Stem cell expression of the AML1/ETO fusion protein induces a myeloproliferative disorder in mice

Abstract

The t(8;21)(q22;q22) translocation, present in 10 -15% of acute myeloid leukemia (AML) cases, generates the AML1͞ETO fusion protein.To study the role of AML1͞ETO in the pathogenesis of AML, we used the Ly6A locus that encodes the well characterized hematopoietic stem cell marker, Sca1, to target expression of AML1͞ETO to the hematopoietic stem cell compartment in mice. Whereas germ-line expression of AML1͞ETO from the AML1 promoter results in embryonic lethality, heterozygous Sca1 ؉/AML1-ETO ires EGFP (abbreviated Sca ؉/AE ) mutant mice are born in Mendelian ratios with no apparent abnormalities in growth or fertility. Hematopoietic cells from Sca ؉/AE mice have markedly extended survival in vitro and increasing myeloid clonogenic progenitor output over time. Sca ؉/AE mice develop a spontaneous myeloproliferative disorder with a latency of 6 months and a penetrance of 82% at 14 months. These results reinforce the notion that the phenotype of murine transgenic models of human leukemia is critically dependent on the cellular compartment targeted by the transgene. This model should provide a useful platform to analyze the effect of AML1͞ETO on hematopoiesis and its potential cooperation with other mutations in the pathogenesis of leukemia.T he t(8;21)(q22;q22) translocation is one of the most commonly detected karyotypic abnormalities in acute myeloid leukemia (AML). This genetic alteration is found in up to 40% of de novo AML cases of the French-American-British M2 subtype, and in 12-15% of AML cases overall (1, 2). In many instances of AML, t(8;21) is the sole cytogenetic abnormality, suggesting that the translocation plays a key role in transformation. The t(8;21) translocation fuses sequences from the AML1 (RUNX1, CBFA2, and PEBP␣) gene on chromosome 21 to the ETO (MTG8) gene on chromosome 8. This translocation results in production of the AML1͞ETO protein, an in-frame fusion of the N terminus of AML1 and virtually the entire ETO protein. AML1 is the DNAbinding subunit of core-binding factor (CBF), a multimeric transcription factor complex that includes CBF␤ and additional transcriptional coactivators. Mice lacking either Aml1 or Cbf␤ fail to develop definitive intraembryonic hematopoiesis and die midgestation (3-6). Mice heterozygous for an AML1͞ETO allele knocked into the Aml1 locus have an identical phenotype, providing genetic evidence that the AML1͞ETO fusion protein acts as a dominant inhibitor of CBF activity (7,8).Although it is clear that CBF plays a critical role in hematopoietic development, several lines of evidence suggest that expression of its dominant inhibitor AML1͞ETO is not sufficient to cause AML. AML1͞ETO expression is frequently detectable in peripheral blood cells from t(8;21) AML patients for years into durable remission (9, 10). Conversely, clonotypic AML1͞ETO sequences were identified in DNA retrospectively extracted from neonatal blood samples from 5 of 10 children with t(8;21) AML, preceding the development of AML by 5-10 years in these cases (11). Several strains of AML1͞ETO-express...

AML1-ETO and C-KIT mutation/overexpression in t(8;21) leukemia: Implication in stepwise leukemogenesis and response to Gleevec

“…These data unveil an important biological function of AE, and demonstrate that AE can activate PTK signal for cell proliferation by itself and may thus contribute to disease progression. Therefore, these data seem also to be in agreement with the ability of AE alone in inducing myeloproliferative disorder in mouse when appropriate promoter was used to drive the gene expression (14). In exploring the possible mechanism underlying AE-induced up-regulation of C-KIT, we found in U937-A͞E cells that TGF-␤1, a potent negative regulator of hematopoiesis, was down-regulated only 6 h after specific induction of AE.…”

Review: genetic models of acute myeloid leukaemia

“…This suggested that these fusion proteins are dominant-negative inhibitors of corresponding normal gene function during primitive haematopoietic lineage commitment. In only one exception, where AML-ETO was targeted to the stem cell compartment by Ly6A locus (Fenske et al, 2004) did animals survive and developed a long (>6 months) latency myeloproliferative disease, reinforcing the idea that disease phenotype is dependent of the cellular compartment-targeted and accumulation of mutations.…”

“…As mentioned in the techniques section, in this approach the mutated exogenous cDNA is targeted in-frame directly to a pre-defined locus by homologous recombination in the ES cells and employed in developing knock-in models of the following: AML1-ETO t(8;21) (Yergeau et al, 1997;Okuda et al, 1998;Fenske et al, 2004), PLZF-RARa t(11;17) (He et al, 1999), CBFb-MYH11 inv(16) t(16;16) found in AML subtype FAB M4Eo (that is myelomonocytic differentiation together with increased eosinophilic progenitors) (Castilla et al, 1996), CBFb-GFP (Kundu et al, 2002), MLL-AF9 t(9;11) found in AML FAB M5 (that is myelomonocytic variants) (Dobson et al, 1999) and MLL-lacZ (Dobson et al, 2000). Resultant AML-ETO or CBFb-MYH11 knock-in fusion genes resulted in embryonic lethality owing to lack of definitive embryonic haematopoiesis in a manner remarkably similar to that of the corresponding (AML1 À/À , CBFb À/À ) knockout animals.…”

Oncogenic pathways of AML1-ETO in acute myeloid leukemia: Multifaceted manipulation of marrow maturation

“…Correspondingly, transfection of AML1-ETO into primary human CD34 + hematopoietic progenitors efficiently blocked erythroid lineage commitment, normally denoted by CD36 and GPA upregulation coupled with CD34 and CD13 downregulation [3]. This effect of AML1-ETO was reminiscent of the erythroid blockade uniquely associated with t(8;21)-positive human AMLs and of the erythroid inhibition seen in several murine models of AML1-ETO transformation [51][52][53][54]. AML1-ETO employed a novel mechanism in its inhibition of erythropoiesis, interfering with the acetylation of GATA-1 mediated by p300/CBP.…”