Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2005 Aug;115(8):2159-68.
doi: 10.1172/JCI24225. Epub 2005 Jul 14.

The AML1-ETO fusion gene and the FLT3 length mutation collaborate in inducing acute leukemia in mice

Christina Schessl  1 , Vijay P S Rawat, Monica Cusan, Aniruddha Deshpande, Tobias M Kohl, Patricia M Rosten, Karsten Spiekermann, R Keith Humphries, Susanne Schnittger, Wolfgang Kern, Wolfgang Hiddemann, Leticia Quintanilla-Martinez, Stefan K Bohlander, Michaela Feuring-Buske, Christian Buske
Affiliations

The AML1-ETO fusion gene and the FLT3 length mutation collaborate in inducing acute leukemia in mice

Christina Schessl et al. J Clin Invest. 2005 Aug.

Abstract

The molecular characterization of leukemia has demonstrated that genetic alterations in the leukemic clone frequently fall into 2 classes, those affecting transcription factors (e.g., AML1-ETO) and mutations affecting genes involved in signal transduction (e.g., activating mutations of FLT3 and KIT). This finding has favored a model of leukemogenesis in which the collaboration of these 2 classes of genetic alterations is necessary for the malignant transformation of hematopoietic progenitor cells. The model is supported by experimental data indicating that AML1-ETO and FLT3 length mutation (FLT3-LM), 2 of the most frequent genetic alterations in AML, are both insufficient on their own to cause leukemia in animal models. Here we report that AML1-ETO collaborates with FLT3-LM in inducing acute leukemia in a murine BM transplantation model. Moreover, in a series of 135 patients with AML1-ETO-positive AML, the most frequently identified class of additional mutations affected genes involved in signal transduction pathways including FLT3-LM or mutations of KIT and NRAS. These data support the concept of oncogenic cooperation between AML1-ETO and a class of activating mutations, recurrently found in patients with t(8;21), and provide a rationale for therapies targeting signal transduction pathways in AML1-ETO-positive leukemias.

PubMed Disclaimer

Figures

Figure 1
Figure 1
Schematic diagram and analysis of expression of different constructs. (A) Retroviral constructs for expression of AML1-ETO and of the AML1-ETO-L148D (31, 47), FLT3-LM, and FLT3-LM-KD mutant proteins. The GFP vector served as a control. AE, AML1-ETO; LTR, long-terminal repeat; RHD, runt homology domain; TAF110, TATA-binding protein–associated factor 110; HHR, hydrophobic heptad repeat; ZNF, zinkfinger; TM, transmembrane; JM, juxtamembrane; PTK, protein tyrosine kinase; KI, kinase insert. (B, C, and E) Western blot analysis of cellular extracts from GP+ E86 and NIH 3T3 cells transfected with the different constructs (the molecular mass is indicated). Kasumi cells served as a positive control. (D) α-pTyr plot demonstrating phosphorylation of FLT3-LM and FLT3-WT but not of FLT3-LM-KD. (F) FACS analysis of Ba/F3 cells transduced with the FLT3 constructs. (G) Growth of IL-3–dependent Ba/F3 cells infected with the different constructs. (H and I) Flow cytometry and RT-PCR analysis of cells coexpressing FLT3-LM/YFP and AML1-ETO/GFP, isolated from a representative leukemic mouse. FL, FLT3 ligand; PB, peripheral blood.
Figure 2
Figure 2
Analyses of CFU-S frequencies. (A) Primary BM cells retrovirally transduced with GFP, AML1-ETO, AML1-ETO-L148D, FLT3-LM, or FLT3-LM-KD vectors or with combinations of the different vectors were isolated by FACS 48 hours after infection and injected into lethally irradiated mice to assess initial (day 0) CFU-S numbers. CFU-S frequency per 1 × 105 initiating BM cells was determined in 3 independent experiments. The number of analyzed mice and the P value compared with the GFP control are indicated. (B) CFU-S frequency of primary BM cells infected with GFP or with both AML1-ETO and FLT3-LM and treated with the inhibitor PKC412 for 48 hours compared with untreated controls.
Figure 3
Figure 3
Survival of transplanted mice. Survival curve of mice transplanted with BM cells expressing AML1-ETO (n = 9), FLT3-LM (n = 9), or GFP (n = 12), of mice transplanted with marrow cells coexpressing AML1-ETO and FLT3-LM (n = 11), and of secondarily transplanted mice (n = 5).
Figure 4
Figure 4
Immunophenotype and morphology of hematopoietic cells recovered from leukemic mice. The plots show representative FACS profiles from BM cells in comparison with cells from GFP control animals, with indication of the proportion of positive cells within the GFP+/YFP+ compartment. The photographs show cytospin preparations (H&E; magnification, ×630) from peripheral blood (A, right image; B and C) and from BM (A, left image). (A) AML with a dominant mast cell population (marked by arrows) (mouse no. 16). (B) B-ALL (mouse no. 17). (C) T-ALL (mouse no. 20).
Figure 5
Figure 5
Histological analyses of leukemic mice. (A–F) Histological analyses of AML (mouse no. 14). Original magnifications: A–C, ×200; inset in C, ×1,000; D and right side of E, ×250; inset in D, ×650; left side of E, ×400; F, ×400. (G–O) Histological analyses of AML with a dominant mast cell population (mouse no. 16). (G–L) Primary recipient. (M–O) Secondary recipient. Original magnifications: G and M, ×100; H, J, and N, ×200; I, K, and O, ×400; L, ×600. Mast cells with metachromatic granulation in the Giemsa stain are indicated by an arrow. MPO, myeloperoxidase; CAE, N-acetyl-chloroacetate esterase.
Figure 6
Figure 6
Analysis of proviral integrations. (A) Southern blot analysis of genomic DNA from different primary mice and a secondary recipient to detect clonal proviral integrations. DNA was digested with EcoRI, which cuts once in the proviral sequence, and blots were hybridized to a GFP/YFP probe. The mouse numbers are indicated (corresponding to those in Table 2). S, spleen; PB, peripheral blood; sec. of 21, second mouse of 1° mouse no. 21. (B) Bubble PCR analyses of retroviral integration sites in diseased mice. The bands (A–I) were isolated, subcloned, and sequenced. A description of the PCR products is given in Table 3. Asterisk indicates resolution as 2 unique bands after subcloning and sequencing of integration sites.
Figure 7
Figure 7
Expression of lymphoid antigens on 52 samples of patients with AML1-ETO–positive AML, determined by immunophenotyping. Samples were defined as negative for expression of cytoplasmic antigens when less than 10% of the cells stained with the antibody, and as negative for expression of surface antigens when less than 20% of the cells stained with the antibody (shaded areas).
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Look AT. Oncogenic transcription factors in the human acute leukemias. Science. 1997;278:1059–1064. - PubMed
    1. Rowley JD. The role of chromosome translocations in leukemogenesis. Semin. Hematol. 1999;36:59–72. - PubMed
    1. Gilliland DG, Tallman MS. Focus on acute leukemias. Cancer Cell. 2002;1:417–420. - PubMed
    1. Schnittger S, et al. Analysis of FLT3 length mutations in 1003 patients with acute myeloid leukemia: correlation to cytogenetics, FAB subtype, and prognosis in the AMLCG study and usefulness as a marker for the detection of minimal residual disease. Blood. 2002;100:59–66. - PubMed
    1. Gilliland DG. Molecular genetics of human leukemias: new insights into therapy [review] Semin. Hematol. 2002;39:6–11. - PubMed

Publication types

MeSH terms

Substances