Transformation from committed progenitor toleukaemia stem cell initiated by MLL–AF9Andrei V. Krivtsov1, David Twomey1,5, Zhaohui Feng2, Matthew C. Stubbs1, Yingzi Wang1, Joerg Faber1,Jason E. Levine1,2, Jing Wang2, William C. Hahn3,5, D. Gary Gilliland4,6, Todd R. Golub2,5,6& Scott A. Armstrong1,2Leukaemias and other cancers possess a rare population of cellscapable of the limitless self-renewal necessary for cancer initiationand maintenance1–7. Eradication of these cancer stem cells isprobably a critical part of any successful anti-cancer therapy,and may explain why conventional cancer therapies are ofteneffective in reducing tumour burden, but are only rarely curative.Given that both normal and cancer stem cells are capable of self-renewal, the extent to which cancer stem cells resemble normaltissue stem cells is a critical issue if targeted therapies are to bedeveloped. However, it remains unclear whether cancer stem cellsmust be phenotypically similar to normal tissue stem cells orwhether they can retain the identity of committed progenitors.Here we show that leukaemia stem cells (LSC) can maintain theglobal identity of the progenitor from which they arose whileactivating a limited stem-cell- or self-renewal-associated pro-gramme. We isolated LSC from leukaemias initiated in committedgranulocyte macrophage progenitors through introduction of theMLL–AF9 fusion protein encoded by the t(9;11)(p22;q23). TheLSC were capable of transferring leukaemia to secondary recipientmice when only four cells were transferred, and possessed animmunophenotype and global gene expression profile very similarto that of normal granulocyte macrophage progenitors. However,a subset of genes highly expressed in normal haematopoietic stemcells was re-activated in LSC. LSC can thus be generated fromcommitted progenitors without widespread reprogramming ofgene expression, and a leukaemia self-renewal-associated signa-ture is activated in the process. Our findings define progressionfrom normal progenitor to cancer stem cell, and suggest thattargeting a self-renewal programme expressed in an abnormalcontext may be possible.Some fusion proteins encoded by translocations found in humanleukaemias including those involving the mixed lineage leukaemia(MLL) gene have been reported to impart leukaemia stem cellproperties on committed haematopoietic progenitors8–10.Weexpressed an MLL–AF9 fusion protein in highly purified interleukin(IL)-7R2Lin2Sca-12c-KitþCD34þFcgRII/IIIhigranulocyte macro-phage progenitors (GMP)11and injected them into sublethallyirradiated syngeneic recipient mice (Supplementary Fig. 1). Within80 days, animals transplanted with murine stem cell virus(MSCV)-MLL–AF9 transduced GMP developed an oligoclonalacute myelogenous leukaemia (AML), similar to other MLL fusionleukaemia models (Supplementary Fig. 2 and SupplementaryTa bl e 1 )9,12–15. To assess the frequency of leukaemia-initiating cells,we injected sublethally irradiated syngeneic recipient mice with 20 to104bone-marrow cells taken from leukaemic mice. All mice thatreceived 500 or more leukaemic cells died of AML. Half of the micethat received 100 cells, and none of the mice that received 20 cellssuccumbed to AML (Supplementary Table 1). Therefore, the calcu-lated frequency of leukaemia-initiating cells in bone marrow isapproximately 1:150. Thus MLL–AF9 induces self-renewal andleukaemia from prospectively isolated GMP.Retroviral or knock-in models of MLL-fusion-induced leukaemiasdemonstrate the presence of IL-7R2Lin2Sca-12c-KitþCD34þ-FcgRII/IIIþGMP-like leukaemic cells (L-GMP) (Fig. 1a)9,14.Wereasoned that these L-GMP might contain LSC. To determinewhether the L-GMP population contains LSC, we sorted Linþ,IL27R2Lin2Sca-12c-Kit2, or L-GMP cells from mice that developedAML (Fig. 1a). Ex vivo growth of these cells in cytokine-supplementedLETTERSFigure 1 | Leukaemic-GMP are enriched for leukaemia stem cells.a, Progenitor analysis of MLL–AF9 leukaemias. L-GMP were prospectivelyisolated for injection to secondary recipients or gene expression analysis.Further characterization of leukaemias can be found in SupplementaryFigs 1 and 2. b, Survival curves for mice injected with limiting dilution ofL-GMP show that four cells can transfer leukaemia. The experiment wasrepeated three times with similar results. Detailed numbers can be found inSupplementary Table 1. c, Survival curves for three mice injected with 5,000or 100,000 L-GMP propagated in culture (AKLG cells) demonstrate therequirement for .5,000 cells to initiate leukaemia.1Division of Hematology/Oncology, Children’s Hospital,2Department of Pediatric Oncology, and3Medical Oncology, Dana Farber Cancer Institute, and4Division of Hematology,Brigham and Women’s Hospital, Harvard Medical School, Boston, Massachusetts 02115, USA.5The Broad Institute of Harvard and MIT, Cambridge, Massachusetts 02142, USA.6Howard Hughes Medical Institute, Chevy Chase, Maryland 20815, USA.Vol 442|17 August 2006|doi:10.1038/nature04980818© 2006 Nature Publishing Groupmethylcellulose revealed that L-GMP possessed higher blast colony-forming activity than other populations (Supplementary Fig. 3).Transplantation of purified L-GMP into sublethally irradiated recipi-ents was used to assess leukaemogenesis in vivo.Eachofthemicetransplanted with 20 to 5 £ 103L-GMP developed AML that wasphenotypically identical to the primary disease (Fig. 1b, Supplemen-tary Fig. 4). One of six mice injected with four L-GMP died of AML(Fig. 1b). Therefore, the L-GMP population contains leukaemia-initiating cells with a frequency of approximately 1:6.In addition, a high frequency of leukaemia-initiating cells in theL-GMP was confirmed by plating in cytokine-supplemented methyl-cellulose. Single colonies were isolated after 5 days and injected intorecipient mice. Fifteen out of 30 mice receiving cells from singleL-GMP derived colonies died of AML within 70 days (data notshown). To further characterize the stem-cell-like properties ofL-GMP, we assessed their potential to generate differentiated pro-geny. L-GMP were easily propagated in liquid culture containingIL-3, a well-described characteristic of murine MLL-fusion-inducedleukaemias12. However, after conversion to liquid culture themajority of the cells demonstrated a more differentiated, lineageþKit2immunophenotype (Supplementary Fig. 5). This more differ-entiated phenotype was seen in conjunction with an increase in thenumber of cells needed to initiate leukaemia