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Proc Natl Acad Sci U S A. Jul 3, 2007; 104(27): 11406–11411.
Published online Jun 18, 2007. doi:  10.1073/pnas.0610477104
PMCID: PMC1892786
Medical Sciences

Lenalidomide inhibits the malignant clone and up-regulates the SPARC gene mapping to the commonly deleted region in 5q− syndrome patients

Abstract

Myelodysplastic syndromes (MDSs) are a group of hematopoietic stem cell disorders characterized by ineffective hematopoiesis and peripheral blood cytopenias. Lenalidomide has dramatic therapeutic effects in patients with low-risk MDS and a chromosome 5q31 deletion, resulting in complete cytogenetic remission in >60% of patients. The molecular basis of this remarkable drug response is unknown. To gain insight into the molecular targets of lenalidomide we investigated its in vitro effects on growth, maturation, and global gene expression in isolated erythroblast cultures from MDS patients with del(5)(q31). Lenalidomide inhibited growth of differentiating del(5q) erythroblasts but did not affect cytogenetically normal cells. Moreover, lenalidomide significantly influenced the pattern of gene expression in del(5q) intermediate erythroblasts, with the VSIG4, PPIC, TPBG, activin A, and SPARC genes up-regulated by >2-fold in all samples and many genes involved in erythropoiesis, including HBA2, GYPA, and KLF1, down-regulated in most samples. Activin A, one of the most significant differentially expressed genes between lenalidomide-treated cells from MDS patients and healthy controls, has pleiotropic functions, including apoptosis of hematopoietic cells. Up-regulation and increased protein expression of the tumor suppressor gene SPARC is of particular interest because it is antiproliferative, antiadhesive, and antiangiogenic and is located at 5q31-q32, within the commonly deleted region in MDS 5q− syndrome. We conclude that lenalidomide inhibits growth of del(5q) erythroid progenitors and that the up-regulation of SPARC and activin A may underlie the potent effects of lenalidomide in MDS with del(5)(q31). SPARC may play a role in the pathogenesis of the 5q− syndrome.

Keywords: gene expression profiling, myelodysplastic syndromes, microarray, erythropoiesis, osteonectin

The myelodysplastic syndromes (MDSs) are a heterogeneous group of hematopoietic malignancies characterized by blood cytopenias, ineffective hematopoiesis, and a hypercellular bone marrow (BM) (1). The MDSs are preleukemic conditions in which transformation into acute myeloid leukemia (AML) occurs in ≈30–40% of cases (1). Unless an allogeneic stem cell transplantation can be offered, MDS is generally considered to be an incurable condition, and responses to chemotherapy are infrequent and short-lasting.

The 5q− syndrome is the most distinct entity among MDSs (2, 3) with a strong genotype–phenotype relationship (4). Lenalidomide (Revlimid, CC-5013) has recently emerged as the drug of choice for treatment of this patient category, as well as for other low-risk MDS patients with del(5q) (5, 6). The initial phase I/II study showed that treatment with lenalidomide abrogated the transfusion need in 83% of low-risk MDS patients with del(5q) and that cytogenetic remissions were frequently observed (5). Recently, a large phase II follow-up study of 148 patients confirmed these promising results, showing erythroid responses and transfusion independency in 76% and 67% of the patients, respectively, and a complete cytogenetic remission in 45% of the patients (6).

Lenalidomide and other immunomodulatory drugs have diverse mechanisms of action with implications in cancer (7), including inhibition of angiogenesis (8, 9) and cell adhesion (10, 11), modulation of cytokines (12, 13), growth inhibition and induction of apoptosis (14, 15), and stimulation of T cells and NK cells (1618). However, the precise mode of action and molecular targets of lenalidomide in MDS are unknown.

The del(5q) is the most common chromosomal abnormality in MDS, occurring at a frequency of 10–15% (19, 20). Del(5q) also occurs in AML (20) and in several other cancers (2123). The del(5q) aberration is associated with a favorable outcome when it occurs as an isolated cytogenetic aberration in low-risk MDS (5q− syndrome) (4, 24). In high-risk MDS and de novo AML, however, a del(5q) is associated with a poor prognosis (24). There is compelling evidence to suggest that the cell of origin in 5q− syndrome is a pluripotent hematopoietic stem cell and that 5q deletions represent an early event in MDS pathogenesis (25, 26).

The commonly deleted region (CDR) or critical region of gene loss of the 5q− syndrome, identified by Boultwood and colleagues (27, 28), is a 1.5-megabase region at 5q31–q32 flanked by the marker D5S413 and the GLRA1 gene. The CDR contains 44 genes (27), including several that are known to act as tumor suppressors (24, 27), for example the secreted protein acidic and rich in cysteine (SPARC/osteonectin/BM-40) gene (27, 29).

The aim of this study was to investigate the direct effects of lenalidomide on isolated differentiating erythroblasts from MDS patients with del(5)(q31) and from healthy controls. Gene expression profiling was used to gain insight into the mode of action of lenalidomide and to identify the molecular targets of this drug.

Results

Lenalidomide Inhibits Growth of del(5q) Hematopoietic Progenitors From MDS Patients.

Lenalidomide, titrated up to doses of 500 μM, did not inhibit the proliferation of BM mononuclear cells from healthy subjects (n = 3) during 4 days of culture, as measured by using incorporation of [3H]thymidine (30) (data not shown). CD34+ cells isolated from the BM of 13 MDS del(5q) patients (Table 1) and 10 healthy controls were cultured by using a method developed to study the generation of erythroblasts (31). After CD34+ cell separation, at day 0 the median percentage of del(5q) cells as measured by FISH was 99% (range 81–99). During the first week of erythroblast culture, the proportion of del(5q) cells remained almost constant, whereas the proportion decreased during the second week because of an outgrowth of cytogenetically normal cells (data not shown). This outgrowth of normal cells was in line with previous findings from our group (32) and with observations from a clinical study (33). Next, we evaluated the specific effect of lenalidomide during days 0–14. By correlating the proliferation index to the proportion of 5q31-deleted cells as determined by FISH, we could estimate the proliferation of the normal cells and the malignant clone independently, as previously described (32). The addition of lenalidomide significantly inhibited the proliferation of cells carrying the 5q31 deletion (P = 0.04 at day 14) (Fig. 1A) but had no inhibitory effects on the proliferation of cells from healthy controls or on the cytogenetically normal cells in the MDS cultures (Fig. 1 B and C).

Table 1.
Characteristics of MDS patient cultures
Fig. 1.
Lenalidomide inhibits cell growth in MDS del(5q) cells. Erythroblast cultures were performed by using cells from MDS del(5q) patients (n = 13) and healthy controls (n = 10). (A–C) Increase of del(5q) cells (A) from MDS patients, cytogenetically ...

CD34+ cells from three non-del(5q) MDS patients (Table 1) were cultured to assess the effects of lenalidomide in these cells. Only one of three samples showed inhibition of cell growth by day 14 (falling within the 25th–75th percentile range of the inhibition seen in the 5q− cultures; data not shown). In the non-del(5q) patient with abnormal karyotype (trisomy 8), there was no difference in cell growth of the trisomy 8 clone (monitored by FISH; data not shown) compared with the normal cells without trisomy 8.

Furthermore, CD34 mononuclear cells (MNC) isolated from two MDS 5q− patients were cultured to assess the effects of lenalidomide on nonerythroid cells. At day 0, <1% of the cells were erythroblasts, as determined by morphology. In the cultures there was a clear inhibition of cell growth of the 5q− cells by day 7 (34% and 65%), markedly more pronounced compared with the cytogenetically normal cells in the same culture (calculated by using the proliferation index and FISH data from days 0 and 7, as described above).

Lenalidomide Reduces the Proportion of Erythroid Cells.

To determine the proportion of erythroid and myeloid cells, FACS analysis was performed on cultured cells at day 7 (MDS 5q−, n = 5; control, n = 3) and day 14 (MDS 5q−, n = 11; control, n = 9). The standard antibody panel included markers of early and late erythroid [CD36 and glycophorin-A (GPA), respectively], and myeloid (CD33) differentiation. At day 7, the CD34+ progenitors had differentiated into intermediate erythroblasts, as previously described (32, 34), and the lenalidomide-treated cells showed a phenotype similar to that of the untreated cells (Fig. 1 D and F). However, in the presence of Epo during the second week, the proportion of mature erythroid cells expressing GPA increased significantly more in cells derived from healthy controls than in cells from MDS del(5q) patients (P < 0.001; Fig. 1 E and G). Focusing on the effects of lenalidomide, treated MDS cells at day 14 showed less erythroid differentiation compared with untreated MDS cells (Fig. 1E).

Analysis of Accessory Cells in the Cultures.

Because lenalidomide has been suggested to act by means of its effect on the BM environment (7), we investigated the lineages of cells at day 14 (MDS 5q−, n = 5; control, n = 7) by applying a broader antibody panel including CD3 (T cells), CD19 (B cells), CD56 (NK cells), CD42b/CD61 (megakaryocytes), and CD14 (monocytes). No significant differences in the proportions of these cells were seen between cultured cells from MDS patients and healthy controls or between treated and untreated cells. The number of accessory cells was low. Three percent or fewer of cells were CD3-, CD19-, or CD56-positive. The median expression of CD42b/CD61 and CD14 was 0.9% (range 0.1–6.7) and 1.9% (range 0.2–10.1), respectively. Furthermore, no adherent cells were seen in the culture flasks at any time point. The broader antibody panel also was applied to a limited number of day 7 culture samples (MDS 5q−, n = 1; control, n = 2) with comparable results.

Lenalidomide Effects on Global Gene Expression Levels.

To identify molecular targets of lenalidomide, we next investigated the gene expression profiles of the lenalidomide-treated and untreated cultured erythroid progenitors from MDS del(5q) patients and from healthy controls. Gene expression profiling was performed on day 7 intermediate erythroblasts from MDS del(5q) patients (n = 9) and healthy controls (n = 8). At day 7, a median of 98% of the MDS cells still carried the 5q deletion; therefore, any observed differences in gene expression levels would be restricted to the clonal cells. For each gene, the expression changes induced by lenalidomide were obtained by paired analysis, comparing each lenalidomide-treated MDS or healthy control sample with the corresponding untreated sample.

Several genes were significantly down-regulated by lenalidomide, including genes involved in erythropoiesis, such as HBA2, HBB, SPTA1, GYPA, GYPB, ALAS2, and KLF1 [supporting information (SI) Table 2]. Many genes were significantly up-regulated by the treatment with lenalidomide, with four genes up-regulated by >2-fold in all MDS del(5q) and all healthy control samples analyzed: SPARC, VSIG4, PPIC, and TPBG (SI Table 2). SPARC showed an average 4.1-fold (range 2.4–8.1) up-regulation in the MDS patients and 4.8-fold (range 3.2–9.5) in the healthy controls (SI Table 2 and Fig. 2A).

Fig. 2.
Increase of SPARC gene expression by treatment with lenalidomide. (A) Expression levels of the SPARC gene in intermediate erythroblasts from day 7 of culture (MDS 5q−, n = 9; control, n = 8). (B) SPARC immunofluorescent staining of cytocentrifuged ...

Of the 44 genes mapping within the CDR of the 5q− syndrome, 41 were represented on the Affymetrix arrays, and SPARC was the only one whose expression levels were significantly increased with lenalidomide treatment (Fig. 2C).

Genes differentially expressed between lenalidomide-treated cells of MDS patients and those of healthy controls were identified by using B statistics. Activin A (B value = 2.66, adjusted P value = 0.047) was one of the most significant differentially expressed genes, with an average up-regulation of 4.4-fold (range 2.3–7.8) in the MDS patients and 1.9-fold (range 1.4–3.0) in the healthy controls.

Pathway analysis found the following pathways that contained genes significantly deregulated in response to lenalidomide: extracellular matrix (ECM) interactions (P = 0.0007), hematopoietic cell lineages (P = 0.0008), and the focal adhesion pathway (P = 0.004).

Expression Levels of SPARC and activin A in Non-del(5q) Cells and in CD34 MNC.

Real-time quantitative PCR was used to evaluate the expression levels of the SPARC and activin A genes in lenalidomide-treated and untreated cultured erythroid progenitors at day 7 from two MDS patients without a del(5q). Lenalidomide treatment increased the expression of both SPARC (9.3-fold and 5.5-fold compared with untreated) and activin A (12.6-fold and 4.3-fold compared with untreated) in these MDS patients.

To assess the effects of lenalidomide on SPARC and activin A expression in nonerythroid cells, real-time quantitative PCR was used to evaluate the expression levels of these genes in the lenalidomide-treated and untreated cultured CD34 MNC at days 2 and 7 from two MDS 5q− patients. At day 2, lenalidomide treatment increased the expression of both SPARC (8.1-fold and 3.1-fold compared with untreated) and activin A (2.5-fold and 1.9-fold compared with untreated) in the two samples. At day 7, lenalidomide treatment increased the expression of both SPARC (2.5-fold and 5.8-fold compared with untreated) and activin A (2.6-fold and 2.5-fold compared with untreated) in the two samples.

Confirmation of Gene Expression Data.

The microarray-generated gene expression data of the SPARC gene and of three other deregulated genes, VSIG4, LRP11, and activin A, were validated by using real-time quantitative PCR (Fig. 3). The concordance between the expression levels obtained with Affymetrix chips and with real-time quantitative PCR was high (correlation range 0.86–0.96), indicating a good level of agreement between the two assays. SPARC immunofluorescent staining of cytocentrifuged cells from day 7 of culture, corresponding to the cells analyzed with gene expression profiling, showed an increased expression of SPARC in the lenalidomide-treated samples (Fig. 2B).

Fig. 3.
Confirmation of gene expression data. Comparison of the expression ratios obtained from real-time quantitative PCR (open bars) and Affymetrix experiments (filled bars) for selected genes.

Discussion

The immunomodulatory drug lenalidomide has dramatic therapeutic effects in MDS patients with del(5)(q31) (5). We present evidence that lenalidomide inhibits growth of MDS del(5q) progenitors and does not affect the growth of normal cells. We investigated the gene expression profiles of the lenalidomide-treated and untreated cultured cells from MDS del(5q) patients and from healthy controls to identify molecular targets of this drug. Lenalidomide significantly affected the expression of several important genes, including the tumor suppressor gene SPARC.

Lenalidomide had no inhibitory effect on normal CD34+ progenitors or cytogenetically normal progenitors from MDS del(5q) BM. In contrast, lenalidomide significantly inhibited growth of the erythroblast del(5q) clone and nonerythroid CD34 MNC with del(5q). Enhanced sensitivity of cells with the del(5q) to lenalidomide also has been observed in certain cell lines, in which the effects are mainly restricted to growth inhibition and cell cycle arrest (35). Most recently, Verhelle et al. (36) showed that lenalidomide inhibits the proliferation of malignant B cells while expanding normal CD34+ progenitor cells. Lenalidomide did not have a general inhibitory effect on cells from other types of MDS, because only one of three non-del(5q) MDS erythroblast cultures was inhibited by lenalidomide. Moreover, lenalidomide did not inhibit trisomy 8 erythroblasts. These findings may have relevance to the clinical observation that MDS patients with del(5q) often develop severe neutropenia and thrombocytopenia when given doses of lenalidomide that are relatively nontoxic in patients with multiple myeloma or solid tumors (37). One explanation for this apparent toxicity could be that a median of 99% of the hematopoietic stem cells in MDS patients with del(5q) are part of the malignant clone (25). The potent inhibition of the del(5q) progenitor cells by lenalidomide, in combination with a prolonged time period to reconstitute the hematopoiesis with the limited number of normal hematopoietic stem cells left, could explain the transient peripheral cytopenias often observed after the use of this drug (5).

The proportion of mature GPA-positive erythroblasts at day 14 was higher in cells from healthy controls compared with cells from MDS patients. In the MDS del(5q) cultures, lenalidomide reduced the proportion of erythroid cells observed after the addition of Epo during the second week.

The identification of the molecular targets of drug treatments in hematological malignancies can shed light on the molecular basis of the disease. For example, the molecular basis of the hypereosinophilic syndrome was identified after this disorder was found to be responsive to imatinib mesylate (38). Microarray-based gene expression profiling is a powerful technology that can identify deregulated genes/gene pathways in cancer and after drug treatment.

Gene expression profiling experiments were performed at day 7, when a median of 98% of the MDS cells possessed the del(5q) as determined by FISH. Any observed differences in gene expression levels would therefore be restricted to the malignant cells. Many genes were deregulated as a result of addition of lenalidomide to the cultured cells. Lenalidomide consistently up-regulated four genes (VSIG4, PPIC, TPBG, and SPARC) in the cultured cells from all of the MDS patients and healthy controls studied. VSIG4 has been shown to be hypermethylated in gastric cancer (39) and is expressed in dendritic cells and activated macrophages (40, 41). PPIC (cyclophilin C) is a member of the cyclophilin family that plays a role in protein folding and binds the immunosuppressive drug cyclosporin A (4244). Interestingly, PPIC maps to 5q23 and is involved in the degradation of the genome during apoptosis (45). TPBG is a tumor-associated antigen and is considered a potential target for the immunotherapy of cancer (46). TPBG expression is associated with poor prognosis in gastric and colorectal cancer (47, 48).

The up-regulation of SPARC is of particular interest because of its location at 5q31-q32, within the CDR of the 5q− syndrome (27). The principal function of SPARC is the regulation of ECM interactions (49, 50). Interestingly the ECM interaction pathway was found to be the most significantly deregulated by lenalidomide in the present study. Genes up-regulated by lenalidomide in this pathway include collagen type 6 A3, laminin beta-2 and integrin beta-1.

In addition, SPARC is antiproliferative, antiadhesive, and antiangiogenic (50, 51), which are the recognized effects of immunomodulatory drugs (7, 11). SPARC is a potent antiangiogenic protein because it blocks VEGF- and FGF-2-induced proliferation of endothelial cells (52, 53). SPARC also functions as a tumor suppressor in several human cancers. For example, decreased SPARC expression has been described in many cancers, including ovarian cancer (54), and in primary leukemia cells and cell lines derived from AML patients with rearrangements involving the mixed lineage leukemia (MLL) gene (55). In vitro, SPARC inhibits growth of several cell lines, including AML–MLL cell lines (55).

SPARC has been shown to stimulate the TGF-β signaling pathway (56), and two genes in this pathway, activin A and activin A receptor, were found to be significantly deregulated in response to treatment with lenalidomide in MDS patient samples in the present study. The activins are known to have effects on many physiological processes including cell proliferation, cell death, differentiation, and immune responses (57). Activin A also has an antitumorigenic effect, inhibiting proliferation of cells from several human cancers (58, 59). Interestingly, p27KIP1 and GATA-1 are potential downstream molecules in activin A-induced differentiation and apoptosis pathways in chronic myelogenous leukemia cells (60). In view of the differential expression of activin A between MDS patients and healthy controls, activin A is a good candidate for a target gene whose functions may be directly or indirectly responsible for some of the hematological effects of lenalidomide in the treatment of MDS.

In the 5q− syndrome, no point mutation has yet been described in SPARC or any other of the 43 candidate genes mapping within the CDR, and it now seems probable that haploinsufficiency is the mechanism involved (24, 27, 61). The SPARC gene maps within the CDR of the 5q− syndrome; therefore patients with the 5q− syndrome have one copy of the gene deleted and one copy retained. The expression levels of the SPARC gene in the untreated intermediate erythroblasts from MDS del(5q) patients were ≈50% of those observed in untreated intermediate erythroblasts from healthy controls, consistent with a single allele loss. Three MDS del(5q) cases showed very low SPARC expression levels of <20%. The increased adhesive properties caused by low SPARC expression may lead to growth advantage and preferential proliferation of the 5q− clone in the BM of MDS patients. Importantly, of all of the 44 genes in the CDR, SPARC was the only one whose expression levels were significantly increased with lenalidomide treatment in cells from healthy controls and from MDS del(5q) patients. SPARC expression levels were also increased by lenalidomide in cells from MDS patients without the del(5q). These data suggest that lenalidomide treatment results in a similar pattern of SPARC expression in normal and MDS cells. However, the important difference is that the lenalidomide-induced increase of SPARC gene expression corrects a deficiency present only in the 5q− cells, making this a plausible mechanism of its action. Clearly lenalidomide acts directly or indirectly to cause transcriptional up-regulation of SPARC. Restoration of normal SPARC levels by lenalidomide may represent at least part of the explanation for the specific inhibition of del(5q) cells demonstrated by this study in vitro. Interestingly, although exogenous SPARC inhibits the growth of AML–MLL blasts by inhibiting cell cycle progression from G1 to S phase, it does not inhibit growth of normal hematopoietic progenitors (55), a finding concordant with our results demonstrating that lenalidomide has no inhibitory effect on normal progenitors.

Lenalidomide down-regulated several genes in cells from both MDS patients and healthy controls, including many genes involved in erythropoiesis, such as α- and β-globin, spectrin, glycophorin A and B, ALAS2, and KLF1.

We conclude that lenalidomide selectively inhibits growth of MDS del(5q) progenitors and significantly up-regulates the SPARC gene at 5q31-q32. The major effects of SPARC, such as growth inhibition, antiadhesion, and antiangiogenesis, mirror the known effects of lenalidomide. We suggest that modulation of SPARC gene expression plays a key role in the mechanism of action of lenalidomide in MDS with del(5q). The localization of the SPARC gene to the CDR of the 5q− syndrome is intriguing, and, in relation to the findings of the present study, we suggest that SPARC may play a role in the molecular pathogenesis of the 5q− syndrome.

Materials and Methods

Study Subjects.

BM samples were taken from 15 MDS patients with a karyotype involving del(5)(q31), three MDS patients with a karyotype not involving del(5)(q31), and 10 healthy individuals. Informed consent was obtained from all subjects, and the study was approved by the Ethical Committee for Research at the Karolinska Institutet. All MDS del(5q) patients had BM blasts below 5%. Patient characteristics are shown in Table 1.

Study Drug.

Lenalidomide (Celgene, Warren, NJ) was dissolved in 10% dimethyl sulfoxide. Stock solution was stored at −20°C. After being thawed, the stock solution was protected from light and kept at room temperature for a maximum of 2 weeks. On the basis of data on multiple myeloma (14) as well as unpublished data on AML cell lines and MDS (62), we used a 10 μM concentration of lenalidomide in our experiments. This concentration is similar to concentrations in subsequent studies (35, 63).

Cells and Cultures.

BM mononuclear cells were isolated by using Lymphoprep (Axis-Shield, Oslo, Norway) density gradient, and CD34+ progenitor cells were separated by using a MACS magnetic labeling system (Miltenyi Biotec, Bergisch Gladbach, Germany), according to the manufacturers' protocols. The purity of CD34+ cells isolated with this system was assessed previously and shown to be >95% (31).

The CD34+ cells were cultured according to a method developed to study the generation of erythroblasts (31). Briefly, CD34+ cells were cultured for 14 days in Iscove's medium (Sigma, Saint Louis, MI) supplemented with BIT 9500 serum substitute (StemCell Technologies, Vancouver, BC, Canada) plus 10 ng/ml recombinant human (rh)IL-3, 10 ng/ml rhIL-6, and 25 ng/ml rh-stem cell factor (SCF) (Biosource Europe, Nivelles, Belgium). Cells were cultured at a concentration of 0.1 × 106 cells per milliliter in two positions: (i) untreated and (ii) treatment with 10 μM lenalidomide. Medium, including cytokines and lenalidomide as above, was replenished every second day to maintain the same cell concentration. At day 7, there was a complete change of medium. Epo at 2 units/ml (Roche, Basel, Switzerland) was added during the second week.

The CD34 MNC isolated from two MDS del(5q) patients were cultured for 7 days at 0.5 × 106 cells/ml in RPMI medium 1640 GlutaMAX (GIBCO/BRL, Paisley, U.K.) with 10% FBS. Cells were cultured in the presence or absence of 10 μM lenalidomide. Medium containing lenalidomide was replenished every second day to maintain cells at the original concentration.

FISH.

Cells were cytocentrifuged at days 0, 7, and 14. The slides were pretreated with pepsin and fixed with formaldehyde/MgCl2. To detect deletions of 5q31, the LSI EGR1/D5S721, D5S23 Dual Color Probe (Abbott-Vysis, Downers Grove, IL) was used; LSI EGR1 detects deletions of 5q31, and LSI D5S721, D5S23 detects 5p15.2 and served as an internal control. Probes were applied as recommended by the manufacturer.

For the CD34 MNC isolated from two MDS del(5q) patients, FISH for 5q31 was performed as above at days 0 and 7. Morphology at day 0 was performed with May–Grünwald–Giemsa staining.

FACS.

FACS phenotyping was performed at day 14 and, if cell counts allowed, at day 7. Antibodies directed against the following surface markers were used: CD34 (Becton Dickinson, San Jose, CA), CD36 (Immunotech, Marseille, France), GPA (DAKO, Copenhagen, Denmark), CD13 (DAKO), CD33 (Becton Dickinson), CD3 (Becton Dickinson), CD19 (DAKO), CD56 (Becton Dickinson), CD14 (Becton Dickinson), CD42b (DAKO), and CD61 (DAKO). Analyses were performed with a FACSCalibur operating with the CellQuest Pro software (Becton Dickinson).

Statistical Analysis.

A Mann–Whitney U test was used to compare different groups regarding an increase of the cell counts or proportion of positive cells determined by FACS analysis. P < 0.05 was considered statistically significant.

Affymetrix Experiments.

Gene expression profiling was performed on cultured erythroid progenitors at day 7. Both untreated and lenalidomide-treated cells from MDS del(5q) patients (n = 9) and healthy controls (n = 8) were analyzed. Cultured cells were resuspended in TRIzol (Invitrogen), and total RNA was extracted according to the protocol supplied by the manufacturer. An aliquot of the RNA samples was conserved for evaluation of quality by using a Bioanalyzer 2100 (Agilent Technologies, Palo Alto, CA). For each sample, 50 ng of total RNA was amplified and labeled with the Two-Cycle cDNA Synthesis and the Two-Cycle Target Labeling and Control Reagent packages (Affymetrix, Santa Clara, CA) according to the manufacturer's recommendations. Ten micrograms of biotin-labeled fragmented cRNA was hybridized to GeneChip Human Genome U133 Plus 2.0 arrays (Affymetrix), covering >47,000 transcripts representing 39,000 human genes. Hybridization occurred at 45°C for 16 h in a Hybridization Oven 640 (Affymetrix). Chips were then washed and stained in a Fluidics Station 450 (Affymetrix) and scanned with a GeneChip Scanner 3000 (Affymetrix).

Microarray Data Analysis.

Cell intensity calculation and scaling was performed by using GeneChip operating software. Data analysis was performed by GeneSpring 7.3 (Agilent Technologies) and the R environment for statistical computing (64) using the Affy and Limma packages (65). Quality control was performed within the GeneChip operating software after scaling the signal intensities of all arrays to a target of 100. Scale factors, background levels, percentage of present calls, 3′/5′ GAPDH ratio, and intensities of spike hybridization controls were within the acceptable range for all samples. Affymetrix CEL files were preprocessed with Robust MultiChip Analysis (66). For each gene, the normalized intensity in each lenalidomide-treated MDS or healthy control sample was paired with the normalized intensity in the corresponding untreated sample and analyzed with Limma. Genes differentially expressed between lenalidomide-treated and untreated samples and between MDS patients and healthy controls also were identified by using Limma. Genes that were significantly differentially expressed (P < 0.001) between lenalidomide-treated and untreated MDS samples were mapped to the KEGG pathway database (Kyoto Encyclopedia of Genes and Genomes, available at www.genome.jp/kegg/pathway.html) by using DAVID Bioinformatic Resources (http://niaid.abcc.ncifcrf.gov). Pathway deregulation was confirmed by using a list of genes with P < 0.05.

Real-Time Quantitative PCR.

Real-time quantitative PCR was used to validate microarray expression data for selected genes (SPARC, VSIG4, LRP11, and activin A). In addition, real-time quantitative PCR was used to evaluate the expression levels of the SPARC and activin A genes in lenalidomide-treated and untreated cultured erythroid progenitors from two MDS patients without a del(5q) and in lenalidomide-treated and untreated cultured CD34 MNC from two MDS del(5q) patients. The expression level of the ABL1 gene was used to normalize for differences in input cDNA. Predeveloped TaqMan Assays were used (Assays-on-Demand; Applied Biosystems, Foster City, CA), and reactions were run on a LightCycler 480 real-time PCR system (Roche Diagnostics, Lewes, U.K.). Each sample was performed in triplicate, and a reverse-transcriptase negative control was tested to exclude any contaminating DNA amplification. The expression ratio between each lenalidomide-treated sample and the corresponding untreated sample was calculated by using the ΔΔCT method (67).

Immunofluorescent Staining of the SPARC Protein.

Cytocentrifuged cells were acquired from day 7 of culture, the same time point used for gene expression profiling. Samples from two MDS 5q− patients and two healthy controls were selected. Cells from the AML cell lines ME-1, KG1a, and MV4–11, with strong, intermediate, and low SPARC expression, respectively, were used as controls (55). The cells were fixed in 4% paraformaldehyde and permeabilized with 0.1% saponin containing 0.5% BSA. The cells were incubated for 60 min with an anti-SPARC antibody (clone ON1–1; Zymed Laboratories, South San Francisco, CA) at a concentration of 10 μg/ml, and subsequently for 30 min with an FITC-conjugated anti-IgG1 antibody (DAKO). Antibody incubation was performed in the presence of saponin and BSA in the concentrations listed above. The stained slides were treated with Vectashield mounting medium (Vector Laboratories, Peterborough, U.K.) containing DAPI for nuclear staining.

Supplementary Material

Supporting Table:

Acknowledgments

We thank all of the medical staff that voluntarily donated BM for this project and Åsa-Lena Dackland for expert assistance with FACS analyses. This work was supported by grants from the Swedish Cancer Society (to E.H.-L.) and the Leukaemia Research Fund of the United Kingdom (to A.P., H.C., J.S.W., and J.B.).

Abbreviations

AML
acute myeloid leukemia
CDR
commonly deleted region
ECM
extracellular matrix
MDS
myelodysplastic syndromes
MNC
mononuclear cells.

Footnotes

The authors declare no conflict of interest.

This article is a PNAS Direct Submission.

This article contains supporting information online at www.pnas.org/cgi/content/full/0610477104/DC1.

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