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The Wnt/β-Catenin Pathway Is Required for the Development of Leukemia Stem Cells in AML

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Science  26 Mar 2010:
Vol. 327, Issue 5973, pp. 1650-1653
DOI: 10.1126/science.1186624

A Pathway to Leukemia

Leukemia is initiated and maintained by a small number of self-renewing cells called leukemia stem cells (LSCs), which share properties with hematopoietic stem cells (HSCs), the self-renewing cells that produce healthy blood cells. Wang et al. (p. 1650) studied mouse models of acute myelogenous leukemia (AML), a disease that is often refractory to existing therapies. Activation of the Wnt/β-catenin signaling pathway was required for efficient oncogene-mediated conversion of HSCs into LSCs. This pathway is among the most well studied signaling pathways in cell biology, setting the stage for testing of β-catenin signaling antagonists in preclinical models of AML.

Abstract

Leukemia stem cells (LSCs) are capable of limitless self-renewal and are responsible for the maintenance of leukemia. Because selective eradication of LSCs could offer substantial therapeutic benefit, there is interest in identifying the signaling pathways that control their development. We studied LSCs in mouse models of acute myelogenous leukemia (AML) induced either by coexpression of the Hoxa9 and Meis1a oncogenes or by the fusion oncoprotein MLL-AF9. We show that the Wnt/β-catenin signaling pathway is required for self-renewal of LSCs that are derived from either hematopoietic stem cells (HSC) or more differentiated granulocyte-macrophage progenitors (GMP). Because the Wnt/β-catenin pathway is normally active in HSCs but not in GMP, these results suggest that reactivation of β-catenin signaling is required for the transformation of progenitor cells by certain oncogenes. β-catenin is not absolutely required for self-renewal of adult HSCs; thus, targeting the Wnt/β-catenin pathway may represent a new therapeutic opportunity in AML.

Acute myelogenous leukemia (AML) is the most common acute leukemia in adults, and most patients are not cured with current therapies. Only a small subset of AML cells are capable of extensive proliferation and self-renewal (1). Such cells are referred to as leukemia stem cells (LSCs) because they share properties, including extensive self-renewal potential, with normal stem cells. LSCs are being studied as potential therapeutic targets (2, 3), and the signaling pathways that control their development and survival are of particular interest. The Wnt/β-catenin pathway is active in certain human leukemias (47) and in normal hematopoietic stem cells (HSCs) (810). Although β-catenin is not required for self-renewal of adult HSC (11, 12), it is unclear whether β-catenin is required for LSC development and maintenance in AML. We have studied well-characterized mouse models of AML to explore whether β-catenin is necessary for LSC development from either HSC or more differentiated committed myeloid progenitor cells.

Homeobox (Hox) genes have been implicated in the regulation of normal stem cell self-renewal (13, 14), and enforced coexpression of Hoxa9 and Meis1a in mouse bone marrow leads to rapid AML development (15). Previous studies demonstrated that leukemogenic fusion proteins, such as those involving the mixed lineage leukemia (MLL) protein (for example, MLL-AF9), can transform non–self-renewing granulocyte/macrophage progenitors (GMP) and activate genes encoding Hox proteins such as HoxA9 and Meis1 (1618). To determine whether a combination of Hoxa9 and Meis1a (HoxA9/M) can transform GMP, we cotransduced sorted Lin c-Kit+ Sca-1 + (KLS) cells, which are enriched for HSC, or Lin c-Kit+ Sca-1 FcRγ+ CD34+ (GMP) cells with viruses encoding HoxA9 (MSCV-HoxA9-GFP) and Meis1a (MSCV-Meis1a-puro), and used either a bulk approach (15, 18, 19) or a single-cell approach (fig. S1). When single cells were expanded in vitro up to 6 weeks, no differences were observed between KLS cells transduced with HoxA9/M (KLS-HoxA9/M), GMP transduced with HoxA9/M (GMP-HoxA9/M), or GMP transduced with MLL-AF9 (GMP-MLLAF9) in proliferation, cell-cycle profile, apoptosis levels, gross morphology, or immunophenotype (fig. S1). However, there was a proliferative defect upon extended culture of GMP-HoxA9/M cells. When cells were injected into recipient mice, 13 of 14 (93%) mice that received GMP-MLLAF9 cells developed AML and 14 of 20 (70%) mice that received KLS-HoxA9/M cells developed AML, whereas only 1 of 23 mice that received GMP-HoxA9/M cells developed AML (fig. S2). These differences in leukemogenic potential could not be ascribed to differential expression of HoxA9 and Meis1 (fig. S3).

To characterize the in vivo defect in HoxA9/M–transduced GMP, we monitored green fluorescent protein–positive (GFP+) cells and their immunophenotypes in recipient mice at multiple timepoints after transplantation. By 18 hours, KLS-HoxA9/M and GMP-HoxA9/M cells homed to the bone marrow with similar efficiency (fig. S2). By 8 days, both cell types displayed heterogeneity of expression of the stem cell marker, c-Kit (c-Kithigh and c-Kitlow) (Fig. 1A). KLS-HoxA9/M cells sustained the two c-Kit populations through 1 month, until ultimately a lethal leukemia developed (Fig. 1A). Conversely, at 1 month after transplantation, GMP-HoxA9/M retained only the c-Kitlow population that was eventually lost from bone marrow (Fig. 1A).

Fig. 1

β-catenin is activated in developing leukemia cells and L-GMP derived from KLS-HoxA9/M and GMP-MLLAF9. (A) Immunophenotypic analyses of GFP+ cells from bone marrow of mice transplanted with pre-leukemia KLS-HoxA9/M or GMP-HoxA9/M cells at 8 days, 1 month, or 4 months (leukemia) after transplantation. (B) The heat map displays the top 30 probe sets for genes that show increased expression in the HSC population (KLS cells) and L-GMP by use of comparative marker selection and permutation testing (200 probe sets passed a cutoff of P < 0.002). Expression of Ptgs-1 (Cox-1) and Ptger1 (prostaglandin E receptor 1) is shown. (C) Immunoblot analysis for active (dephosphorylated) β-catenin in normal GMP or L-GMP derived from GMP-MLLAF9 or KLS-HoxA9/M mediated leukemias. (D) Immunofluorescence assessment for active β-catenin in c-Kithigh or c-Kitlow populations isolated from mice 1 month after injection of pre-leukemia KLS-HoxA9/M cells. Red, active β-catenin; blue, Hoechst; merge, active β-catenin/Hoechst.

To determine whether the c-Kithigh and c-Kitlow populations that were identified 1 month after injection of KLS-HoxA9/M cells are functionally distinct, we used in vivo limiting dilution analysis to measure the frequency of leukemia-initiating cells (LICs) in these two populations. LICs are defined as cells isolated before the development of clinically evident leukemia that can initiate leukemia in subsequent recipient mice. A high frequency of LIC was observed in the c-Kithigh population. In contrast, c-Kitlow cells were inefficient at inducing leukemia (fig. S4). We next assessed LSC activity in the fully developed HoxA9/M–induced leukemias. Limiting dilution analysis demonstrated that the Gr1–/(low)c-Kithigh population was over 100-fold enriched for LSC as compared with that of the Gr1+c-Kithigh cells (figs. S4 and S5) and had an immunophenotype similar to MLL-AF9 LSC (L-GMP) (18, 20) (fig. S6). Thus, maintenance of the c-Kithigh population is associated with leukemia development, and cellular heterogeneity is found throughout the process of leukemia development. The inability of GMP-HoxA9/M cells to induce leukemia prompted us to search for a self-renewal pathway active in LSC that might be deficient in the GMP-HoxA9/M cells.

We previously demonstrated that Lin Sca-1 c-Kithigh FcRγ+ CD34+ cells (termed L-GMP) are enriched for LSC in the MLL-AF9 AML model and that L-GMP are globally similar to normal cells at a mid-myeloid stage of development but express a subset of genes normally highly expressed in HSC (18). Given that the LSC population in HoxA9/M–driven leukemia described here has a similar immunophenotype, we reasoned that genes highly expressed in MLL-AF9 L-GMP, HoxA9/M L-GMP, and HSC but not in GMP might pinpoint genes that are important for leukemic self-renewal. Supervised analysis of gene expression data demonstrated a group of genes that are highly expressed in normal and leukemia stem cells and expressed at lower levels in normal myeloid progenitors (Fig. 1B). Prostaglandin-endoperoxide synthase 1 (Ptgs1) (also known as Cycloxygenase-1 or Cox-1) and one of the prostaglandin receptors (Ptger1) were up-regulated in both GMP-MLLAF9–derived and KLS-HoxA9/M–derived L-GMP, which was confirmed through quantitative ploymerase chain reaction (Fig. 1B and fig. S6). Because previous studies have highlighted a critical connection between prostaglandin synthesis and the Wnt/β-catenin pathway (2124), we measured β-catenin activation. An antibody specific for activated β-catenin demonstrated activity in GMP-MLLAF9–derived and KLS-HoxA9/M–derived L-GMPs but not in normal GMP, as assessed by means of immunoblot or immunofluorescence (Fig. 1C and fig. S7). Also, β-catenin activity was found in GMP-MLLAF9–derived and KLS-HoxA9/M–derived cells grown in vitro but not in GMP-HoxA9/M–derived cells (fig. S7). Activated/nuclear β-catenin is found in c-Kit high but not c-Kitlow cells isolated from mice 1 month after injection with KLS-HoxA9/M cells (Fig. 1D).

Given the activation pattern of β-catenin in GMP-MLLAF9–derived and KLS-HoxA9/M–derived leukemias and its minimal activation in GMP-HoxA9/M–derived cells, we hypothesized that the inability of HoxA9/M to transform GMP might be due to its inability to sufficiently activate β-catenin. To test this, we transduced GMP-HoxA9/M cells with a retrovirus encoding a constitutively active form of β-catenin (βcat*) (Fig. 2A). Transplantation of GMP-HoxA9/M cells transduced with βcat* produced AML similar to KLS-HoxA9/M cells (Fig. 2B). Expression of βcat* in GMP cells did not induce leukemia (Fig. 2B).

Fig. 2

Constitutively active β-catenin cooperates with HoxA9/M to induce AML from GMP cells. (A) Constitutively active βcat* was transduced into pre-leukemia KLS-HoxA9/M or GMP-HoxA9/M cells, and protein levels were assessed by means of immunoblot analysis. (B) Survival curves of mice receiving KLS or GMP cells transduced with active βcat* (controls), GMP-HoxA9/M, GMP-HoxA9/M cells transduced with active βcat*, or KLS-HoxA9/M. 5 × 105 cells were transplanted into sublethally irradiated recipients (n = 10 recipients in each group). P was determined by using the log-rank test.

We next tested the effect of indomethacin (Indo), a reversible COX inhibitor that blocks activation of the Wnt pathway by suppressing β-catenin expression (23). Exposure of LIC-enriched c-Kithigh cells to indomethacin reduced β-catenin levels in vitro (Fig. 3A), and mice treated with Indo showed a dramatic reduction in the c-Kithigh population after 7 days of treatment (Fig. 3B). We performed a limiting dilution assay using flow-sorted GFP+ cells from bone marrow of control and Indo-treated mice, which demonstrated a 100-fold decrease in LIC after indo treatment (Fig. 3C). Furthermore, Indo treatment of a fully developed MLL-AF9 leukemia partially suppressed β-catenin (fig. S8) and reduced LSC frequency (Fig. 3D). Thus, indomethacin treatment can suppress the LIC compartment that is critical for the development of AML and also influences established LSC, suggesting that AML is dependent on β-catenin signaling. However, the therapeutic effect is greater in situations of minimal disease (Fig. 3C) versus more extensive disease (Fig. 3D).

Fig. 3

Pharmacological inhibition of β-catenin impairs LSC function. (A) Immunoblot analysis of β-catenin levels in c-Kithigh cells sorted from bone marrow at 1 month after injection of mice with KLS-HoxA9/M cells, which were subsequently grown in methylcellulose with and without exposure to indomethacin for 3 weeks (19, 26). (B) Immunophenotypic analysis of bone marrow GFP+ cells after injection of KLS-HoxA9/M cells and treatment with either vehicle or indomethacin (repeated with 2 independent clones) for 7 days. Treatment began at day 3 after injection, and mice were sacrificed at day 10 after transplantation (19, 23). (C and D) Survival curves of mice (five mice for each group) injected with the indicated number (10 to 104) of GFP+ marrow cells sorted from control or Indo-treated mice that had received pre-leukemia KLS-HoxA9/M cells (C) or leukemic GMP-MLLAF9 cells (D) (repeated with two independent clones).

Next, we examined the ability of HoxA9/M to induce leukemia from β-catenin–deficient KLS cells using bone marrow from βcatloxP/loxP mice (25). We transduced βcatloxP/loxP KLS cells with HoxA9/M, sorted single cells (KLS-HoxA9/M βcatloxP/loxP), and subsequently transduced the cells with a retroviral vector encoding either Cre recombinase or an empty vector. Cells transduced with Cre recombinase (KLS-HoxA9/M βcat−/−) were devoid of wild-type (WT) β-catenin (fig. S9). These cells were injected into recipient mice, and we monitored expansion of KLS-HoxA9/M βcat loxP/loxP, KLS-HoxA9/M βcat−/−, and control cells in bone marrow. KLS-HoxA9/M βcatloxP/loxP cells expanded efficiently in mouse bone marrow (Fig. 4A) and ultimately produced leukemia. However, loss of β-catenin led to impaired expansion, and KLS-HoxA9/M βcat−/− cells ultimately lost their ability to expand in bone marrow (Fig. 4A). βcat* rescued the proliferative defect of KLS-HoxA9/M βcat−/− cells (fig. S9). Additionally, βcatWT KLS-HoxA9/M cells, βcatWT KLS-HoxA9/M cells expressing cre recombinase, and KLS-HoxA9/M βcatloxP/loxP cells induced AML in all recipients with similar latencies, whereas KLS-HoxA9/M βcat−/− cells were dramatically impaired in their ability to initiate AML (Fig. 4B). Three mice injected with KLS-HoxA9/M βcat−/− cells did ultimately develop AML; however, in the two cases that were captured before the mouse succumbed to the disease there was incomplete excision of β-catenin (fig. S9). Finally, a similar experiment demonstrated a dramatic reduction in the ability of MLL-AF9 to induce leukemia from GMP (Fig. 4C).

Fig. 4

Deletion of β-catenin impairs the development of AML induced by KLS-HoxA9/M and GMP-MLLAF9. (A) GFP+ cells in bone marrow of mice injected with floxed β-catenin (βcatloxp/loxp) or β-catenin-deficient (βcat−/−) KLS-HoxA9/M cells at 18 hours, 1 month, or 4 months after transplantation. HoxA9/M-βcatloxP/loxP KLS cells were transduced with a retroviral vector encoding Cre recombinase to generate βcat−/− KLS-HoxA9/M cells or an empty vector to generate control βcatloxp/loxp KLS-HoxA9/M cells. After selection, 1 × 106 infected cells were transplanted into sublethally irradiated recipients (n = 5 recipients in each group). Mice were sacrificed at the indicated time points to assess for GFP+ cells. (B) Survival curves of mice transplanted with βcat−/− KLS-HoxA9/M cells, βcatloxp/loxp KLS-HoxA9/M cells, and empty vector or Cre-infected WT KLS-HoxA9/M (KLS cells were sorted from WT C57BL/6 mice and transformed with HoxA9/M so as to assess the effect of Cre on KLS-HoxA9/M leukemia development; n = 10 mice for each group). (C) Survival curves of mice transplanted with βcat−/− GMP-MLLAF9 cells and βcatloxp/loxp GMP-MLLAF9 cells (n = 10 mice for each group). P was determined by using the log-rank test.

We have shown that β-catenin is required for Hox-mediated transformation of HSC and MLL-AF9–mediated transformation of committed progenitor cells. Our data also indicate that the lack of β-catenin activation in normal GMP limits the ability of Hox genes to efficiently transform committed progenitor cells. This is in contrast to MLL-AF9, which activates sufficient β-catenin to transform GMP. Overall, these findings support the concept that activation or maintenance of HSC-associated developmental pathways in downstream myeloid cells is an important component of AML development. Given that β-catenin activity is crucial for HSC survival during fetal development, but not for adult HSC survival/proliferation (9, 11, 12), targeting the β-catenin pathway may be an attractive therapeutic avenue in this disease.

Supporting Online Material

www.sciencemag.org/cgi/content/full/327/5973/1650/DC1

Materials and Methods

Figs. S1 to S9

References

References and Notes

  1. Materials and methods are available as supporting material on Science Online.
  2. We thank D. Fisher, X. He, and H. Widlund for plasmid pcDNA3.1-βcat-*-Flag; D. Williams for helpful discussion and advice; and M. Smith for administrative assistance. This work was supported by the NIH grants 5P01CA66996 (to S.A.A.) and 5R01HL048801 (to L.I.Z.), the American Cancer Society (RSG-09-068-01), and the Harvard Stem Cell Institute. S.A.A. is a Leukemia and Lymphoma Society Scholar. Microarray data was submitted to the National Center for Biotechnology Information Gene Expression Omnibus (GEO) GSE20377. L.I.Z. is a founding member of and stockholder in Fate Therapeutics, a biotechnology company that is developing stem cell therapeutics and that has acquired a patent covering the use of prostaglandin E2 in hematopoietic stem cell modulation and tissue regeneration. W.G. and T.E.N. are paid consultants for Fate Therapeutics.
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