Essential role of Jun family transcription factors in PU.1 knockdown–induced leukemic stem cells Ulrich Steidl 1 , Frank Rosenbauer 1,2 , Roel G W Verhaak 3 , Xuesong Gu 4 , Alexander Ebralidze 1 , Hasan H Otu 4,5 , Steffen Klippel 1 , Christian Steidl 6 , Ingmar Bruns 7 , Daniel B Costa 1 , Katharina Wagner 1 , Manuel Aivado 4 , Guido Kobbe 7 , Peter J M Valk 3 , Emmanuelle Passegue ´ 8 , Towia A Libermann 4 , Ruud Delwel 3 & Daniel G Tenen 1 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. The transcription factor PU.1 (encoded by Sfpi1) is indispensable for myelomonocytic differentiation during normal hematopoiesis 1 . Several reports suggest that reduced function of PU.1 might also have a central role in AML, a disease entity characterized by dis- turbed myeloid development 2,3 . Recent experimental evidence pro- poses a model of AML pathogenesis in which two major molecular events are required for the development of malignant cells. One causes a differentiation arrest, and a second confers self-renewal properties on the cells, thereby ultimately leading to the formation of a pool of leukemic stem cells (LSCs) 4 . We have shown previously that the transcriptional control of Sfpi1 gene expression is mediated by a distal upstream regulatory element (URE) that is highly conserved among multiple species, including mice and humans 5,6 . We also demonstrated that knockout of this distal enhancer of Sfpi1, which reduces PU.1 expression levels by 80% in the bone marrow, leads to the development of AML in mice 7,8 . The course of disease in- cludes a preleukemic phase with an accumulation of immature myelomonocytic cells in the marrow but still normal peripheral blood counts, followed by a leukemic phase with high numbers of malignant immature cells in the blood and marrow. However, the molecular mechanisms underlying the malignant transformation are poorly understood. A widely used method to uncover oncogenic pathways is the examination of tumor cells, but this approach is hampered by at least two conceptual challenges: first, the bulk of tumor cells appear at the final stage of disease and hence are likely to show many nonspecific alterations beside the primary oncogenic events that lead to cancerous stem cell formation, and second, an adequate cellular control popula- tion is not available. This is particularly true for AML, in which a developmental block is a hallmark of the disease and results in a bulk tumor population of immature cells that cannot be easily compared with normal blood or bone marrow cells 9 . In this study, we carried out genome-wide transcriptional analysis of HSCs isolated from mice in which PU.1 was knocked down (referred to as ‘PU.1-knockdown mice’) at the preleukemic stage to identify prospective pathways that lead to LSC development. We delineate a transcriptional pattern in HSCs that precedes leukemic transformation and demonstrate that dysregulation of distinct identi- fied targets is essential for LSC function, a finding that could be therapeutically useful in treatment of this disease. Received 24 July; accepted 7 September; published online 15 October 2006; doi:10.1038/ng1898 1 Harvard Institutes of Medicine, Harvard Medical School and Harvard Stem Cell Institute, Boston, Massachusetts 02115, USA. 2 Max-Delbru ¨ ck-Center for Molecular Medicine, 13125 Berlin, Germany. 3 Department of Hematology, Erasmus University Medical Center, 3015GE Rotterdam, The Netherlands. 4 Beth Israel Deaconess Medical Center Genomics Center and Bioinformatics Core and Beth Israel Deaconess Medical Center, Boston, Massachusetts 02215, USA. 5 Department of Genetics and Bioengineering, Yeditepe University, Istanbul 34755, Turkey. 6 Department of Hematology and Oncology, University of Goettingen, 37075 Goettingen, Germany. 7 Department of Hematology, Oncology and Clinical Immunology, University of Duesseldorf, 40225 Duesseldorf, Germany. 8 Developmental and Stem Cell Biology Program, University of California, San Francisco, California 94314, USA. Correspondence should be addressed to D.G.T. (dtenen@bidmc.harvard.edu). NATURE GENETICS VOLUME 38 [ NUMBER 11 [ NOVEMBER 2006 1269 ARTICLES © 2006 Nature Publishing Group http://www.nature.com/naturegenetics