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.gene expression profiling ͉ myelodysplastic syndromes ͉ microarray ͉ erythropoiesis ͉ osteonectin
Low-risk myelodysplastic syndromes (MDS), including refractory anemia and sideroblastic anemia, are characterized by increased apoptotic death of erythroid progenitors. The signaling pathways that elicit this pathologic cell death in MDS have, however, remained unclear. Treatment with erythropoietin in combination with granulocyte colony-stimulating factor (G-CSF) may synergistically improve the anemia in patients with MDS, with a concomitant decrease in the number of apoptotic bone marrow precursors. Moreover, we have previously reported that G-CSF inhibits Fas-induced caspase activation in sideroblastic anemia (RARS). The present data demonstrate that almost 50% of erythroid progenitor cells derived from patients with MDS exhibit spontaneous release of cytochrome c from mitochondria with ensuing activation of caspase-9, whereas normal erythroid progenitors display neither of these features. G-CSF significantly inhibited cytochrome c release and suppressed apoptosis, most noticeably in cells from patients with sideroblastic anemia. Furthermore, inhibition of caspase-9 suppressed both spontaneous and Fas-mediated apoptosis of erythroid progenitors in all low-risk MDS cases studied. We propose that the increased sensitivity of MDS progenitor cells to death receptor stimulation is due to a constitutive activation of the mitochondrial axis of the apoptotic signaling pathway in these cells. These studies yield a mechanistic explanation for the beneficial clinical effects of growth factor administration in patients with MDS, and provide a model for the study of growth factor-mediated suppression of apoptosis in other bone marrow disorders. IntroductionThe myelodysplastic syndromes (MDS) constitute a heterogeneous group of clonal stem cell disorders characterized by ineffective hematopoiesis, various degrees of pancytopenia, and a risk of progression to acute myeloid leukemia. 1 Low-risk myelodysplastic syndromes, including refractory anemia (RA) and RA with ringed sideroblasts (RARS), are defined as MDS with a low probability of progression to leukemia and with a relatively favorable outcome. 2 Anemia and transfusion dependency are the main clinical problems for these patients.Increased apoptosis of bone marrow precursors is a hallmark of MDS, [3][4][5] and is thought to underlie the ineffective hematopoiesis evidenced in individuals with MDS. Indeed, apoptosis mediated via the death receptor, Fas, and its ligand has been suggested to serve as an important pathogenic mechanism in MDS. [6][7][8][9] However, a clear-cut correlation between the level of expression of these apoptosis regulators and the degree of bone marrow apoptosis, or cytopenia, has not been demonstrated. In addition to extrinsic, death receptor-mediated induction of apoptosis, intrinsic signaling pathways that depend on mitochondrial events, including the release of apoptogenic factors such as cytochrome c, also exist. 10,11 Interestingly, signs of mitochondrial pathology are commonly seen in MDS, including the characteristic accumulation of iron in...
IntroductionThe low-risk myelodysplastic syndromes (MDSs) refractory anemia (RA) and RA with ringed sideroblasts (RARS) are characterized by profound anemia and transfusion dependency, and a relatively low risk of progression to acute myeloid leukemia. 1,2 In RARS, the anemia is mirrored by hyperplastic but severely ineffective erythropoiesis due to increased apoptosis of erythroid progenitors. 3 The erythropoiesis of RA patients is also inadequate with apoptotic features, but may range from hypo-to hyperplastic, and shows no or few ringed sideroblasts. [4][5][6] The pathogenesis of RA anemia seems to be more heterogeneous, including T-cellmediated bone marrow failure in a subset of patients. 7,8 We have recently demonstrated that the erythroid apoptosis of low-risk MDS is initiated at a very early stage of stem cells and is associated with mitochondrial release of cytochrome c with subsequent activation of caspase-9 and effector caspase-3. Importantly, in RARS, granulocyte colony-stimulating factor (G-CSF) inhibits spontaneous release of cytochrome c, loss of mitochondrial membrane potential, and caspase activation, and restores erythroid proliferation. 9,10 Iron is predominantly stored in ferritin within cells. The multiple forms, or isoferritins, that can be found in human tissues are composed of variable proportions of 2 subunits: L-ferritin (light) and H-ferritin (heavy), encoded by genes located on chromosomes 11 and 19, respectively. Since free iron is potentially harmful to the cell, it is sequestered and detoxified to the less soluble ferric form by ferroxidase activity. H-ferritin (HF) exerts most of its ferroxidase activity in the cytosol. 11,12 Recently, a novel mitochondrial ferritin gene (MtF) was reported. This intronless gene contains a mitochondrial localization signal and is expressed in the mitochondrial matrix. It exhibits more than 75% sequence identity to the HF gene. 13,14 Mature erythroblasts from patients with X-linked sideroblastic anemia and RARS, in contrast to normal erythroblasts, express MtF 14 ; however, it is still unknown at which stage of erythroid differentiation this abnormal expression appears.The main task for the mitochondrion is to produce energy (adenosine triphosphate [ATP]). This occurs in the respiratory chain, consisting of 5 multiprotein enzyme complexes (I-V) and 2 15 Cytochrome c is closely associated with complex IV (cytochrome c oxidase) and has a major function to mediate the electron transport between complex III and IV. However, cytochrome c is also a key player in the regulation of apoptosis. 16,17 Erythroid differentiation requires activation of the erythropoietin (Epo) receptor followed by activation of the Jak-Stat pathway. This process is modulated by a complex network of transcription factors via activation of a set of target genes. GATA-1 plays a crucial role in erythroid development, and can switch the common lymphoid progenitors and granulocyte/monocyte progenitors toward megakaryocyte/erythrocyte lineage. 18,19 Epo induces globin gene expression an...
Treatment with granulocyte colony-stimulating factor plus erythropoietin may improve haemoglobin levels in patients with ringsideroblastic anaemia (RARS) and reduce bone marrow apoptosis. We studied bone marrow from 10 RARS patients, two of whom were also investigated after successful treatment. Mononuclear, erythroid and CD34+ cells were analysed with regard to proliferation, apoptosis, clonogenic capacity and oncoprotein expression, in the presence or absence of Fas-agonist, Fas-blocking antibody 2 and caspase-3 inhibitor. During culture, RARS bone marrow cells showed higher spontaneous apoptosis (P < 0.05) and caspase activity (P < 0.05)) than bone marrow cells from healthy donors. Eight out of nine patients had reduced growth of erythroid colony-forming units (CFU-E) (< 10% of control) and granulocyte-macrophage CFU (CFU-GM) (< 50% of control) from CD34+ cells. Fas ligation increased apoptosis and decreased colony growth equally in RARS and controls, but caused significantly more caspase activation in RARS (P < 0.01). Fas-blocking antibody showed no significant inhibitory effect on spontaneous apoptosis or ineffective haematopoiesis, as measured using phosphatidylserine exposure, the terminal deoxynucleotide transferase-mediated dUTP-biotin nick-end labelling technique, caspase activity or clonogenic growth. Caspase inhibition reduced apoptosis, increased proliferation and enhanced erythroid colony growth from CD34+ cells in RARS, but showed no effect on normal cells. CFU-E improved > 1000% after successful treatment. Thus, erythroid apoptosis in RARS is initiated at the CD34+ level and growth factor treatment may improve stem cell function. Enhanced caspase activation at the stem cell level, albeit not mediated through endogenous activation of the Fas receptor, contributes to the erythroid apoptosis in RARS.
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