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CANCER STEM CELLS |
aInstitute of Molecular and Cell Biology, Singapore;
bDepartment of Pediatric Science, Graduate School of Medicine, University of Tokyo, Tokyo, Japan;
cOncology Research Institute, National University of Singapore, Singapore;
dRIKEN, Research Center for Allergy and Immunology, Yokohama, Japan;
eHoward Hughes Medical Institute, Skirball Institute of Biomolecular Medicine, New York University School of Medicine, New York, New York, USA
Key Words. AML1 • Runx • c-Kit • FLT3 • Senescence
Correspondence: Yoshiaki Ito, M.D. Institute of Molecular and Cell Biology, 61 Biopolis Drive, Singapore 138673. Telephone: 65-6586-9646; Fax: 65-6779-1117; e-mail: itoy{at}imcb.a-star.edu.sg
Received on January 25, 2007;
accepted for publication on August 28, 2007.
First published online in STEM CELLS EXPRESS September 6, 2007.
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ABSTRACT |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionDisclosure of Potential...AcknowledgmentsReferences |
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Disclosure of potential conflicts of interest is found at the end of this article.
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INTRODUCTION |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionDisclosure of Potential...AcknowledgmentsReferences |
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The RUNX1/AML1 gene encodes the DNA binding 
subunit of the Runt domain transcription factor PEBP2/CBF. The non-DNA binding β subunit (PEBP2β/CBFβ encoded by CBFB) of this heterodimeric transcription factor is essential for the function of RUNX1 (nomenclature given in [2]). RUNX1 and CBFB are critical for the generation and maintenance of hematopoietic stem cells (HSC) and are frequently targeted in human leukemia [3–5]. Chromosomal translocation, the most pervasive causative alteration in human leukemia, frequently involves RUNX1 and CBFB and generates chimeric genes that inhibit the activity of wild-type RUNX1 in a dominant-negative manner [6, 7]. Point mutations of RUNX1 have also been implicated in human leukemias. Biallelic mutations of RUNX1 are frequently found in the acute myeloid leukemia (AML) M0 subtype, and monoallelic mutations cause Familial platelet disorder with predisposition to acute myelogenous leukemia (FPD/AML), sporadic leukemia, and myelodysplastic syndrome [8, 9]. These RUNX1 point mutants are nonfunctional as transcription factors. Therefore, loss-of-function of RUNX1 is considered to be a common underlying mechanism for RUNX1-related leukemias.
However, ample evidence suggests that RUNX1 alteration per se does not readily induce leukemia and that additional genetic changes, or a "second hit," are required for full-blown leukemia. Activation of the RAS pathway is one of the most common genetic changes cooperating with RUNX1 alteration in human leukemias. The RAS gene family and its upstream factor genes, type III receptor tyrosine kinases such as c-KIT and FLT3, are frequently mutated in RUNX1-related leukemias [5, 10–12].
Our mouse model for FPD/AML, which uses the BXH2 mouse system to identify second-hit mutations cooperating with alternations in Runx1, has also suggested a synergy between Runx1 insufficiency and Ras activation [11]. The BXH2 mouse strain harbors an ecotropic murine leukemia retrovirus that functions as an insertional mutagen to induce myeloid leukemia within a year in more than 90% of the animals [13]. When Runx1 is heterozygously disrupted in BXH2 mice, which we refer to as BXH2-Runx1+/–, the latency of leukemia onset is shortened, suggesting that Runx1+/– status makes the mouse more leukemia-prone [11]. The retroviral integration, in turn, can be used as a tag to identify oncogenes or tumor suppressor genes, making this mouse model an excellent system to identify genes that cooperate with Runx1 insufficiency to promote leukemogenesis. One of the genes most frequently targeted by the retrovirus in BXH2 mice is Nf-1, a negative regulator of the proto-oncogene Ras through its GTPase-activating protein activity. Interestingly, BXH2-Runx1+/– mice showed a greater incidence of integration into Nf-1 compared with BXH2-Runx1+/+ mice. Furthermore, c-Kit and RasGrp1, both of which activate the Ras pathway, were preferentially affected in BXH2-Runx1+/– mice [11], suggesting that activation of the RAS pathway is the most common genetic event cooperating with RUNX1 alteration. The precise mechanism of this cooperation, however, remains elusive.
Here, we show that Runx1 inactivation changes the size and properties of hematopoietic stem/progenitor cells, thereby promoting survival of a leukemia-initiating clone carrying oncogenic Ras. Our findings have uncovered a novel role of RUNX1 in hematopoietic stem/progenitor cells and provide a profound insight into the development of leukemia.
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MATERIALS AND METHODS |
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Retroviral Transduction
XhoI and NotI sites within the murine stem cell virus (MSCV)-internal ribosome entry site (IRES)-green fluorescent protein (GFP) (MIG) retroviral vector were used for plasmid construction. Further information for plasmid construction is given in the supplemental online Methods. Culture of the Phoenix-Eco packaging cell line, preparation of retrovirus supernatant, and infection of bone marrow cells were performed as previously described [11].
Bone Marrow Transplantation
Bone marrow cells (2 x 105) transfected with the MIG retroviral vector were transplanted intravenously into C57BL/6 mice sublethally irradiated (5 Gy). Transfection efficiency of MIG vector, which is indicated by GFP positivity, was approximately 20% for all transfectants. The recipient mice were monitored weekly for complete blood cell counts and GFP positivity in peripheral blood from 3 weeks after transplantation. Necropsies of diseased mice were carried out as previously described [11].
Flow Cytometric Analysis
Flow cytometric analysis was performed using a FACSVantage instrument as previously described [11]. All monoclonal antibodies were purchased from BD Pharmingen (San Diego, http://www.bdbiosciences.com/index_us.shtml) (supplemental online Methods). For side population analysis, 106 cells, from which red blood cells had been removed by lysis, in 1 ml of prewarmed (37°C) Dulbecco's modified Eagle's medium with 2% fetal bovine serum (FBS) were incubated with 5 µg/ml Hoechst 33342 (Sigma-Aldrich) at 37°C for 90 minutes. The cells were resuspended in cold Hanks' balanced salt solution with 2% FBS and 10 mM HEPES buffer and analyzed immediately.
In Vitro Cell Culture Assays
c-Kit+ GFP+ bone marrow cells transfected with MIG vector were sorted and subjected to in vitro assays: liquid culture, long-term culture-initiating cell (LTC-IC) assay, and colony-forming unit-culture assay. The details of these experiments are available in the supplemental online Methods.
Quantitative Real-Time Polymerase Chain Reaction
The c-Kit+ GFP+ fraction of bone marrow cells transduced with MIG vector was sorted by fluorescence-activated cell sorting (FACS) directly into TRIZOL LS Reagent (Invitrogen, Carlsbad, CA, http://www.invitrogen.com) to isolate RNA. cDNAs were synthesized by Expand Reverse Transcriptase (Roche Diagnostics, Mannheim, Germany, http://www.roche-applied-science.com) and were subjected to reverse transcription polymerase chain reaction (RT-PCR) and real-time PCR. The details of these experiments are given in the supplemental online Methods.
Apoptosis Assay
Cells (105) in binding buffer (BD Pharmingen) were stained with Annexin V-PE (65875X; BD Pharmingen) and 7-aminoactinomycin D (7-AAD) (A9400; Sigma-Aldrich) for 15 minutes at room temperature in the dark and analyzed immediately by flow cytometry.
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RESULTS |
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In our BXH2 mouse leukemia model, peripheral blood samples were periodically collected, and white blood cell counts (WBC) were monitored. When leukemias with Nf-1 integration developed, we designed primers specific for the particular integration site and examined the Nf-1 integration retrospectively, using the stored blood samples. This allowed us to determine the behavior of the clones carrying the Nf-1 integration during the latent period. In an example of Nf-1 integration in a Runx1+/+ background (Fig. 1A, lower panel), a strong band representing the Nf-1 integration was detected at 45 weeks, corresponding to the onset of full-blown leukemia. Unexpectedly, this mouse also showed a smaller band at 31 weeks. Sequencing of this PCR product revealed that there was another Nf-1 integration event approximately 330 base pairs upstream of the 45-week integration site. The band at 31 weeks was not detected in the later blood samples or at the time of leukemia onset. Corresponding to this additional band, the peripheral WBC was transiently elevated at 31 weeks (Fig. 1A, upper panel).
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Runx1 Insufficiency Synergizes with Activated N-RAS In Vitro
To verify the cooperativity of Runx1 haploinsufficiency and Nf-1 inactivation, we carried out in vitro experiments using cell culture system. Since Nf-1 inactivation is difficult to recapitulate experimentally, human N-RAS mutations were introduced instead. Using the MIG (MSCV-IRES-enhanced green fluorescence protein [EGFP]) retrovirus vector (Fig. 2
A), wild-type (WT) or oncogenic human N-RAS (G12V and G12S) was introduced into mouse bone marrow cells from Runx1+/+, +/–, or –/– mice (Fig. 2B). N-RAS G12V has the strongest activity among reported mutants [20], and N-RAS G12S was identified in a patient's leukemia cells with inv [16], which generates the PEBP2β/CBFβ-SMMHC chimeric protein. When cells were cotransfected with ERK1, the protein was more strongly phosphorylated in cells with N-RAS G12V than with N-RAS G12S (Fig. 2C).
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B) and subjected to the experiments. In liquid culture, N-RAS G12V and G12S transfectants showed higher proliferative capacity than mock transfectants in the Runx1+/+ background in 7-day cultures (Fig. 2D). This capacity was further enhanced in the Runx1+/– background (Fig. 2D). The same trends were observed in colony assays using semisolid medium (Fig. 2E). The number of colonies per number of plated cells did not show substantial differences, but the cell number per colony in N-RAS G12V or G12S transfectants in the Runx1+/– background was clearly higher than in the Runx1+/+ background. Furthermore, the number of colonies demonstrated clear difference when Runx1–/– cells were used instead of Runx1+/– cells (Fig. 2F). These data indicate that the capacity of oncogenic N-RAS to stimulate proliferation of bone marrow cells was profoundly elevated in the Runx1insufficient background, which includes both Runx1+/– and Runx1–/– status.
Runx1 Insufficiency and Oncogenic N-RAS Show Synergism In Vivo
To further experimentally confirm the cooperativity between Runx1 insufficiency and oncogenic N-RAS, we carried out bone marrow transplantation assay using the above oncogenic N-RAS transfectants. In this experiment, we compared Runx1+/+ and Runx1–/– cohorts. Runx1–/– bone marrow cells are generated by Cre-recombinase-mediated knockout of Runx1 (we refer to this conditional knockout as Runx1–/– here). None of the 20 mice transplanted with mock or wild-type N-RAS transfectants showed any hematological abnormalities for 12 months observation period regardless of the Runx1 status. In clear contrast, the recipient mice transplanted with oncogenic N-RAS transfectants, including both N-RAS G12V and N-RAS G12S, developed AML-like disease as early as 25 days after transplantation (Fig. 2G), as previously reported [21]. Of these, the recipient mice transplanted with Runx1–/– cells carrying oncogenic N-RAS showed significantly shorter latency for the disease onset compared with those with Runx1+/+ cells (p = .0082; Fig. 2G). Spleen weight of the disease mice transplanted with oncogenic N-RAS Runx1–/– transfectants was much higher than those with oncogenic N-RAS Runx1+/+ transfectants (p = .0001; Fig. 2H), suggesting that the proliferation capacity of Runx1–/– abnormal cells are higher than that of Runx1+/+ abnormal cells. Immunophenotypic analysis of GFP+ leukemia-like cells also showed a clear difference between Runx1–/– and Runx1+/+ cohorts (supplemental online Table 1). Although the leukemia-like cells were primarily myeloid lineage cells, Runx1–/– abnormal cells were also positive for lymphoid marker CD8 antigen, whereas those of Runx1+/+ abnormal cells were not (Fig. 2I). This suggests that lineage restriction of the cells is abrogated in Runx1–/– background. Collectively, it is clear that this mouse experiment recapitulated the synergism between the genetic changes frequently observed in human AML, namely activated RAS pathway and RUNX1 insufficiency.
Inactivation of Runx1 Increases, and Oncogenic N-RAS Decreases, the c-Kit+ Stem/Progenitor Compartment
In the midst of the above experiment to study the synergism between Runx1 insufficiency and oncogenic N-RAS, we noticed drastic changes in the percentage of c-Kit+ GFP+ cell fraction among the transfectants. As shown in the FACS profiles (Fig. 3A), Runx1+/– bone marrow cells have a larger c-Kit+ stem/progenitor compartment than the Runx1+/+ bone marrow cells (mock: 53% vs. 75.9%). In contrast, the introduction of N-RAS G12V and G12S into Runx1+/+ bone marrow cells dramatically reduced the c-Kit+ fraction, and N-RAS G12V had a stronger effect than G12S (Fig. 3A).
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Stem/Progenitor Cells Are Increased in Runx1-Deficient Mice
To confirm the increase of stem/progenitor cells in Runx1 insufficiency, we investigated the frequency of HSC in Runx1 conditional knockout mice. The HSC fraction is usually defined immunophenotypically as c-Kit+ Sca-1+ lineage marker-negative (Lin–) cells (KSL cells) [22]. Consistent with previous reports using Runx1 conditional knockout mice [23, 24], the frequency of KSL cells in Runx1–/– bone marrow was twofold higher than that in Runx1+/+ cells (Fig. 4A). The progenitor fraction, defined as c-Kit+ Lin–, a slightly differentiated cell compared with genuine stem cells, also increased in the Runx1–/– background (61.8% vs. 53.6%, data not shown).
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The increase in stem cells was also confirmed by an LTC-IC assay [26]. The number of colony-forming cells scored after more than 30 days culture on OP9 stromal cells, which support the maintenance of HSC in vitro, is considered to reflect the HSC frequency. The plating efficiency of Runx1–/– cells was approximately 2.5 times higher than that of Runx1+/+ cells, suggesting that Runx1–/– cells have a higher number of HSC than Runx1+/+ cells (Fig. 4C). In addition, colonies from Runx1–/– bone marrow cells contained approximately twice as many cells as were formed by Runx1+/+ cells (Fig. 4D).
In summary, three independent HSC assays indicated that the loss of Runx1 increases the numbers of HSC in vivo, implicating Runx1 as a negative regulator of stem cell numbers. It is also of note that not only HSC but also the progenitor fraction increased in Runx1–/– bone marrow, as suggested by the higher percentage of c-Kit+ Lin– cells and the larger colony size in the LTC-IC assay (Fig. 4D).
Runx1 Deficiency Enhances the Survival of Bone Marrow Cells
To elucidate the mechanism of the increase of the stem/progenitor fraction in Runx1-deficient cells, we examined whether the increase was related to a reduction in cell death. Total bone marrow cell populations were separated into Annexin V+ 7-AAD– and Annexin V+ 7-AAD+ fractions, representing apoptotic and dead cells, respectively (Fig. 5A). In mock transfectants, the Runx1+/+ cell population contained 16.7% apoptotic and 11.4% dead cells, a total of 28.1% nonviable cells. In contrast, the Runx1–/– cell population contained 12.5% apoptotic and 3.5% dead cells, a total of 16% nonviable cells (Fig. 5A), little more than half the total nonviable cells in the Runx1+/+ population. Similarly, dead cells detected by propidium iodide (PI) instead of 7-AAD in the c-Kit+ stem/progenitor fraction were considerably reduced in Runx1–/– cells compared with Runx1+/+ cells (Fig. 5B).
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Oncogenic N-RAS Induces Senescence and Apoptosis That Are Suppressed by Runx1 Deficiency
Oncogenic activation of N-RAS in the cell primarily results in hyperproliferation (Figs. 2, 6D). However, uncontrolled cell proliferation appears to be sensed by the cell and triggers mechanisms to counteract proliferation. Cellular senescence and apoptosis are part of such cellular fail-safe mechanisms [32]. Oncogenic Ras induces senescence through activation of the p19Arf pathway [33]. Indeed, N-RAS G12V and G12S, but not N-RAS WT, induced p19Arf expression in the c-Kit+ fraction of Runx1+/+ mice very strongly compared with mock transfectants (Fig. 6A). To examine the induction of senescence by oncogenic N-RAS, we measured several recently reported senescence-related biomarkers, p16Ink4a, p15Ink4b, Dec1, and DcR2 [34]. Of these genes, Dec1 and p16Ink4a were induced by N-RAS G12V and G12S (Fig. 6A). Therefore, one of the consequences of activated N-RAS expression in c-Kit+ stem/progenitor cells appears to be the induction of senescence.
Oncogenic Ras also induces apoptosis mediated by p19Arf. Therefore, apoptosis should be stimulated in oncogenic N-RAS transfectants. Indeed, the nonfractionated Runx1+/+ bone marrow cells harboring oncogenic N-RAS showed a subtle increase in apoptotic and dead cells (Fig. 5A). Moreover, the PI+ dead cell fraction in the c-Kit+ population showed a prominent increase by expression of oncogenic N-RAS (Fig. 5B), suggesting that oncogene-induced apoptosis was triggered preferentially in stem/progenitor cells.
In Runx1–/– cells, oncogene-induced senescence and apoptosis appeared to be suppressed. Oncogenic N-RAS-induced elevation of p19Arf, p16Ink4a, and Dec1 expression in the Runx1+/+ background was mildly suppressed in Runx1–/– c-Kit+ cells (Fig. 6). This suppression is probably due to the upregulation of Bmi-1 in Runx1–/– cells since Bmi-1 suppresses the expression of p16Ink4a and p19Arf. In addition, a potent antiapoptotic gene, Bcl-2, was elevated in Runx1–/– cells. Therefore, senescence and apoptosis are expected to be significantly suppressed in Runx1-deficient cells. In fact, in addition to the changes in marker gene expression, we observed a clear reduction of oncogenic N-RAS-induced cell death represented by PI+ cells in Runx1–/– c-Kit+ stem/progenitor cells (Fig. 5B).
Interestingly, activated N-RAS alone increased the expression of Bmi-1 in both Runx1+/+ and Runx1–/– cells (Fig. 6A). This elevation may be a driving force of proliferation by oncogenic N-RAS and may be at least partially responsible for the synergism between Runx1 insufficiency and activated N-RAS. Similarly, upregulation of Bcl-2 by oncogenic N-RAS was further enhanced in the Runx1–/– background and could contribute to the synergism between Runx1 insufficiency and oncogenic N-RAS (Fig. 6A).
Oncogenic N-RAS-Induced Differentiation Is Blocked by Runx1 Insufficiency
In addition to apoptosis and senescence, oncogenic Ras also induces differentiation, in particular, toward the myeloid lineage in hematopoietic cells [35, 36]. Oncogenic N-RAS drove Runx1+/+ bone marrow cells to differentiate into Gr-1high Mac-1high hyperlobulated myeloid cells compared with mock transfectants, which showed modest differentiation toward both granulocytes and macrophages (Fig. 7). In contrast, this differentiation was blocked in the Runx1–/– background, as was most clearly indicated by the lack of Gr-1+ cells in FACS profiles. It has been reported that this oncogenic Ras-induced differentiation is at least in part due to the suppression of proliferation by upregulation of p21Waf-1 [36, 37]. Indeed, we observed the elevation of p21Waf-1 expression in oncogenic N-RAS transfectants, and, consistent with the block of myeloid cell differentiation in morphology and FACS profiles, the elevated p21Waf-1 expression was suppressed in the Runx1–/– background (Fig. 6A). These data suggest that Runx1 insufficiency suppresses oncogenic N-RAS-induced myeloid cell development.
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Runx1 Is Induced by Oncogenic N-RAS
Runx1 appears to be required for inducing senescence, apoptosis, and cell differentiation in response to oncogenic N-Ras. We therefore investigated the behavior of Runx1 itself when an oncogenic event arises in cells. Upon expression of oncogenic N-RAS, Runx1 was transcriptionally activated (Fig. 6C), suggesting that Runx1 plays a central role in a variety of cellular responses to oncogenic stimuli (Fig. 6D).
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DISCUSSION |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionDisclosure of Potential...AcknowledgmentsReferences |
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The changes in stem/progenitor cells due to Runx1 impairment appear to contribute to leukemogenesis by preventing the leukemia-initiating clone from being eliminated by the fail-safe mechanisms normally triggered by oncogenic stimuli. This mechanism of Runx1 insufficiency in supporting oncogenic events appears to be consistent with the fact that RAS pathway-activating mutations are the most common cooperating genetic alterations in human RUNX1-related leukemias. The early disappearance of an Nf-1 integrated clone in a BXH2-Runx1+/+ mouse (Fig. 1A) may reflect activation of the oncogene-induced fail-safe machinery. Surprisingly, in our BXH2 mouse leukemia model, such transient peaks in WBC, probably due to oncogene-induced initial proliferation followed by disappearance due to detrimental effects, were frequently observed (Fig. 1B). More surprisingly, a similar spontaneous improvement even without any treatment is also observed in human subjects of juvenile myelomonocytic leukemia (JMML) with RAS mutations [43]. As majority of JMMLs are caused by either NF-1 or RAS mutations, our observation in the mouse model appears to recapitulate a natural course of human leukemia development. Furthermore, a mutation in RUNX1 has been found in a case of JMML patient who did not undergo spontaneous remission [44]. Therefore, oncogene-induced apoptosis, senescence, and differentiation seem to be taking place frequently in abortive leukemogenic events, and the impairment of these protective mechanisms at the initial stage due to, for example, loss-of-function mutation of RUNX1 may be a relatively common phenomenon in the development of leukemia.
The features observed in Runx1–/– stem/progenitor cells are shared by cells carrying another, more pervasive RUNX1-related genetic alteration, the chimeric gene RUNX1-ETO. p14ARF/p19Arf is suppressed by RUNX1-ETO [45], and BCL-2 is upregulated in cells carrying RUNX1-ETO [27]. The suppression of p14ARF and upregulation of BCL-2 have been confirmed in human t(8;21) leukemia samples carrying RUNX1-ETO [45, 46]. Therefore, the attenuation of oncogene-induced adverse effects or disruption of the fail-safe program in the stem/progenitor cells due to an altered RUNX1 gene seems to be a more general mechanism of leukemogenesis in RUNX1-related leukemias.
In this study, we observed that Runx1 insufficiency suppresses oncogenic N-RAS-induced adverse effects (i.e., apoptosis, senescence, and differentiation). RUNX1 insufficiency appears to disrupt the fail-safe mechanism normally active in the stem/progenitor cells, thereby contributing to the development of leukemia. In other words, our observations shed light on the hitherto unrecognized physiological role of RUNX1: maintenance of the fail-safe mechanism to protect hematopoietic stem/progenitor cell pool from oncogenic insult. Investigation of the detailed mechanism of this newly identified role of RUNX1 will be an important subject for further studies. This fail-safe mechanism and its disruption in oncogenesis seems fundamental but has not been fully recognized to date. Loss-of-function of RUNX1, one of the most common causes of leukemia, may serve as a model to elucidate the details of this newly emerging oncogenic mechanism, which may be potentially widespread in other types of cancers. Gaining a better understanding of this mechanism might lead to new strategies to treat cancer stem cell-associated resistance to chemotherapy, which has been the subject of intense discussion in recent years.
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DISCLOSURE OF POTENTIAL CONFLICTS OF INTEREST |
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ACKNOWLEDGMENTS |
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REFERENCES |
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, p < .05, compared with Runx1–/– mock transfectant. (G): Survival curve of the mice transplanted with oncogenic N-RAS transfectants. Significant difference between Runx1+/+ and Runx1–/– cohorts is shown as P value. *, This cohort consists of the recipients of mock Runx1+/+ transfectants (n = 6), those of mock Runx1–/– transfectants (n = 6), those of wild-type N-RAS Runx1+/+ transfectants (n = 4), and those of wild-type N-RAS Runx1–/– transfectants (n = 4). (H): Spleen weight of the leukemia-like mice. Unpaired Student's t test shows significant difference between Runx1+/+ and Runx1–/– cohorts. (I): Representative immunophenotypic profiles for abnormal cells from the leukemia-like mice transplanted with Runx1+/+ G12V or Runx1–/– G12V transfectants. Analyzed tissues are spleen and liver, respectively. The expression of GFP is shown along the x-axis, and the y-axis shows the expression of cell-surface markers specified over the each profile. Abbreviations: CFU-C, colony-forming unit-culture; EGFP, enhanced green fluorescent protein; GFP, green fluorescent protein; hrs, hours; IRES, internal ribosome entry site; LTR, long terminal repeat; MSCV, murine stem cell virus; PI, propidium iodide; WT, wild-type.
