The translocation (8;21), generating the AML1-ETO fusion protein, is one of the most frequent chromosomal abnormalities associated with acute myelogenous leukemia (AML). To elucidate its role in oncogenesis, bone marrow (BM) cells were infected with a retroviral vector carrying AML1-ETO and transplanted into mice. In contrast to previous transgenic mouse models, we show that AML1-ETO directly stimulates granulopoiesis, suppresses erythropoiesis, and impairs the maturation of myeloid, B, and T lymphoid cells in vivo. To determine the significance of earlier findings that expression of the tumor suppressor ICSBP is often downregulated in AML myeloblasts, AML1-ETO was introduced into BM cells derived from mice lacking the interferon regulatory factor ICSBP. Our findings demonstrate that AML1-ETO synergizes with an ICSBP deficiency to induce myeloblastic transformation in the BM, reminiscent of AML.

Mice lacking transcription factor interferon consensus sequence binding protein (ICSBP) develop a syndrome similar to human chronic myeloid leukemia and are immunodeficient. In order to define the molecular mechanisms responsible for the cellular defects of ICSBP−/− mice, we used bone marrow‐derived macrophages (BMM) to identify genes deregulated in the absence of ICSBP. Here, we report that disabled‐2 (Dab2), a signal phosphoprotein, is transcriptionally up‐regulated and accumulates in the cytoskeleton/membrane fraction of ICSBP−/− BMM. Moreover, our results revealed Dab2 as a novel IFN‐γ‐response gene. Both ICSBP and the Ets‐transcription factor PU.1 bind to the Dab2 promoter, whereby ICSBP represses PU.1‐induced Dab2 promoter transactivation in vitro. Notably, repression of Dab2 expression by ICSBP is also found in myeloid progenitors. Overexpression of Dab2 leads to accelerated cell adhesion and spreading, accompanied by enhanced actin fiber formation. Furthermore, cell adhesion induces transient Dab2 phosphorylation and its translocation to the cytoskeletal/membrane fraction. Our results identify a novel role of Dab2 as an inducer of cell adhesion and spreading, and strongly suggest that the up‐regulation of Dab2 contributes to the hematopoietic defect seen in ICSBP−/− mice.

Using retroviral vectors encoding enhanced green fluorescent protein (EGFP), we addressed to what extent expression of retroviral transgenes in hematopoietic cells depends on the multiplicity of infection (MOI) and on the halflife of the encoded protein. We show that an elevation of the MOI not only elevates the frequency of transduced cells, but also increases transgene expression levels and reduces interanimal variability in vivo (hematopoietic cells of C57BL/6J mice analyzed 13 weeks after transplantation). This suggests that the MOI has to be carefully controlled and should be adapted as desired for clinical studies when evaluating vector performance in preclinical models. The impact of protein stability is demonstrated by comparing vectors expressing EGFP or a destabilized variant with a C-terminal PEST-sequence, d2EGFP. The loss of expression

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The t(12;21) translocation, generating the TEL/AML1 fusion protein, is the most common genetic lesion in childhood cancer. Using a bone marrow transplantation model, we demonstrate that TEL/AML1 expression impinges on normal hematopoietic differentiation, leading to the in vivo accumulation and persistence of an early progenitor compartment with a Sca1 þ /Kit hi /CD11b þ phenotype and an increased self-renewal capacity, as documented by replating assays in vitro. Differentiation of these cells is not blocked, but the frequency of mature blood cells arising from TEL/AML1-transduced progenitors is low. Impaired differentiation is prominently observed in the pro-B-cell compartment, resulting in an proportional increase in early progenitors in vivo, consistent with the t(12;21) ALL phenotype. Despite the accumulation of both multipotent and B-cell progenitors in vivo, no leukemia induction was observed during an observation period of over 1 year. These results are consistent with findings in twins with concordant ALL, showing that TEL/AML1 generates a preleukemic clone in utero that persists for several years in a clinically covert fashion. Furthermore, our studies showed that the pointed domain of TEL/AML1, which recruits transcriptional repressors and directs oligomerization with either TEL/ AML1 or wild-type TEL, was essential for the observed differentiation impairment and could not be replaced with another oligomerization domain.

Mef2c is a MADS (MCM1-agamous IntroductionHematopoiesis is a tightly regulated system in which mature blood cells are constantly replenished from hematopoietic stem cells in the bone marrow, and which responds quickly to foreign invasion or environmental stress. The molecular pathways controlling normal and stress-related hematopoiesis are often targets of oncogenic lesions, the accumulation of which leads to leukemia. In particular, transcriptions factors that play pivotal roles in regulating the differentiation of myeloid cells into active mediators of the innate immune system-granulocytes, macrophages (developing through monocytes), and dendritic cells-are frequently deregulated in acute myeloid leukemia (AML). 1,2 These include the Ets-family member PU.1, the Runt transcription factor RUNX1, the interferon-regulatory factor-8 (IRF-8), and the basic leucine zipper protein (bZIP) factors C/EBP␣, c-JUN, and JUNB. Extensive cross-talk exists between these transcription factors-most likely enabling a quick and expedient response to stress signals, but also ensuring the return to homeostasis. Understanding the complex controls of monocytic and granulocytic differentiation not only provides insight into the innate immune response but also into AML.Recent studies have revealed a role of myocyte enhancer factor 2 (MEF2) transcription factors in acute leukemia. The gene encoding Mef2d has been identified at the breakpoint of a variant t(1;19) in acute lymphoblastic leukemia (ALL), [3][4][5] and the gene encoding Mef2c has recently been shown to be up-regulated in leukemic stem cells of MLL-associated leukemia (Krivtsov et al 6 and M.S., A.S., M. Forster, A.E., M.A., R. Delwel, J. Löhler, R. Slany, E.N.O., and C.S., manuscript submitted), which is associated with myelomonocytic or monocytic phenotypes. 7 Interestingly, both genes have been found as targets of retroviral integration sites in leukemia induced by murine leukemia virus. 8 The Mef2 proteins belong to a distinct class of the MADS (MCM1-agamous-deficient serum response factor) family of transcription factors, and were originally identified as important regulators of myogenic differentiation. 9 There are 4 vertebrate MEF2 genes, which are expressed in distinct but overlapping patterns during embryogenesis. Similar to other MADS domain proteins, MEF2 proteins associate with a variety of transcription cofactors to control specific sets of downstream target genes-and their activity is profoundly influenced by developmental cues and signals from the extracellular environment through several transcriptional and posttranslational controls. 10 The importance of Mef2c in the development of skeletal, cardiac, and smooth muscle is well documented, 9 and more recently, its role in craniofacial and bone development has been revealed. 11,12 Accumulating evidence also supports the importance of MEF2 transcription factors in neuronal differentiation and survival 13,14 and in mediating T-cell receptor-mediated apoptosis and cytokine expression 15,16 ; however, their role in n...

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Site-specific recombination in genetically modified cells can be achieved by the activity of Cre recombinase from bacteriophage P1. Commonly an expression vector encoding Cre is introduced into cells; however, this can lead to undesired side-effects. Therefore, we tested whether cell-permeable Cre fusion proteins can be directly used for lox-specific recombination in a cell line tailored to shift from red to green fluorescence after loxP-specific recombination. Comparison of purified recombinant Cre proteins with and without a heterologous 'protein transduction domain' surprisingly showed that the unmodified Cre recombinase already possesses an intrinsic ability to cross the membrane border. Addition of purified recombinant Cre enyzme to primary bone marrow cells isolated from transgenic C/EBPalpha(fl/fl) mice also led to excision of the 'floxed' C/EBPalpha gene, thus demonstrating its potential for in vivo applications. We conclude that Cre enyzme itself or its intrinsic membrane-permeating moiety are attractive tools for direct manipulation of mammalian cells.