRX, by chromosomal translocations resulting in the juxtaposition of genetic elements and formation of HRX fusion genes is one of the most common genetic alterations in human acute leukemia (52). These translocations occur in approximately 10% of acute lymphoid leukemias (ALLs), 5% of acute myeloid leukemias (AMLs), and 85% of topoisomerase II inhibitor-related secondary leukemias in adults. Furthermore, these translocations are present in half of all the de novo leukemias in children younger than 1 year (26).HRX, also referred to as ALL-1, MLL-1, or HTRX (13, 18, 52, 60), is a ubiquitously expressed 3,969-amino-acid nuclear protein (28) with unknown biologic function. HRX shares at least two regions of strong homology with the similarly sized Drosophila Trx: a series of centrally located zinc finger-like domains and a carboxy-terminal stretch of 210 amino acids. In Drosophila, Trx controls body segment patterning as a positive transcriptional regulator of the homeotic selector genes of the Antennapedia and bithorax complexes (7). Studies with transgenic mice have shown that the function of Hrx in mice has features in common with that of Trx in Drosophila. Hrx has been demonstrated to be required for proper segment identity and to function positively as a regulator of Hox gene expression in Hrx heterozygous and homozygous deficient mice (58).To date, more than 26 differen...

MLL fusion partners AF4 and AF9 interact at subnuclear foci

“…MLL also interacts with GADD34, which in turn interacts with the SWI/SNF complex through the SNF5 protein. 70 Viewed together, a network of interactions involving MLL and its fusion partners begins to emerge (Figure 7). Given that no one common function or domain has been identified in all the MLL fusion partners in nearly 10 years of study, we propose a new hypothesis based on the interactions we and others have discovered to explain the fusion partner's role in Figure 6 The MLL portion of MLL-AF4 directs MLL-AF4 expression, but retains the ability to bind to AF9.…”

MLL: How complex does it get?

“…These domains do not recognize a specific sequence but rather a structure of cruciform or bent DNA. The MLL AT-hook region was shown to interact with SET protein (not to be confused with SET domain), protein phosphatase 2A (PP2A), and the proapoptotic protein GADD34 [Adler et al, 1997;Adler et al, 1999]. MLL fusion proteins, but not the wild-type MLL, inhibit GADD34-induced apoptosis suggesting that this may be an important factor in MLL-associated leukemias [Adler et al, 1999].…”

“…The MLL AT-hook region was shown to interact with SET protein (not to be confused with SET domain), protein phosphatase 2A (PP2A), and the proapoptotic protein GADD34 [Adler et al, 1997;Adler et al, 1999]. MLL fusion proteins, but not the wild-type MLL, inhibit GADD34-induced apoptosis suggesting that this may be an important factor in MLL-associated leukemias [Adler et al, 1999]. It is unclear what role the AThooks play in MLL gene regulation, but it is possible that they aid in the targeting of MLL to specific genomic region by recognizing these DNA structures.…”

Activation ofGadd34 by diverse apoptotic signals and suppression of its growth inhibitory effects by apoptotic inhibitors

“…Overexpression of Gadd34 leads to a decrease in cell growth [4] and was shown to enhance IRinduced apoptosis [27]. The predicted Gadd34 protein product has a region of similarity with ICP34.5, a herpes simplex virus protein known to inhibit apoptosis of virally infected cells [4].…”