C/EBPγ deregulation results in differentiation arrest in acute myeloid leukemia
Meritxell Alberich-Jordà, … , Ruud Delwel, Daniel G. Tenen
Meritxell Alberich-Jordà, … , Ruud Delwel, Daniel G. Tenen
Published November 19, 2012
Citation Information: J Clin Invest. 2012;122(12):4490-4504. https://doi.org/10.1172/JCI65102.
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Research Article Oncology

C/EBPγ deregulation results in differentiation arrest in acute myeloid leukemia

Abstract

C/EBPs are a family of transcription factors that regulate growth control and differentiation of various tissues. We found that C/EBPγ is highly upregulated in a subset of acute myeloid leukemia (AML) samples characterized by C/EBPα hypermethylation/silencing. Similarly, C/EBPγ was upregulated in murine hematopoietic stem/progenitor cells lacking C/EBPα, as C/EBPα mediates C/EBPγ suppression. Studies in myeloid cells demonstrated that CEBPG overexpression blocked neutrophilic differentiation. Further, downregulation of Cebpg in murine Cebpa-deficient stem/progenitor cells or in human CEBPA-silenced AML samples restored granulocytic differentiation. In addition, treatment of these leukemias with demethylating agents restored the C/EBPα-C/EBPγ balance and upregulated the expression of myeloid differentiation markers. Our results indicate that C/EBPγ mediates the myeloid differentiation arrest induced by C/EBPα deficiency and that targeting the C/EBPα-C/EBPγ axis rescues neutrophilic differentiation in this unique subset of AMLs.

Authors

Meritxell Alberich-Jordà, Bas Wouters, Martin Balastik, Clara Shapiro-Koss, Hong Zhang, Annalisa DiRuscio, Hanna S. Radomska, Alexander K. Ebralidze, Giovanni Amabile, Min Ye, Junyan Zhang, Irene Lowers, Roberto Avellino, Ari Melnick, Maria E. Figueroa, Peter J.M. Valk, Ruud Delwel, Daniel G. Tenen

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Figure 4

Downregulation of C/EBPγ in murine C/EBPα-KO LKS cells restores neutrophilic differentiation in cell culture.

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Downregulation of C/EBPγ in murine C/EBPα-KO LKS cells restores neutroph...
(A) Western blot analysis on 293T cells transfected with or without C/EBPγ-HA in combination with shRNA-expressing constructs. sh, shRNA targeting C/EBPγ. Blots were stained with HA and HSP90 (loading control) antibodies. (B) LKS cells from pI:pC-treated CebpaFlox/Flox cre+ (KO) mice were transduced with either NSC#1 or C/EBPγ shRNA#1 (sh#1). Cebpg mRNA expression was determined 2 days after infection. The y axis indicates relative Cebpg levels expressed as percentage of Gapdh plus SD in 2 independent mice, P = 0.0042. (C) C/EBPα-KO LKS cells were infected with NSC#1 or C/EBPγ shRNA#1 (sh#1) and seeded in semi-solid medium in the presence of puromycin. 12 days after culture, individual colonies were picked up and cell morphology was analyzed. Immature colonies contained blast and myeloblast; macrophage colonies contained mainly monocytes and macrophages; mix colonies contained immature cells, monocyte-macrophages, and neutrophils; and neutrophilic colonies had only neutrophils. Original magnification, ×40. (D) Percentage of the distinct colony types determined by cell morphology analysis in individual colonies from NSC#1- or sh#1-infected C/EBPα-KO LKS cells 12 days after culture. 3 independent experiments are shown (LKS#1, 2, and 3). (E) Quantitative RT-PCR was performed in NSC#1- and sh#1-infected C/EBPα-KO LKS cells 12 days after semi-solid culture (cultures shown in D). Expression of Cebpg, neutrophil elastase (Ela2), Mpo, Cepbe, and G-CSF–R (Csf3r) was determined in 2 independent mice. The y axis indicates relative expression in 1 representative mouse as percentage of Gapdh plus SD.