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The RUNX1/ETO fusion gene is usually found in Acute Myeloid Leukaemia (AML) of the FAB M2 subtype, in the granulocytic lineage (Rowley 1984; Brunning and McKenna 1994). This fusion gene is often found in early adulthood, and is

associated with a good prognosis (Choi et al. 2006). Secondary mutations occurring in other genes are important in the development of AML (Elagib and Goldfarb 2007).

The Runx1 gene (or AML1, CBFA2, PEBP2αB) is essential for the development

of the haematopoietic system during embryogenesis (Okuda et al. 1996; Wang et al. 1996b), and is one of the most common targets of chromosomal translocation in human leukaemias (Look 1997; Speck and Gilliland 2002). Three Runx splice variants are possible, and all contain a ‘Runt homology domain’. The two longer isoforms (numbers 1 and 2) also have a transactivation domain that is absent on the shorter isoform (Meyers et al. 1993; Bae et al. 1994; Takahashi et al. 1995). This short form may still have a regulatory role, as it binds more efficiently to regulatory regions of DNA, but does not initiate transcription following binding (Meyers et al. 1993; Bae et al. 1994; Licht 2001). Runx1 combines with CBFβ to form a heterodimer that co-operates with other transcription factors and is thus able to regulate a range of haematopoietic lineage- specific genes (Lutterbach and Hiebert 2000). The RUNX-1/CBF-β heterodimer may

regulate gene expression by recruiting proteins with histone acetyl transferase (HAT) activity, thus acetylating nearby histone tails and in turn switching on gene expression (reviewed Licht 2001).

The human and murine Eight Twenty-One (ETO) gene (also known as ‘Myeloid Translocation Gene 8’ (MTG8)) belongs to a family of three myeloid transforming genes (Gamou et al. 1998; Kitabayashi et al. 1998) and was identified as the partner of AML-1 in the t(8;21) fusions (Miyoshi et al. 1993; Erickson et al. 1996). Much information about the co-repressor function of ETO has been gleaned from studies of the repressive activity of AML1/ETO (Hiebert et al. 2001). The zinc-finger domains of ETO (NHR2 and NHR4) are required for AML1/ETO repression of transcription and suggest a co-repressor role for ETO (Lenny et al. 1995). Numerous co-repressor binding partners have been identified, including nuclear receptor co-repressor (N-CoR) (Wang et al. 1998), the silencing mediator of retinoic acid and thyroid hormone receptors (SMRT) (Chen and Evans 1995; Gelmetti et al. 1998), mSin3A and HDAC1 (Gelmetti et al. 1998; Wang et al. 1998; Lutterbach et al. 1998a), as well as the promyelocytic leukaemia zinc-finger (PLZF) and Growth factor independence-1 (Glf1) (Melnick et al. 2000). The NHR2 domain mmediates dimerisation, and multiple ETO domains are required for maximal repression (Lutterbach et al. 1998b)

MTG8 also plays an important role in development of the gastro-intestinal system as indicated by gene-targeting in mice in which inactivation of the MTG8 gene resulted in reduced viability of heterozygotes, evidently stemming from gross disruption of the gut architecture, including absence of the midgut in many cases (Calabi et al. 2001).

1.2.3.1 RUNX1-ETO Breakpoint

The RUNX1-ETO translocation, t(8;21)(q22;q22), is one of the most common in acute myeloid leukaemia, and comprises the N-terminal end of the RUNX-1 protein, and the c-terminus of the ETO protein (Miyoshi et al. 1991, and see Figure 1.3). Where Runx1 is involved in acute myeloid leukaemia translocations, the Runt homology domains are usually intact, but the transactivation domain is often replaced (Nucifora and Rowley 1995; Speck and Gilliland 2002).

It is thought that AML1-ETO fusion leads to the development of leukaemias because the RUNX1 transcriptional activator, after fusion with ETO, becomes a transcriptional repressor of the RUNX1 activation targets (reviewed in Elagib and Goldfarb 2007), that is, those required for haematopoiesis and differentiation (Sakakura et al. 1994; Gelmetti et al. 1998). RUNX1/ETO may also selectively bind co-repressor proteins (eg. N-CoR, SMRT) with deacetylating activities and then prevent the binding of proteins that stimulate acetyl transferase activity, resulting in the repression of target genes (Wang et al. 1999). This binding appears to be regulated by the ETO domain (Gelmetti et al. 1998). However it is becoming clear that rather than causing the indiscriminate inhibition of RUNX-1 targets, specific genes, such as G-CSF, are upregulated by RUNX1-ETO (Shimizu et al. 2000). Similarly microarray studies demonstrate that a range of genes can be upregulated or downregulated (Shimada et al. 2000).

1.2.3.2 Neutralisation of the RUNX1-ETO Fusion Gene

AML is conventionally treated with chemotherapies, such as a combination of an anthracycline (eg. daunorubicin or idarubicin) and cytarabine (Lowenberg et al. 1999). As permanent remission is rare after this treatment alone (Cassileth et al. 1988), it is usually followed with consolidating treatments. Depending on the patient’s prognosis this may consist of further chemotherapy if the prognosis is good, or

Figure 1.3 Domains of the RUNX1/ETO protein

Figure 1.3 - AML1/ETO (RUNX1/ETO) promiscuously targets and blocks the activities of RXR/RAR and other hematopoietic transcription factors. Different functional domains of the AML1/ETO fusion protein are indicated as follows: the Runt DNA-binding domain (DBD) of the AML1 moiety; a region that shares homology with TAF110 and other related TAF proteins; a heptad repeat of hydrophobic amino acids (HHR); a region that shares homology withDrosophilanervy protein; and a C-terminal region

containing 2 nonclassical zinc fingers. Positions of indicated transcription factors relative to AML1/ETO do not reflect regions of interaction with the fusion protein. From Petrie and Zelent (2007).

allogeneic or autologous stem cell transplantation for patients with a poor prognosis (Appelbaum et al. 2000).

Alternatively, as abnormal recruitment and altered activity of histone deacetylases (HDAC’s) are a common theme in acute myeloid leukaemias (Minucci et al. 2001), the development of various HDAC inhibitors (HDACi) offers the potential for differentiation therapy in a range of acute myeloid leukaemias. However, while this is able to revert the malignant phenotype in some cases (Marks et al. 2000), inhibition of the enzymatic activity of the HDAC complex on its own is not sufficient to restore the ability of AML cells to differentiate (Minucci et al. 2001). Combination with retinoic acid is emerging as the superior option, even in non-APL cases of AML (ie. those with the RUNX1-ETO translocation) (Ferrara et al. 2001).

In spite of these developments, no specific and effective therapy has thus far been found for AML, yet molecular therapies have achieved some promising results. The RUNX1-ETO fusion mRNA has successfully been targeted in vitro with hammerhead ribozymes, resulting in protein knockdown (Szyrach et al. 2001), and inhibition of cell growth (Kozu et al. 2000). Similarly the use of siRNAs targeted against the RUNX1-ETO selectively downregulated the fusion mRNA, without effect on normal RUNX1 (Heidenreich et al. 2003). This resulted in increased sensitivity to differentiation agents and reduced clonogenicity of the cells (Heidenreich et al. 2003). Microarrays showed that these effects were accompanied by upregulation of antiproliferative genes and genes associated with differentiation, but down-regulation of genes associated with drug resistance and a poor prognosis (Dunne et al. 2006). Finally, the transient delivery of the siRNA molecules proved sufficient to delay tumour formation in vivo (Martinez Soria et al. 2009).