Block of C/EBPα function by phosphorylation in acute myeloid leukemia with FLT3 activating mutations

Beth Israel Deaconess Medical Center/Harvard Medical School, Boston, MA 02115, USA.
Journal of Experimental Medicine (Impact Factor: 13.91). 02/2006; 203(2):371-381.
Source: PubMed

ABSTRACT Mutations constitutively activating FLT3 kinase are detected in ∼30% of acute myelogenous leukemia (AML) patients and affect downstream pathways such as extracellular signal–regulated kinase
(ERK)1/2. We found that activation of FLT3 in human AML inhibits CCAAT/enhancer binding protein α (C/EBPα) function by ERK1/2-mediated phosphorylation, which may explain the differentiation block of leukemic blasts. In MV4;11 cells,
pharmacological inhibition of either FLT3 or MEK1 leads to granulocytic differentiation. Differentiation of MV4;11 cells was
also observed when C/EBPα mutated at serine 21 to alanine (S21A) was stably expressed. In contrast, there was no effect when serine 21 was mutated to
aspartate (S21D), which mimics phosphorylation of C/EBPα. Thus, our results suggest that therapies targeting the MEK/ERK cascade or development of protein therapies based on transduction
of constitutively active C/EBPα may prove effective in treatment of FLT3 mutant leukemias resistant to the FLT3 inhibitor therapies.

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    • "This phosphorylation induces a conformational change in CEBPA such that the transactivation domains of two CEBPA molecules within a dimer move further apart. Activation of FLT3 in human AML appears to block the phosphorylation of CEBPA at serine 21 which may explain the differentiation block of blasts in leukemias with activated FLT3 (Radomska et al., 2006). Additional in vitro reports exist on various CEBPA phosphorylation sites (Figure 1). "
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    ABSTRACT: The current paradigm on leukemogenesis indicates that leukemias are propagated by leukemic stem cells. The genomic events and pathways involved in the transformation of hematopoietic precursors into leukemic stem cells are increasingly understood. This concept is based on genomic mutations or functional dysregulation of transcription factors in malignant cells of patients with acute myeloid leukemia (AML). Loss of the CCAAT/enhancer binding protein-alpha (CEBPA) function in myeloid cells in vitro and in vivo leads to a differentiation block, similar to that observed in blasts from AML patients. CEBPA alterations in specific subgroups of AML comprise genomic mutations leading to dominant-negative mutant proteins, transcriptional suppression by leukemic fusion proteins, translational inhibition by activated RNA-binding proteins, and functional inhibition by phosphorylation or increased proteasomal-dependent degradation. The PU.1 gene can be mutated or its expression or function can be blocked by leukemogenic fusion proteins in AML. Point mutations in the RUNX1/AML1 gene are also observed in specific subtypes of AML, in addition to RUNX1 being the most frequent target for chromosomal translocation in AML. These data are persuasive evidence that impaired function of particular transcription factors contributes directly to the development of human AML, and restoring their function represents a promising target for novel therapeutic strategies in AML.
    Oncogene 11/2007; 26(47):6829-37. DOI:10.1038/sj.onc.1210765
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    • "For example, activation of the FLT3 receptor phosphorylates, P85 and Shc, in lymphoid Ba/F3 cells but not in fibroblastic NIH3T3 cells (Dosil et al, 1993; Zhang et al, 1999). Activation of ERK1/2 and decreased expression of C/EBPa was detected in FLT3/ITD patient samples, but not in FLT3/ITD-transfected murine 32Dcl3 or 503 cell lines in some other studies (Zheng et al, 2002; Radomska et al, 2006). "
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    ABSTRACT: Activating mutation of FLT3 by internal tandem duplications (ITDs) in the juxtamembrane region is the most common molecular aberration found in acute myeloid leukaemia (AML). In this study, a lentiviral vector containing two promoters achieved consistent and efficient co-expression of FLT3/ITD and GFP in transduced human CD34(+) haematopoietic stem/progenitor cells (HSPCs). When cultured in medium containing stem cell factor, thrombopoietin and FLT3 ligand (FL), FLT3/ITD-transduced cells demonstrated enhanced self-renewal and survival potential, unaffected by the withdrawal of FL. These cells retained a CD34(+)CD38(-/dim) immunophenotype, typical of HSPCs. Compared to cells transduced with a vector expressing GFP alone, FLT3/ITD-transduced HSPCs had a higher fraction of cells in active cell cycle. FLT3/ITD-transduced HSPCs were more sensitive to the induction of cytotoxicity by CEP-701, a selective FLT3 inhibitor, indicating a rapid 'addiction' to signalling through this oncogenic pathway. The FLT3/ITD-transduced HSPCs showed increased expression of Pim-1, c-Myc and Cyclin D3 (CCND3), each of which may contribute to the altered genetic programme instituted by FLT3/ITD signalling. Taken together, these results indicate that FLT3/ITD mutations may contribute to leukaemic transformation of normal HSPCs by prolonging survival, promoting proliferation and partially blocking differentiation. CEP-701 may act as a potent therapeutic agent for AML stem cells harbouring FLT3/ITD mutations.
    British Journal of Haematology 05/2007; 137(1):64-75. DOI:10.1111/j.1365-2141.2007.06525.x
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    ABSTRACT: CCAAT enhancer binding protein alpha (C/EBP alpha) is the founding member of a family of basic region/leucine zipper (bZIP) transcription factors and is a master regulator of granulopoiesis. It is expressed at high levels throughout myeloid differentiation and binds to the promoters of multiple myeloid-specific genes at different stages of myeloid maturation. Profound hematopoietic abnormalities occur in mice nullizygous for C/EBP alpha including a selective early block in the differentiation of granulocytes. Mutations in C/EBP alpha are present in a subset of patients with AML presenting with a normal karyotype. These mutations can result in the expression of a 30 kDa dominant negative C/EBP alpha isoform, which contributes to loss of C/EBP alpha function. The molecular basis for this observation remains unknown. In addition to phosphorylation, C/EBP alpha is modified, post-translationally by a small ubiquitin-related modifier (SUMO) at a lysine residue (K159), which lies within the growth inhibitory region of the C/EBP alpha protein. Sumoylation at K159 in the C/EBP alpha protein prevents association of the SWI/SNF chromatin remodeling complex with C/EBP alpha, thereby hampering transactivation. In this review, the functional implications of post-translational modification, particularly sumoylation, of C/EBP alpha in normal granulopoiesis and leukemia are considered.
    Blood Cells Molecules and Diseases 07/2008; 41(1):77-81. DOI:10.1016/j.bcmd.2008.02.011
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