A dominant-negative mutant of C/EBPα, associated with acute myeloid leukemias, inhibits differentiation of myeloid and erythroid progenitors of man but not mouse

Abstract

The CCAAT/enhancer binding protein alpha (C/EBP␣) is an essential transcription factor for granulocytic differentiation. C/EBP␣ mutations are found in approximately 8% of acute myeloid leukemia (AML) patients. Most of these mutations occur in the N-terminal coding region, resulting in a frame shift and the enhanced translation of a dominant-negative 30-kDa protein, which may be responsible for the differentiation block observed in AML. To test this hypothesis, we introduced a cDNA encoding an Nterminal mutated C/EBP␣ (mut10) into primary hematopoietic progenitors using a retroviral vector. Expression of mut10 in human CD34 ؉ cord blood cells dramatically inhibited differentiation of both myeloid and erythroid lineages. Immunohistochemical analysis demonstrated coexpression of both myeloid and erythroid markers in the immature transformed cells. Surprisingly, mut10 did not block myelocytic differentiation in murine progenitors but did alter their differentiation kinetics and clonogenicity. Experiments were performed to confirm that the differential effect of mut10 on murine and human progenitors was not due to species-specific differences in C/EBP␣ protein sequences, expression levels, or inefficient targeting of relevant cells. Taken together, our results underline the intrinsic differences between hematopoietic controls in mouse and human and support the hypothesis that mutations in CEBPA are critical events in the disruption of myeloid differentiation in AMLs. IntroductionThe hallmark of acute leukemia is the clonal, neoplastic proliferation of immature hematopoietic cells. The observed inhibition or complete arrest in differentiation can conceivably be due to an indirect mechanism, in which genetic mutations that disrupt cell cycle controls support proliferation at the expense of differentiation, and/or due to genetic abnormalities that directly interfere with the differentiation program. In support of the latter hypothesis, growing evidence suggests that the transcription factors that play pivotal roles in lineage-specific differentiation may be important targets of transformation in acute leukemias. 1 The basic zipper (B-ZIP) transcription factor CCAAT/enhancer binding protein ␣ (C/EBP␣) and the ETS-family transcription factor PU.1 are 2 major regulators of myeloid differentiation. 2,3 Unlike PU.1, which governs transcription of a wide spectrum of myeloid-specific genes found in both early hematopoietic stem cells and mature monocytes and granulocytes, C/EBP␣ regulation has a more specific function in granulopoiesis. Indeed, C/EBP␣ knockout mice show a selective block in the differentiation of granulocytes, including neutrophils and eosinophils. 4 Furthermore, the constitutive expression of C/EBP␣ in myeloid progenitors results in the induction of granulocytic development and partial inhibition of monocytic and erythroid development. [5][6][7] Several independent studies have demonstrated mutations in the CEBPA gene encoding C/EBP␣ in approximately 8% of acute myeloid leukemia (AML). [8][9][10][11] These m...

Enforced Expression of NUP98-HOXA9 in Human CD34+ Cells Enhances Stem Cell Proliferation

“…One third of week 5 cobblestone areas derived from NUP98-HOXA9 -transduced CD34 + cells were able to generate secondary week 5 cobblestone areas following repassage onto fresh MS-5, whereas no control cobblestone area had this capacity (right ). cells expressing a dominant-negative C/EBPa (39). The clinical relevance is highlighted by reports of dominant-negative C/EBPa mutations in AML patients leading to a block in myeloid differentiation (40).…”

A Comparison of Two Methods for Screening CEBPA Mutations in Patients with Acute Myeloid Leukemia

“…3 Most of the frameshifting N-terminal mutations cause enhanced translation of a dominant-negative 30-kDa protein, which may be responsible for the differentiation block observed in acute myeloid leukemias expressing this protein. 3 Mutations in the CEBPA gene are among the most common mutations in AML, particularly in patients with French-American-British (FAB) subtypes M1 and M2. 4 -6 The majority of leukemias with CEBPA mutations have a normal karyotype.…”

“…The other group of mutations consists of truncating out-of-frame insertions or deletions in the N-terminus, resulting in premature termination of the normal 42-kDa protein. 3 Most of the frameshifting N-terminal mutations cause enhanced translation of a dominant-negative 30-kDa protein, which may be responsible for the differentiation block observed in acute myeloid leukemias expressing this protein. 3 Mutations in the CEBPA gene are among the most common mutations in AML, particularly in patients with French-American-British (FAB) subtypes M1 and M2.…”

Investigating human leukemogenesis: from cell lines to in vivo models of human leukemia

“…A mutant isoform of C/EBPa associated with human AML blocked myeloid differentiation in CD34 þ human cells, whereas its expression in mouse cells induced a decrease in clonogenicity, but no differentiation block. 62 Together, these observations highlight that species-specific differences do exist in the mechanisms of malignant transformation; therefore, caution should be exercised when extrapolating results from mouse models to the human situation. 61,63 They also underscore the need for experimental models where primary human cells are used as a substrate for transformation, to gain an appreciation of the elements of the human leukemogenic process, from the functional contributions of individual oncogenes, to the sequence of molecular lesions required for the generation of LSCs.…”