Role of GATA-1 and HSP70 in the Dyserythropoiesis of Early Myelodysplastic Syndromes.

Abstract 3823

Poster Board III-759

Myelodysplastic syndromes (MDS) are heterogeneous hematopoietic stem cell disorders characterized by a hypercellular dysplastic bone marrow (BM) with peripheral blood cytopenias, mainly anemia. Early MDS with less than 10% BM blasts which belong in most cases to low and intermediate-1 (int-1) risk groups according to the International Prognostic Scoring System (IPSS), usually demonstrate dyserythropoiesis. The growth of erythroid progenitors is altered, with increased caspase activation leading to excessive cell death, and cellular dysplasia characterized, in liquid culture of CD34⁺-derived erythroid progenitors, by a delayed expression of the glycophorin A (GPA) marker. The erythroid/megakaryocytic transcription factor GATA-1, required for erythroid commitment, is a target protein for proteases of the caspase family. Accordingly, caspase-3 is activated and GATA-1 is cleaved when erythroid cells undergo apoptosis, e.g. when starved from cytokines or exposed to Fas receptor agonists. PARP-1, Acinus and Lamin B are also cleaved in this setting. On the other hand, the limited activation of caspase-3 that is required for the normal maturation of basophilic to polychromatophilic erythroblasts does not lead to GATA-1 cleavage. We have shown previously that, during normal erythroid differentiation, the ATP-dependent chaperone protein HSP70 re-localized from the cytoplasm to the nucleus to protect GATA-1 from caspase-mediated proteolysis. The present study was undertaken to determine whether alteration of this pathway could play a role in the dyserythropoiesis observed in 22 patients with MDS. In cultured MDS erythroblasts, we observed that concomitantly to the burst of caspase activation, GATA-1 expression spontaneously decreased despite the presence of cytokines. The addition of pan-caspase inhibitors, i.e. zVAD or qVD rescued the expression of GATA-1, suggesting that GATA-1 downregulation depended on caspase-mediated proteolysis. Immuno-fluorescence or immunoblot experiments (n=11) demonstrated a defect in the nuclear relocalization of HSP70 in MDS erythroblasts when caspases were activated. On the contrary to the observations made in normal cells, HSP70 did not co-localize with GATA-1 or activated caspase-3 in MDS cell nuclei. Ectopic expression, through retroviral transduction of MDS progenitors (n=8), of a μGATA-1 mutant protein uncleavable by caspases, induced GATA-1 target mRNAs (PBGDe, GPA, EPO-R, b- and g-globins), and rescued erythroid differentiation with an increased number of morphologically differentiated cells and a GPA up-regulation. μGATA-1 had no effect on the apoptotic phenotype. These results indicate that the re-localization of HSP70 to the nucleus in differentiating erythroblasts is altered in MDS. This contributes to dyserythropoiesis through GATA-1 proteolysis. Ongoing studies explore the pathways involved in HSP70 nuclear distribution to identify which of these pathways is deregulated in MDS cells.