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Logo of nihpaAbout Author manuscriptsSubmit a manuscriptHHS Public Access; Author Manuscript; Accepted for publication in peer reviewed journal;
 
Leukemia. Author manuscript; available in PMC 2010 August 3.
Published in final edited form as:
PMCID: PMC2914549
NIHMSID: NIHMS222020

PML-RARα Initiates Leukemia by Conferring Properties of Self-Renewal to Committed Promyelocytic Progenitors

Abstract

Acute promyelocytic leukemia (APL) is characterized by hyperproliferation of promyelocytes, progenitors that are committed to terminal differentiation into granulocytes, making it an ideal disease in which to study the transforming potential of less primitive cell types. We utilized a murine model of APL in which the PML-RARα oncogene is expressed from the endogenous Cathepsin G promoter to test the hypothesis that leukemia stem cell activity resides within the differentiated promyelocyte compartment. We prospectively purified promyelocytes from transgenic mice at various stages of disease and observed that PML-RARα-expressing promyelocytes from young preleukemic mice had acquired properties of self-renewal both in vitro and in vivo. Progression to acute leukemia was associated with an expansion of the promyelocyte compartment at the expense of other stem, progenitor and terminally differentiated populations. Leukemic promyelocytes exhibited properties of self-renewal, and were capable of engendering leukemia in secondary recipient mice. These data indicate that PML-RARα alone can confer properties of self-renewal to committed hematopoietic progenitors prior to the onset of disease. These findings are consistent with the hypothesis that cancer stem cells may arise from committed progenitors that lack stem cell properties, provided that the initiating mutation in cancer progression activates programs that confer properties of self-renewal.

Keywords: self-renewal, progenitor, promyelocyte, APL

Introduction

The cancer stem cell (CSC) hypothesis posits the existence of a rare population of tumor cells that is responsible for propagation and maintenance of a tumor phenotype. Furthermore, similar to normal developmental hierarchies, it has been proposed that CSC, but not their clonogenic progeny, alone possess properties of long-term self-renewal. Thus, identification and characterization of these leukemia-initiating cells could translate into the development of more effective treatment modalities.

There is strong experimental support for the existence of CSC in human acute myeloid leukemias, initially from experiments using a NOD-SCID xenotransplantation model of human acute myeloid leukemia (AML) cells (13). These studies indicated that the leukemia stem cells (LSCs) are rare cells contained within the more primitive CD34+ CD38 population, resembling transformed hematopoietic stem cells (HSCs). LSC activity was demonstrated by these investigators in all AML subtypes tested, but were not demonstrated in acute promyelocytic leukemia associated with expression of the PML-RARα fusion as a consequence of an acquired t(15;17). These observations suggest either that LSC were not present in this differentiated subtype of leukemia, or were resident in another hematopoietic compartment.

Although the cell of origin for leukemia stem cells is not known, studies in mice have indicated that hematopoietic stem cells may be targets of transformation in leukemogenesis (48). This is an attractive hypothesis in that normal tissue stem cells have inherent long-term self-renewal potential that is requisite for tumorigenesis. However, a recent body of evidence suggests that more mature progenitor cells, that normally lack any potential for self-renewal, may be an origin of leukemia stem cells. In this model, committed progenitors re-acquire properties of self-renewal mediated by the respective leukemia oncogene (9, 10). For example, myeloid progenitors of the granulocyte-monocyte lineage (GMPs) may acquire leukemia-initiating potential in mice after retroviral transduction of the leukemia associated fusion proteins MLL-ENL (8), MOZ-TIF2 (11), or 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 lack self-renewing potential.

These models are not mutually exclusive - leukemia stem cells may potentially arise either from the stem cell or from the progenitor compartment. However, hematopoietic progenitors in the myeloid lineage are short lived, and AML, like most cancers, requires multiple mutations. Thus, for a leukemia stem cell to arise from a committed progenitor, one must posit that the initiating mutation itself confers self-renewal potential to a committed progenitor. The retroviral transduction models noted above do not explicitly address this possibility. Tumors that arise in this context are typically mono- or oligoclonal, suggesting that secondary mutations may be involved, and that integration site effects may also contribute to the disease phenotype.

To better understand the role of leukemia oncogenes in committed progenitor populations, we took advantage of a mouse model of leukemia developed by Ley and colleagues (13) in which (i) PML-RARα is expressed from the endogenous murine cathepsin G promoter that obviates contribution to phenotype from retroviral integration sites, (ii) leukemia develops in a terminally differentiating hematopoietic compartment, the promyelocyte, that has no potential for self-renewal and (iii) there is a long latency before development of leukemia in which animals are phenotypically normal, allowing for the analysis of the consequences of PML-RARα expression during the earliest stages of malignant transformation.

Our studies demonstrate that the acquisition of self-renewal capability in the promyelocyte compartment is mediated by PML-RARα as an initiating step in the pathogenesis of APL, providing further evidence that committed progenitors can in fact possess leukemia-initiating activity. Furthermore, the use of a knock-in model in which the PML-RARα fusion is expressed from the endogenous Cathepsin G promoter allows for transplantation of syngeneic tissues to demonstrate the leukemogenic potential of transformed committed progenitors, providing a platform for addressing mechanisms of the regulation of self-renewal capability in the leukemia stem cell compartment.

Materials and Methods

Mice

Cathepsin-G-PML-RARα knock-in mice (13) were backcrossed at least eight generations into the C57BL/6 background, and this strain was used for all subsequent experiments. Genotyping was performed as described previously utilizing genomic tail DNA as a PCR template (13, 14). All animals were housed in microisolator cages under pathogen-free conditions and all experiments were conducted with the ethical approval of the Children’s Hospital Animal Care and Use Committee. Animals were observed on a weekly basis for the development of hematopoietic malignancy and were sacrificed when moribund. Automated total and differential blood cell counts were obtained using a Hemavet 950 (Drew Scientific, CT, USA). Upon sacrifice, spleen weights were recorded, and all organs were collected and stored in 10% neutral buffered formalin (Sigma). Single cell suspensions were prepared from spleen and bone marrow and cells were subsequently frozen in 10% dimethylsulfoxide (Sigma)/90% FBS for further analysis.

Histopathologic Analysis

Tissues were fixed for at least 72 hours in 10% neutral buffered formalin (Sigma), dehydrated in ethanol, cleared in xylene, and infiltrated with paraffin on an automated processor (Leica Bannockburn, IL, USA). Four micron thick tissue sections were placed on charged slides, deparaffinized with xylene, rehydrated through graded alcohol washes, and stained with hematoxylin and eosin (H&E).

Flow cytometry and purification of hematopoietic stem and progenitor populations

Cells were stained for immunophenotype analysis using the following antibodies: APC-conjugated Gr-1, PE-conjugated Mac-1, APC-conjugated CD34, and PE-conjugated c-kit. Analysis was performed using a FACScalibur cytometer (Beckton Dickinson, Mountain View, CA) and CELLQuest software. A minimum of 10,000 viable cells was analyzed by gating on 7-AAD negative populations. Multiparameter flow cytometry was used to isolate hematopoietic stem cells (HSCs) and myeloid progenitors as described previously (15). Briefly, HSCs were distinguished as LinSca-1+c-kit+ (LSK fraction), common myeloid progenitors (CMPs) as LinSca-1c-kit+CD34+FcγRII/IIIlo, granulocyte-monocyte progenitors (GMPs) as LinSca-1c-kit+CD34+FcγRII/IIIhi, and megakaryocyte-erythrocyte progenitors (MEPs) as LinSca-1c-kit+CD34FcγRII/IIIlo. For promyelocyte and mature granulocyte identification, staining was performed as described with the following changes: Sca-1 antibody was added to the lineage depletion cocktail, and Gr-1 antibody was excluded from the lineage depletion cocktail. Promyelocytes were distinguished as c-kit+CD34+Gr-1+ and mature granulocytes as c-kitCD34Gr-1+. Dead cells were excluded from all analyses by propidium-iodide staining. Cells were sorted into 30% FBS/PBS for subsequent assays. Analysis and purification was performed using a FACSAria cytometer (Becton Dickinson, Mountain View, CA) and FACSDiva or FlowJo (Treestar, San Carlos, CA) software programs.

Quantitative real-time PCR

RNA was extracted from cells using the RNeasy Micro or Mini kits (Qiagen) per the manufacturer’s instructions, incorporating a DNAse1 digest to remove any contaminating genomic DNA. cDNA was prepared from RNA using Taqman® reverse transcription reagents (Applied Biosystems). Taqman® gene expression assays were used to analyze expression of the following murine genes: cathepsin G, neutrophil elastase, gelatinase B, myeloperoxidase, and Gapdh. Detection of expression of the PML-RARα fusion transcript was performed using a custom made Taqman probe specific to the fusion junction and flanking primers. PCR reactions were performed and analyzed using the 7300 Real Time PCR System and SDS Software (Applied Biosystems). Expression values were normalized to GAPDH.

Colony forming assays and bone marrow transplantation

Purified populations of stem or progenitor cells were plated in MethoCult GF M3434 methylcellulose culture medium (StemCell Technologies, Vancouver, BC, Canada). For initial platings of purified populations, 500 HSCs, 1000 CMPs or GMPs, and 10,000 promyelocytes were seeded in duplicate cultures. Colonies were counted after 7–9 days. Individual colonies or pooled colonies from an entire culture dish were harvested for cytospin preparations and subsequent staining with Wright-Giemsa. Serial replating was performed every 7 days by amalgamating all cells from duplicate dishes and re-seeding of new duplicate cultures at a density of 10,000 cells per plate. For ATRA differentiation experiments, cells were plated in M3434 media as described in the presence or absence of 1 μM ATRA (Sigma-Aldrich). Cells were harvested after 7 days and stained with Wright-Giemsa. For transplantation, sorted promyelocytes or mature granulocytes from healthy and leukemic PR/+ mice were resuspended in Hank’s Balanced Salt Solution. Cells were injected via the lateral tail vein into sub-lethally irradiated (552 rad) syngeneic recipient mice (4–6 weeks of age). For in vivo reconstitution assays, 50,000 promyelocytes from healthy PR/+ animals were combined with 500,000 whole bone marrow cells from wildtype mice and injected into lethally irradiated (552rad × 2) syngeneic recipient mice.

Microarray analysis

Total RNA was prepared from sorted promyelocytes isolated from pooled bone marrow samples from wild type, healthy PR/+, or leukemic PR/+ animals using the RNeasy micro kit (Qiagen). Samples were prepared in triplicate for each genotype. Linear amplification, biotin labeling, and fragmentation of amplified cDNA was carried out using the Ovation RNA Amplification System V2 and the FL-Ovation cDNA Biotin Module V2 (NuGen Technologies, San Carlos, CA) exactly according to the manufacturer’s instructions. Labeled probes were hybridized to Affymetrix (San Jose, CA) GeneChip Mouse Genome 430A 2.0 arrays. Raw gene expression values were processed with the robust multi-array analysis (RMA) (16) algorithm using BioConductor software (17).

Statistical Analyses

Statistical significance of differences in parameters measured between wildtype, healthy PR/+ and leukemic PR/+ animals was assessed using a two-tailed unpaired t-test.

Results

PML-RARα confers properties of self-renewal to hematopoietic progenitors

We first assessed the effect of expression of PML-RARα from the endogenous cathepsin G promoter on hematopoietic stem and progenitor compartments in bone marrow derived from 6–8 week old heterozygous transgenic mice prior to the development of leukemia (“Healthy PR/+”). We used multiparameter flow cytometry to analyze the hematopoietic stem cell compartment (LinSca1+Kit+, LSK cells), common myeloid progenitors (CMP), granulocyte monocyte progenitor (GMP) or megakaryocyte-erythrocyte progenitor (MEP) populations (15) (see Materials and Methods). We observed no differences in the proportion of these stem and progenitor compartments when comparing PR/+ with wild type littermate controls (Figures 1a–b, shown graphically in c). We also tested the LSK, CMP, and GMP compartments for the expression of PML-RARα and found low levels of expression of the transgene in earlier stem and progenitor compartments. In that the fusion is expressed from the endogenous cathepsin G locus, these findings suggest that the Cathepsin G promoter is active, albeit at much lower levels, in other compartments within the myeloid lineage in addition to the promyelocyte compartment (See Supplementary Figure 1). These findings confirm and expand on previously reported data indicating a minimal phenotype in transgenic animals prior to the development of leukemia (13).

Figure 1
PML-RARα expression alters stem and progenitor compartments in the leukemic state. Whole bone marrow from wild type (wt), healthy PR/+ or leukemic PR/+ animals was sorted into hematopoietic stem cell (LSK fraction), common myeloid progenitor (CMP), ...

We then tested each of these populations of cells, respectively, for serial replating potential in methylcellulose in the absence of stroma, an in vitro surrogate for self-renewing potential (4, 5, 8, 11, 18, 19). LSK, CMP or GMP derived from healthy PR/+ mice showed serial replating potential, whereas cells derived from wildtype mice did not (Figure 2a). Colonies were blast-like in appearance, with small compact size and rounded edges (Figure 2bi), and contained c-kit+Gr-1+Mac-1+ cells with immature myeloid morphology (Figure 2bii, 2biii and 2c). This is in contrast to the typical granulocyte-monocyte (GM) colony morphology containing differentiated granulocytic and monocytic cell types observed in plates seeded with wildtype cells (Figure 2biv, 2bv, and 2bvi). Morphology was indistinguishable between colonies formed from an initial plating of LSKs, CMPs, or GMPs (data not shown). These findings indicated that PML-RARα could confer certain properties of self-renewal to early progenitor populations in vitro, even in the absence of frank leukemia.

Figure 2
PML-RARα confers in vitro self-renewal ability to bone marrow progenitors. (a) Numbers of colonies observed per 10,000 cells plated in serial replating assays from either wild type (left) or healthy PR/+ (right) animals (mean ± SEM). LSKs, ...

Prospective identification and purification of promyelocyte progenitors from mouse whole bone marrow

Although we observed functional changes in the LSK, CMP, and GMP compartments in healthy PR/+ mice, we were most interested in assessing the effects of PML-RARα expression in more differentiated myeloid lineage cells, including promyelocytes and terminally differentiated granulocytes. We developed a novel multiparameter flow cytometry strategy with c-Kit, CD34 and Gr-1 staining that enabled prospective purification of these cells (Figure 3a). After lineage and Sca-1 depletion, mature granulocytes were contained within the c-KitCD34Gr-1+ fraction of the bone marrow, whereas promyelocytes were found within the c-Kit+CD34+Gr-1+ fraction (Figure 3a–b). Sorted granulocytes displayed characteristic morphologic features, including multi-lobed nuclei, while sorted promyelocytes were more immature in appearance, with a higher nuclear to cytoplasmic ratio and the presence of brightly staining azurophilic granules (Figure 3b, arrows). The promyelocytic identity of the purified population was further confirmed by quantitative RT-PCR analysis of the expression of primary and secondary granule proteins associated with granulocytic maturation (Figure 3c). As would be expected, promyelocytes expressed the primary granule genes for neutrophil elastase (ELA2), cathepsin G (CTSG), and myeloperoxidase (MPO). In contrast, promyelocytes did not express gelatinase B (GelB), which encodes a secondary granule protein associated with the later myelocyte stage of granulocyte development (20). This expression profile was specific for the promyelocyte compartment, as granulocyte-monocyte progenitors (GMPs) expressed markedly lower levels of myeloperoxidase and cathepsin G (Supplementary Figure 2). These data were corroborated by genome-wide expression analysis of purified promyelocytes performed using Affymetrix microarrays (Figure 3d).

Figure 3
Purified populations of granulocytes and promyelocyte progenitors can be obtained using multiparameter flow cytometry. (a) Sorting scheme for isolation and purification of mature granulocytes and promyelocyte progenitors from a wildtype mouse. (b) Cytospin ...

PML-RARα confers properties of self-renewal to promyelocytes

We utilized our flow cytometric approach to purify and compare promyelocytes and mature granulocytes from bone marrow derived from wild type or PR/+ animals prior to development of leukemia. While there were no significant differences in peripheral blood counts between wild type and PR/+ animals in this model (data not shown), there was a subtle increase in granulocytes in the bone marrow of healthy PR/+ animals compared to wild type (12.25% vs. 3.58%, Figs 4a–b, shown graphically in d). The number of promyelocytes was also modestly increased (1.27% vs. 0.46%, Figure 4a–b, shown graphically in d), although none of these differences reached statistical significance. Expression of PML-RARα did not alter the phenotype of the promyelocyte compartment, as granule protein expression profiles were virtually indistinguishable between wildtype and healthy PR/+ animals (Figure 4e).

Figure 4
Promyelocytes expressing PML-RARα are expanded in the leukemic state. Lineage/Sca-1 depleted bone marrow cells from wt (a), healthy PR/+ (b), and leukemic PR/+ (c) mice were gated into c-kit+CD34+ and c-kitCD34 fractions (left ...

However, despite the similarity in granule protein expression profiles between wildtype and PML-RARα-expressing promyelocytes, a striking finding was observed in serial replating assays in the absence of stromal support: promyelocytes derived from PR/+ animals, but not wildtype animals, demonstrated serial replating activity in the absence of stroma (Figure 5a). There was no replating activity in granulocytes derived from either genotype, which may be due to the fact that the expression level of PML-RARα is significantly reduced in the granulocyte compartment as compared to the promyelocyte compartment (Figure 5b). These in vitro data suggested that PML-RARα expression in the promyelocyte compartment may lead to enhanced self-renewal, as was observed in earlier progenitor compartments. To test this hypothesis in vivo, we purified promyelocytes from healthy PR/+ animals and transplanted 50,000 of these cells with helper bone marrow into syngeneic recipient animals. We then monitored for the expression of PML-RARα in the peripheral blood of these animals over the course of 16 weeks. As shown in Figure 5c, PML-RARα expression was observed in the peripheral blood of transplanted mice at all time points tested, indicating long-term reconstitution of recipient animals with PML-RARα-expressing promyelocytes. These findings suggest that this terminally differentiating population of cells had acquired potential for self-renewal prior to the development of leukemia as a consequence of expression of PML-RARα.

Figure 5
Promyelocytes expressing PML-RARα display in vitro and in vivo properties of self-renewal. (a) Numbers of colonies after serial replating of purified promyelocytes from either wildtype or healthy PR/+ animals (mean ± SEM). (b) Quantitative ...

PML-RARα alters hematopoietic stem, progenitor, and granulocyte compartments in the leukemic state

PR/+ animals ultimately develop a phenotype that is very similar to human acute promyelocytic leukemia, with a long latency that is associated with the acquisition of secondary mutations (13, 21). We assayed the effects of leukemic transformation on the stem and progenitor compartments using multiparameter flow cytometry, and observed a marked reduction in the number of LSKs, CMPs, and MEPs compared to healthy PR/+ or wild type controls, with a concomitant expansion of the GMP compartment (Figure 1a–c, shown graphically in d). Furthermore, in leukemic animals there was a dramatic expansion of the promyelocyte compartment that comprised 25.42% of the cells after lineage depletion, as compared to 1.27% in healthy PR/+ animals and 0.46% of wild type animals (Figure 4a–d, p<0.0001). The expansion of the promyelocyte compartment was coupled with a reduction in the number of mature granulocytes to normal levels when compared to healthy PR/+ mice (Figure 4d). Promyelocytes derived from leukemic animals retained their morphology and expression of primary granule proteins as determined by cytospin and quantitative RT-PCR analyses (Figure 4e and data not shown).

Leukemic promyelocytes are enriched for leukemia-initiating activity and are retinoic acid responsive

Given the significant expansion observed in the promyelocyte compartment of leukemic PR/+ animals, we hypothesized that this population of cells harbored leukemia-initiating activity. To test this hypothesis, we sorted promyelocytes and granulocytes from leukemic and healthy PR/+ animals and transplanted these purified populations into sublethally irradiated syngeneic recipient mice. As shown in Figure 6, promyelocytes from leukemic donor mice were able to transfer disease in vivo. Mice receiving 50,000 flow-purified leukemic promyelocytes succumbed to a rapidly fatal acute leukemia with a median disease latency of 42 days (Figure 6a). The disease recapitulated the leukemic phenotype observed in primary animals with hypercellular marrow predominated by myeloid cells that were c-Kit+Gr1+Mac-1+ (Figure 6b–c), with infiltration of liver and spleen (Figure 6b–c). Limit dilution transplant assays with purified promyelocytes indicated that the frequency of leukemia initiating cells was ~1:100 within the leukemic promyelocyte compartment (Figure 6d). In contrast, no disease was observed when purified granulocytes derived from these same animals were transplanted. These data indicate that promyelocytes, but not granulocytes, have efficient leukemia-initiating activity, and that leukemia stem cells in this murine model of disease can be demarcated within the terminally differentiating myeloid compartment. Of note, these promyelocytic leukemia-initiating cells were responsive to differentiation agent therapy with all-trans-retinoic acid (ATRA) (Figure 6e). After 7 days in methylcellulose culture, untreated leukemic promyelocytes retained their immature morphology, while the same cells treated with 1μM ATRA showed signs of differentiation and apoptosis. Thus, it can be inferred that ATRA is capable of reprogramming promyelocytic leukemia stem cells to terminally differentiated cells that are incapable of self-renewal, and accounts in part for the disease-remitting activity of ATRA.

Figure 6
Leukemic promyelocytes are enriched for leukemia-initiating activity. (a) Kaplan-Meier survival curves for recipient mice transplanted with 50,000 leukemic promyelocytes (blue), 50,000 leukemic granulocytes (black), or 50,000 non-leukemic promyelocytes ...

Discussion

The cancer/leukemia stem cell theory has recently been called into question (22), based in large part on criticism of the xenograft transplantation model that has been used to lay the foundation for this area of investigation in cancer research (13). Subsequent studies have extended the body of evidence that rare cells within the leukemic population drive tumor formation, and that these cells could in fact be transformed progenitors (8, 11, 12). A caveat to these analyses is that they rely on either human to mouse xenograft or retroviral transduction of purified cell populations and subsequent transplantation into secondary recipient animals. Thus, they confer disease through non-physiologic conditions, and cannot address questions regarding the pathologic initiation and development of disease due to progressive oncogenic insults. Here we have addressed these issues by assessing the leukemia stem cell theory using a model system of disease that expresses an oncogene in the appropriate developmental compartment without the need for retroviral transduction, thus more closely mimicking the human pathologic condition.

We selected acute promyelocytic leukemia as a model to characterize leukemia stem cells for several reasons. First, we were able utilize a well-characterized knock-in mouse model of the disease in which expression of PML-RARα is targeted to the promyelocyte compartment. Second, APL represents a relatively more differentiated form of acute leukemia as compared to other subtypes, and is therefore an attractive model in which to test the possibility that more differentiated progenitors can possess leukemia-initiating activity. Of note, APL (FAB subtype M3) was the only AML subtype that was unable to transfer disease to NOD-SCID recipient mice, regardless of the fraction of cells transplanted (2, 3). This lends further support to the notion that the leukemia-initiating population within APL is most likely not derived from an HSC. Finally, APL is the only leukemia with a clinically proven differentiation therapy in the form of all-trans retinoic acid. Therefore, the effects of a known successful treatment for the disease could be tested specifically on the leukemia-initiating population.

Our results demonstrate that in acute promyelocytic leukemia, a committed progenitor, the promyelocyte, has the capacity to transfer disease upon transplantation, and therefore possesses leukemia stem cell properties. This data supports the notion that leukemia stem cells need not be derived from hematopoietic stem cells, and that indeed a committed progenitor can be transformed into a cell capable of maintaining the leukemic clone. The hallmark features of a leukemia stem cell are the ability to both self-renew and to differentiate into all cell types of the primary disease. Experiments by others have indicated that PML-RARα may possess the potential to confer self-renewal, but these assays were solely conducted in vitro and relied on retroviral transduction of PML-RARα into stem and progenitor populations (23, 24). Here, we have demonstrated that progenitor populations from PML-RARα expressing animals do in fact possess the capacity to self-renew. Importantly, this ability is conferred to progenitor populations in the absence of frank leukemia; the mice used in these experiments were roughly 8 weeks of age, and appeared perfectly healthy. Despite the lack of leukemic transformation, PML-RARα-expressing promyelocytes displayed properties of self-renewal both in vitro and in vivo, a striking finding considering promyelocytes are thought to be post-mitotic cells that are committed to terminal differentiation into granulocytes, cells that normally survive only hours to days once in circulation. This suggests that PML-RARα may confer self-renewal ability to progenitor populations normally lacking this capacity as an initiating step in the process of leukemogenesis. Indeed, analysis of the stem and myeloid progenitor compartments of PR/+ animals revealed no differences in the frequencies of these populations in the healthy state, suggesting that despite the capacity for self-renewal, these cells do not yet possess the requisite pro-survival or proliferative advantage required for leukemogenesis.

The fact that APL is a disease of promyelocytes prompted us to carefully explore this more differentiated stage of granulocyte development. Our results demonstrate that the shift from the healthy to leukemic state is accompanied by a massive expansion of the promyelocyte (c-kit+CD34+Gr-1+) compartment. Furthermore, this compartment is capable of transferring disease to secondary recipient animals, and is highly enriched for leukemia initiating activity as determined by limiting dilution transplants. These leukemic promyelocytes possess true leukemia stem cell properties, as defined by self-renewal and differentiation. Their capacity to serially transplant disease indicates that they must possess self-renewal ability in order to perpetuate the tumor. Additionally, flow cytometric and histologic analyses of tissues from diseased animals confirm that the leukemic animals succumbed to a disease that recapitulated that of the primary donor.

These results suggest a model for the development of APL in which PML-RARα plays a key role in initiating disease by conferring self-renewal, one of the requisite features of leukemia stem cells, to committed progenitors (See Supplementary Figure 3). Although under normal physiologic conditions of myeloid development the hematopoietic stem cell is the only cell capable of self-renewal in the pathway of differentiation leading to the formation of a mature granulocyte, upon expression of PML-RARα, promyelocytic progenitors acquire the ability to self-renew. The sustained maintenance of these progenitors “primes” them for the acquisition of additional mutations that will lead to frank leukemia. Upon acquisition of additional mutations that confer proliferative and/or pro-survival advantage within the promyelocyte compartment, cooperativity between these secondary mutations and PML-RARα leads to massive expansion of promyelocyte progenitors, and acute leukemia results.

It is important to note that our results do not formally address the target cell of transformation in APL in which the initial PML-RARα mutation occurs, a question that remains unanswered in the APL field (25, 26). Interesting results obtained by Lane and Ley (27) suggest that proteolytic processing of PML-RARα by neutrophil elastase is an important event for leukemic transformation (28) and therefore, PML-RARα may be expressed in early stem and progenitor compartments, but may not be active or oncogenic until it is cleaved by enzymes that are only present later in myeloid differentiation. Further analysis in both murine models and human patient samples will be needed to clarify this debate.

These data provide several provocative insights into the contribution of the PML-RARα oncogene to leukemogenesis. First, this experimental system enables analysis of the physiologic consequences of oncogene expression in a committed progenitor prior to the onset of leukemia, and an ability to monitor changes with disease progression. Second, these findings help to understand how a hematopoietic progenitor that is committed to differentiation and cell death may be a target for transformation. In the context of multiple mutations that are required for leukemogenesis, it would be imperative that the initiating mutation enables self-renewal potential that would allow for acquisition of secondary mutations that confer the overt leukemia phenotype. PML-RARα expression meets these criteria in conferring long-term self-renewing potential to promyelocytes that are normally short lived. Third, it is remarkable that expression of a single leukemia oncogene – PML-RARα – apparently confers such properties to a cell that is primarily distinguished by its short half-life with terminal differentiation.

The identification and characterization of cancer stem cells has been catalyzed by the notion that current chemotherapeutics fail to eradicate cancer stem cells, leading to eventual disease relapse (reviewed in (10). Therefore, there is increasing interest in identifying biologically distinct features of cancer stem cells that distinguish them from other cell types, including normal stem cells, in order to design better therapies that specifically target cancer stem cells. Recent data from hematologic malignancies provide evidence that differences do exist between normal HSCs and leukemia-initiating cells, providing hope that this strategy will be fruitful in eventually identifying targeted therapeutics that can eradicate leukemia stem cells (2931). In the case of APL, our data suggests that the leukemia-initiating promyelocytes are susceptible to differentiation therapy mediated by all-trans retinoic acid therapy. However, particularly for the sub-population of patients who develop eventual resistance to ATRA therapy, it is important to develop effective alternative therapies.

Supplementary Material

Suppl Figure 1

Suppl Figure 2

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Supplementary Figure Legends

Acknowledgments

We would like to thank Maricel Gozo and Dena Leeman for technical support, as well as Stefan Fröhling, Claudia Scholl and all the members of the Gilliland laboratory for helpful discussions and comments regarding this manuscript.

Footnotes

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