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Cancer Research 66, 9967, October 15, 2006. doi: 10.1158/0008-5472.CAN-06-1346
© 2006 American Association for Cancer Research
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Cell, Tumor, and Stem Cell Biology

Role of SPA-1 in Phenotypes of Chronic Myelogenous Leukemia Induced by BCR-ABL–Expressing Hematopoietic Progenitors in a Mouse Model

Kohei Kometani1, Misayo Aoki1, Shin Kawamata3, Yoriko Shinozuka1, Takumi Era3, Masafumi Taniwaki2, Masakazu Hattori1 and Nagahiro Minato1

1 Department of Immunology and Cell Biology, Graduate School of Biostudies, Kyoto University; 2 Department of Hematology and Oncology, Kyoto Prefectural University of Medicine, Kyoto, Japan; and 3 Laboratory for Stem Cell Biology, RIKEN Center for Developmental Biology, Kobe, Japan

Requests for reprints: Nagahiro Minato, Department of Immunology and Cell Biology, Graduate School of Biostudies, Kyoto University, Sakyo-ku, Kyoto 606-8501, Japan. Phone: 81-75-753-4659; Fax: 81-75-753-4403; E-mail: minato{at}imm.med.kyoto-u.ac.jp.


   Abstract
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 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
SPA-1 is a negative regulator of Rap1 signal in hematopoietic cells, and SPA-1-deficient mice develop myeloproliferative disorders (MPD) of long latency. In the present study, we showed that the MPDs in SPA-1–/– mice were associated with the increased hematopoietic stem cells expressing LFA-1 in bone marrow and their premature mobilization to spleen with extensive extramedullary hematopoiesis, resembling human chronic myelogenous leukemia (CML). We further showed that human BCR-ABL oncogene caused a partial down-regulation of endogenous SPA-1 gene expression in mouse hematopoietic progenitor cells (HPC) and immature hematopoietic cell lines. Although both BCR-ABL-transduced wild-type (wt) and SPA-1–/– HPC rapidly developed CML-like MPD when transferred to severe combined immunodeficient mice, the latter recipients showed significantly increased proportions of BCR-ABL+ Lin– c-Kit+ cells compared with the former ones. Serial transfer experiments revealed that spleen cells of secondary recipients of BCR-ABL+ wt HPC failed to transfer MPD to tertiary recipients due to a progressive reduction of BCR-ABL+ Lin– c-Kit+ cells. In contrast, SPA-1–/– BCR-ABL+ Lin– c-Kit+ cells were sustained at high level in secondary recipients, and their spleen cells could transfer MPD to tertiary recipients, a part of which rapidly developed blast crisis. Present results suggest that endogenous SPA-1 plays a significant role in regulating expansion and/or survival of BCR-ABL+ leukemic progenitors albeit partial repression by BCR-ABL and that Rap1 signal may represent a new molecular target for controlling leukemic progenitors in CML. (Cancer Res 2006; 66(20): 9967-76)


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Rap1 is a member of Ras family of small GTPases serving as a molecular switch integrating a wide range of stimuli. Activation of Rap1 is induced by specific guanine nucleotide exchange factors coupled with various receptors, although it is negatively regulated by GTPase-activating proteins (1). Rap1 signal plays an important role in controlling cell adhesion via integrins, cadherins, and possibly other adhesion molecules and thereby regulates diverse function of many cellular systems (2). Rap1 signal also regulates activation of mitogen-activated protein kinases (MAPK), including extracellular signal-regulated kinase (ERK) and p38MAPK, depending on cell contexts (3–5). Accumulating evidence indicates that Rap1 signal is involved in various aspects of malignancy. For instance, loss-of-function mutations of DOCK4 gene encoding a Rap1 activator were found in certain human tumor cells, and it was revealed that the resulting defect in Rap1 activation was responsible for impaired intercellular adherence junctions and aggressive invasion of the tumor cells in vivo (6). It was also reported that genetic polymorphism of SPA-1 (also called SIPA-1) gene in hosts was a major factor determining the efficiency of lung metastasis of mammary tumor cells in a mouse model (7). Consistently, prostate cancers with metastasis in humans showed a significantly higher SPA-1 gene expression than those without metastasis (7).

Chronic myelogenous leukemia (CML) represents leukemia of hematopoietic stem cells (HSC). The balance between self-renewal and differentiation of normal HSC is tightly controlled through interaction with specific hematopoietic microenvironments called niche (8). However, leukemic HSCs produce massive numbers of leukemic committed progenitors and terminally differentiated leukocytes in a dysregulated manner, and this chronic phase of CML is eventually followed by blast crisis resembling acute leukemia (9). The vast majority of human CML is caused by BCR-ABL fusion gene resulting from a chromosomal translocation, t(9;22)(q34;q11). BCR-ABL oncoprotein constitutively activates diverse signaling pathways for cell proliferation, survival, and gene expression mostly through its tyrosine kinase activity (9). Thus, a specific BCR-ABL kinase inhibitor, imatinib mesylate, may rapidly reduce the massive peripheral leukemia cell burden (10, 11). However, recent studies indicate that BCR-ABL+ leukemic HSCs resist imatinib mesylate, being called residual disease (11–13). It has been also shown that BCR-ABL fails to confer self-renewing leukemic potential to committed and differentiated hematopoietic cells unlike certain oncogenes for acute leukemia (14), although blast crisis may arise in these cells (15). These results strongly suggest that CML genesis in humans may involve complex interactions of BCR-ABL with intrinsic host factors in normal HSC (9, 14).

We previously reported that SPA-1-deficient mice developed a spectrum of myeloproliferative disorders (MPD) of long latency and suggested that SPA-1 might contribute to hematopoietic homeostasis by negatively regulating Rap1 activation (16, 17). Recent reports also indicated that BCR-ABL induced constitutive Rap1 activation in hematopoietic cells (18, 19) and that this in part mediated enhanced proliferation, survival, and cell adhesion (20). These results prompted us to investigate possible role of endogenous SPA-1 in phenotypes of CML caused by BCR-ABL+ hematopoietic progenitor cells (HPC) in a mouse model. We provide evidence that endogenous SPA-1 plays a significant role in controlling expansion and/or survival of BCR-ABL+ leukemic progenitors as well as blast crisis in vivo.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Mice. SPA-1-deficient (–/–) mice of 129 background were reported previously (16). They were backcrossed to C57BL/6 and BALB/c mice at least 10 generations. A cohort of 20 C57BL/6 SPA-1–/– mice and their control littermates was routinely monitored by hemogram analysis for 20 months. When they became moribund with signs of MPD, they were sacrificed for detailed hematologic analysis. For BCR-ABL gene transduction experiments, 4- to 8-week-old BALB/c SPA-1–/– mice with no hematologic abnormalities were used. C.B-17/Scid [severe combined immunodeficient (SCID)] mice were purchased from CLEA Japan, Inc. (Tokyo, Japan). All the mice were maintained in a specific pathogen-free condition in the Institute of Laboratory Animals, Graduate School of Medicine, Kyoto University (Kyoto, Japan).

Cells and culture. Ba/F3 (pro-B) and FDC-P2 (immature myeloid) cell lines were maintained in RPMI 1640 supplemented with 10% FCS, 50 µmol/L 2-mercaptoethanol, antibiotics, and 100 units/mL interleukin (IL)-3. Plat-E cells were kindly provided by Dr. Kitamura (Tokyo University, Tokyo, Japan) and maintained in DMEM supplemented with 10% FCS, antibiotics, 1 µg/mL puromycin (Sigma, St. Louis, MO), and 10 µg/mL blastocidin (Funakoshi, Tokyo, Japan).

Immunoblotting, immunoprecipitation, and pull-down assay. Cells were lysed in lysis buffer [150 mmol/L NaCl, 50 mmol/L Tris-HCl (pH 7.6), 0.5% Triton X-100, 1 mmol/L phenylmethylsulfonyl fluoride, 1 mmol/L Na3VO4, 10 mmol/L NaF, 2 µg/mL leupeptin, 2 µg/mL aprotinin] and subjected to immunoblotting and immunoprecipitation as described previously (16). Antibodies included anti-Abl (8E9; BD Biosciences, San Jose, CA), anti-Rap1 (121; Santa Cruz Biotechnology, Santa Cruz, CA), anti-Ras (Upstate Biotechnology, Lake Placid, NY), anti-SPA-1 (16), and anti–glyceraldehyde-3-phosphate dehydrogenase (GAPDH; 6C5; Chemicon, Temecula, CA). Rap1GTP was determined by pull-down assay as before (16), and RasGTP was detected using Ras Activation Assay kit (Upstate Biotechnology).

Cell adhesion and migration assay. Cells labeled with 2',7'-bis-(2-carboxyethyl)-5(and-6)carboxy-fluorescein (Molecular Probes, Eugene, OR) at 37°C for 20 minutes were plated onto microtiter plates that had been coated with 7.5 µg/mL fibronectin (Sigma) and blocked with 3% bovine serum albumin (BSA). After incubation at 37°C for 30 minutes, the wells were washed and the remaining fluorescence was measured using a fluorescence reader (ARVO, Perkin-Elmer, Wellesley, MA). Migration assays were done using Transwell chambers, Chemotaxicell, of 5-µm pore size (Kurabo, Osaka, Japan). Transwell filters were coated with 1 µg/mL fibronectin, and the cells (1 x 105 in 300 µL) in migration medium (0.5% BSA/Iscove's medium) were added into the upper chamber. Four hours later, the cells migrated in lower chamber were counted.

Retroviral infection and transplantation of bone marrow cells. Retroviral plasmids, pMSCV IRES GFP (MIG), MIG containing p210 BCR-ABL (MIG210), and MIG containing p210 BCR-ABL kinase-dead (KD) mutant (MIG210KD), were reported previously (21). Recombinant retrovirus was produced by transfecting the retroviral plasmids into Plat-E packaging cells using LipofectAMINE 2000 (Invitrogen, Carlsbad, CA). Ba/F3 and FDC-P2 cells were infected with the supernatants containing MIG210 or MIG210KD retrovirus, and GFP+ cells were sorted. The sorted cells were further transfected with pcDL-SR{alpha}-myc-SPA-1 (20 µg) and pSV2-neo (2 µg) using electroporation followed by selection with G418 (Nacalai Tesque, Kyoto, Japan). Bone marrow cells were harvested from 4- to 12-week-old normal and SPA-1–/– BALB/c mice that had been primed with 150 mg/kg 5-fluorouracil (5-FU; Kyowa Hakko Kogyo, Tokyo, Japan) and further enriched for HPC by depleting lineage marker-positive cells using a cocktail of antibodies (anti-Thy1, anti-B220, anti-Gr-1, anti-Mac-1, and anti-Ter119 antibodies) and anti-rat IgG-coated magnetic beads (Dynabeads M-450, Dynal, Oslo, Norway) or by cell sorting using FACSVantage (BD Biosciences). The Lin– HPCs were cultured in complete RPMI 1640 containing 10 ng/mL IL-6, 10 ng/mL IL-11, 10 ng/mL Flt-3 ligand, and 50 ng/mL stem cell factor (SCF; Genzyme, Minneapolis, MN) for overnight. The cells were then infected with retroviral supernatants by spinoculation (1,800 x g, 60 minutes, 32°C) followed by culture in the same medium. The infection procedure was repeated twice, and the infected HPCs were injected i.v. into 2.75 Gy {gamma}-ray-irradiated SCID mice (3 x 104 cells per head). For serial transplantation, splenocytes from recipient mice were injected i.v. to 2.75 Gy {gamma}-ray-irradiated SCID mice (5 x 106 cells per head).

Flow cytometric analysis. Multicolor flow cytometric analysis was done using FACSCalibur (BD Biosciences) as before (16). Antibodies included phycoerythrin (PE)-conjugated or allophycocyanin-conjugated anti-CD3 (145-2C11), anti-B220 (RA3-6B2), anti-Thy1.2 (55-2.1), anti-Gr-1 (RB6-8C5), anti-Mac-1 (M1/70), anti-Ter119, anti-c-Kit (2B8; BD Biosciences), and PE-conjugated anti-rat IgG (Southern Biotechnology Associates, Birmingham, AL).

Hematologic analysis. Peripheral blood leukocyte counts were monitored by using an automated cell counter (Nihon Kohden, Tokyo, Japan). Blood and spleen cells were lysed for RBC, cytospun, and stained with May-Giemsa solution (Muto Pure Chemicals, Tokyo, Japan). Splenocytes treated with corcemid (Karyo Max, Life Technologies, Grand Island, NY) were subjected to spectral karyotyping (SKY) analysis using standard procedures.

Reverse transcription-PCR and Southern blotting. Total RNA was extracted using ISOGEN (Nippon Gene, Tokyo, Japan) and treated with DNase I (Invitrogen) followed by cDNA synthesis using SuperScript III (Invitrogen). PCR primers were as follows: BCR-ABL, 5'-cacagcattccgctgaccatca-3' (forward) and 5'-gcttcacaccattccccattgt-3' (reverse); SPA-1, 5'-tgaaagacagcagcagtcctc-3' (forward) and 5'-ctgccagcttccgacataatc-3' (reverse); Rap1, 5'-cgtgagtacaagctagtggtcctt-3' (forward) and 5'-atttatctgtctgaccaggtcat-3' (reverse); and GAPDH, 5'-gaacggatttggccgtattg-3' (forward) and 5'-gatgatgacccttttggctc-3' (reverse). Southern and Northern blotting analyses were done as described before (16) using full-length SPA-1, Rap1, Ig heavy chain JH, and EGFP cDNA probes.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
MPDs of SPA-1–/– mice are associated with increased LFA-1+ HSC in bone marrow and their premature mobilization to spleen. The vast majority of SPA-1–/– mice of C57BL/6 background developed MPD, and all died during the 2nd year of age, whereas none of control littermates showed signs of MPD. As summarized in Table 1 , 9 of 20 C57BL/6 SPA-1–/– mice developed granulocyte-dominant CML-like MPD with or without blastic cells, 6 mice developed B-cell leukemia mostly of B1 cell phenotype, a mouse T-cell leukemia (T-ALL), and 2 mice developed undefined MPD. Two mice developed severe anemia with no apparent evidence of overt MPD. They showed marked bilateral hydronephrosis most likely due to diabetes insipidus (22) and were diagnosed as renal anemia. Irrespective of leukemia phenotypes, most of the diseased mice revealed increased Lin– c-Kit+ Sca-1+ (LKS) cells enriched for HSC in bone marrow compared with age-matched control littermates (Fig. 1A ). Furthermore, the SPA-1–/– mice also accumulated significant numbers of LKS cells in spleen, whereas they were rarely detected in spleen of controls (Fig. 1A). The younger (5-month old) C57BL/6 SPA-1–/– mice with no signs of MPD showed only slight increase in LKS cells in bone marrow (Fig. 1A) and undetectable LKS cells in spleen (data not shown). The numbers of Lin– c-Kit+ Sca-1– (LKS–) cells enriched for the committed progenitors were also increased markedly in spleen of diseased SPA-1–/– mice, whereas their average number in bone marrow was comparable with that of control mice albeit increased LKS cells (Fig. 1B). These results together with marked splenomegaly (Table 1) suggested extensive extramedullary hematopoiesis in SPA-1–/– mice.


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  		Table 1. Pleiotropic MPD in C57BL/6 SPA-1–/– mice

 

Figure 1
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  		Figure 1. Increased LFA-1+ LKS cells and their premature mobilization to spleen in C57BL/6 SPA-1–/– mice. A and B, 5-month-old C57BL/6 SPA-1–/– mice with no hematologic abnormalities (gray circles) and ≥12-month old C57BL/6 SPA-1–/– mice with overt MPD (closed circles; also see Table 1) were sacrificed along with age-matched control littermates (open circles). The proportions of LKS (A) and LKS– (B) cells were analyzed by using FACSCalibur, and the total LKS and LKS– cell numbers in each tissue were calculated. Bars, mean values. C, bone marrow (BM) and spleen cells from leukemic SPA-1–/– and control mice were four-color analyzed with antibodies for Lin– marker cocktail, c-Kit, Sca-1, and {alpha}L or ß1-integrin subunits. Left, representative profiles for expression of {alpha}L and ß1 subunits of integrins in bone marrow LKS fraction. Solid and fine lines, profiles of SPA-1–/– and control mice, respectively, and the proportions of positive fractions are indicated. Solid areas, control IgG staining. Right, numbers of {alpha}L– (open circles) and {alpha}L+ (closed circles) LKS cells in bone marrow and spleen. LKS cells were practically undetectable in control spleen. N.D., not detected; NS, statistically not significant.

 
The abnormal appearance of LKS cells in spleen of SPA-1–/– mice prompted us to investigate the expression of integrins on LKS cells in bone marrow. Although only a minor proportion of LKS cells expressed LFA-1 ({alpha}L) in bone marrow of control mice, the proportions of LFA-1+ LKS cells in bone marrow of SPA-1–/– mice with overt MPD were increased (Fig. 1C, left). Consistently, the numbers of LFA-1+ LKS cells in bone marrow were significantly increased in diseased SPA-1–/– mice, whereas those of LFA-1– LKS cells remained comparable with control mice (Fig. 1C, right). The vast majority of LKS cells in the spleen of SPA-1–/– mice also showed LFA-1 expression (Fig. 1C, right). On the other hand, essentially all the LKS cells exhibited ß1-integrin expression in both SPA-1–/– and control mice (Fig. 1C, left). The results collectively suggested that preferential expansion of LFA-1+ HSC and their premature mobilization out of bone marrow might underlie the MPD of late onset in SPA-1–/– mice irrespective of leukemia phenotypes.

BCR-ABL down-regulates endogenous SPA-1 gene expression and constitutively activates Rap1. The clinical features of MPD in the majority of SPA-1–/– mice, such as granulocyte-dominant leukocytosis and marked splenomegaly, were reminiscent of human CML, and we first investigated possible effects of BCR-ABL, a major cause of human CML, on endogenous SPA-1 expression in HPCs. HPCs (Lin– population) were sorted from bone marrow of normal BALB/c mice primed with 5-FU and infected with MIG retrovirus containing p210 BCR-ABL (MIG210) or empty retrovirus followed by culture in the presence of bone marrow stroma cells for a week. The GFP+ Lin– cells were then resorted, and SPA-1 gene transcripts were examined by semiquantitative reverse transcription-PCR (RT-PCR). As shown in Fig. 2A , GFP+ (BCR-ABL+) HPC showed significantly reduced SPA-1 transcripts compared with control HPC, whereas Rap1 transcripts remained unchanged. We also transplanted MIG210-infected HPC into irradiated SCID mice, and GFP+ Lin– c-Kit+ cells were sorted from the spleen cells of recipients that developed CML-like MPD. Again, the sorted GFP+ HPC exhibited reduced SPA-1 transcripts compared with the corresponding Lin– c-Kit+ population in bone marrow of control SCID mice (Fig. 2B). Because it was technically unfeasible to detect SPA-1 protein in primary BCR-ABL+ Lin– HPC due to limited cell numbers, we extended analysis to immature hematopoietic cell lines. Expression of BCR-ABL in both FDC-P2 and Ba/F3 cells caused a significant decrease in endogenous SPA-1 protein, whereas a KD mutant of BCR-ABL hardly affected it (Fig. 2C). SPA-1 transcripts were also reduced by BCR-ABL, whereas the half-life of SPA-1 protein tended to be rather increased (Supplementary Fig. S1), indicating that reduced SPA-1 transcripts were directly related to reduced expression of SPA-1 protein. Consistently, BCR-ABL expression resulted in marked activation of Rap1 along with Ras and ERK (Fig. 2C). Although total Rap1 protein expression was enhanced in these cell lines for unknown reasons, the strong Rap1 activation could not be ascribed to the effect as shown by activation indices (Fig. 2C). To support this, BCR-ABL-induced Rap1 activation was abrogated completely by SPA-1 overexpression with increased Rap1 level being unaffected at all (Fig. 2D). Although expression of SHIP and p27Kip1 was also repressed by BCR-ABL as reported, it was unaffected by SPA-1 overexpression either (Fig. 2D). Functionally, BCR-ABL expression enhanced integrin-dependent adhesion to fibronectin and transmigration of Ba/F3 in the absence of IL-3, and the effect was again inhibited strongly by SPA-1 overexpression, confirming the role of Rap1 signaling (Fig. 2E). Because direct estimation of Rap1 activation in primary HPC was unfeasible, we did adhesion assay using GFP+ Lin– c-Kit+ cells sorted from spleen cells of recipients with MIG210/wild-type (wt) and SPA-1–/– HPC. BCR-ABL+ wt HPC showed significantly enhanced adhesion compared with normal Lin– c-Kit+ cells, whereas phorbol 12-myristate 13-acetate (PMA) induced equivalent adhesion in both (Fig. 2F). Furthermore, BCR-ABL+ SPA-1–/– HPC showed even more enhanced adhesion than BCR-ABL+ wt HPC (Fig. 2F). These results suggested that BCR-ABL partially, if not completely, repressed endogenous SPA-1 gene expression and contributed to constitutive Rap1 activation in primary HPC, although reduced SPA-1 protein in primary BCR-ABL+ HPC remained to be confirmed.


Figure 2
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  		Figure 2. BCR-ABL partially represses endogenous SPA-1 gene expression and induces constitutive Rap1 activation. A, sorted HPCs (Lin–) of normal BALB/c mice were infected with either MIG210 or empty MIG and cultured in the presence of PA6 stroma cells for a week. GFP+ Lin– cells were resorted and analyzed for expression of indicated transcripts by RT-PCR at varying cycles. B, MIG210/wt HPCs were transferred into irradiated SCID mice. Three weeks later, GFP+ Lin– c-Kit+ cells were sorted from spleen of recipients and analyzed for expression of indicated transcripts by RT-PCR. As a control, sorted Lin– c-Kit+ cells in bone marrow of normal SCID mice were used. Bottom, relative ratios of SPA-1 to GAPDH signals. C, FDC-P2 and Ba/F3 cells were infected with MIG210, MIG210KD, or empty MIG, and GFP+ cells were sorted. Expression of indicated proteins in randomly selected GFP+ clones was examined by immunoblotting. Rap1GTP and RasGTP were assessed by pull-down assay. Bottom, relative Rap1 activation indices (Rap1GTP to total Rap1) with reference to vector control. D, FDC-P2 cells infected with MIG210 were further transfected with Myc-tagged SPA-1 cDNA, and expression of indicated proteins was examined by immunoblotting. Bottom, relative Rap1 activation indices. E, Ba/F3 and FDC-P2 cells transduced with MIG, MIG210, MIG210KD, or MIG210 plus SPA-1 cDNA were assayed for adhesion to fibronectin (FN)-coated dishes (left) and transmigration via fibronectin-coated filters (right). Black columns, in a group, anti-VLA4/5 antibodies were included in the culture. Columns, mean of triplicate determinations; bars, SE. F, MIG210/wt and SPA-1–/– HPCs were transferred into irradiated SCID mice, and 3 weeks later, GFP+ Lin– c-Kit+ cells were sorted from spleen. The sorted cells and Lin– c-Kit+ cells from normal SCID bone marrow were assayed for adhesion to fibronectin-coated dishes in the absence (black columns) or presence (white columns) of PMA. Columns, mean of triplicate determinations; bars, SE.

 
CML induced by MIG210/SPA-1–/– HPC is associated with increased leukemic progenitors. To directly examine possible role of endogenous SPA-1 in BCR-ABL-induced CML genesis, we next compared phenotypes of CML induced by MIG210/wt and SPA-1–/– HPC in vivo. For these experiments, SPA-1–/– mice of BALB/c background were used because BALB/c mice were reported to develop CML by BCR-ABL more reproducibly than C57BL/6 mice (23). Lin– bone marrow cells were sorted from young SPA-1–/– and wt littermates primed with 5-FU, infected with MIG210 in vitro, and transplanted into 2.75 Gy {gamma}-ray-irradiated SCID mice (3 x 104 cells per head). Infection efficiencies as judged by expression of GFP were equivalent (17.4% for wt HPC versus 14.7% for SPA-1–/– HPC on average). All the eight recipients of MIG210/wt HPC rapidly developed CML with marked increase in granulocytes as well as massive splenomegaly and became moribund mostly within 3 weeks (Fig. 3A ). Eight of 10 recipients with MIG210/SPA-1–/– HPC also developed CML, whereas the rest of mice developed acute leukemia of B cell and macrophage lineages (data not shown). The mean survival time, leukocytosis, and splenomegaly were largely comparable between the two groups (Fig. 3A). It was noted, however, that spleen cells of recipients with MIG210/SPA-1–/– HPC contained significantly increased proportions of BCR-ABL+ (GFP+) Lin– c-Kit+ cells compared with those with MIG210/wt HPC (Fig. 3B). Similar results were obtained in bone marrow cells (Supplementary Fig. S2A). Total Lin– proportions were also increased in recipients with MIG210/SPA-1–/– HPC. Consistently, peripheral blood of the formers contained considerable proportions of immature erythroid and/or myeloid cells, whereas peripheral blood of the latters showed almost exclusive expansion of granulocytes (Fig. 3C). To examine whether the difference was related to the efficacies of initial lodgment of transferred MIG210/HPC in hematopoietic tissues, we assessed GFP+ CFU-C numbers in spleen and bone marrow of the recipients 24 hours after the cell transfer. No significant difference in GFP+ CFU-C numbers was observed in both tissues between the two groups (Supplementary Fig. S2B). Although not shown, none of the recipients with SPA-1–/– HPC harboring an empty MIG caused MPD during the observation period for 4 months. The results suggested that BCR-ABL+ SPA-1–/– progenitors showed significantly enhanced expansion and/or survival in vivo compared with control BCR-ABL+ progenitors.


Figure 3
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  		Figure 3. Increased proportions of BCR-ABL+ Lin– c-Kit+ leukemic progenitor cells in spleen of recipients with MIG210/SPA-1–/– HPC. A, 3 x 104 MIG210-infected wt ({circ}) and SPA-1–/– (bullet) Lin– bone marrow cells were transferred into irradiated SCID mice, and the recipients were sacrificed when they became moribund. Expected survival days, total blood leukocyte numbers (WBC), and spleen weights are plotted. Bars, mean values. Lines, mean values of control SCID mice. B, spleen cells from recipients were multicolor analyzed with GFP versus anti-Mac-1, anti-Gr-1, anti-Ter119, and anti-c-Kit antibodies as well as the mixtures of lineage markers (anti-Thy1, B220, Gr-1, Mac-1, and Ter119) using FACSCalibur, and proportions of various types of hematopoietic cells in GFP+ population were determined. Bars, mean values. C, peripheral blood was lysed for RBC and cytospun followed by Giemsa staining. Arrowheads, immature or blastic cells. Aliquots of the peripheral blood were three-color analyzed for GFP, anti-Mac-1, and anti-Gr-1 antibodies, and the profiles in GFP+ fractions are indicated. Note the significant Mac-1– Gr-1– population in recipients with MIG210/SPA-1–/– HPC.

 
Prolonged serial transfer of CML followed by blast crisis in recipients with MIG210/SPA-1–/– HPC. The comparison of long-term courses of MPD, however, was hampered in this model because of "acute" lethality of recipients in part due to pulmonary distresses, including hemorrhage. We therefore extended analysis to serial transfer experiments. Spleen cells from primary recipients of both groups were capable of transferring characteristic CML to secondary recipients within 3 weeks at 5 x 106 cells per head. Those from secondary recipients with MIG210/wt HPC, however, failed to transfer MPD to tertiary recipients, except for a recipient that developed thymic lymphoma after quite a long latency (16 weeks; Fig. 4 ). As shown in Fig. 5A , the spleen cells of secondary recipients (W3.1 and W3.2) showed reduced proportions of leukemic progenitors (GFP+ Lin– c-Kit+) compared with the primary recipient (W3), suggesting their progressive exhaustion in vivo. Consistently, peripheral blood of secondary recipients almost exclusively consisted of mature granulocytes (Fig. 5B). In contrast, spleen cells from all the secondary recipients with MIG210/SPA-1–/– HPC could transfer lethal MPD to tertiary recipients with short latency (3-7.5 weeks; Fig. 4). Spleen cells of secondary recipients (K7.1-K7.3) retained comparable with or even greater proportions of leukemic progenitors than those of primary recipient (Fig. 5A), and the peripheral blood contained significant immature or blastic cells (Fig. 5B). It was confirmed that total spleen cells from primary recipients with MIG210/SPA-1–/– HPC showed increased amounts of Rap1GTP compared with those with MIG210/wt HPC (Fig. 5C).


Figure 4
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  		Figure 4. Serial transfer experiments reveal prolonged survival of BCR-ABL+ SPA-1–/– HPC in vivo. Spleen cells from primary recipients of MIG210/wt HPC (W3 and W4) and MIG210/SPA-1–/– HPC (K7) were transferred into 2.75 Gy {gamma}-ray-irradiated SCID mice at 5 x 106 cells per head. All the secondary recipients developed CML-like MPD in 2 to 3 weeks, and their spleen cells were further transferred into tertiary recipients. Tertiary recipients of W3 and W4 remained healthy for >20 weeks, except for one, which developed thymic lymphoma 16 weeks later. All the tertiary recipients of K7 developed MPD within 3 to 7.5 weeks. A group of them (K7. 3.1 and K7.3.2) developed B-lineage lymphoblasts with systemic lymphadenopathy, and the spleen or LN cells could transfer the leukemia infinitely thereafter. Cross mark, leukemic death. Peripheral leukocyte number (WBC, x10–3/µL) and spleen weight (mg) of each recipient before death or sacrifice.

 

Figure 5
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  		Figure 5. BCR-ABL+ SPA-1–/– HPC sustained in secondary recipients developed blast crisis in tertiary recipients. A, spleen cells from primary and secondary recipients with MIG210/wt HPC (W3, W3.1, and W3.2) and MIG210/SPA-1–/– HPC (K7, K7.1, K7.2, and K7.3) were three-color analyzed with GFP, anti-c-Kit antibody, and mixture of Lin– markers. Boxed, proportions of Lin– c-Kit+ cells in GFP+ populations. B, peripheral blood of secondary (W3.1 and K7.3) and tertiary (K7.1.1 and K7.3.1) recipients was lysed for RBC and stained with Giemsa solution. Arrowheads, immature or blastic leukocytes. K7.1.1 showed many immature erythroid cells, and K7.3.1 did homogenous B220+ lymphoblastic cells. C, Rap1GTP in total spleen cells from recipients with MIG210/wt HPC (W1, W2, and W3) and MIG210/SPA-1–/– HPC (K1, K2, and K7) mice that developed overt CML was determined by pull-down assay. Bone marrow cells from normal SCID mice served as a control. D, DNAs were extracted from K7 (CML), K7.3 (CML), and K7.3.1.2 (B-cell leukemia) mice, and Southern blotting was done with GFP and JH cDNA probes. Arrowheads, clonal bands. Karyotypes of spleen cells from K7.3.1.2.1 mice with B-cell leukemia were analyzed with a SKY method. Chromosomal translocations were detected in four of five metaphase cells, t(2;5) in three and t(3;6) in a cell (data not shown). Although not shown, no translocation was detected in spleen of preceding mouse (K.7.3.1.2).

 
One of the tertiary recipients with MIG210/SPA-1–/– HPC (K7.1.1) developed MPD with marked increase in immature erythroid cells in addition to granulocytes, whereas others (K7.3.1 and K7.3.2) developed a massive expansion of B220+ lymphoblastic cells (Fig. 5B). The latters could be transferred serially to normal recipients as such (Fig. 4) and diagnosed as B-cell leukemia. To investigate the origin of B-cell leukemia, we traced their clonality by examining retroviral integration sites. Spleen cells of primary K7 recipient showed many integration sites, among which three major clones predominated in secondary recipient with CML (K7.3; Fig. 5D). In spleen cells of a quaternary recipient (K7.3.1.2), in which GFP+ cells predominantly consisted of blastic B cells, two of the three integration sites were conserved (Fig. 5D). Consistently, Southern blot analysis revealed two predominant Ig heavy chain gene rearrangement bands (Fig. 5D), strongly suggesting that at least two leukemic B-cell clones were derived from original CML progenitors. Karyotype analysis showed no gross abnormality in leukemic cells of K7.3 and K7.3.1.2 recipients (data not shown), whereas those of the next recipient (K7.3.1.2.1) revealed chromosomal translocations (Fig. 5D). The results suggested that persistent BCR-ABL+ SPA-1–/– progenitors were prone to blast crisis in vivo.


   Discussion
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Gene-targeting studies in mice have shown that deficiencies in particular genes result in chronic MPD resembling human CML, including ICSBP (24) and JunB (25). ICSBP promotes differentiation of committed G/M progenitors to macrophage lineage at the cost of their proliferation (26), whereas JunB strongly suppresses self-renewing activity of long-term HSC in part by enhancing p16INK4a expression (27). Although these results suggest that deregulated control of HSC or HPC proliferation may eventually lead to CML-like MPD, abnormal expansion of self-renewing HSC pool per se may not necessarily result in overt MPD (28–30). In humans, the vast majority of CML is caused by BCR-ABL fusion gene, and studies using mouse models have indicated that BCR-ABL expression alone is sufficient to cause CML (31, 32). BCR-ABL induces activation of many signaling pathways, such as Ras/ERK, phosphatidylinositol 3-kinase, and signal transducers and activators of transcription 5, mostly via the constitutive kinase activity, causing clonal expansion of HSC and their differentiated progenies (9). Nonetheless, accumulating evidence also suggests that the transformation of HSC involves complex interaction of BCR-ABL with intrinsic host factors controlling self-renewal and differentiation of normal HSC (9, 14). We previously reported that SPA-1–/– mice developed a spectrum of MPD through deregulated Rap1 activation in HPC (16). Recent reports further indicated that BCR-ABL induced constitutive Rap1 activation in both human and mouse hematopoietic cells (18). In present study, we investigated possible role of endogenous SPA-1 in phenotypes of CML induced by BCR-ABL+ HPC.

Expansion and premature mobilization to periphery of LFA-1+ HSC underlie MPD of late onset in SPA-1–/– mice. Present study confirmed that SPA-1 deficiency resulted in pleiotropic leukemia of long latency in C57BL/6 genetic background, indicating that the different leukemia phenotypes were not attributed to mixed genetic background in previous study (16). Two dominant phenotypes were CML-like MPD and B-cell leukemia, although the features tended to be mixed in some mice. The majority of the latter showed a B1 cell phenotype (B220+ CD5+ CD11a+), and this may be related to constitutive activation of self-reactive B1 cells (4). Despite the phenotypic differences, diseased SPA-1–/– mice commonly showed an increase in the numbers of LKS cells not only in bone marrow but also in spleen. It was noteworthy that LFA-1+ LKS cells, which comprised a minor population in normal bone marrow, were preferentially increased in bone marrow of the SPA-1–/– mice, and the vast majority of LKS cells in the spleen were also LFA-1+. Clone-sort analysis revealed that LFA-1+ LKS cells developed colonies in the presence of SCF and IL-3 in vitro earlier than LFA-1– LKS cells,4 implicating that they represented multipotent progenitors at more advanced stages. It is reported that self-renewing capacity with restrained differentiation of normal HSC is maintained by interaction with bone marrow niche, and their retention in niche depends on a controlled balance of different adhesion molecules, including N-cadherin, and migratory integrins, such as LFA-1 (29, 33). Thus, it was suggested that accelerated differentiation and expansion of LFA-1+ LKS cells as well as their premature mobilization out of bone marrow underlay the development of MPD with extensive extramedullary hematopoiesis in SPA-1–/– mice.

Down-regulation of endogenous SPA-1 gene expression and constitutive Rap1 activation by BCR-ABL. To investigate the role of SPA-1 in BCR-ABL-induced CML, we first examined the effect of BCR-ABL on endogenous SPA-1 gene expression in HPC. It was revealed that expression of BCR-ABL in primary HPC caused significant, if not complete, reduction in endogenous SPA-1 gene transcripts. Due to limited cell numbers, SPA-1 protein levels in primary HPC remained to be investigated. However, BCR-ABL caused a significant decrease in SPA-1 protein in immature hematopoietic cell lines in a kinase activity-dependent manner in parallel with reduced SPA-1 transcripts. Although it was reported that E6TP1 protein, a SPA-1 family member, was rapidly degraded by human papillomavirus E6 oncoprotein (34), BCR-ABL did not reduce the half-life of SPA-1 protein at all. Thus, it was suggested that BCR-ABL expression caused down-regulation of SPA-1 protein via transcriptional repression in the cell lines. BCR-ABL represses the expression of potential tumor suppressors, such as SHIP (35), p27Kip1 (36), and ICSBP (37), and SPA-1 may be added to this list. Consistently, BCR-ABL-transduced cell lines exhibited a constitutive activation of Rap1 in the absence of cytokines, and this was abrogated by forced overexpression of SPA-1. Although it was reported that BCR-ABL induced Rap1 activation through phosphorylation of CrkL and recruitment of C3G (19), present results suggested that down-regulation of SPA-1 might also contribute to the BCR-ABL-induced Rap1 activation. The effects of BCR-ABL on cell adhesion have been a matter of argument (38–42). Recent reports, however, indicated that BCR-ABL enhanced basal cell adhesion in the absence of cytokines, whereas it rather inhibited IL-3-induced cell adhesion (20, 43). Present results showed that BCR-ABL enhanced integrin-mediated cell adhesion in both primary HPC and cell lines and that SPA-1 overexpression abolished the enhanced cell adhesion concomitantly with abrogation of Rap1 activation. Thus, it was suggested strongly that Rap1 signal might take a part in alteration of adhesive and migratory behavior of BCR-ABL+ HPC.

SPA-1 deficiency affects phenotypes of BCR-ABL-induced CML: prolonged survival of leukemic progenitors in vivo and rapid blast crisis. Although primary recipients with MIG210/wt and SPA-1–/– HPC developed largely comparable CML-like MPD characterized by marked granulocytosis and splenomegaly, it was revealed that the latter recipients contained significantly increased BCR-ABL+ Lin– c-Kit+ cells in both spleen and bone marrow compared with the former ones. Furthermore, the proportions of BCR-ABL+ Lin– c-Kit+ cells were sustained in secondary recipients with MIG210/SPA-1–/– HPC, whereas they were significantly exhausted in secondary recipients with MIG210/wt HPC. The development of CML depends on the presence of leukemic stem/progenitor cells, and consistently, spleen cells from secondary recipients with MIG210/SPA-1–/– HPC were able to transfer MPD to tertiary recipients, whereas those with MIG210/wt HPC failed to do so. Several explanations may be considered for the effects. First, BCR-ABL+ SPA-1–/– progenitors might home to and lodge in the hematopoietic microenvironments more efficiently following i.v. administration. Rap1 signal was shown to enhance homing and transendothelial migration of normal leukocytes via RapL-mediated integrin activation (44). Present results, however, indicated that comparable numbers of BCR-ABL+ CFU-Cs were recovered from the bone marrow and spleen of recipients with MIG210/wt and SPA-1–/– HPC 24 hours after the cell injection. Thus, it seemed unlikely that enhanced initial lodgment of MIG210/SPA-1–/– HPC to either bone marrow or spleen was a major cause. Second, SPA-1–/– BCR-ABL+ progenitors might show enhanced proliferation and/or survival in vivo. It was reported recently that BCR-ABL-induced Rap1 activation contributed to ERK and Akt activation via B-Raf/MAPK/ERK kinase 1, causing enhanced proliferation and reduced apoptosis in myeloid cells (20). However, ERK activation in present BCR-ABL+ cell lines was affected only barely, if any, by SPA-1 overexpression,5 and possible role of ERK activation remained to be seen. On the other hand, it was reported that quiescence of normal HSC was maintained by interaction with bone marrow niche stroma cells (33). Present results revealed that SPA-1–/– mice with spontaneous MPD showed increased LFA-1+ HPC and their accelerated immobilization to periphery causing vigorous extramedullary hematopoiesis. Thus, it might be possible that accelerated displacement of BCR-ABL+ SPA-1–/– HPC from niche contributed to their enhanced expansion in vivo.

Blast crisis to acute leukemia is a hallmark of human CML. Although it was recapitulated using serial transfer in mouse model, it was rather infrequent and occurred only after long latency (45, 46). In the present study, however, a portion of tertiary recipients with MIG210/SPA-1–/– HPC rapidly developed blastic B-cell leukemia, whereas those with MIG210/wt HPC rarely did. It was confirmed that the blastic B-cell clones were derived from original leukemic clones in primary recipients bearing CML. It was reported recently that a portion of transgenic mice conditionally expressing BCR-ABL gene in HSC developed blast crisis preferentially in B-cell lineage following CML (32), and thus, B-cell preference for crisis could be characteristic of murine model. Gross chromosomal translocations were found to develop only later in serial transfer of blastic leukemia, and therefore, they were considered to reflect the progression of leukemia rather than a primary cause of crisis. Possible genetic alterations responsible for blast crisis, if any, remained to be seen. In any case, these results suggested that BCR-ABL+ SPA-1–/– HPCs were also prone to blast crisis.

It is becoming evident that control of leukemic stem cells is crucial for potential eradication of human CML (47). Thus far, our attempts to overexpress SPA-1 in primary BCR-ABL+ HPC to overcome Rap1 signal failed because of technical difficulty of cotransducing two large genes in the same HPC, and experiments using mice conditionally expressing SPA-1 transgene in HPC are currently under way. Nonetheless, Rap1 signal may represent a potential target to control leukemic stem cells in human CML.


   Acknowledgments
 
Grant support: Grant-in-Aid for Scientific Research from the Ministry of Education, Science, Culture, Sports, and Technology of Japan.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

We thank Dr. O. Witte for providing us plasmids, Drs. K. Katagiri and T. Kinashi (Kansai Medical University, Osaka, Japan) for technical assistance on cell adhesion assay, and Dr. Y. Tanaka (Kyoto University) for proofreading the article.


   Footnotes
 
Note: Supplementary data for this article are available at Cancer Research Online (http://cancerres.aacrjournals.org/).

4 Unpublished observation. Back

5 K. Kometani, M. Hattori, and N. Minato, unpublished observation. Back

Received 4/13/06. Revised 7/19/06. Accepted 8/23/06.


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 Introduction
 Materials and Methods
 Results
 Discussion
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