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Essential role of Jun family transcription factors in PU.1 knockdown–induced leukemic stem cells

Abstract

Knockdown of the transcription factor PU.1 (encoded by Sfpi1) leads to acute myeloid leukemia (AML) in mice. We examined the transcriptome of preleukemic hematopoietic stem cells (HSCs) in which PU.1 was knocked down (referred to as 'PU.1-knockdown HSCs') to identify transcriptional changes preceding malignant transformation. Transcription factors c-Jun and JunB were among the top-downregulated targets. Restoration of c-Jun expression in preleukemic cells rescued the PU.1 knockdown–initiated myelomonocytic differentiation block. Lentiviral restoration of JunB at the leukemic stage led to loss of leukemic self-renewal capacity and prevented leukemia in NOD-SCID mice into which leukemic PU.1-knockdown cells were transplanted. Examination of human individuals with AML confirmed the correlation between PU.1 and JunB downregulation. These results delineate a transcriptional pattern that precedes leukemic transformation in PU.1-knockdown HSCs and demonstrate that decreased levels of c-Jun and JunB contribute to the development of PU.1 knockdown–induced AML by blocking differentiation and increasing self-renewal. Therefore, examination of disturbed gene expression in HSCs can identify genes whose dysregulation is essential for leukemic stem cell function and that are targets for therapeutic interventions.

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Figure 1: Examination of the phenotypic stem cell compartment and identification of LSCs in PU.1-knockdown mice.
Figure 2: Expression profiling in PU.1-knockdown HSCs.
Figure 3: Quantitative real-time RT-PCR and RNA slot blotting corroborate differential gene expression.
Figure 4: PU.1 directs activity of the Junb promoter.
Figure 5: Restoration of c-Jun expression partially rescues myelomonocytic differentiation.
Figure 6: Restoration of JunB expression in leukemic cells of PU.1-knockdown mice inhibits leukemogenesis.
Figure 7: PU.1 and JunB expression in individuals with AML.

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Acknowledgements

We thank K. Martens for excellent assistance with mouse husbandry, M. Joseph for help with array hybridization, T. Dajaram and C. Hetherington for quantitative real-time RT-PCR analysis and J. Tigges and V. Toxavidis for expert assistance with multicolor flow cytometry and high-speed cell sorting. U.S. thanks S. Steidl for invaluable support and advice. U.S. also thanks R. Kronenwett and R. Haas for long-term support and mentorship. This work was supported by US National Institutes of Health grant CA41456 to D.G.T. and by fellowships of the Dr. Mildred Scheel Foundation for Cancer Research to U.S. (D/03/41221) and the Lymphoma Research Foundation to F.R.

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Correspondence to Daniel G Tenen.

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Supplementary information

Supplementary Fig. 1

Principal component analysis separates PU.1 knockdown from wild-type HSC on the basis of their gene expresson profiles. (PDF 14 kb)

Supplementary Fig. 2

Gene expression data of wild-type and PU.1 knockdown HSC measured by Affymetrix Mouse Genome 430 2.0 arrays. (PDF 20 kb)

Supplementary Fig. 3

Direct interaction pathway analysis. (PDF 187 kb)

Supplementary Fig. 4

Restoration of c-Jun expression rescues colony formation upon GM-CSF stimulation. (PDF 19 kb)

Supplementary Fig. 5

Flow cytometric analysis shows engraftment of GFP lentivirus-transduced PU.1 knockdown cells upon transplantation into NOD-SCID mice. (PDF 25 kb)

Supplementary Fig. 6

JunB and PU.1 expression in individuals with AML. (PDF 122 kb)

Supplementary Table 1

Selection of up- and downregulated genes in PU.1 knockdown HSC. (PDF 74 kb)

Supplementary Table 2

Oligonucleotides used for PCR and EMSA. (PDF 26 kb)

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Steidl, U., Rosenbauer, F., Verhaak, R. et al. Essential role of Jun family transcription factors in PU.1 knockdown–induced leukemic stem cells. Nat Genet 38, 1269–1277 (2006). https://doi.org/10.1038/ng1898

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