Identification of Defects in the Transcriptional Program during Differentiation of CD34+Cells Selected from Patients with Both, Low- and High-Risk Myelodysplastic Syndromes.

The development of MDS is suggested to follow a multistep pathogenesis and is characterized by accumulation of molecular defects of the hematopoietic stem/progenitor cells. To detect alterations within the transcriptional program in MDS derived CD34+ cells during lineage-specific differentiation, CD34+ bone marrow cells were selected from healthy individuals (n=3) and patients with low-risk (IPSS, n=3) or high-risk (n=4) MDS and stimulated in vitro with EPO, TPO or G/GM-CSF to induce lineage-specific differentiation. Lineage-determined cells were harvested and if necessary purified by immunomagnetic beads at days 4, 7 and 11 for gene expression profiling. Gene expression was analyzed by oligonucleotide microarrays (HG-U133A, Affymetrix, Santa Clara, CA). The experiments were done in triplicates for each of the time points and each of the conditions. First, we identified 260 genes with a significant expression pattern associated with normal lineage-specific differentiation. These continuously up- or down-regulated genes are considered to be part of a specific genetic program of normal hematopoietic cells during lineage-specific differentiation. In MDS, 57% of these genes showed a different expression from the normal transcriptional pattern. Thirteen of 24 genes up-regulated during normal erythropoiesis were opponently expressed in MDS containing putative new erythro-specific genes like two GTPase activator proteins, RAP1GA1 and ARHGAP8, which regulate small Rho GTPases. Fourteen of 22 continuously up-regulated genes during normal granulopoietic development displayed a significantly different expression in MDS containing the putative candidate desmocollin 2, a gene which is involved in intercellular cell-adhesion. Delta-like 1 (DLK1) is known to be overexpressed in stem cells from patients with myelodysplastic syndrome. The role of DLK1 in normal hematopoiesis is still not defined. We found DLK1 with increasing expression during normal megakaryopoiesis but reverse expression during megakaryopoiesis in MDS. Interestingly, in erythropoiesis from both, high- and low risk MDS we found overexpression of Bladder cancer overexpressed (BLOV1) and Apoptosis inhibitor 5 (API5, which acts as a cellular survival factor by inhibiting apoptosis after growth factor withdrawal). These genes are not expressed in normal erythropoiesis. Furthermore, we identified the gene for a novel v-maf-like protein F, MafF-like (v-maf: musculoaponeurotic fibrosarcoma oncogene homolog F) to be significantly downregulated exclusively in low-risk MDS. MafF belongs to a basic leucine-zipper(bZIP)-transcription factor family normally involved in multiple physiological processes including hematopoiesis and stress responses. Our data provide the first comprehensive transcriptional analysis of differentiating human CD34+ cells derived from normal individuals compared to MDS. It gives new insights to understand the alteration of differentiation and proliferation of MDS derived CD34+ cells. In particular, the study could identify the gene encoding for the MafF-like protein that acts as a transcriptional regulator of normal hematopoiesis to be significantly down-regulated in low-risk MDS.