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ORIGINAL ARTICLE PML–Rara Initiates Leukemia by Conferring Properties of Self-Renewal to Committed Promyelocytic Progenitors

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ORIGINAL ARTICLE PML–Rara Initiates Leukemia by Conferring Properties of Self-Renewal to Committed Promyelocytic Progenitors Leukemia (2009) 23, 1462–1471 & 2009 Macmillan Publishers Limited All rights reserved 0887-6924/09 $32.00 www.nature.com/leu ORIGINAL ARTICLE PML–RARa initiates leukemia by conferring properties of self-renewal to committed promyelocytic progenitors S Wojiski1,2, FC Guibal3, T Kindler1,2, BH Lee1,2, JL Jesneck4,5, A Fabian1,2, DG Tenen3,6 and DG Gilliland1,2,6 1Division of Hematology, Brigham and Women’s Hospital, Boston, MA, USA; 2Department of Genetics, Howard Hughes Medical Institute, Harvard Medical School, Boston, MA, USA; 3Department of Hematology/Oncology, Harvard Institutes of Medicine, Boston, MA, USA; 4Cancer Program, Broad Institute of Harvard and MIT, Cambridge, MA, USA; 5Department of Pediatric Oncology, Dana-Farber Cancer Institute, Harvard Medical School, Boston, MA, USA and 6Harvard Stem Cell Institute, Harvard University, Boston, MA, USA Acute promyelocytic leukemia (APL) is characterized by the leukemia stem cells (LSCs) are rare cells contained within hyperproliferation of promyelocytes, progenitors that are the more primitive CD34 þ CD38À population, resembling committed to terminal differentiation into granulocytes, making it an ideal disease in which to study the transforming potential transformed hematopoietic stem cells (HSCs). LSC activity was of less primitive cell types. We utilized a murine model of APL in shown by these investigators in all AML subtypes tested, but which the PML–RARa oncogene is expressed from the were not shown in acute promyelocytic leukemia (APL) endogenous cathepsin G promoter to test the hypothesis that associated with the expression of the PML–RARa fusion as a leukemia stem cell (LSC) activity resides within the differen- consequence of an acquired t(15;17). These observations tiated promyelocyte compartment. We prospectively purified suggest that either LSCs were not present in this differentiated promyelocytes from transgenic mice at various stages of disease and observed that PML–RARa-expressing promyelo- subtype of leukemia, or were resident in another hematopoietic cytes from young preleukemic mice had acquired properties of compartment. self-renewal both in vitro and in vivo. Progression to acute Although the cell of origin for LSCs is not known, studies in leukemia was associated with an expansion of the promyelo- mice have indicated that HSCs may be targets of transformation cyte compartment at the expense of other stem, progenitor and in leukemogenesis.4–8 This is an attractive hypothesis in that terminally differentiated populations. Leukemic promyelocytes normal tissue stem cells have inherent long-term self-renewal exhibited properties of self-renewal, and were capable of engendering leukemia in secondary recipient mice. These data potential that is requisite for tumorigenesis. However, a recent indicate that PML–RARa alone can confer properties of self- body of evidence suggests that more mature progenitor cells, renewal to committed hematopoietic progenitors before that normally lack any potential for self-renewal, may be an the onset of disease. These findings are consistent with the origin of LSCs. In this model, committed progenitors reacquire hypothesis that cancer stem cells may arise from committed properties of self-renewal mediated by the respective leukemia progenitors that lack stem cell properties, provided that the oncogene.9,10 For example, myeloid progenitors of the granu- initiating mutation in cancer progression activates programs that confer properties of self-renewal. locyte-monocyte lineage (GMPs) may acquire leukemia-initia- Leukemia (2009) 23, 1462–1471; doi:10.1038/leu.2009.63; ting potential in mice after retroviral transduction of the 8 11 published online 26 March 2009 leukemia-associated fusion proteins MLL-ENL, MOZ-TIF2 or Keywords: self-renewal; progenitor; promyelocyte; APL MLL-AF9.12 These leukemia-initiating progenitors can be distinguished from their normal counterparts in that they share certain characteristics of normal HSCs, including properties of self-renewal and the capacity to differentiate into progeny that Introduction lack self-renewing potential. These models are not mutually exclusive; LSCs may poten- The cancer stem cell hypothesis posits the existence of a rare tially arise either from the stem cell or from the progenitor population of tumor cells that is responsible for propagation and compartment. However, hematopoietic progenitors in the maintenance of a tumor phenotype. Furthermore, similar to myeloid lineage are short lived, and AML, like most cancers, normal developmental hierarchies, it has been proposed that requires multiple mutations. Thus, for an LSC to arise from a cancer stem cell, but not their clonogenic progeny, alone committed progenitor, one must posit that the initiating possess properties of long-term self-renewal. Thus, identification mutation itself confers self-renewal potential to a committed and characterization of these leukemia-initiating cells could progenitor. The retroviral transduction models noted above do translate into the development of more effective treatment not explicitly address this possibility. Tumors that arise in this modalities. context are typically mono- or oligoclonal, suggesting that There is strong experimental support for the existence of secondary mutations may be involved, and that integration site cancer stem cell in human acute myeloid leukemias (AMLs), effects may also contribute to the disease phenotype. initially from experiments using a NOD-SCID xenotransplanta- To better understand the role of leukemia oncogenes in tion model of human AML cells.1–3 These studies indicated that committed progenitor populations, we took advantage of a mouse model of leukemia developed by Westervelt and Correspondence: Dr DG Gilliland, Professor of Medicine, Harvard colleagues13 in which (i) PML–RARa is expressed from the Medical School and Brigham and Women’s Hospital, Division of endogenous murine cathepsin G promoter that obviates con- Hematology, 1 Blackfan Circle, Boston, MA 02115, USA. E-mail: [email protected] tribution to phenotype from retroviral integration sites, (ii) Received 21 February 2009; accepted 24 February 2009; published leukemia develops in a terminally differentiating hematopoietic online 26 March 2009 compartment, the promyelocyte, that has no potential for Self-renewal of PML–RARa-expressing promyelocytes S Wojiski et al 1463 self-renewal, and (iii) there is a long latency before development (MEPs) as LinÀSca-1Àc-Kit þ CD34ÀFcgRII/IIIlo. For promyelo- of leukemia in which animals are phenotypically normal, cyte and mature granulocyte identification, staining was carried allowing for the analysis of the consequences of PML–RARa out as described with the following changes: Sca-1 antibody was expression during the earliest stages of malignant transformation. added to the lineage depletion cocktail, and Gr-1 antibody was Our studies show that the acquisition of self-renewal excluded from the lineage depletion cocktail. Promyelocytes capability in the promyelocyte compartment is mediated by were distinguished as c-Kit þ CD34 þ Gr-1 þ and mature PML–RARa as an initiating step in the pathogenesis of APL, granulocytes as c-KitÀCD34ÀGr-1 þ . Dead cells were excluded providing further evidence that committed progenitors can in from all analyses by propidium-iodide staining. Cells were fact possess leukemia-initiating activity. Furthermore, the use of sorted into 30% fetal bovine serum/phosphate-buffered a knock-in model in which the PML–RARa fusion is expressed saline for subsequent assays. Analysis and purification was from the endogenous cathepsin G promoter allows for performed using an FACSAria cytometer (Becton Dickinson) and transplantation of syngeneic tissues to show the leukemogenic FACSDiva or FlowJo (Treestar, San Carlos, CA, USA) software potential of transformed committed progenitors, providing a programs. platform for addressing mechanisms of the regulation of self-renewal capability in the LSC compartment. Quantitative real-time PCR (RT-PCR) RNA was extracted from cells using the RNeasy Micro or Mini Materials and methods Kits (Qiagen, Valencia, CA, USA) per the manufacturer’s instructions, incorporating a DNAse1 digest to remove any Mice contaminating genomic DNA. cDNA was prepared from RNA Cathepsin-G-PML–RARa knock-in mice13 were backcrossed at using Taqman reverse transcription reagents (Applied Biosys- least eight generations into the C57BL/6 background, and this tems, Foster City, CA, USA). Taqman gene expression assays strain was used for all subsequent experiments. Genotyping was were used to analyze the expression of the following murine performed as described previously utilizing genomic tail DNA genes: cathepsin G, neutrophil elastase, gelatinase B, myeloper- as a PCR template.13,14 All animals were housed in micro- oxidase and glyceraldehyde 3-phosphate dehydrogenase. isolator cages under pathogen-free conditions, and all experi- Detection of expression of the PML–RARa fusion transcript ments were conducted with the ethical approval of the was carried out using a custom made Taqman probe specific to Children’s Hospital Animal Care and Use Committee. Animals the fusion junction and flanking primers. PCR reactions were were observed on a weekly basis for the development of performed and analyzed using the 7300 Real Time PCR System hematopoietic malignancy and were killed when moribund. and SDS Software (Applied Biosystems). Expression values were Automated total and differential blood cell counts were normalized to GAPDH.
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