The t(8;21)-encoded AML1-ETO chimeric product is believed to be causally involved in up to 15% of acute myelogenous leukemias through an as yet unknown mechanism. To directly investigate the role of AML1-ETO in leukemogenesis, we used gene targeting to create anAML1-ETO “knock-in” allele that mimics the t(8;21). Unexpectedly, embryos heterozygous for AML1-ETO(AML1-ETO/+) died around E13.5 from a complete absence of normal fetal liver–derived definitive hematopoiesis and lethal hemorrhages. This phenotype was similar to that seen following homozygous disruption of either AML1 orCBFβ. However, in contrast to AML1- or CBFβ-deficient embryos, fetal livers from AML1-ETO/+ embryos contained dysplastic multilineage hematopoietic progenitors that had an abnormally high self-renewal capacity in vitro. To further document the role of AML1-ETO in these growth abnormalities, we used retroviral transduction to express AML1-ETO in murine adult bone marrow–derived hematopoietic progenitors. AML1-ETO–expressing cells were again found to have an increased self-renewal capacity and could be readily established into immortalized cell lines in vitro. Taken together, these studies suggest that AML1-ETO not only neutralizes the normal biologic activity of AML1 but also directly induces aberrant hematopoietic cell proliferation.

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The t(8;21)-encoded AML1-ETO chimeric product is believed to be causally involved in up to 15% of acute myelogenous leukemias through an as yet unknown mechanism. To directly investigate the role of AML1-ETO in leukemogenesis, we used gene targeting to create anAML1-ETO “knock-in” allele that mimics the t(8;21). Unexpectedly, embryos heterozygous for AML1-ETO(AML1-ETO/+) died around E13.5 from a complete absence of normal fetal liver–derived definitive hematopoiesis and lethal hemorrhages. This phenotype was similar to that seen following homozygous disruption of either AML1 orCBFβ. However, in contrast to AML1- or CBFβ-deficient embryos, fetal livers from AML1-ETO/+ embryos contained dysplastic multilineage hematopoietic progenitors that had an abnormally high self-renewal capacity in vitro. To further document the role of AML1-ETO in these growth abnormalities, we used retroviral transduction to express AML1-ETO in murine adult bone marrow–derived hematopoietic progenitors. AML1-ETO–expressing cells were again found to have an increased self-renewal capacity and could be readily established into immortalized cell lines in vitro. Taken together, these studies suggest that AML1-ETO not only neutralizes the normal biologic activity of AML1 but also directly induces aberrant hematopoietic cell proliferation.

To define the leukemic potential of AML1-ETO, we recently generated a mouse strain with a conditional loxP-stop-loxP (LSL)-AML1-ETO knock-in allele that can be activated in hematopoietic cells by Cre-mediated recombination. Expression of AML1-ETO under these conditions readily induced the immortalization of multipotential hematopoietic progenitors, but failed to induce overt leukemia. Induction of secondary mutations with the alkylating agent ENU, however, resulted in the development of an acute leukemia similar to human t(8;21)-AML. Immortal AML1-ETO expressing non-leukemic cells grew as growth factor-dependent cell lines, where as the murine AML1-ETO expressing leukemias grew as growth factor-independent cell lines. This suggested that alterations in growth factor signaling pathways may cooperate with AML1-ETO to induce full transformation. Consistent with this interpretation, we have recently demonstrated that ~50% of core-binding factor leukemias have activating mutations in NRAS or KRAS. To directly assess the cooperativity of AML1-ETO and oncogenic Ras, we crossed the AML1-ETO / Mx1-Cre mice with a strain containing a LSL-KrasG12D knock-in allele. Expression of these two oncoproteins was induced simultaneously by Cre-mediated deletion of the upstream transcriptional stop cassettes. Importantly, expression levels for both genes depended on their respective endogenous regulatory sequences. As reported by others, activation of the LSL-KrasG12D allele alone resulted in the development of a lethal myeloproliferative disease (MPD), with all mice dying within 120 days (n=17). Triple transgenic mice (LSL-AML1-ETO / Mx1-Cre / LSL-KrasG12D, n=14) also died within 120 days of activation of both conditional alleles. Like the Mx1-Cre / KrasG12D only mice, the triple transgenic mice developed an MPD characterized by leukocytosis, anemia, splenomegaly and hepatic periportal leukocyte infiltrates. Surprisingly, no significant differences were noted in the frequency, latency, or phenotype of the MPD that developed in mice expressing either KrasG12D alone or both AML1-ETO and KrasG12D. Moreover, no AML was observed in the AML1-ETO and KrasG12D expressing mice. We further examined the cooperativity between these two oncoproteins by expressing a human NRASG12D cDNA through an MSCV-based retroviral vector that coexpressed a Cre-GFP fusion protein (MSCV-NRASG12D-IRES-Cre-GFP). Transduced bone marrow cells were transplanted into lethally irradiated mice and the recipients monitored for the development of disease. Expression of mutant NRASG12D by itself efficiently induced the development of a rapidly fatal MPD and a T-cell lymphoma/leukemia in the majority of transplanted mice. However, we again were unable to obtain any evidence for collaboration between NRASG12D and AML1-ETO. The clinical presentation as well as the morphology and immunophenotype of the myeloproliferative process were essentially identical between NRASG12D and AML1-ETO / NRASG12D induced disease. The only difference observed was a reduced incidence of T-cell disease in the AML1-ETO expressing mice, explicable by the previously published finding that AML1-ETO is toxic to developing T-cells. The results demonstrate that despite the frequent occurrence of RAS mutations in AML1-ETO expressing human AML, the coexpression of these two oncoproteins in mice using the experimental approaches described fails to induce an acute myeloid leukemia. These data suggest that these two oncogenic alterations require additional genetic lesions to induce acute leukemia.