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

Andrea Pellagatti*, Martin Ja¨ dersten†, Ann-Mari Forsblom†, Helen Cattan*, Birger Christensson‡, Emma K. Emanuelsson†, Mats Merup†, Lars Nilsson§¶, Jan Samuelssonʈ, Birgitta Sander‡, James S. Wainscoat*, Jacqueline Boultwood*, and Eva Hellstro¨ m-Lindberg†**

*Leukaemia Research Fund Molecular Haematology Unit, Nuffield Department of Clinical Laboratory Sciences, John Radcliffe Hospital, Oxford OX3 9DU, United Kingdom; †Division of Hematology, Department of Medicine, Karolinska Institutet, SE-141 86 Stockholm, Sweden; ‡Department of Pathology, Karolinska University Hospital Huddinge, SE-141 86 Stockholm, Sweden; §Hematopoietic Stem Cell Laboratory, Lund Strategic Research Center for Stem Cell Biology and Cell Therapy, Lund University, SE-221 84 Lund, Sweden; ¶Department of Hematology, Lund University Hospital, SE-221 00 Lund, Sweden; and ʈDepartment of Medicine, South Hospital, SE-118 83 Stockholm, Sweden

Edited by George Klein, Karolinska Institutet, Stockholm, Sweden, and approved May 7, 2007 (received for review November 27, 2006) Myelodysplastic syndromes (MDSs) are a group of hematopoietic a large phase II follow-up study of 148 patients confirmed these stem cell disorders characterized by ineffective hematopoiesis and promising results, showing erythroid responses and transfusion peripheral blood cytopenias. Lenalidomide has dramatic therapeu- independency in 76% and 67% of the patients, respectively, and a tic effects in patients with low-risk MDS and a 5q31 complete cytogenetic remission in 45% of the patients (6). deletion, resulting in complete cytogenetic remission in >60% of Lenalidomide and other immunomodulatory drugs have diverse patients. The molecular basis of this remarkable drug response is mechanisms of action with implications in (7), including unknown. To gain insight into the molecular targets of lenalido- inhibition of angiogenesis (8, 9) and cell adhesion (10, 11), modu- mide we investigated its in vitro effects on growth, maturation, lation of cytokines (12, 13), growth inhibition and induction of and global in isolated erythroblast cultures from (14, 15), and stimulation of T cells and NK cells (16–18). MDS patients with del(5)(q31). Lenalidomide inhibited growth of However, the precise mode of action and molecular targets of differentiating del(5q) erythroblasts but did not affect cytogenet- lenalidomide in MDS are unknown. ically normal cells. Moreover, lenalidomide significantly influenced The del(5q) is the most common chromosomal abnormality in the pattern of gene expression in del(5q) intermediate erythro- MDS, occurring at a frequency of 10–15% (19, 20). Del(5q) also blasts, with the VSIG4, PPIC, TPBG, activin A, and SPARC occurs in AML (20) and in several other (21–23). The up-regulated by >2-fold in all samples and many genes involved in del(5q) aberration is associated with a favorable outcome when it erythropoiesis, including HBA2, GYPA, and KLF1, down-regulated Ϫ in most samples. Activin A, one of the most significant differen- occurs as an isolated cytogenetic aberration in low-risk MDS (5q tially expressed genes between lenalidomide-treated cells from syndrome) (4, 24). In high-risk MDS and de novo AML, however, MDS patients and healthy controls, has pleiotropic functions, a del(5q) is associated with a poor prognosis (24). There is Ϫ including apoptosis of hematopoietic cells. Up-regulation and compelling evidence to suggest that the cell of origin in 5q increased protein expression of the tumor suppressor gene SPARC syndrome is a pluripotent hematopoietic stem cell and that 5q is of particular interest because it is antiproliferative, antiadhesive, deletions represent an early event in MDS pathogenesis (25, 26). and antiangiogenic and is located at 5q31-q32, within the com- The commonly deleted region (CDR) or critical region of gene monly deleted region in MDS 5q؊ syndrome. We conclude that loss of the 5qϪ syndrome, identified by Boultwood and colleagues lenalidomide inhibits growth of del(5q) erythroid progenitors and (27, 28), is a 1.5-megabase region at 5q31–q32 flanked by the that the up-regulation of SPARC and activin A may underlie the marker D5S413 and the GLRA1 gene. The CDR contains 44 genes potent effects of lenalidomide in MDS with del(5)(q31). SPARC may (27), including several that are known to act as tumor suppressors play a role in the pathogenesis of the 5q؊ syndrome. (24, 27), for example the secreted protein acidic and rich in cysteine (SPARC/osteonectin/BM-40) gene (27, 29). gene expression profiling ͉ myelodysplastic syndromes ͉ microarray ͉ The aim of this study was to investigate the direct effects of erythropoiesis ͉ osteonectin lenalidomide on isolated differentiating erythroblasts from MDS patients with del(5)(q31) and from healthy controls. Gene expres- he myelodysplastic syndromes (MDSs) are a heterogeneous sion profiling was used to gain insight into the mode of action of Tgroup of hematopoietic malignancies characterized by blood lenalidomide and to identify the molecular targets of this drug. 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 Ϸ Author contributions: A.P. and M.J. contributed equally to this work; J.S.W., J.B., and E.H.-L. 30–40% of cases (1). Unless an allogeneic stem cell transplan- designed research; A.P., M.J., A.-M.F., B.C., E.K.E., M.M., L.N., J.S., and B.S. performed tation can be offered, MDS is generally considered to be an research; A.P., M.J., H.C., J.S.W., J.B., and E.H.-L. analyzed data; and A.P., M.J., J.S.W., J.B., incurable condition, and responses to chemotherapy are infrequent and E.H.-L. wrote the paper. and short-lasting. The authors declare no conflict of interest. The 5qϪ syndrome is the most distinct entity among MDSs (2, This article is a PNAS Direct Submission. 3) with a strong genotype–phenotype relationship (4). Lenalido- Freely available online through the PNAS open access option. mide (Revlimid, CC-5013) has recently emerged as the drug of Abbreviations: AML, acute myeloid leukemia; CDR, commonly deleted region; ECM, extra- choice for treatment of this patient category, as well as for other cellular matrix; MDS, myelodysplastic syndromes; MNC, mononuclear cells. low-risk MDS patients with del(5q) (5, 6). The initial phase I/II **To whom correspondence should be addressed: E-mail: [email protected]. study showed that treatment with lenalidomide abrogated the This article contains supporting information online at www.pnas.org/cgi/content/full/ transfusion need in 83% of low-risk MDS patients with del(5q) and 0610477104/DC1. that cytogenetic remissions were frequently observed (5). Recently, © 2007 by The National Academy of Sciences of the USA

11406–11411 ͉ PNAS ͉ July 3, 2007 ͉ vol. 104 ͉ no. 27 www.pnas.org͞cgi͞doi͞10.1073͞pnas.0610477104 Downloaded by guest on October 1, 2021 Table 1. Characteristics of MDS patient cultures Patient no. Age, yr Sex FAB WHO Transfused Karyotype

del(5q) erythroblast MDS 19 58 F RA 5qϪ Yes 46,XX,del(5)(q13q33) MDS 25 82 F RA 5qϪ Yes 46,XX,del(5)(q13q33) MDS 29 92 F RA RCMD Yes 46,XX,del(5)(q13q33),del(11)(q14) MDS 32 39 F RA 5qϪ Yes 46,XX,del(5)(q13q33) MDS 38 76 F RA 5qϪ Yes 46,XX,del(5)(q13q33) MDS 41 83 F RA 5qϪ Yes 46,XX,del(5)(q13q33) MDS 43 71 F RA 5qϪ Yes 46,XX,del(5)(q15) MDS 50 75 F RA 5qϪ Yes 46,XX,del(5)(q13q33) MDS 76 84 M RA RA Yes 46,XY,del(5)(q13q33),i(21)(q10) MDS 83 80 F RA RCMD Yes 46,XX,del(5)(q13q33),del(11)(q13) MDS 88 77 F RA RCMD Yes 46,XX,del(5)(q14q34),t(11;16)(p13;q23),del(13)(q13q21) MDS 89 84 M RA CMML Yes 46,XY,del(5)(q14q34) MDS 92 76 F RA 5qϪ No 46,XX,del(5)(q13q33) non-del(5q) erythroblast MDS 21 68 M RAEB RAEB I Yes 47XY Tri 8 MDS 27 75 F RAEB RAEB I Yes 46XX MDS 74 70 F RARS RCMD-RS Yes 46XX del(5q) CD34Ϫ MNC MDS 22 81 M RA RA Yes 46XY 5q- (breakpoint includes 5q31), 20q- MDS 29 92 F RA RCMD Yes 46,XX,del(5)(q13q33),del(11)(q14)

FAB, French–American–British classification; WHO, World Health Organization classification; F, femaile; RA, refractory anemia; M, male; RCMD, refractory anemia with multilineage dysplasia; CMML, chronic myelomonocytic leukemia; RAEB, refractory anemia with excess blasts; RARS, refractory anemia with ringed sideroblasts.

Results The addition of lenalidomide significantly inhibited the prolifera- ϭ MEDICAL SCIENCES Lenalidomide Inhibits Growth of del(5q) Hematopoietic Progenitors tion of cells carrying the 5q31 deletion (P 0.04 at day 14) (Fig. 1A) From MDS Patients. Lenalidomide, titrated up to doses of 500 ␮M, but had no inhibitory effects on the proliferation of cells from did not inhibit the proliferation of BM mononuclear cells from healthy controls or on the cytogenetically normal cells in the MDS ϭ cultures (Fig. 1 B and C). healthy subjects (n 3) during 4 days of culture, as measured by ϩ using incorporation of [3H]thymidine (30) (data not shown). CD34 cells from three non-del(5q) MDS patients (Table 1) CD34ϩ cells isolated from the BM of 13 MDS del(5q) patients were cultured to assess the effects of lenalidomide in these cells. Only one of three samples showed inhibition of cell growth by day (Table 1) and 10 healthy controls were cultured by using a method 14 (falling within the 25th–75th percentile range of the inhibition developed to study the generation of erythroblasts (31). After ϩ seen in the 5qϪ cultures; data not shown). In the non-del(5q) CD34 cell separation, at day 0 the median percentage of del(5q) patient with abnormal karyotype (trisomy 8), there was no differ- cells as measured by FISH was 99% (range 81–99). During the first ence in cell growth of the trisomy 8 clone (monitored by FISH; data week of erythroblast culture, the proportion of del(5q) cells re- not shown) compared with the normal cells without trisomy 8. mained almost constant, whereas the proportion decreased during Furthermore, CD34Ϫ mononuclear cells (MNC) isolated from the second week because of an outgrowth of cytogenetically normal two MDS 5qϪ patients were cultured to assess the effects of cells (data not shown). This outgrowth of normal cells was in line lenalidomide on nonerythroid cells. At day 0, Ͻ1% of the cells were with previous findings from our group (32) and with observations erythroblasts, as determined by morphology. In the cultures there from a clinical study (33). Next, we evaluated the specific effect of was a clear inhibition of cell growth of the 5qϪ cells by day 7 (34% lenalidomide during days 0–14. By correlating the proliferation and 65%), markedly more pronounced compared with the cytoge- index to the proportion of 5q31-deleted cells as determined by netically normal cells in the same culture (calculated by using the FISH, we could estimate the proliferation of the normal cells and proliferation index and FISH data from days 0 and 7, as described the malignant clone independently, as previously described (32). above).

Fig. 1. Lenalidomide inhibits cell growth in MDS del(5q) cells. Erythroblast cultures were performed by using cells from MDS del(5q) patients (n ϭ 13) and healthy controls (n ϭ 10). (A–C) Increase of del(5q) cells (A) from MDS patients, cytogenetically normal cells from MDS del(5q) patients (B), and cells from healthy controls (C). Significant inhibition of cell expansion was seen in del(5q) cells (P ϭ 0.04 at day 14), whereas no effect was seen in normal cells. (D and E) Flow cytometry analysis of cultured cells from MDS 5qϪ patients at day 7 (n ϭ 5) (D) and day 14 (n ϭ 11) (E) showed that, during the second week after the addi- tion of erythropoietin, lenalidomide decreased the generation of erythroid cells (expressing CD36 and GPA) and thereby increased the proportion of myeloid cells (expressing CD33). (F and G) The phenotypes of cells from healthy controls at day 7 (n ϭ 3) (F) and day 14 (n ϭ 9) (G) were similar in treated and untreated samples. At day 14, the proportion of mature erythroblast expressing GPA was higher in cultured cells from healthy controls compared with cells from MDS del(5q) patients (P Ͻ 0.001). The boxes represent the 25th, 50th, and 75th percentiles; bars correspond to the 10th and 90th percentiles; and all samples below the 10th or above the 90th percentiles are shown as points.

Pellagatti et al. PNAS ͉ July 3, 2007 ͉ vol. 104 ͉ no. 27 ͉ 11407 Downloaded by guest on October 1, 2021 Lenalidomide Reduces the Proportion of Erythroid Cells. To deter- mine the proportion of erythroid and myeloid cells, FACS analysis was performed on cultured cells at day 7 (MDS 5qϪ, n ϭ 5; control, n ϭ 3) and day 14 (MDS 5qϪ, n ϭ 11; control, n ϭ 9). The standard panel included markers of early and late erythroid [CD36 and glycophorin-A (GPA), respectively], and myeloid (CD33) differentiation. At day 7, the CD34ϩ 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 Ͻ 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). Fig. 2. Increase of SPARC gene expression by treatment with lenalidomide. (A) Expression levels of the SPARC gene in intermediate erythroblasts from day Analysis of Accessory Cells in the Cultures. Because lenalidomide has 7 of culture (MDS 5qϪ, n ϭ 9; control, n ϭ 8). (B) SPARC immunofluorescent been suggested to act by means of its effect on the BM environment staining of cytocentrifuged MDS del(5q) cells from day 7 of culture, corre- (7), we investigated the lineages of cells at day 14 (MDS 5qϪ, n ϭ sponding to the cells analyzed with gene expression profiling. Nuclei are 5; control, n ϭ 7) by applying a broader antibody panel including stained with DAPI. (C) Effects of lenalidomide treatment on the expression CD3 (T cells), CD19 (B cells), CD56 (NK cells), CD42b/CD61 levels of the genes mapping to the CDR of the 5qϪ syndrome (41 of 44 are Ϫ (megakaryocytes), and CD14 (monocytes). No significant differ- represented on the Affymetrix arrays) in the 5q erythroblasts. The average ences in the proportions of these cells were seen between cultured proportional change after lenalidomide treatment is shown. The SPARC gene is highlighted in red. 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 genes significantly deregulated in response to lenalidomide: extra- median expression of CD42b/CD61 and CD14 was 0.9% (range cellular matrix (ECM) interactions (P ϭ 0.0007), hematopoietic cell 0.1–6.7) and 1.9% (range 0.2–10.1), respectively. Furthermore, no lineages (P ϭ 0.0008), and the focal adhesion pathway (P ϭ 0.004). 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 Expression Levels of SPARC and activin A in Non-del(5q) Cells and in Ϫ ϭ ϭ culture samples (MDS 5q , n 1; control, n 2) with CD34؊ MNC. Real-time quantitative PCR was used to evaluate the 7 comparable results. expression levels of the SPARC and activin A genes in lenalido- mide-treated and untreated cultured erythroid progenitors at day Lenalidomide Effects on Global Gene Expression Levels. To identify 7 from two MDS patients without a del(5q). Lenalidomide treat- molecular targets of lenalidomide, we next investigated the gene ment increased the expression of both SPARC (9.3-fold and 5.5-fold expression profiles of the lenalidomide-treated and untreated cul- compared with untreated) and activin A (12.6-fold and 4.3-fold tured erythroid progenitors from MDS del(5q) patients and from compared with untreated) in these MDS patients. healthy controls. Gene expression profiling was performed on day To assess the effects of lenalidomide on SPARC and activin A 7 intermediate erythroblasts from MDS del(5q) patients (n ϭ 9) expression in nonerythroid cells, real-time quantitative PCR was and healthy controls (n ϭ 8). At day 7, a median of 98% of the MDS used to evaluate the expression levels of these genes in the cells still carried the 5q deletion; therefore, any observed differ- lenalidomide-treated and untreated cultured CD34Ϫ MNC at days ences in gene expression levels would be restricted to the clonal Ϫ cells. For each gene, the expression changes induced by lenalido- 2 and 7 from two MDS 5q patients. At day 2, lenalidomide mide were obtained by paired analysis, comparing each lenalido- treatment increased the expression of both SPARC (8.1-fold and mide-treated MDS or healthy control sample with the correspond- 3.1-fold compared with untreated) and activin A (2.5-fold and ing untreated sample. 1.9-fold compared with untreated) in the two samples. At day 7, Several genes were significantly down-regulated by lenalidomide, lenalidomide treatment increased the expression of both SPARC including genes involved in erythropoiesis, such as HBA2, HBB, (2.5-fold and 5.8-fold compared with untreated) and activin A SPTA1, GYPA, GYPB, ALAS2, and KLF1 [supporting information (2.6-fold and 2.5-fold compared with untreated) in the two samples. (SI) Table 2]. Many genes were significantly up-regulated by the treatment with lenalidomide, with four genes up-regulated by Confirmation of Gene Expression Data. The microarray-generated Ͼ2-fold in all MDS del(5q) and all healthy control samples ana- gene expression data of the SPARC gene and of three other lyzed: SPARC, VSIG4, PPIC, and TPBG (SI Table 2). SPARC deregulated genes, VSIG4, LRP11, and activin A, were validated by showed an average 4.1-fold (range 2.4–8.1) up-regulation in the using real-time quantitative PCR (Fig. 3). The concordance be- MDS patients and 4.8-fold (range 3.2–9.5) in the healthy controls tween the expression levels obtained with Affymetrix chips and with (SI Table 2 and Fig. 2A). real-time quantitative PCR was high (correlation range 0.86–0.96), Of the 44 genes mapping within the CDR of the 5qϪ syndrome, indicating a good level of agreement between the two assays. 41 were represented on the Affymetrix arrays, and SPARC was the SPARC immunofluorescent staining of cytocentrifuged cells from only one whose expression levels were significantly increased with day 7 of culture, corresponding to the cells analyzed with gene lenalidomide treatment (Fig. 2C). expression profiling, showed an increased expression of SPARC in Genes differentially expressed between lenalidomide-treated the lenalidomide-treated samples (Fig. 2B). cells of MDS patients and those of healthy controls were identified by using B statistics. Activin A (B value ϭ 2.66, adjusted P value ϭ Discussion 0.047) was one of the most significant differentially expressed genes, The immunomodulatory drug lenalidomide has dramatic thera- with an average up-regulation of 4.4-fold (range 2.3–7.8) in the peutic effects in MDS patients with del(5)(q31) (5). We present MDS patients and 1.9-fold (range 1.4–3.0) in the healthy controls. evidence that lenalidomide inhibits growth of MDS del(5q) pro- Pathway analysis found the following pathways that contained genitors and does not affect the growth of normal cells. We

11408 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0610477104 Pellagatti et al. Downloaded by guest on October 1, 2021 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 (cyclo- philin C) is a member of the family that plays a role in and binds the immunosuppressive drug cyclosporin A (42–44). Interestingly, PPIC maps to 5q23 and is involved in the degradation of the genome during apoptosis (45). TPBG is a tumor-associated and is considered a potential target for the of cancer (46). TPBG expression is associated with poor prognosis in gastric and (47, 48). The up-regulation of SPARC is of particular interest because of its location at 5q31-q32, within the CDR of the 5qϪ syndrome (27). The principal function of SPARC is the regulation of ECM interactions (49, 50). Interestingly the ECM interaction pathway Fig. 3. Confirmation of gene expression data. Comparison of the expression was found to be the most significantly deregulated by lenalidomide ratios obtained from real-time quantitative PCR (open bars) and Affymetrix in the present study. Genes up-regulated by lenalidomide in this experiments (filled bars) for selected genes. pathway include collagen type 6 A3, laminin beta-2 and integrin beta-1. In addition, SPARC is antiproliferative, antiadhesive, and anti- investigated the gene expression profiles of the lenalidomide- angiogenic (50, 51), which are the recognized effects of immuno- treated and untreated cultured cells from MDS del(5q) patients and modulatory drugs (7, 11). SPARC is a potent antiangiogenic from healthy controls to identify molecular targets of this drug. protein because it blocks VEGF- and FGF-2-induced proliferation Lenalidomide significantly affected the expression of several im- of endothelial cells (52, 53). SPARC also functions as a tumor portant genes, including the tumor suppressor gene SPARC. ϩ suppressor in several human cancers. For example, decreased Lenalidomide had no inhibitory effect on normal CD34 pro- SPARC expression has been described in many cancers, including genitors or cytogenetically normal progenitors from MDS del(5q) (54), and in primary leukemia cells and cell lines BM. In contrast, lenalidomide significantly inhibited growth of the MEDICAL SCIENCES Ϫ derived from AML patients with rearrangements involving the erythroblast del(5q) clone and nonerythroid CD34 MNC with mixed lineage leukemia (MLL) gene (55). In vitro, SPARC inhibits del(5q). Enhanced sensitivity of cells with the del(5q) to lenalido- growth of several cell lines, including AML–MLL cell lines (55). mide also has been observed in certain cell lines, in which the effects SPARC has been shown to stimulate the TGF-␤ signaling are mainly restricted to growth inhibition and cell cycle arrest (35). pathway (56), and two genes in this pathway, activin A and activin Most recently, Verhelle et al. (36) showed that lenalidomide inhibits A receptor, were found to be significantly deregulated in response the proliferation of malignant B cells while expanding normal ϩ to treatment with lenalidomide in MDS patient samples in the CD34 progenitor cells. Lenalidomide did not have a general present study. The activins are known to have effects on many inhibitory effect on cells from other types of MDS, because only one physiological processes including cell proliferation, cell death, dif- of three non-del(5q) MDS erythroblast cultures was inhibited by ferentiation, and immune responses (57). Activin A also has an lenalidomide. Moreover, lenalidomide did not inhibit trisomy 8 antitumorigenic effect, inhibiting proliferation of cells from several erythroblasts. These findings may have relevance to the clinical human cancers (58, 59). Interestingly, p27KIP1 and GATA-1 are observation that MDS patients with del(5q) often develop severe potential downstream molecules in activin A-induced differentia- neutropenia and thrombocytopenia when given doses of lenalido- tion and apoptosis pathways in chronic myelogenous leukemia cells mide that are relatively nontoxic in patients with multiple myeloma (60). In view of the differential expression of activin A between or solid tumors (37). One explanation for this apparent toxicity MDS patients and healthy controls, activin A is a good candidate could be that a median of 99% of the hematopoietic stem cells in for a target gene whose functions may be directly or indirectly MDS patients with del(5q) are part of the malignant clone (25). The responsible for some of the hematological effects of lenalidomide potent inhibition of the del(5q) progenitor cells by lenalidomide, in in the treatment of MDS. combination with a prolonged time period to reconstitute the In the 5qϪ syndrome, no point mutation has yet been described hematopoiesis with the limited number of normal hematopoietic in SPARC or any other of the 43 candidate genes mapping within stem cells left, could explain the transient peripheral cytopenias the CDR, and it now seems probable that haploinsufficiency is the often observed after the use of this drug (5). mechanism involved (24, 27, 61). The SPARC gene maps within the The proportion of mature GPA-positive erythroblasts at day 14 CDR of the 5qϪ syndrome; therefore patients with the 5qϪ was higher in cells from healthy controls compared with cells from syndrome have one copy of the gene deleted and one copy retained. MDS patients. In the MDS del(5q) cultures, lenalidomide reduced The expression levels of the SPARC gene in the untreated inter- the proportion of erythroid cells observed after the addition of Epo mediate erythroblasts from MDS del(5q) patients were Ϸ50% of during the second week. those observed in untreated intermediate erythroblasts from The identification of the molecular targets of drug treatments in healthy controls, consistent with a single allele loss. Three MDS hematological malignancies can shed light on the molecular basis of del(5q) cases showed very low SPARC expression levels of Ͻ20%. the disease. For example, the molecular basis of the hypereosino- The increased adhesive properties caused by low SPARC expression philic syndrome was identified after this disorder was found to be may lead to growth advantage and preferential proliferation of the responsive to imatinib mesylate (38). Microarray-based gene ex- 5qϪ clone in the BM of MDS patients. Importantly, of all of the 44 pression profiling is a powerful technology that can identify dereg- genes in the CDR, SPARC was the only one whose expression levels ulated genes/gene pathways in cancer and after drug treatment. were significantly increased with lenalidomide treatment in cells Gene expression profiling experiments were performed at day 7, from healthy controls and from MDS del(5q) patients. SPARC when a median of 98% of the MDS cells possessed the del(5q) as expression levels were also increased by lenalidomide in cells from determined by FISH. Any observed differences in gene expression MDS patients without the del(5q). These data suggest that lena- levels would therefore be restricted to the malignant cells. Many lidomide treatment results in a similar pattern of SPARC expression genes were deregulated as a result of addition of lenalidomide to the in normal and MDS cells. However, the important difference is that

Pellagatti et al. PNAS ͉ July 3, 2007 ͉ vol. 104 ͉ no. 27 ͉ 11409 Downloaded by guest on October 1, 2021 the lenalidomide-induced increase of SPARC gene expression were cultured in the presence or absence of 10 ␮M lenalidomide. corrects a deficiency present only in the 5qϪ cells, making this a Medium containing lenalidomide was replenished every second plausible mechanism of its action. Clearly lenalidomide acts directly day to maintain cells at the original concentration. or indirectly to cause transcriptional up-regulation of SPARC. Restoration of normal SPARC levels by lenalidomide may repre- FISH. Cells were cytocentrifuged at days 0, 7, and 14. The slides were sent at least part of the explanation for the specific inhibition of pretreated with pepsin and fixed with formaldehyde/MgCl2.To del(5q) cells demonstrated by this study in vitro. Interestingly, detect deletions of 5q31, the LSI EGR1/D5S721, D5S23 Dual Color although exogenous SPARC inhibits the growth of AML–MLL Probe (Abbott-Vysis, Downers Grove, IL) was used; LSI EGR1 blasts by inhibiting cell cycle progression from G1 to S phase, it does detects deletions of 5q31, and LSI D5S721, D5S23 detects 5p15.2 not inhibit growth of normal hematopoietic progenitors (55), a and served as an internal control. Probes were applied as recom- finding concordant with our results demonstrating that lenalido- mended by the manufacturer. mide has no inhibitory effect on normal progenitors. For the CD34Ϫ MNC isolated from two MDS del(5q) patients, Lenalidomide down-regulated several genes in cells from both FISH for 5q31 was performed as above at days 0 and 7. Morphology MDS patients and healthy controls, including many genes involved at day 0 was performed with May–Gru¨nwald–Giemsa staining. in erythropoiesis, such as ␣- and ␤-globin, spectrin, glycophorin A and B, ALAS2, and KLF1. FACS. FACS phenotyping was performed at day 14 and, if cell We conclude that lenalidomide selectively inhibits growth of counts allowed, at day 7. directed against the following MDS del(5q) progenitors and significantly up-regulates the SPARC surface markers were used: CD34 (Becton Dickinson, San Jose, gene at 5q31-q32. The major effects of SPARC, such as growth CA), CD36 (Immunotech, Marseille, France), GPA (DAKO, inhibition, antiadhesion, and antiangiogenesis, mirror the known Copenhagen, Denmark), CD13 (DAKO), CD33 (Becton Dickin- effects of lenalidomide. We suggest that modulation of SPARC son), CD3 (Becton Dickinson), CD19 (DAKO), CD56 (Becton gene expression plays a key role in the mechanism of action of Dickinson), CD14 (Becton Dickinson), CD42b (DAKO), and lenalidomide in MDS with del(5q). The localization of the SPARC CD61 (DAKO). Analyses were performed with a FACSCalibur gene to the CDR of the 5qϪ syndrome is intriguing, and, in relation operating with the CellQuest Pro software (Becton Dickinson). to the findings of the present study, we suggest that SPARC may play a role in the molecular pathogenesis of the 5qϪ syndrome. Statistical Analysis. A Mann–Whitney U test was used to compare different groups regarding an increase of the cell counts or pro- Materials and Methods portion of positive cells determined by FACS analysis. P Ͻ 0.05 was Study Subjects. BM samples were taken from 15 MDS patients with considered statistically significant. a karyotype involving del(5)(q31), three MDS patients with a karyotype not involving del(5)(q31), and 10 healthy individuals. Affymetrix Experiments. Gene expression profiling was performed Informed consent was obtained from all subjects, and the study was on cultured erythroid progenitors at day 7. Both untreated and approved by the Ethical Committee for Research at the Karolinska lenalidomide-treated cells from MDS del(5q) patients (n ϭ 9) and Institutet. All MDS del(5q) patients had BM blasts below 5%. healthy controls (n ϭ 8) were analyzed. Cultured cells were Patient characteristics are shown in Table 1. resuspended in TRIzol (Invitrogen), and total RNA was extracted according to the protocol supplied by the manufacturer. An aliquot Study Drug. Lenalidomide (Celgene, Warren, NJ) was dissolved in of the RNA samples was conserved for evaluation of quality by 10% dimethyl sulfoxide. Stock solution was stored at Ϫ20°C. After using a Bioanalyzer 2100 (Agilent Technologies, Palo Alto, CA). being thawed, the stock solution was protected from light and kept For each sample, 50 ng of total RNA was amplified and labeled with at room temperature for a maximum of 2 weeks. On the basis of the Two-Cycle cDNA Synthesis and the Two-Cycle Target Labeling data on multiple myeloma (14) as well as unpublished data on AML and Control Reagent packages (Affymetrix, Santa Clara, CA) cell lines and MDS (62), we used a 10 ␮M concentration of according to the manufacturer’s recommendations. Ten micro- lenalidomide in our experiments. This concentration is similar to grams of biotin-labeled fragmented cRNA was hybridized to Ge- concentrations in subsequent studies (35, 63). neChip U133 Plus 2.0 arrays (Affymetrix), cov- ering Ͼ47,000 transcripts representing 39,000 human genes. Cells and Cultures. BM mononuclear cells were isolated by using Hybridization occurred at 45°C for 16 h in a Hybridization Oven 640 Lymphoprep (Axis-Shield, Oslo, Norway) density gradient, and (Affymetrix). Chips were then washed and stained in a Fluidics CD34ϩ progenitor cells were separated by using a MACS magnetic Station 450 (Affymetrix) and scanned with a GeneChip Scanner labeling system (Miltenyi Biotec, Bergisch Gladbach, Germany), 3000 (Affymetrix). according to the manufacturers’ protocols. The purity of CD34ϩ cells isolated with this system was assessed previously and shown to Microarray Data Analysis. Cell intensity calculation and scaling was be Ͼ95% (31). performed by using GeneChip operating software. Data analysis The CD34ϩ cells were cultured according to a method developed was performed by GeneSpring 7.3 (Agilent Technologies) and the to study the generation of erythroblasts (31). Briefly, CD34ϩ cells R environment for statistical computing (64) using the Affy and were cultured for 14 days in Iscove’s medium (Sigma, Saint Louis, Limma packages (65). Quality control was performed within the MI) supplemented with BIT 9500 serum substitute (StemCell GeneChip operating software after scaling the signal intensities of Technologies, Vancouver, BC, Canada) plus 10 ng/ml recombinant all arrays to a target of 100. Scale factors, background levels, human (rh)IL-3, 10 ng/ml rhIL-6, and 25 ng/ml rh-stem cell factor percentage of present calls, 3Ј/5Ј GAPDH ratio, and intensities of (SCF) (Biosource Europe, Nivelles, Belgium). Cells were cultured spike hybridization controls were within the acceptable range for all at a concentration of 0.1 ϫ 106 cells per milliliter in two positions: samples. Affymetrix CEL files were preprocessed with Robust (i) untreated and (ii) treatment with 10 ␮M lenalidomide. Medium, MultiChip Analysis (66). For each gene, the normalized intensity in including cytokines and lenalidomide as above, was replenished each lenalidomide-treated MDS or healthy control sample was every second day to maintain the same cell concentration. At day paired with the normalized intensity in the corresponding untreated 7, there was a complete change of medium. Epo at 2 units/ml sample and analyzed with Limma. Genes differentially expressed (Roche, Basel, Switzerland) was added during the second week. between lenalidomide-treated and untreated samples and between The CD34Ϫ MNC isolated from two MDS del(5q) patients were MDS patients and healthy controls also were identified by using cultured for 7 days at 0.5 ϫ 106 cells/ml in RPMI medium 1640 Limma. Genes that were significantly differentially expressed (P Ͻ GlutaMAX (GIBCO/BRL, Paisley, U.K.) with 10% FBS. Cells 0.001) between lenalidomide-treated and untreated MDS samples

11410 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0610477104 Pellagatti et al. Downloaded by guest on October 1, 2021 were mapped to the KEGG pathway database (Kyoto Encyclopedia Immunofluorescent Staining of the SPARC Protein. Cytocentrifuged of Genes and Genomes, available at www.genome.jp/kegg/ cells were acquired from day 7 of culture, the same time point used pathway.html) by using DAVID Bioinformatic Resources (http:// for gene expression profiling. Samples from two MDS 5qϪ patients niaid.abcc.ncifcrf.gov). Pathway deregulation was confirmed by and two healthy controls were selected. Cells from the AML cell using a list of genes with P Ͻ 0.05. lines ME-1, KG1a, and MV4–11, with strong, intermediate, and low SPARC expression, respectively, were used as controls (55). The Real-Time Quantitative PCR. Real-time quantitative PCR was used to cells were fixed in 4% paraformaldehyde and permeabilized with validate microarray expression data for selected genes (SPARC, 0.1% saponin containing 0.5% BSA. The cells were incubated for VSIG4, LRP11, and activin A). In addition, real-time quantitative 60 min with an anti-SPARC antibody (clone ON1–1; Zymed PCR was used to evaluate the expression levels of the SPARC and Laboratories, South San Francisco, CA) at a concentration of 10 activin A genes in lenalidomide-treated and untreated cultured ␮g/ml, and subsequently for 30 min with an FITC-conjugated erythroid progenitors from two MDS patients without a del(5q) and in lenalidomide-treated and untreated cultured CD34Ϫ MNC anti-IgG1 antibody (DAKO). Antibody incubation was performed from two MDS del(5q) patients. The expression level of the ABL1 in the presence of saponin and BSA in the concentrations listed gene was used to normalize for differences in input cDNA. Pre- above. The stained slides were treated with Vectashield mounting developed TaqMan Assays were used (Assays-on-Demand; Ap- medium (Vector Laboratories, Peterborough, U.K.) containing plied Biosystems, Foster City, CA), and reactions were run on a DAPI for nuclear staining. LightCycler 480 real-time PCR system (Roche Diagnostics, Lewes, U.K.). Each sample was performed in triplicate, and a reverse- We thank all of the medical staff that voluntarily donated BM for this transcriptase negative control was tested to exclude any contami- project and Åsa-Lena Dackland for expert assistance with FACS anal- nating DNA amplification. The expression ratio between each yses. This work was supported by grants from the Swedish Cancer Society lenalidomide-treated sample and the corresponding untreated sam- (to E.H.-L.) and the Leukaemia Research Fund of the United Kingdom ple was calculated by using the ⌬⌬CT method (67). (to A.P., H.C., J.S.W., and J.B.).

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