Survivin Isoforms and HSP90 Are Downregulated in Erythropoiesis during Lineage Specific Differentiation of MDS Hematopoietic Progenitor Cells.

Abstract

Survivin has recently been shown to critically regulate erythroid versus megakaryocytic differentiation in mouse hematopoietic stem cells. Its role in human hematopoiesis has not been fully elucidated yet. To answer the question whether dysregulation of expression of survivin and its protein stabilisator HSP90 contributes to the ineffective erythropoiesis in myelodysplastic syndrome (MDS), we have generated an in vitro model of MDS lineage-specific hematopoietic differentiation by culturing CD34+ cells from healthy donors (n=7) and MDS patients (low risk: RA/n=6, RARS/n=3; high risk: RAEB/n=4, RAEB-T/n=2) with EPO, TPO and GCSF. Cell harvest was at days 0, 4, 7 and 11. Expression analysis of survivin splicing variants (wt, 2a, d3ex) and of HSP90 was done by real time RT-PCR (qPCR) for each lineage at each time point. Furthermore, epigenetic analysis of key regulatory genes by methylation specific PCR and qPCR to detect DNMT1 (maintenance methylation) and DNMT3a and 3b (de novo methylation) expression was performed for each of the experimental conditions. RNA expression of all survivin isoforms and of HSP90 was continously upregulated during normal erythropoiesis. During late megakaryopoiesis of normal hematopoietic stem cells a significant downregulation was seen with elevated expression values for survivin wt and survivin 2b during early thrombopoiesis. In contrast, during MDS erythropoiesis a significant downregulation of expression of all survivin isoforms and HSP90 during late erythropoiesis was observed. Lower expression for survivin wt, survivin 2b and HSP90 was seen during early MDS megakaryopoiesis. Promotor methylation analyisis revealed MDS specific aberrant methylation for survivin, particularly during MDS high risk erythropoiesis (p=0.02). However, expression of DNMT1 was only elevated during erythropoiesis and in low risk MDS. A significant inverse correlation between RNA expression of DNMT enzymes and survivin splicing variants could not be detected. In conclusion our data support the essential role for survivin in regulation of erythropoietic versus megakaryopoietic differentiation of hematopoietic precursor cells. The dysregulation of survivin expression during MDS erythropoiesis may therefore account for the ineffective erythropoiesis known in MDS. Furthermore, downregulation of survivin (protein) stabilisator HSP90 might additionally contribute to this effect. Although an inverse correlation with overexpression of DNMTs and downregulation of survivin splicing variants was not seen, the aberrant survivin promoter methylation during MDS erythropoiesis indicates that epigenetic dysregulation results in low survivin expression.