Genetic manipulation of AML1-ETO–induced expansion of hematopoietic precursors in a Drosophila model

Abstract

Among mutations in human Runx1/AML1 transcription factors, the t(8;21)(q22;q22) genomic translocation that creates an AML1-ETO fusion protein is implicated in etiology of the acute myeloid leukemia. To identify genes and components associated with this oncogene we used Drosophila as a genetic model. Expression of AML1-ETO caused an expansion of hematopoietic precursors in Drosophila, which expressed high levels of reactive oxygen species (ROS). Mutations in functional domains of the fusion protein suppress the proliferative phenotype. In a genetic screen, we found that inactivation of IntroductionRUNX genes encode highly conserved Runt domain transcription factors (AML/RUNX proteins), which play critical roles during blood cell development in metazoan species. [1][2][3][4] Mutations that disrupt the activity of AML1/Runx1 lead to hematopoietic disorders including myelodysplastic syndrome and acute myeloid leukemia (AML). 2,5 AML is the most frequent and debilitating form of leukemia with approximately a quarter million new cases diagnosed each year worldwide. The t(8;21) (q22;q22) chromosomal translocation that creates the AML1-ETO fusion product is present in 12%-15% of all cases of AML patients. The M2 subtype of AML carrying the AML1-ETO fusion protein is characterized by excessive production of granulocyte precursors. 6 Under normal conditions, AML1/RUNX1 (also known as core binding factor-␣, CBF␣) in association with CBF␤ binds the enhancer core sequence, TGT/CGGT, to activate tissue-specific expression of a number of hematopoietic genes. 4 ETO (also known as MTG8) functions as a transcriptional repressor by recruiting a protein complex consisting of histone deacetylases (HDACs), the nuclear receptor co-receptor (N-CoR), mSin3A and the silencing mediator of retinoid and thyroid hormone receptor (SMRT). 7 ETO function has been implicated in the Notch pathway, 8 and it interacts with SON and TCF4. 9,10 The (8,21)(q22;q22) chromosomal aberration produces a potent oncogenic fusion protein consisting of most of the Runt domain of AML1 and nearly the complete sequence of ETO. 11 The general view among investigators in the field is that the AML1-ETO fusion product acts as a constitutive repressor form of AML1. 12,13 It has also been shown that this fusion protein has other activities in addition to interfering with AML1 function. 14,15 For example, AML1-ETO knock-in mouse embryos contain highly proliferative multilineage hematopoietic progenitors that are not found when either AML1 or CBF␤ is disrupted. 12,14 Evidently, the full range of AML1-ETO target genes as well as components involved in the pathway, and their relevance to AML pathogenesis is not completely understood.To further our understanding of AML pathogenesis, additional animal models that allow in vivo genetic analysis are needed to complement studies in the existing mouse models. To this end, a Drosophila model is valuable due to the availability of facile genetic screens, a nearly complete collection of mutants, and advanced genetic technologies. T...

Myeloid leukemia factor is a conserved regulator of RUNX transcription factor activity involved in hematopoiesis

“…In mammals, RUNX1 acts mostly as a brake on blood cell progenitor proliferation, and decreased RUNX1 dosage, as well as MDS/AML-associated mutations or translocations affecting RUNX1, tend to promote aberrant self-renewal (34). Interestingly, RUNX1-ETO also promotes hematopoietic progenitor cell expansion in Drosophila (14,35), and both WT1 overexpression and activation of the Notch signaling pathway have been linked to RUNX1-ETO-induced AML (36,37). Given these similarities, characterizing the function of LZ in the control of crystal cell number may have broader implications.…”

Notch cooperates with Lozenge/Runx to lock haemocytes into a differentiation programme

“…Munoz-Alonso et al, 2012;Palomero et al, 2006;Pierce et al, 2004;Zanet et al, 2005). Other targets include Hnf4, which was previously implicated in causing haemocyte expansion downstream of AML (Runx)-ETO (Sinenko et al, 2010), and several genes involved in cell motility (e.g. CLIP-120), as well as other conserved genes with unknown functions, such as CG32369 (LONRF1-3).…”

Genetic dissection of leukemia-associated IDH1 and IDH2 mutants and D-2-hydroxyglutarate in Drosophila

“…Wg is also expressed in crystal cells, which strongly express hml. 36,68 In larvae expressing Idh-R195H in hemocytes, the pool of Wg1 hemocytes was expanded, and a majority of these Wg1 hemocytes weakly expressed hml ( Figure 5B), suggesting an expansion of prohemocytes.68 Nevertheless, a subpopulation of Wg1 cells strongly expressed hml, suggesting that crystal cells were also expanded. We confirmed that mature crystal cells were also increased in larvae expressing Idh-R195H using the crystal cell-specific C4 marker ( Figure 5C).…”

“…[32][33][34][35] In fact, introduction of oncogenes such as AML1-ETO fusion and the hyperactive fly homolog of JAK2 can also block differentiation or stimulate proliferation of Drosophila blood cells. [36][37][38][39][40][41] We reasoned that Drosophila could provide insight into the function of IDH mutations from human cancer and could be used as a model to reveal genetic interactions that mutant IDH and D-2HG depend on to drive their biological phenotypes.Drosophila contains a single NADP 1 -dependent IDH called Idh (CG7176; note the lower-case "dh" for fly Idh, in contrast to all capital letters for human IDH1 and IDH2). Idh is 73% and 66% identical at the protein level to human IDH1 and IDH2, respectively (see supplemental Figure 1 on the Blood Web site).…”

“…Wg is also expressed in crystal cells, which strongly express hml. 36,68 In larvae expressing Idh-R195H in hemocytes, the pool of Wg1 hemocytes was expanded, and a majority of these Wg1 hemocytes weakly expressed hml ( Figure 5B), suggesting an expansion of prohemocytes.…”