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.

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 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 = 0.04 at day 14), whereas no effect was seen in normal cells. (D and E) Flow cytometry analysis of cultured cells from MDS 5q− patients at day 7 (n = 5) (D) and day 14 (n = 11) (E) showed that, during the second week after the addition 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 = 3) (F) and day 14 (n = 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 < 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.

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 MDS del(5q) cells from day 7 of culture, corresponding to the cells analyzed with gene expression profiling. Nuclei are stained with DAPI. (C) Effects of lenalidomide treatment on the expression levels of the genes mapping to the CDR of the 5q− syndrome (41 of 44 are represented on the Affymetrix arrays) in the 5q− erythroblasts. The average proportional change after lenalidomide treatment is shown. The SPARC gene is highlighted in red.

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.