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AML1–ETO downregulates the granulocytic differentiation factor C/EBPα in t(8;21) myeloid leukemia

Nature Medicine volume 7, pages 444–451 (2001)Cite this article

Abstract

The transcription factor CCAAT/enhancer binding protein α, or C/EBPα, encoded by the CEBPA gene, is crucial for the differentiation of granulocytes. Conditional expression of C/EBPα triggers neutrophilic differentiation, and Cebpa knockout mice exhibit an early block in maturation. Dominant-negative mutations of CEBPA have been found in some patients with acute myeloid leukemia (AML), but not in AML with the t(8;21) translocation which gives rise to the fusion gene RUNX1–CBF2T1 (also known as AML1–ETO) encoding the AML1–ETO fusion protein. RUNX1–CBF2T1 positive-AML blasts had eight-fold lower CEBPA RNA levels and undetectable C/EBPα protein levels compared with other subgroups of AML patients. Conditional expression of RUNX1–CBF2T1 in U937 cells downregulated CEBPA mRNA, protein and DNA binding activity. AML1–ETO appears to suppress C/EBPα expression indirectly by inhibiting positive autoregulation of the CEBPA promoter. Conditional expression of C/EBPα in AML1–ETO-positive Kasumi-1 cells results in neutrophilic differentiation. We suggest that restoring C/EBPα expression will have therapeutic implications in RUNX1–CBF2T1-positive leukemias.

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Figure 1: CEBPA mRNA is selectively downregulated in RUNX1–CBF2T1+ leukemia blasts.
Figure 2: C/EBPα protein expression and DNA binding are highly reduced in primary RUNX1–CBF2T1+ leukemia blasts.
Figure 3: Conditional expression of AML1-ETO downregulates CEBPA expression and inhibits C/EBPα function.
Figure 4: AML1–ETO inhibits CEBPA autoregulation in myeloid cells.
Figure 5: Restoration of C/EBPα expression in RUNX1–CBF2T1+ cells restores granulocytic differentiation.

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References

  1. Birkenmeier, E.H. et al. Tissue-specific expression, developmental regulation, and genetic mapping of the gene encoding CCAAT/enhancer binding protein. Genes Dev. 3, 1146–1156 (1989).

    Article  CAS  Google Scholar 

  2. Cao, Z., Umek, R.M. & McKnight, S.L. Regulated expression of three C/EBP-isoforms during adipose conversion of 3T3-L1 cells. Genes Dev. 5, 1538–1552 (1991).

    Article  CAS  Google Scholar 

  3. Scott, L.M., Civin, C.I., Roth, P. & Friedman, A.D. A novel temporal expression pattern of three C/EBP-family members in differentiating myelomonocytic cells. Blood 80, 1725–1735 (1992).

    CAS  Google Scholar 

  4. Flodby, P., Barlow, C., Kyleford, H., Ährlund-Richter, L. & Xanthopoulos, G. Increased hepatic cell proliferation and lung abnormalities in mice deficient in CCAAT/enhancer binding protein alpha. J. Biol. Chem. 271, 24753–24760 (1996).

    Article  CAS  Google Scholar 

  5. Chandrasekaran, C. & Gordon, J.I. Cell lineage-specific and differentiation-dependent patterns of CCAAT/enhancer binding protein α expression in the gut epithelium of normal and transgenic mice. Proc. Natl. Acad. Sci. USA 90, 8871–8875 (1993).

    Article  CAS  Google Scholar 

  6. Swart, G.W.M., van Groningen, J.J.M., van Ruissen, F., Bergers, M. & Schalkwijk, J. Transcription factor C/EBPα: novel sites of expression and cloning of the human gene. J. Biol. Chem. 378, 373–379 (1997).

    CAS  Google Scholar 

  7. Zhang, D.E. et al. Absence of granulocyte colony-stimulating factor signaling and neutrophil development in CCAAT enhancer binding protein α-deficient mice. Proc. Natl. Acad. Sci. USA 94, 569–574 (1997).

    Article  CAS  Google Scholar 

  8. Radomska, H.S. et al. CCAAT/enhancer binding protein α is a regulatory switch sufficient for induction of granulocytic development from bipotential myeloid progenitors. Mol. Cell. Biol . 18, 4301–4314 (1998).

    Article  CAS  Google Scholar 

  9. Wang, X., Scott, E., Sawyers, C.L. & Friedman, A.D. C/EBPα bypasses granulocyte-stimulating factor signals to rapidly induce PU.1 gene expression, stimulate granulocytic differentiation, and limit proliferation in 32D cl3 myeloblasts. Blood 94, 560–571 (1999).

    CAS  Google Scholar 

  10. Pabst, T. et al. Dominant negative mutations of CEBPA, encoding CCAAT/enhancer binding protein-α (C/EBPα), in acute myeloid leukemia. Nature Genet. 27, 263–270 (2001).

    Article  CAS  Google Scholar 

  11. Osato, M. et al. Biallelic and heterozygous point mutations in the Runt domain of the AML1/PEBP2αB gene associated with myeloblastic leukemias. Blood 93, 1817–1824 (1999).

    CAS  Google Scholar 

  12. Nucifora, G. et al. Detection of DNA rearrangements in the AML1 and ETO loci and of an AML1/ETO fusion mRNA in patients with t(8;21) acute myeloid leukemia. Blood 81, 883–888 (1993).

    CAS  Google Scholar 

  13. Song, W.J. et al. Haploinsufficiency of CBFA2 causes familial thrombocytopenia with propensity to develop acute myelogenous leukemia. Nature Genet. 23, 166–175 (1999).

    Article  CAS  Google Scholar 

  14. Erickson, P. et al. Identification of breakpoint in t(8;21) acute myelogenous leukemia and isolation of a fusion transcript, AML1/ETO, with similarity to Drosophila segmentation gene, runt. Blood 80, 1825–1831, 1992.

    CAS  Google Scholar 

  15. Miyoshi, H.T. et al. The t(8;21) translocation in acute myeloid leukemia results in production of an AML1-MTG8 fusion transcript. EMBO J. 12, 2715–2721 (1993).

    Article  CAS  Google Scholar 

  16. Okuda, T., van Deursen, J., Hiebert, S.W., Grosveld, G. & Downing, J.R. AML1, the target of multiple chromosomal translocations in human leukemia, is essential for normal fetal liver hematopoiesis. Cell, 84, 321–330 (1996).

    Article  CAS  Google Scholar 

  17. Miyoshi, H. et al. t(8;21) breakpoints on chromosome 21 in acute myeloid leukemia are clustered within a limited region of a single gene. AML1. Proc. Natl. Acad. Sci. USA 88, 10431–10434 (1991).

    Article  CAS  Google Scholar 

  18. Frank, R. et al. The AML1/ETO fusion protein blocks transactivation of the GM-CSF promoter by AML1B. Oncogene, 11, 2667–2674 (1995).

    CAS  Google Scholar 

  19. Nuchprayoon, L. et al. PEBP2/CBF, the murine homolog of the human myeloid AML1 and PEBP2β/CBF β proto-oncoproteins, regulates the murine myeloperoxidease and neutrophil elastase genes in immature myeloid cells. Mol. Cell. Biol. 14, 5558–5568 (1994).

    Article  CAS  Google Scholar 

  20. Zhang, D.E. et al. CCAAT enhancer-binding protein (C/EBP) and AML1 (CBFα2) synergistically activate the macrophage colony-stimulating factor receptor promoter. Mol. Cell. Biol. 16, 1231–1240 (1996).

    Article  CAS  Google Scholar 

  21. Petrovick, M.S. et al. Multiple functional domains of AML1: PU.1 and C/EBPα synergize with different regions of AML1. Mol. Cell. Biol. 18, 3915–3925 (1998).

    Article  CAS  Google Scholar 

  22. Zhang, D.E. et al. Identification of a region which directs the monocytic activity of the colony-stimulating factor 1 (macrophage colony-stimulating factor) receptor promoter and binds PEBP2/CBF (AML1). Mol. Cell. Biol. 14, 8085–8095 (1994).

    Article  CAS  Google Scholar 

  23. Rhoades, K.L. et al. Synergistic up-regulation of the myeloid-specific promoter for the macrophage colony-stimulating factor receptor by AML1 and the t(8;21) fusion protein may contribute to leukemogenesis. Proc. Natl. Acad. Sci. USA 93, 11895–11900 (1996).

    Article  CAS  Google Scholar 

  24. Yergeau, D.A. et al. Embryonic lethality and impairment of hematopoiesis in mice heterozygous for an AML1–ETO fusion gene. Nature Genet. 15, 303–306, 1997.

    Article  CAS  Google Scholar 

  25. Klampfer, L., Zhang, J., Zelenetz, A.O., Uchida, H. & Nimer, S.D. The AML1/ETO fusion protein activates transcription of BCL-2. Proc. Natl. Acad. Sci. USA 93, 14059–14064 (1996).

    Article  CAS  Google Scholar 

  26. Frank, R.C., Sun, X., Berguido, F.J., Jakubowiak, A. & Nimer, S.D. The t(8;21) fusion protein, AML1/ETO transforms NIH3T3 cells and activates AP-1. Oncogene 18, 1701–1710 (1999).

    Article  CAS  Google Scholar 

  27. Rhoades, K.L. et al. Analysis of the role of AML1–ETO in leukemogenesis using an inducible transgenic mouse model. Blood 96, 2108–2115 (2000).

    CAS  Google Scholar 

  28. Meyers, S., Lenny, N. & Hiebert, S.W. The t(8;21) fusion protein interferes with AML-1B-dependent transcriptional activation. Mol. Cell. Biol. 15, 1974–1982 (1995).

    Article  CAS  Google Scholar 

  29. Westendorf, J.J. et al. The t(8;21) fusion product, AML-1-ETO, associates with C/EBPα, inhibits C/EBPα–dependent transcription, and blocks granulocytic differentiation. Mol. Cell. Biol. 18, 322–333 (1998).

    Article  CAS  Google Scholar 

  30. Asou, H. et al. Establishment of a human acute myeloid leukemia cell line (Kasumi-1) with 8;21 chromosome translocation. Blood 77, 2031–2036 (1991).

    CAS  Google Scholar 

  31. Sundstrom, C. & Nilsson, K. Establishment and characterization of a human histiocytic lymphoma cell line (U-937). Int. J. Cancer 17, 565–577 (1976).

    Article  CAS  Google Scholar 

  32. Smith, L.T., Hohaus, S., Gonzalez, D.A., Dziennis, S.E. & Tenen, D.G. PU.1 (Spi-1) and C/EBPα regulate the granulocyte colony-stimulating factor receptor promoter in myeloid cells. Blood 88, 1234–1247 (1996).

    CAS  Google Scholar 

  33. Timchenko, N. et al. Autoregulation of the human C/EBPα gene by stimulation of upstream stimulatory factor binding. Mol. Cell. Biol. 15, 1192–1202 (1995).

    Article  CAS  Google Scholar 

  34. Christy, R.J., Kaestner, K.H., Geiman, D.E. & Lane, M.D. CCAAT/enhancer binding protein gene promoter: binding of nuclear factors during differentiation of 3T3-L1 preadipocytes. Proc. Natl. Acad. Sci. USA 88, 2593–2597 (1991).

    Article  CAS  Google Scholar 

  35. Legraverend, K., Antonson, P., Flodby, P. & Xanthopoulos, K.G. High level activity of the mouse CCAAT/enhancer binding protein (C/EBPα) gene promoter involves autoregulation and several ubiquitous transcription factors. Nucleic Acids Res. 21, 1735–1742 (1993).

    Article  CAS  Google Scholar 

  36. Rabbits, T.H. Chromosomal translocations in human cancer. Nature 372, 143–149 (1994).

    Article  Google Scholar 

  37. Tenen, D.G., Hromas, R., Licht, J.D. & Zhang, D.E. . Transcription factors, normal myeloid development, and leukemia. Blood 90, 489–519 (1997).

    CAS  Google Scholar 

  38. Miyamoto, T., Weissman, J.L., and Akashi, K. AML1/ETO-expressing nonleukemic stem cells in acute myelogenous leukemia with 8;21 chromosomal translocation. Proc. Natl. Acad. Sci. USA 97, 7521–7526 (2000)

  39. Tang, Q.Q., Jiang, M.S., and Lane, M.D. Repressive effect of Sp1 on the C/EBPα gene promoter: role in adipocyte differentiation. Mol. Cell. Biol. 19, 4855–4865 (1999).

    Article  CAS  Google Scholar 

  40. Jiang, M.S. et al. Derepression of the C/EBPα gene during adipogenesis: identification of AP-2α as a repressor. Proc. Natl. Acad. Sci. USA 95, 3467–3471 (1998).

    Article  CAS  Google Scholar 

  41. Mink, S., Mutschler, B., Weiskirchen, R., Bister, K. & Klempnauer, K.H. A novel function for Myc: inhibition of C/EBP-dependent gene activation. Proc. Natl. Acad. Sci. USA . 93, 6635–6640 (1996).

    Article  CAS  Google Scholar 

  42. Huettner, C., Paulus, W. & Roggendorf, W. mRNA expression of the immunosuppressive cytokine IL-10 in human gliomas. Am. J. Pathol. 146, 317–322 (1995).

    CAS  PubMed Central  Google Scholar 

  43. Schwaller, J., Pabst, T., Bickel, M., Borisch, B., Fey, M.F. & Tobler, A . Comparative detection and quantitation of human CDK inhibitor mRNA expression of p15INK4B, p16β, p18INK4C, p19INK4D, p21WAF1, p27KIP1 and p57KIP2 by RT-PCR using a polycompetitive internal standard. Br. J. Haematol. 99, 896–900 (1998).

    Article  Google Scholar 

  44. Nordeen, S.K. Luciferase reporter gene vectors for analysis of promoters and enhancers. Biotechniques 6, 454–458 (1988).

  45. Gossen, M. & Bujard, H. Tight control of gene expression in mammalian cells by tetracycline-responsive promoters. Proc. Natl. Acad. Sci. USA 89, 5547–5551 (1992).

    Article  CAS  Google Scholar 

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Acknowledgements

We thank S. Schnittger and W.D. Ludwig for assistance with analysis of patient samples; A. Friedman for the C/EBP-α–ER retrovirus; C. Huettner for the GAPDH competitor; T. Cheng and D. Scadden for CD34-G0 and FACS-sorted bone-marrow cells; G. Grosveld for the U937T (tTA) cell line; S. Hiebert for the RUNX1–CBF2T1 deletion mutants and the CBF2T1 antibody; G. Darlington and D. Gonzalez for CEBPA promoter constructs; E. Stillner for the sequence analysis of the CEBPA promoter; N. Kamada for the Kasumi-1 cell line; and L. Clayton and M. Singleton for valuable suggestions with the manuscript. This work was supported by grants of the Swiss National Science Foundation (#81BS-051911 and SSMBS #1011) (to T.P.) and NIH grant CA72009 (to D.G.T. and D.E.Z.) and HL56745 (to D.G.T.).

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Authors and Affiliations

  1. Hematology/Oncology Division, Harvard Institutes of Medicine, Harvard Medical School, Boston, Massachusetts, USA

    Thomas Pabst, Beatrice U. Mueller, Nari Harakawa & Daniel G. Tenen

  2. Department of Medicine III, Grosshadern, and Clinical Cooperative Group Acute Myeloid Leukemia of the National Research Center for Environment and Health (GSF), Munich, Germany

    Claudia Schoch, Torsten Haferlach, Gerhard Behre & Wolfgang Hiddemann

  3. The Scripps Research Institute, La Jolla, California, USA

    Dong-Er Zhang

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Pabst, T., Mueller, B., Harakawa, N. et al. AML1–ETO downregulates the granulocytic differentiation factor C/EBPα in t(8;21) myeloid leukemia. Nat Med 7, 444–451 (2001). https://doi.org/10.1038/86515

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