Mesenchymal cells regulate the response of acute lymphoblastic leukemia cells to asparaginase (original) (raw)
ASNS expression in leukemic lymphoblasts and its relation to presenting characteristics. To determine the level of ASNS expression in leukemic lymphoblasts, we used a gene expression profiling database, obtained via Affymetrix U133A gene arrays, that included cell samples from 288 patients with newly diagnosed childhood ALL. The median signal intensity for ASNS (probe 205047_s_at) was 265.4 (range, 8.8–2229.1). Comparison of ASNS expression levels (categorized as lower, middle, and higher) according to presenting clinicobiologic features (Table 1) failed to demonstrate significant relationships with most of these variables, including age and leukocyte count. However, expression levels were significantly higher in cases of T-lineage ALL compared with other subtypes (P < 0.0001). Within the B-lineage ALL group, patients with the _TEL-AML1_ gene or that were hyperdiploid (>50 chromosomes) were more likely to have lower ASNS expression levels than were patients without these features (P = 0.033 and P <0.0001, respectively). Similar relationships were observed when ASNS expression was analyzed as a continuous variable: higher ASNS levels in T-lineage ALL cases (P < 0.0001) and lower levels TEL-AML1 (P = 0.003) and hyperdiploid cases (P < 0.0001; data not shown).
Correlation between ASNS expression with presenting clinicobiologic features
Expression of ASNS in MSCs. Leukemic lymphoblasts isolated from bone marrow rapidly die in standard culture media, but thrive on layers of bone marrow MSCs (25–30), suggesting their dependence on survival factors produced within this microenvironment. We therefore studied ASNS expression levels in bone marrow–derived MSCs. We first determined ASNS gene expression in bone marrow MSCs immortalized by enforced expression of the human telomerase reverse transcriptase (TERT) gene. These cells are indistinguishable from primary MSCs: they grow at a normal rate, they do not form tumors in immunodeficient mice, they can differentiate to osteoblasts and chondrocytes, and they support normal and leukemic hematopoiesis (31). The mean of 2 signal intensity measurements performed with the same gene array used to study ALL cells was 5,164.2, a value nearly 20 times higher than the median values determined in leukemic blasts (Figure 1A). We next used real-time PCR to compare ASNS expression in immortalized MSCs to that of primary MSCs (obtained from 9 healthy donors and 13 patients with ALL in complete remission) and primary leukemic cells obtained at diagnosis from 35 children with ALL. As shown in Figure 1B, ASNS gene expression levels in primary MSCs were variable but overall much higher than those measured in ALL samples. ASNS expression in primary MSCs was on average 23 times higher than in primary ALL cells. ASNS levels in 3 B-lineage ALL cell lines (RS4;11, 380, and REH) were higher than those of most primary ALL samples but lower than those of MSCs (Figure 1B). The cell line that most resemble primary ALL cells in terms of ASNS expression was RS4;11, as also shown by semiquantitative PCR and Western blotting (Figure 1, C and D). In contrast to what was previously reported for ALL cells (20, 32), we did not detect changes in ASNS expression by RT-PCR or Western blotting in MSCs exposed to asparaginase (1.0–5.0 IU/ml) for up to 48 hours (data not shown).
Expression of ASNS in ALL cells and bone marrow–derived MSCs. (A) Expression of ASNS mRNA in 288 samples of primary ALL cells compared with that of _TERT_-immortalized MSCs (T-MSCs; mean of 2 measurements denoted by circles) by Affymetrix GeneChip arrays. Boxes denote interquartile range; whiskers denote range; lines denote median. *Cases lacking known genetic abnormalities. (B) Expression of ASNS mRNA in ALL cell lines, primary ALL samples and primary MSCs as measured by real-time RT-PCR. Primary MSCs were obtained from the bone marrow of 9 healthy donors (Donor MSCs) and 13 patients with ALL undergoing therapy and in complete remission (ALL-CR MSCs). Shown is ASNS expression relative to that of the TBP gene. (C) ASNS mRNA expression in primary ALL cell samples, the RS4;11 ALL cell line, and primary MSCs by semiquantitative RT-PCR; signals obtained with _TERT_-immortalized MSCs are also shown. β-Actin was used as a control. (D) Western blot analysis of ASNS protein expression in ALL cell lines and _TERT_-immortalized MSCs. After probing with the anti-ASNS antibody, the membrane was stripped and reprobed with an anti-actin antibody.
MSCs protect ALL cells from asparaginase cytotoxicity. To determine whether coculture with MSCs could affect the sensitivity of ALL cells to asparaginase, we performed experiments with the 3 B-lineage ALL cell lines. For each cell line, we first determined the concentration of asparaginase that would cause 50% killing after 2 days of culture in cultures without MSCs. Then we used this concentration to treat the cell lines in parallel cultures performed with and without _TERT_-immortalized MSCs. As shown in Figure 2, the number of viable leukemic cells recovered after exposure to asparaginase for 2 days in 10 experiments with the 3 cell lines was consistently higher when cultures were performed in the presence of MSCs. To determine whether primary MSCs could also protect ALL cells from asparaginase cytotoxicity, we performed similar experiments using primary MSCs (obtained from 9 healthy individuals and 13 children with ALL in complete remission) instead of _TERT_-immortalized MSCs. As shown in Figure 2, primary MSCs also protected ALL cells from asparaginase cytotoxicity, regardless of whether they were obtained from ALL patients or healthy donors. There was no significant relation between protective capacity and MSCs levels of ASNS expression measured by real-time PCR (data not shown).
MSCs protect ALL cells from asparaginase cytotoxicity. The ALL cell lines 380, REH, and RS4;11 were cultured with and without MSCs for 48 hours in the presence of the concentration of asparaginase that causes 50% killing in 2 days of culture, as previously determined for each cell line in experiments without MSCs (1.0 IU/ml for 380 and REH; 0.001 IU/ml for RS4;11). Values are mean ± SD of cell killing obtained in cultures with MSCs relative to that obtained in parallel cultures without MSCs. Ten experiments were performed for each ALL cell line with _TERT_-immortalized MSCs; MSCs from 9 healthy donors and 13 patients with ALL in complete remission were also tested for each ALL cell line. Each experiment consisted of 4 measurements.
To ensure that the residual leukemic cells detected in cultures with MSCs were capable of proliferation, we performed experiments with the 3 cell lines in which we collected all residual cells after culture, washed out the asparaginase, and plated the cells in fresh medium in the absence of MSCs. After 7 days, the number of cells recovered from these cultures increased by 5- to 21-fold, indicating that the cells remaining on the MSC layers after asparaginase treatment were indeed viable and capable of proliferation. Reportedly, ASNS expression in ALL cells is upregulated after contact with mouse macrophages (33), but we did not detect increased levels of ASNS by Western blotting in RS4;11, 380, or REH cells after contact with MSCs (data not shown), ruling this out as the mechanism underlying the resistance of ALL cells to asparaginase that we observed.
Expression of ASNS in MSCs is associated with their capacity to support leukemic lymphoblasts. Taken together, the preceding results suggested that the protective effect of MSCs on ALL cells was related to their high levels of ASNS expression. To test this possibility, we decreased the levels of ASNS expression in immortalized MSCs using RNA interference and sought to determine whether this downregulation affected the capacity of the cells to protect ALL cells from asparaginase cytotoxicity. When the cells were transduced with a siRNA sequence targeted to ASNS, expression of the enzyme was markedly and stably decreased, whereas a scrambled sequence had no effect (Figure 3, A and B). Although it lacked any effect on the rate of cell growth, morphology, or confluence pattern of MSCs, ASNS downregulation profoundly altered the cells’ ability to protect ALL cells from asparaginase cytotoxicity. As shown in Figure 3C, asparaginase killed significantly more leukemic cells when the MSCs were transduced with the target siRNA sequence rather than the scrambled control sequence.
Downregulation of ASNS expression in MSCs by RNA interference decreases their capacity to protect ALL cells from asparaginase cytotoxicity. (A) Expression of ASNS mRNA in _TERT_-immortalized MSCs transduced with an _ASNS_-targeted siRNA construct (Tar) compared with that of cells transduced with a scrambled control construct (Scr) and that of nontransduced MSCs (Non) by semiquantitative RT-PCR. β-Actin was used as a control. (B) Expression of ASNS protein in the same cells by Western blotting. After probing with the anti-ASNS antibody, the membrane was stripped and reprobed with an anti-actin antibody. (C) Cytotoxicity of asparaginase against the ALL cell lines REH and RS4;11 was measured in coculture with MSCs transduced with the target siRNA construct or the scrambled control construct. Cytotoxicity was measured after 48 hours of exposure to 1 IU/ml and 0.001 IU/ml asparaginase for REH and RS4;11, respectively. Each symbol indicates the results of 1 experiment (mean of 2 measurements). Bars correspond to the median of 8 experiments.
We next investigated whether enforced expression of ASNS in MSCs using a retroviral vector (Figure 4A) could augment their protective capacity. Although it did not produce changes in the growth rate or appearance of MSCs, stable ASNS overexpression significantly enhanced the cells’ capacity to protect ALL cells from asparaginase cytotoxicity, as shown by experiments in which ALL cells were cultured on either MSCs overexpressing ASNS or cells transduced with a control vector lacking the ASNS gene (Figure 4B).
Upregulation of ASNS expression in MSCs increases their capacity to protect ALL cells from asparaginase cytotoxicity. (A) Expression of ASNS protein in _TERT_-immortalized MSCs transduced with an ASNS retroviral vector (ASNS) compared to that of cells transduced with an empty control vector (Mock) and that of nontransduced MSCs by Western blotting. After probing with the anti-ASNS antibody, the membrane was stripped and reprobed with an anti-actin antibody. (B) Cytotoxicity of asparaginase on the ALL cell lines REH and RS4;11 was measured in coculture with MSCs transduced with either ASNS or empty vector. Cytotoxicity was measured after 48 hours of exposure to 1 IU/ml and 0.001 IU/ml asparaginase for REH and RS4;11, respectively. Each symbol indicates the results of 1 experiment (mean of 2 measurements). Bars correspond to the median of 8 experiments.
The third line of evidence supporting our central hypothesis came from studies with MSC clones defined by different levels of endogenous ASNS expression. These clones were derived from single-cell sorting of the _TERT_-immortalized MSCs used in the previous experiments. After screening the clones’ ASNS expression, we identified one clone (referred to as clone B) that exhibited lower levels of ASNS (Figure 5A). Comparison of the protective capacity of this clone with clone A, whose ASNS expression level was similar to findings in unsorted MSCs, revealed a lower protective capacity for clone B (Figure 5B). We then asked whether increasing ASNS expression in clone B would restore its protective capacity. Transduction with the retroviral vector containing the ASNS construct described above led to robust and stable ASNS expression in the otherwise deficient MSCs (Figure 5A). Figure 5B shows the results of experiments comparing the protective capacity of the MSCs defective for endogenous ASNS after transduction with the _ASNS_-containing vector or an empty control vector: enforced expression of ASNS significantly improved the capacity of these MSCs to protect ALL cells from asparaginase cytotoxicity.
MSC clones with different levels of endogenous ASNS expression have different capacities to protect ALL cells from asparaginase cytotoxicity. (A) Different levels of expression of ASNS mRNA in _TERT_-immortalized unsorted MSCs and in 2 MSC clones (clones A and B) by semiquantitative RT-PCR. Expression of ASNS transcripts in clone B after retroviral transduction of ASNS or empty vector is also shown. β-Actin was used as a control. (B) Cytotoxicity of asparaginase against the ALL cell lines REH and RS4;11 was measured in coculture with MSCs with different levels of ASNS expression. Cytotoxicity was measured after 48 hours of exposure to 1 IU/ml and 0.001 IU/ml asparaginase for REH and RS4;11, respectively. Each symbol indicates the results of 1 experiment (mean of 2 measurements). Bars correspond to the median of 8 experiments.
Asparagine secretion by MSCs is determined by their expression of ASNS. Results of the previous experiments indicated that the mechanism underlying the protective capacity of MSCs relies on their high levels of ASNS expression. Since ASNS catalyzes asparagine biosynthesis, we postulated that MSCs mitigate the effect of asparaginase through the secretion of asparagine. In experiments with the RS4;11 cell line, addition of asparagine to the culture medium decreased the sensitivity of leukemic cells to asparaginase (Figure 6A). Measurement of the production of asparagine by MSCs showed that these cells do indeed secrete this amino acid in their milieu. Moreover, levels of asparagine in culture supernatants collected from MSCs after 24 hours of culture were directly related to levels of ASNS expression in the cells: in supernatants of MSCs treated with the siRNA target sequence, the amino acid was nearly undetectable (Figure 6B). In contrast, secretion of serine, glycine, and valine was not reduced by ASNS siRNA treatment (Figure 6B). In accord with these results, we found that culturing RS4;11 on a microporous membrane that prevents contact with MSCs but allows free flow of soluble factors also protected the cells from asparaginase when cultures were performed in the presence of MSCs (Figure 6C). Likewise, the addition of MSC-conditioned medium to cultures of RS4;11 cells reduced asparaginase cytotoxicity, whereas the addition of a mixture of MSC-derived cytokines — IL-1α, IL-1β, IL-3, IL-6, IL-7, IL-11, stem cell factor, and Fms-like tyrosine kinase 3 ligand — instead of MSC-conditioned medium had no effect (Figure 6C).
The protective effects of MSCs against asparaginase cytotoxicity are mediated by asparagine biosynthesis. (A) RS4;11 ALL cells were cultured for 48 hours in the presence of asparaginase (0.001 IU/ml). The effect of adding increasing concentrations of asparagine to the tissue culture medium are shown. Values are mean and SD of 4 measurements. (B) Concentration of asparagine in asparagine-free, FCS-free tissue culture medium (MEM) was measured after 24 hours of culture with MSCs expressing different levels of ASNS. Compared are MSCs with downregulated ASNS by siRNA and MSCs transduced with a scrambled sequence as well as MSCs with upregulated ASNS by retroviral transduction and MSCs transduced with an empty vector. Levels of control amino acids serine, glycine, and valine are also shown. (C) RS4;11 cells were cultured either in the absence of MSCs (Plastic), in direct contact with MSCs, in Transwell inserts suspended over MSCs (no MSC contact), in MSC-conditioned medium (collected after 48 hours of culture; MSC Sup) or with a mixture of MSC-derived cytokines (IL-1α, IL-1β, IL-3, IL-6, IL-7, IL-11, stem cell factor, and Fms-like tyrosine kinase 3 ligand; Cyto). Cytotoxicity was measured after 48 hours of exposure to 0.001 IU/ml asparaginase. Values are mean and SD of 4 measurements.
Effect of MSCs on the sensitivity of primary ALL cells to asparaginase. We next tested whether the ASNS-dependent protective effects of MSCs on leukemic cell lines extend to primary ALL cells. In 28 of the 35 primary B-lineage ALL cases that we studied, asparaginase cytotoxicity when leukemic cells were cultured on MSCs with low ASNS expression levels due to siRNA treatment was significantly higher than that of cells transduced with a scrambled control construct (P < 0.05; Figure 7A). Conversely, in 31 of the 35 cases, cytotoxicity was significantly decreased when the leukemic cells were cultured on MSCs overexpressing ASNS after retroviral transduction compared with cells transduced with an empty vector. These results indicate that ASNS expression in MSCs is an important determinant of the asparaginase sensitivity of primary ALL cells.
Expression of ASNS in MSCs determines the susceptibility of primary ALL cells to asparaginase. (A) Primary ALL cells, obtained from the diagnostic bone marrow of 35 children with ALL, were exposed for 48 hours to asparaginase (1.0 IU/ml) in the presence of MSCs with ASNS expression downregulated by siRNA or upregulated by ASNS transduction. Results were compared to those of parallel cultures using MSCs transduced with a scrambled siRNA construct or empty vector, respectively. Values are mean ± SD cell killing obtained in cultures with each MSC type relative to that of its corresponding control (indicated by the dashed line). Differences between the test and control cultures were significant (P < 0.05, Student’s t test) in all samples except 1–6 and 23 in cultures with MSCs underexpressing ASNS and 5 and 14–16 in cultures with MSCs overexpressing ASNS. (B) The mean cytotoxicity ratio obtained in cultures with MSCs overexpressing ASNS were subtracted from those with MSCs underexpressing ASNS to derive a protection index. Figure shows the relation between this protection index and genetic subtype in the 35 ALL samples. P = 0.068, hyperdiploid cases; *Cases lacking known genetic abnormalities, P = 0.023; Wilcoxon rank-sum test. (C) Relationship between protection index and ASNS transcript expression in ALL cells by real-time PCR. _r_2 by regression analysis is shown.
Using the combined data from the above experiments, we derived a MSC protection index in each case by subtracting the mean cytotoxicity ratio obtained in cultures with MSCs with upregulated ASNS expression after transduction with the ASNS vector from that of MSC cultures in which ASNS expression was downregulated by siRNA. Figure 7B shows the relation between this index and the genetic subclassification of the 35 ALL cases studied. Of note, 4 of the 5 hyperdiploid (>50 chromosomes) cases studied had an index below the median (P = 0.068, Wilcoxon rank-sum test), whereas 7 of 9 cases lacking known genetic abnormalities had an index above the median (P = 0.023). Susceptibility to the protective effects of MSCs was unrelated to the leukemic cells’ immunophenotypic classification (early pre-B, pre-B, or transitional B; data not shown) or to the levels of ASNS expression in ALL cells as measured by gene expression array in 28 cases and by real-time PCR in all 35 cases (Figure 7C). We also assessed whether expression of amino acid transporter A1 and A2, 2 molecules that have previously been shown to participate in asparagine transport (34, 35), was related to ALL cell responses to the protective effect of MSCs. Transcripts encoding both molecules were detectable by microarray in all 28 cases studied, but there was no relation to leukemic cell responses to MSC protection (data not shown).