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[Cancer Research 65, 3290-3298, April 15, 2005]
© 2005 American Association for Cancer Research

Cell and Tumor Biology

Defective p38 Mitogen-Activated Protein Kinase Signaling Impairs Chemotaxic but not Proliferative Responses to Stromal-Derived Factor-1{alpha} in Acute Lymphoblastic Leukemia

Linda J. Bendall1, Rana Baraz1, Julius Juarez1, Wei Shen1 and Kenneth F. Bradstock2

1 Westmead Institute for Cancer Research, Westmead Millennium Institute, University of Sydney and 2 Department of Hematology, Westmead Hospital, Westmead, New South Wales, Australia

Requests for reprints: Linda J. Bendall, Westmead Institute for Cancer Research, Westmead Millennium Institute, University of Sydney, Darcy Road, Westmead, NSW 2145, Australia. Phone: 61-2-9845-9069; Fax: 61-2-9845-9102; E-mail: linda_bendall{at}wmi.usyd.edu.au.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The chemokine stromal-derived factor-1{alpha} (SDF-1{alpha}) regulates leukemic cell motility and proliferation; however, the importance of these functions in the growth and dissemination of leukemia is unclear. We examined SDF-1{alpha}–mediated responses of cells from 27 cases of acute lymphoblastic leukemia (ALL). Although cells from the majority of cases showed chemotactic and proliferative responses to SDF-1{alpha}, a subset of cases did not undergo chemotaxis in response to SDF-1{alpha}, while still demonstrating dependence on SDF-1{alpha} for proliferation in stroma-supported cultures. This chemotactic defect was associated with an absence of phosphorylation of p38 mitogen-activated protein kinase (MAPK) induced by SDF-1{alpha}, and of SDF-1{alpha}–induced augmentation of ß1 integrin–mediated adhesion. Signaling through phosphoinositide 3-kinase and MEK was not affected. No correlation was observed between CXCR4 expression and chemotactic function, in vitro migration into bone marrow stromal layers, and engraftment of leukemic cells in nonobese diabetic/severe combined immunodeficient (NOD/SCID) mice. This study suggests that signaling through p38 MAPK is required for ALL cell chemotaxis but not for proliferation, and that the loss of a chemotactic response to SDF-1{alpha} does not impede engraftment in NOD/SCID mice.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Acute lymphoblastic leukemia (ALL) is the most common form of childhood cancer and a major cause of death in children (1). ALL also accounts for 10% to 20% of cases of acute leukemia in adults between the ages of 15 and 50 years. Although ALL is usually responsive to chemotherapy, ~25% of children and 65% of adults with ALL develop a relapse of their disease (1). Whereas some of these patients can be salvaged with bone marrow transplantation, the majority will die of leukemia. New approaches to the treatment of ALL are necessary to cure the significant proportion of patients for whom current therapies fail. An understanding of the relative importance of cell growth compared with trafficking and dissemination of this disease may provide opportunities for the development of such treatment strategies.

The majority of ALL cases arise from B-cell progenitors in the bone marrow. Leukemic cells from patients with B-cell progenitor ALL utilize the ß1 integrins VLA-4 and VLA-5 to bind to stromal layers (2, 3) and these proteins are important for bone marrow engraftment in the nonobese diabetic/severe combined immunodeficiency (NOD/SCID) mouse model (4, 5). Myeloid cytokines, such as stem cell factor, granulocyte macrophage colony-stimulating factor, and interleukin (IL)-3, can augment the function of ß1 integrins in normal hematopoietic progenitors (6), but B-lineage lymphoid cells do not respond to these cytokines (7). More recently, the chemokine stromal-derived factor-1 (SDF-1) or CXCL12 was also shown to modulate the activity of VLA-4, VLA-5, and LFA-1 in normal hematopoietic progenitors (8) and we showed a similar effect of SDF-1{alpha} in ALL cells (9).

SDF-1{alpha} was initially identified as a pre-B-cell growth-stimulating factor (10) binding a sole seven-transmembrane G protein–coupled receptor, CXCR4. The importance of SDF-1{alpha} and CXCR4 in normal B-cell development is evident from the severe defects in B lymphopoiesis in SDF-1{alpha} and CXCR4 knockout mice (11, 12). SDF-1{alpha} is a powerful chemoattractant for many hematopoietic cells, including CD34+ stem and progenitor cells (13), and has been implicated in the homing and engraftment of these cells in the bone marrow (14). In addition to regulating cell motility, SDF-1{alpha} can also play a role in cell survival (15, 16) and proliferation (17), often in synergy with other growth factors (18). SDF-1{alpha} binding to CXCR4 can induce a number of distinct, measurable events, including receptor internalization, elevation of cytoplasmic Ca2+ levels, activation of phosphoinositide 3-kinase (PI-3K), phosphorylation of MEK/ERK and components of focal adhesion complexes, including paxillin, p130cas, and focal adhesion kinase in many cell types (19–21). Most of these are dependent on Gi proteins and can be inhibited by pertussis toxin (22, 23). In contrast, receptor internalization is pertussis toxin insensitive, mediated by G protein–coupled receptor kinases and ß-arrestin (19, 24). Chemotaxis seems to be dependent on both Gi signaling and receptor internalization (24, 25).

We have previously shown that CXCR4 is highly expressed on all cases of B-cell progenitor ALL and that SDF-1{alpha} contributes to B-cell progenitor ALL cell migration into bone marrow stromal layers in vitro (26), and the homing and engraftment of B-cell progenitor ALL cells in the marrow of NOD/SCID mice (9). Therefore, it seems likely that SDF-1{alpha} will play a similar role in the homing and engraftment of ALL cells as that observed for normal progenitors. However, a substantial study of the role of CXCR4 and adhesion molecules in ALL engraftment in NOD/SCID mice is yet to be reported.

In this study, we examined in vitro SDF-1{alpha} responses of ALL cells from 27 patients. The correlation between in vitro function, bone marrow homing, and the production of overt leukemia in NOD/SCID mice by a subset of these samples was also studied. We did not identify a correlation between ALL cell engraftment, CXCR4 expression, or SDF-1{alpha} responsiveness. However, we identified a group of patients whose leukemic cells engrafted into NOD/SCID mice despite being unresponsive to SDF-1{alpha} in chemotaxis assays. This implies that other major factors are responsible for homing to the bone marrow in these patient samples.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cells. Leukemic blasts were obtained from 27 patients (details are given in Table 1) with B-cell progenitor ALL, after informed consent and institutional ethics committee approval. Mononuclear cells from peripheral blood or bone marrow samples were prepared and cryopreserved as previously described (27). Cell viability of thawed samples was 83 ± 14%. Cell numbers in all methods refer to viable cell number. Normal B-cell progenitors or CD34+ cells were isolated from normal bone marrow by magnetic affinity cell sorting separation. This was done as previously described (28) for CD34+ cell isolation except that QBEND-biotin was substituted with FMC63-biotin for B-cell progenitors. Bone marrow stromal cells were grown from bone marrow mononuclear cells obtained from normal healthy individuals after informed consent and institutional ethics committee approval. Complex Dexter-type stromal layers and bone marrow fibroblasts were established as previously described (28).


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  		Table 1. Clinical information

 
Antibodies and reagents. The following monoclonal antibodies were purchased: anti-CXCR4-PE (12G5), anti-CD49d-PE (9F10), anti-CD49e-PE (lla1), anti-CD29-PE (MAR4), anti-CD13-PE (L138), anti-CD34-PerCP (8G12), anti-CD10-FITC (SS2/36), anti-CD19-APC (SJ25C1), and anti-CD19-PE (4G7; BD PharMingen, Sydney, Australia); anti-phospho-p38 mitogen-activated protein kinase (MAPK), anti-p38 MAPK, anti-phospho-ERK, anti-ERK, anti-phospho-AKT, anti-AKT (Cell Signaling Technologies, Beverly, MA); horseradish peroxidase (HRP)–conjugated swine anti-rabbit immunoglobulins (DAKO, Glostrup, Denmark) and HRP-conjugated goat anti-mouse immunoglobulins (Sigma, St. Louis, MO). All antibodies were used as recommended by the manufacturer. FMC63-biotin was prepared as previously described (29) from material purified from hybridoma culture supernatant on protein G columns. SDF-1{alpha} was purchased from Peprotech (Rocky Hill, NJ) or from Philip Owen (University of British Columbia, Vancouver, British Columbia, Canada). TC14012 was synthesized by Mimotopes (Clayton, Victoria, Australia). Inhibitors of the following signaling molecules were purchased from Calbiochem-Merck KgaA (Darmstadt, Germany): the MEK inhibitor (PD98059), the PI-3K inhibitor (LY294002), and the p38 MAPK inhibitor (SB203500) and its control (SB202474).

Flow cytometry, calcium flux, and CXCR4 internalization. Flow cytometric analysis of cells labeled with directly conjugated monoclonal antibodies was done as previously described (17). For the evaluation of SDF-1{alpha}–mediated Ca2+ flux, 2 x 106 ALL cells were loaded with the Ca2+-sensitive fluorescent dye fura red (1 ng/mL; Molecular Probes, Eugene, OR) in NaCl (145 mmol/L) containing 0.1% bovine serum albumin (BSA), 5 mmol/L glucose, 10 mmol/L HEPES, 5 mmol/L KCl (pH 7.5) for 30 minutes at 37°C, then resuspended in 1 mL of the same buffer containing 1 mmol/L CaCl2. The increase in cytoplasmic Ca2+ concentrations was measured as a decrease in fura red fluorescence. Cells were treated with vehicle alone or with 100 ng/mL of SDF-1{alpha} or 0.5 µmol/L ionomycin (A23187; Sigma) as a positive control. Cells were analyzed by flow cytometry using time as a parameter. Stimulants were added after 40 seconds of analysis to establish a baseline and data were collected for a further 2 minutes. Internalization of CXCR4 following exposure to SDF-1{alpha} was determined by incubating ALL cells with or without SDF-1{alpha} (100 ng/mL) for 30 minutes at 37°C. Cells were then incubated with 100 mmol/L glycine (pH 3.5) for 1 minute, washed with PBS/BSA/azide, labeled with 12G5-PE, and analyzed by flow cytometry. All flow cytometry–based assays were done using a viable cell gate to exclude dead cells from the analysis.

Chemotaxis and migration assays. Chemotaxis and migration assays were done as previously described (17, 30) using Transwell Culture Inserts (Costar, Corning, NY) with a pore size of 5 µm. The chemotactic index was then calculated by dividing the percentage of cells migrated in the presence of SDF-1{alpha} by the percentage of cells migrated in its absence. In some experiments, cells were treated with the indicated agents for 2 hours at 37°C before plating in the assay. A viable cell gate was used to exclude dead cells from the analysis in both starting and migrated cell populations.

Adhesion and proliferation assays. Adhesion assays were done as previously described (31) with cells allowed to adhere for 15 minutes. Where indicated, SDF-1{alpha} was added at 2 µg/mL. For adhesion to fibronectin, 51Cr4O7-labeled adherent cells were lysed in 1% SDS and counted in a TopCount plate reader. The viability of cells in this assay was 88 ± 15% with a median of 95%. For adhesion to bone marrow fibroblasts, the entire remaining adherent layer was collected using trypsin/EDTA, cells labeled with CD10-FITC, CD19-APC, CD34-PerCP, and CD13-PE and analyzed by flow cytometry. The number of adherent B-cell progenitors per fibroblast (identified by CD13 expression and forward and side scatter) was calculated and compared with the number of input cells. For normal samples, analysis was done separately for B-cell progenitors at different stages of maturation as determined by CD34, CD19, and CD10 expression. A viable cell gate was used to exclude dead cells from the analysis. Proliferation assays were done as previously described (17).

Western blotting. To obtain sufficient cells for these experiments, cells for cases 1345, 1338, and 0398 were recovered from the spleen of xenografted mice and cells from case 1578 were expanded in stroma supported culture. ALL cells were washed and resuspended between 15 x 106 and 40 x 106 cells/mL in RPMI containing 0.5% BSA. SDF-1{alpha} was added at a final concentration of 100 ng/mL and the cells incubated for the indicated time periods at 37°C. Stimulation was stopped by the addition of excess cold PBS contain 1 mmol/L Na3VO4. Cell pellets were lysed in 10 mmol/L Tris, 150 mmol/L NaCl (pH 7.5) containing 1 mmol/L EDTA, 4 mmol/L Na3VO4, 10 mmol/L NaF, 4 µg/mL aprotinin, 1 mmol/L phenylmethylsulfonyl fluoride, 0.1 mg/mL leupeptin and protease inhibitor tablet as per manufacturer's instruction at 4°C for 30 minutes and the lysates clarified by centrifugation at 14,000 x g for 10 minutes. Equal protein was loaded in each lane of a 7.5% SDS-PAGE gel and transferred onto nitrocellulose. Phosphorylated and total proteins were detected sequentially on the same membrane using specific primary antibodies, secondary antibodies conjugated to HRP and enhanced chemiluminescence (Perkin-Elmer, Boston, MA). Bands were quantitated by densitometry.

Homing and engraftment of leukemia in nonobese diabetic/severe combined immunodeficiency mice. Mice received 3 Gy total body irradiation 24 hours before administration of leukemic cells via tail vein injection. For homing studies, leukemic cells were labeled with the fluorochrome CFSE (Peprotech) before injection, and labeled cells were identified in the bone marrow of mice by flow cytometry as previously described (9). For engraftment studies, mice received between 2 x 106 and 38 x 106 cells by tail vein injection. They were monitored for signs of leukemia including hind limb paralysis and/or weight loss and were sacrificed when disease was apparent. Bone marrow and spleen were analyzed by flow cytometry and other organs by gross examination at autopsy and histology as previously described (5).

Statistics. Comparisons between two groups were done using Student's t test between multiple groups using ANOVA analysis. Pairwise comparisons between groups were adjusted for multiple comparisons using Bonferroni's method. Linear regression was used to determine correlations between variables.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Chemotactic responses of B-cell progenitor acute lymphoblastic leukemia cells to stromal-derived factor-1{alpha}. We (32) and others (33) have previously reported that ALL cells undergo chemotaxis toward a gradient of SDF-1{alpha}. Recently, ALL samples have been reported to be more sensitive to SDF-1{alpha} than normal CD34+ progenitors (33). However, our dose response experiments revealed heterogeneous responses to SDF-1{alpha} by ALL samples in chemotaxis assays. Cells from ALL cell lines, such as NALM6 and most ALL cases, responded strongly to low doses of SDF-1{alpha}, with maximal responses occurring with <20 ng/mL of SDF-1{alpha}. However, some cases only responded to higher doses of SDF-1{alpha}, in a manner similar to normal CD34+ cells, whereas cells from other cases failed to respond even to high doses of SDF-1{alpha} (Fig. 1A). Overall, in this study, ALL cases showed a mean chemotactic response of 6.7 ± 9.0-fold (range 0.3-37.7, n = 22) over background to 100 ng/mL of SDF-1{alpha} (Fig. 1B). Interestingly, four cases (1345, 0398, 1338, and 1241) completely failed to show any chemotactic response to SDF-1{alpha}. In these cases, there was no statistically significant difference between the number of cells recovered from the lower chamber of wells containing or lacking SDF-1{alpha} and these cases are termed unresponsive. Cells from another four cases (0181, 0407, 0178, and 1901) displayed only minor, although statistically significant, responses with a chemotactic index of between 1.2 and 2.0. CXCR4 expression levels did not explain the varied responses of ALL cells, with no correlation between the chemotactic response to SDF-1{alpha} and CXCR4 expression being detected by regression analysis. This is reminiscent of decreases to SDF-1{alpha} responsiveness during normal B-cell progenitor maturation, which is disproportionate with alterations in CXCR4 expression (34). However, the level of phenotypic maturity, as assessed by CD34 and CD10 expression, of individual leukemic samples did not account for altered levels of responsiveness to SDF-1{alpha} (Table 1; Fig. 1). Similarly, there was no correlation between the chemotactic response of ALL cells to SDF-1{alpha} and cell viability on sample thawing, patient WBC count, or whether the sample was obtained from the bone marrow or peripheral blood. There were no common cytogenetic abnormalities in the unresponsive cases with two having complex cytogenetic defects and two having normal karyotypes. Two of the four patients are still in first complete remission after 50 and 51 months and the remaining patients relapsed after 9 and 20 months and died following repeated disease relapses 26 and 36 months, respectively. All unresponsive samples were obtained from pediatric cases. Pediatric cases were slightly overrepresented with 16 cases compared with 11 adult cases and there was no difference in the overall chemotactic response of samples from pediatric cases compared with those obtained from adult patients.



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  		Figure 1. Analysis of ALL cell CXCR4 expression and chemotactic responses to SDF-1{alpha}. A, ALL cells and CD34+ progenitors were assessed for their chemotactic response to the indicated concentrations of SDF-1{alpha}. Results of chemotaxis assays are expressed as the chemotactic index at each concentration, calculated as described in Materials and Methods. Points, mean of two replicates; bars, SD. Patient numbers or cell type are indicated. B, ALL samples were labeled with 12G5-PE and analyzed by flow cytometry. CXCR4 expression (black dots) is expressed as the mean fluorescence intensity (MFI). The chemotactic response of ALL samples to 100 ng/mL SDF-1{alpha} was assessed in a 3-hour chemotaxis assay. Results are expressed as the chemotaxic index calculated as described in Materials and Methods. The first four patients in this figure are those that failed to respond to SDF-1{alpha} in chemotaxis assays.

 
Stromal-derived factor-1{alpha}–mediated receptor internalization and Ca2+ mobilization. Upstream signaling events mediated by SDF-1{alpha} include the internalization of the receptor, mediated by G protein–coupled receptor kinases and ß-arrestin (19, 35), and Gi protein–mediated mobilization of Ca2+ from internal stores (23, 24). Both of these signal pathways play a role in SDF-1{alpha}–mediated chemotaxis (24). All but 3 (0181, 0483, and 1241) of 17 ALL cases tested showed CXCR4 internalization of >20% following a 30-minute exposure to SDF-1{alpha}, with a mean reduction in surface expression of 52 ± 25% Fig. 2. One (1241) of the three cases that did not internalize CXCR4 had been unresponsive to SDF-1{alpha} in in vitro chemotaxis assays; another (0181) had shown only a very weak chemotactic, although statistically significant, response (chemotactic index = 1.22) and the third case (0483) had a chemotactic index of 2.6. In all 13 cases examined, including three of the four cases that did not undergo chemotaxis toward SDF-1{alpha}, SDF-1{alpha} elicited increases in the cytoplasmic Ca2+ concentration (data not shown).



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  		Figure 2. Internalization of CXCR4 by ALL samples in response to SDF-1{alpha}. A, ALL cells were cultured with (black columns) or without (white columns) 100 ng/mL SDF-1{alpha} for 30 minutes before assessment of CXCR4 expression. The results are given as mean fluorescence intensity. Patient numbers are indicated. The first four patients are those that did not undergo chemotaxis in response to SDF-1{alpha}. *, Patients where internalization was <20%. B, ALL cells were cultured with (thick line) or without (thin line) 100 ng/mL SDF-1{alpha} before assessment of CXCR4 expression. Dotted lines, isotype control. Patient numbers are indicated in the top right-hand corner of each histogram.

 
Stromal-derived factor-1{alpha}–mediated integrin activation in B-cell progenitor acute lymphoblastic leukemia. We have previously shown that baseline adhesion of ALL cells to fibronectin is principally mediated by VLA-5 with a minor contribution of VLA-4 (36), whereas both integrins contributed to a similar degree to ALL cell adhesion to bone marrow fibroblasts (3). SDF-1{alpha} stimulation increased the contribution of VLA-4–mediated adhesion to fibronectin and resulted in an increased overall adhesion (9). The SDF-1{alpha} unresponsive cases showed predominantly VLA-5–mediated adhesion to fibronectin, typical of ALL cells, with one case (0398) having a significant although minor VLA-4–mediated component. In contrast to most ALL cases, the adhesion of these cells to bone marrow fibroblasts was largely VLA-4 independent (data not shown). SDF-1{alpha} increased the adhesion of ALL cells to fibronectin from 30.3 ± 19.7% to 36.1 ± 23.6% (P < 0.006, n = 15) and to bone marrow fibroblasts from 40.8 ± 17.1% to 54.7 ± 23.0% (P < 0.0001, n = 20). However, adhesion was not significantly increased to fibronectin in 7 (1345, 0398, 1338, 1241, 0483, 0085, and 0181) of the 15 samples and to bone marrow fibroblasts in 6 (1345, 0398, 1338, 1241, 1133, and 0535) of the 20 samples examined Fig. 3). The four cases (1345, 0398, 1338, and 1241) that had failed to undergo chemotaxis to SDF-1{alpha} did not increase binding to fibronectin or bone marrow fibroblasts in response to SDF-1{alpha}. No other cases tested were similarly unresponsive in both adhesion assays. All three cases that had not internalized CXCR4 also failed to enhance adhesion to fibronectin in response to SDF-1{alpha}, accounting for two of the remaining three samples that did not modulate ß1 integrin function (0181 and 0483). Overall, there was a weak association between SDF-1{alpha}–mediated chemotaxis and augmentation of ß1 integrin function, although this failed to reach statistical significance. The cases that did not modulate ß1 integrin function in response to SDF-1{alpha} showed similar baseline adhesion to bone marrow fibroblasts to that observed for other ALL cases and normal B-cell progenitors, suggesting that there is no underlying defect in adhesion molecule function.



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  		Figure 3. Adhesive responses of ALL cells to SDF-1{alpha}. The adhesion of ALL cells (A and B) or normal bone marrow B-cell lineage cells (i.e., CD19-positive cells) from four donors (C, CD19+CD34+CD10+ fraction; D, CD19+CD34–CD10+ fraction; and E, CD19+CD34–CD10– fraction) to fibronectin (A) or bone marrow fibroblasts (B-E) was assessed in the presence (black columns) or absence (white columns) of 2 µg/mL SDF-1{alpha} in 15-minute adhesion assays. Patient numbers are indicated and the first four patients are those that did not respond to SDF-1{alpha} in chemotaxis assays. Columns, mean of four replicates; bars, SD. *P < 0.05 comparing control and SDF-1{alpha}–treated cells.

 
We have previously reported that SDF-1{alpha} is involved in the migration of ALL cells into monolayers of bone marrow fibroblasts (32). Eight of the 15 ALL cases tested showed a similar migratory capacity (16.24 ± 8.35%) as normal bone marrow B-cell progenitors at a similar stage of maturation (14.52 ± 4.29%, n = 3). Three cases (0181, 0483, and 1241) showed decreased migratory capacity (3.62 ± 1.43%, P = 0.004 compared with normals). Interestingly, all of these cases also failed to internalize CXCR4, suggesting a defect in G protein–coupled receptor kinases or ß-arrestin function and also failed to modulate ß1 integrin function in response to SDF-1{alpha} as determined by adhesion to fibronectin. Four cases (0398, 1338, 0275, and 0535) showed increased migratory capacity compared with normal B-cell progenitors. One of these (0535, migration = 85.79 ± 2.79, chemotactic index = 37.73 ± 6.07) had shown an exceptionally high response to SDF-1{alpha} in chemotaxis assays; however, two other cases (0398 and 1338, migration = 30.88 ± 3.30 and 20.76 ± 0.81, respectively; P < 0.05 compared with controls) were completely unresponsive to SDF-1{alpha} in chemotaxis assays.

Overall, we identified four patients (1345, 0398, 1338, and 1241) whose cells consistently failed to modulate adhesion molecule function or to undergo chemotaxis in response to SDF-1{alpha}. However, there was no apparent impact of these defects on the migration of these cells into bone marrow fibroblast monolayers. We also observed three cases that failed to internalize CXCR4 following exposure to SDF-1{alpha}. These three cases also failed to up-regulate ß1 integrin function and migrated only poorly through bone marrow stromal layers. However, we could not detect any correlation between migration into stromal layers and expression of CXCR4 or ability to respond to SDF-1{alpha} in chemotaxis assays.

Dependence on stromal-derived factor-1{alpha} for stroma-supported proliferation of B-cell progenitor acute lymphoblastic leukemia cells. The in vitro survival of most cases of ALL is poor, and, as a result, proliferation is low or absent in the majority of cases when cultured in serum-free medium. Therefore, to examine the effect of SDF-1{alpha} on ALL cell proliferation, we used a serum-free culture system that included stromal support and blocked the effect of endogenous SDF-1{alpha} using the specific SDF-1{alpha} antagonist TC14012 (37). We have previously shown that TC14012 inhibits stromal-dependent proliferation of ALL cells in the majority of cases with only marginal effects on ALL cell viability (17). Stromal support increased ALL cell proliferation between 2.4- and 893.6-fold over that observed in the absence of stroma (Fig. 4). TC14012 significantly inhibited the proliferation of five of the six cases tested by a mean of 71.2 ± 9.5%. The remaining case (0336) showed a small, but statistically insignificant decrease in stromal-dependent proliferation in the presence of TC14012. All three of the cases tested that failed to undergo chemotaxis to SDF-1{alpha} (0398, 1338, and 1345) showed a clear dependence on SDF-1{alpha} for stromal-dependent proliferation.



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  		Figure 4. ALL require SDF-1{alpha} for stromal-dependent proliferation. ALL cells were cultured alone (Stroma Free) or in the presence of irradiated bone marrow stromal cells with (Stroma + TC14012) or without (Stroma) the addition of the SDF-1{alpha} antagonist TC14012 for 4 days. Proliferation was assessed following the addition of [3H]thymidine. Columns, mean proliferation of four replicates expressed in cpm; bars, SD. Cultures on stromal layers have been corrected for background counts incorporated into stromal layers cultured without ALL cells [3H]thymidine incorporation into stromal layers in the absence of ALL cells was always <150 cpm (mean 79 ± 39, n = 6). *P < 0.05 compared with proliferation on stromal layers. {dagger}P < 0.05 compared with cells ALL cells cultured alone.

 
Altered signaling through p38MAPK in unresponsive cases. In an attempt to understand why some ALL samples can respond to SDF-1{alpha} in a proliferative manner, yet not undergo chemotaxis or modulate adhesion molecule function in response to SDF-1{alpha}, we examined the signaling events involved in SDF-1{alpha}–mediated ALL cell chemotaxis and proliferation. Using the specific inhibitor of p38 MAPK, SB203500, in chemotaxis assays, we determined that signaling through p38 MAPK was essential for chemotaxis in the pre-B ALL cell line NALM6 and patient ALL cells, whereas signaling through the MEK/ERK and PI-3K/AKT pathways played a lesser but, in some cases, significant role (Fig. 5A). Studies of the phosphorylation of ERK, AKT, and p38 MAPK revealed that in NALM6 cells and ALL cells that were fully responsive to SDF-1{alpha}, all three pathways were activated by SDF-1{alpha} (Fig. 5B). p38 MAPK showed some background phosphorylation but this increased in response to SDF-1{alpha}, reaching a maximum between 2 and 5 minutes and declining thereafter. ERK and AKT had minimal background phosphorylation and were maximally phosphorylated at 2 minutes with ERK phosphorylation declining rapidly and AKT declining more slowly. In contrast, in the three unresponsive cases with sufficient cells to examine (1345, 0398, and 1338), p38 MAPK was not phosphorylated in response to SDF-1{alpha}. Blotting baseline samples from each of these cases on the one gel revealed that phosphorylated p38 MAPK levels were increased, relative to ß-actin, in cases 1345 and 1338 (data not shown). However, no response to SDF-1{alpha} was observed in these cases and no phosphorylation of p38 MAPK could be detected in case 0398 at any time point. Phosphorylation of ERK was observed in cases 1338 and 0398 and the response was present, but more sustained, in case 1345. Phosphorylation of AKT was similar to that observed in fully responsive cases in each case (Fig. 5C). Together, these data suggest that the loss of signaling through the p38 MAPK pathway is likely to be responsible for the failure of chemotaxis in these cases.



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  		Figure 5. ALL cells require signaling through p38 MAPK for chemotactic responses. A, ALL cells, NALM6, or indicated cases were treated with pertussis toxin (PTX), LY294002 (LY), SB203580 (SB), SB202474 (SB C), or PD98059 (PD) for 2 hours before assessment of their chemotactic response to 200 ng/mL SDF-1{alpha}. Columns, chemotactic index; bars, SD of duplicate determinations. SDF-1{alpha}–responsive (B) and SDF-1{alpha}–unresponsive (C) ALL cells were treated with 100 ng/mL SDF-1{alpha} for the indicated time points. Cell lysates were examined using antibodies to phosphorylated p38 MAPK (pp38), total p38 MAPK (p38), phosphorylated ERK (pERK), total ERK (ERK), phosphorylated AKT (pAKT), or total AKT (AKT) by Western blotting. Phosphorylated and total protein for each protein was assessed on the one blot. The intensity of bands was assessed by densitometry and the relative phosphorylation shown by the numbers below the blots. For phosphorylated p38 MAPK from patient 0398, phosphorylated AKT from patient 1578 and phosphorylated ERK from NALM6 cells, no baseline phosphorylation could be detected so all time points were normalized to the maximum response (designated 1). Patient numbers are indicated above each panel.

 
Homing and engraftment of B-cell progenitor acute lymphoblastic leukemia cells in nonobese diabetic/severe combined immunodeficiency mice. SDF-1{alpha} and CXCR4 are crucial for the homing and engraftment of human CD34+ progenitors in NOD/SCID mice (14). We (9) and others (33) have shown CXCR4 to be similarly involved in the homing and engraftment of B-cell progenitor ALL in NOD/SCID mice using CXCR4 down-regulation, pertussis toxin treatment, or anti-CXCR4 antibody coating. Therefore, we compared the homing of 10 cases of ALL with CXCR4 expression and function. Surprisingly, we did not observe a correlation between CXCR4 expression, SDF-1{alpha}–mediated chemotaxis, and the ability of cells to home to the bone marrow (Table 2) by regression analysis. However, of the three cases tested that did not undergo chemotaxis in response to SDF-1{alpha}, two showed a reduced capacity to home to the bone marrow when compared with cases that had responded to SDF-1{alpha} in chemotaxis assays. In cases where homing to the marrow was poor, increased numbers of cells were not detected in the peripheral blood or spleens of the animals, suggesting that the cells were not trapped in the peripheral blood or that they had been removed by the spleen (data not shown).


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  		Table 2. Homing and engraftment of leukemia in NOD/SCID mice

 
We examined samples from 14 patients (Table 1) with ALL for their ability to produce leukemia in NOD/SCID mice. Mice receiving 8 of the 14 samples developed overt leukemia, displaying weight loss and/or hind limb paralysis 16.5 ± 8.2 weeks postinjection (range 3-24 weeks; Table 2). Although mice received variable numbers of cells (range 2 x 106-38 x 106), there was no correlation between cell dose and time to engraftment or the overall engraftment [mean cell dose for engrafted samples of 17.5 x 106 (median 18) cells and for nonengrafted samples 16.7 x 106 (median 15) cells]. At autopsy, leukemia was detected in the bone marrow, spleens, and livers of all mice examined (82 ± 18% and 78 ± 20% human cells in bone marrow and spleen, respectively, at the time of sacrifice). Although there seemed to be a higher expression of CXCR4 on cases that engrafted in NOD/SCID mice (mean fluorescence intensity of 172 ± 168, range 15-477, n = 8 on engrafted samples compared with mean fluorescence intensity of 58 ± 38, range 31-120, n = 5 on nonengrafted samples), this did not reach statistical significance (P = 0.17). Similarly, there was no correlation between SDF-1{alpha}–mediated chemotaxis and engraftment in the mice (chemotactic index of 4.2±5.3, n = 8, for samples that engrafted compared with chemotactic index of 5.7 ± 7.6, n = 4 for those that did not engraft; P = 0.69).

Of the eight samples that engrafted in the NOD/SCID mice, three (0398, 1345, and 1338) had failed to undergo chemotaxis to SDF-1{alpha} and another (0181) had displayed a poor response. We considered that CXCR4 function may have been restored once the cells were in the mice; however, this was not the case because cells recovered from the bone marrow of engrafted animals were similarly unresponsive to SDF-1{alpha} despite high levels of CXCR4 expression (data not shown). The fourth case (1241) that failed to undergo chemotaxis to SDF-1{alpha} did not engraft in NOD/SCID mice. Although mice receiving the cases that did not undergo chemotaxis engrafted significantly more rapidly than mice receiving cells from SDF-1{alpha}–responsive cases (9 ± 7 and 21 ± 6 weeks, respectively; P < 0.05) the numbers are small and the result needs to be confirmed with larger numbers of samples. In addition, two of the unresponsive cases produced leukemic infiltrates in the kidney. Only one other case had kidney involvement. This data shows that chemotactic and adhesive responses to SDF-1{alpha} are not required for the engraftment of ALL cell in NOD/SCID mice, although they may well contribute to engraftment in cases that do undergo chemotaxis to SDF-1{alpha}.


   Discussion
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
There is a significant body of literature demonstrating that signaling through CXCR4 is important for the engraftment of hematopoietic cells in NOD/SCID mice (8, 14, 38). We (9) and others (33) have recently presented evidence for a similar role for CXCR4 in the engraftment of cells from patients with ALL. Ligation of CXCR4 by SDF-1{alpha} regulates a number of cellular responses including chemotaxis, adhesion, proliferation, and survival (8, 10, 13, 18). The contribution of these various functions of the SDF-1{alpha}/CXCR4 axis in ALL cell biology, and to the engraftment of these cells in NOD/SCID mice, is largely unknown. To date, studies examining the role of CXCR4 in hematopoietic cell engraftment have relied on methods that completely block SDF-1{alpha} binding to CXCR4 using monoclonal antibodies (14), down-regulate CXCR4 expression by ligand-mediated desensitization (9, 14) or cytoplasmic retention (39), or block major, multifunctional signaling pathways using pertussis toxin or toxin B (9, 33). The first two methods block all responses to SDF-1{alpha} and the third potentially blocks signaling mediated by a number of ligands, making the assessment of which CXCR4-mediated responses are important impossible. In this study, we have identified a number of ALL patient samples that lack chemotactic and adhesive responses to SDF-1{alpha} while remaining dependent on SDF-1{alpha} for stroma-dependent proliferation. The comparison of these samples with ALL samples that undergo chemotaxis to SDF-1{alpha} provides insights into the relative importance of SDF-1{alpha}–mediated functions in ALL cell biology. They also provide evidence regarding the relative importance of signaling events downstream of CXCR4 in these processes.

In this study, we investigated a range of SDF-1{alpha}–driven responses in cells from a cohort of 27 ALL cases. The chemotactic response of patient ALL cells to SDF-1{alpha} varied considerably, with cells from 18% of the patients tested completely failing to respond in chemotaxis assays and a further 15% only showing minor responses (chemotactic index of <2) to high concentrations (100 ng/mL) of SDF-1{alpha}. Therefore, whereas some cases of ALL show the enhanced chemotaxis previously reported (33), this did not apply to all. Levels of expression of CXCR4 did not predict the chemotactic response of ALL cells to SDF-1{alpha}, with all cases expressing high levels as previously reported (32, 40, 41). Interestingly, the cases that failed to respond to SDF-1{alpha} in chemotaxis assays still required SDF-1{alpha} for stromal-dependent proliferation. This suggests a separation of SDF-1{alpha}–induced chemotactic and proliferative responses in a subpopulation of ALL cases.

Analysis of SDF-1{alpha}–mediated signaling revealed a deficiency in signaling through p38 MAPK in cases that failed to undergo chemotaxis to SDF-1{alpha}, a finding consistent with our ability to block chemotaxis using a p38 MAPK inhibitor and the reported role of p38 MAPK in actin polymerization. Whereas some groups have also reported a role for p38 MAPK in SDF-1{alpha}–mediated chemotaxis in T lymphocytes (42), others have not been able to show this (20, 43). Our current study does not identify the precise point at which the signaling pathway from CXCR4 to p38 MAPK is compromised in our unresponsive cases and it is possible that this varies between the cases tested. Several studies have identified molecules upstream of p38 MAPK as being important for chemotaxis including the Rho GTPases, particularly Cdc42 (33), and the Rap GTPase, Rap2. Indeed, Rap2-deficient B cells have a phenotype similar to that observed in our unresponsive cases with both lacking SDF-1{alpha}–mediated chemotaxis and stimulation of ß1 integrin function but having normal baseline adhesion. The three cases that failed to internalize CXCR4 shared this lack of SDF-1{alpha}–driven integrin activation (indeed one case, 0214, was common to both groups) and showed reduced chemotaxis to SDF-1{alpha}. Internalization of CXCR4 is dependent on ß-arrestin2 and G protein–coupled receptor kinase, and the activity of these proteins is required for maximal chemotactic responses of lymphocytes to SDF-1{alpha} (19, 24, 25). Interestingly, ß-arrestin2 is also reported to be upstream of p38 MAPK (24), although the p38 MAPK pathway can be activated independently of glycogen kinase synthases and ß-arrestin2. Three of the four unresponsive cases internalized CXCR4 normally suggesting a defect downstream of G protein–coupled receptor kinase and ß-arrestin2 upstream of p38 MAPK. The fact that cases that lacked a chemotactic response to SDF-1{alpha} displayed normal or even enhanced migration into stromal layers suggests that other factors can compensate for the lack of SDF-1{alpha}–mediated chemotaxis in these cases.

In contrast to chemotaxis, SDF-1{alpha}–driven proliferative signals are thought to be mediated through MEK/ERK and PI-3K/AKT signaling pathways (44, 45) and survival signals through PI-3K/AKT (46). These pathways were still intact in our unresponsive cases, and inhibitors of MEK or PI-3K had no or partial effects on ALL cell chemotaxis, respectively, findings consistent with those obtained by others in lymphoid cells (24, 47). Overall, it seems that the failure to undergo increased phosphorylation of p38 MAPK results in defects in chemotaxis but not proliferative responses to SDF-1{alpha}.

The successful engraftment of three of the four ALL cases that did not undergo chemotaxis to SDF-1{alpha} provides strong evidence that chemotactic responses to SDF-1{alpha} are not necessary for ALL cell engraftment in NOD/SCID mice and that other chemotactic mechanisms directing the traffic of ALL cells from the circulation to the marrow may exist. The NOD/SCID mouse model of human leukemia provides an excellent representation of the disease in the patient, reflecting both the dissemination of the human disease to extramedullary sites and correlating with the prognosis of the disease in the original patient (48, 49). Therefore, it is possible that our findings using the NOD/SCID mouse model will also apply to the biology of ALL cells in patients. Overall, our data suggest that the role of SDF-1{alpha} in supporting ALL cell survival and proliferation may be more important for the engraftment of ALL cells in NOD/SCID mice than its role in mediating chemotaxis and homing. These findings provide new insights into leukemic cell biology and may enable the development of novel therapeutic protocols aimed at preventing cancer cell proliferation and dissemination, while only minimally affecting normal hematopoietic stem cells in patients with ALL.


   Acknowledgments
 
Grant support: Anthony Rothe Memorial Trust, Cure Cancer Australia Foundation, Westmead Millennium Foundation, and a Faculty of Medicine/Medical Foundation Postgraduate Research Scholarship from the University of Sydney (J. Juarez).

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. Sundreswran Ramanathan and Lyra Pearson for assistance in collating patient information, Dr. Ben Gu for assisting in establishing a flow cytometric assay for the measurement of cytoplasmic calcium levels, and Dr. Richard Lock and Rachael Papa for the in vivo expansion of two of the ALL cases.

Received 9/20/04. Revised 1/12/05. Accepted 2/ 9/05.


   References
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 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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