As described above, several studies have demonstrated disruption of human DS fetal haematopoiesis in the absence of GATA1 mutations (Chou et al., 2008; Roy et al., 2012; Tunstall-Pedoe et al., 2008), thus implicating a direct role of T21. Chromosome 21 harbours several genes that have known roles in haematopoiesis and megakaryopoiesis, and an extra copy of one or more of these genes may result in disruption of haematopoiesis.
38 In view of the difficulties inherent in studying human fetal haematopoiesis, most studies that have investigated the role of these genes have focused on murine haematopoiesis. As it appears likely that the haematological abnormalities seen in DS are due to a combination of genes, several mouse models have been used to investigate the role of T21 in the disruption of haematopoiesis. As discussed above, the genes on Hsa21 are spread across 3 murine chromosomes, with the majority on Mmu16. Several murine models of DS exist which have partial trisomy for various regions of Mmu16 and recapitulate some features of DS, including facial dysmorphism, heart abnormalities and defects in learning and behaviour (Figure 1-3). In addition, a number of haematopoietic defects have been identified.
Figure 1-3 Mouse models of DS
Illustration of Hsa21 and the regions of trisomy contained within the various mouse models of DS. Note that TC1 is trisomic for almost the entire Hsa21 and that Ts1Rhr is only trisomic for the DSCR. Orange region shows the Down syndrome Critical Region (DSCR). Other colours identify regions shared between mouse models. (Adapted from (Malinge et al., 2009))
39 The most widely studied model of DS is the Ts65Dn mouse, which is trisomic for 104 genes on Hsa21 (Reeves et al., 1995). These mice have a macrocytic anaemia and some go on to develop a myeloproliferative disorder (MPD) with thrombocytosis, megakaryocyte proliferation and myelofibrosis (Kirsammer et al., 2008). However, this MPD is only seen in older mice and is not present during the fetal or neonatal period in contrast to the pattern seen in human DS. Similarly, none of the mice developed TAM or ML-DS, indicating that T21 is not sufficient for leukaemia initiation in this murine model. Interestingly, knockdown of the ERG gene, which is on Hsa21, led to resolution of the MPD, whilst knockdown of another haematopoietic gene on Hsa21, RUNX1, had no identifiable effect, strongly implicating increased dosage of ERG in the disruption of haematopoiesis (Ng et al., 2010).
The Ts1cje murine model, which is trisomic for 81 Hsa21 genes also develops macrocytic anaemia, although fails to develop a MPD or leukaemia, even in the context of GATA1 gene knockdown (Carmichael et al., 2009). Similarly, the Tc1 model, which carries an almost complete freely segregating copy of Hsa21, shows macrocytic anaemia, splenomegaly and possible thrombocytosis, but does not develop leukaemia, even with the introduction of GATA1s (Alford et al., 2010).
Overall, whilst these models provide some indication that T21 can disrupt murine haematopoiesis, none faithfully recapitulate the abnormalities seen in fetal haematopoiesis in DS, limiting their value in the investigation of leukaemia initiation.
Despite the failure of mouse models to accurately reproduce the abnormalities seen in DS, many studies have attempted to identify the roles of individual trisomic genes in DS haematopoiesis. The two genes that have received the greatest attention are
40
RUNX1 and ERG, both of which are known oncogenes and are essential regulators
of megakaryopoiesis.
RUNX1 is a particularly compelling candidate gene as it encodes a transcription
factor essential for fetal megakaryopoiesis and HSC maintenance, and is known to be frequently translocated in AML (Okuda et al., 1996). In addition, it has been linked with GATA1 in megakaryopoiesis providing a mechanism by which a GATA1 mutation could initiate leukaemia (Elagib et al., 2003). However, RUNX1 is not overexpressed in either human DS fetal CD34+ cells (Roy et al., 2012; Tunstall- Pedoe et al., 2008), T21 embryonic stem cells (ESC), T21 induced pluripotent stem cells (iPSC) or in ML-DS blasts (Bourquin et al., 2006), questioning its role in DS.
The ETS family of transcription factor genes also plays a key role in HSC maintenance and megakaryopoiesis, with ETS2 as well as ERG, located on Hsa21 (Loughran et al., 2008; Wang et al., 2002). Forced overexpression of either ERG or
ETS2 in an erythroblastoid cell line (K562), resulted in megakaryocytic
differentiation, demonstrating the megakaryocytic bias of these genes (Ge et al., 2008; Rainis et al., 2005). In addition, overexpression of ERG and ETS2 resulted in megakaryocyte expansion in wild type or GATA1 mutant murine FL progenitors (Stankiewicz and Crispino, 2009). Furthermore, ERG overexpression immortalised GATA1s mutant cells and was associated with an upregulation of the JAK/STAT pathway. A recent paper from Shai Izraeli’s group using an ERG/GATA1s double- transgenic mouse provides the strongest evidence for the role of ERG in leukaemia initiation in DS (Birger et al., 2013). The authors showed that ERG overexpression alone resulted in MEP expansion in the FL, as previously described in human DS (Tunstall-Pedoe et al., 2008). Addition of GATA1s led to further expansion of MEP and megakaryocyte progenitors, a gene profile similar to that of human TAM and progression to myeloid leukaemia by 3 months of age. This mouse provides the
41 closest approximation of DS haematopoiesis and strongly implicates trisomy of
ERG in the disruption of haematopoiesis. However, it appears likely that more
detailed studies using human fetal haematopoietic cells are now required to provide further insight into the role of T21 in DS haematopoiesis.