GSK3β down-regulation in Mesenchymal Stem Cells from patients with Myelodysplastic Syndrome.

In addition to neoplastic transformation of hematopoietic progenitors,bone marrow microenvironment damages can contribute to myeloid neoplasms development and maintenance. Several functional and morphological abnormalities of bone marrow mesenchymal stromal cells (BM-MSC) have been described in myeloid neoplasms. Nevertheless, molecular bases of differences between MSCs from normal and leukemic/myelodysplastic bone marrows are still unknown. Deregulation of genes belonging to PI3K/AKT signaling pathway has been described in MSC from different type of cancers. We studied the expression profile of genes belonging to PI3K/AKT signaling pathway in MSCs from 40 patients including 10 de novo AML, 10 de novo MDS, 10 t-MN (therapy-related myeloid neoplasms) and 10 patients with limited stage lymphoma without bone marrow involvement (used as normal control). BM-MSCs were obtained by Ficoll-gradient centrifugation of bone marrow samples and cultured up to 70% confluence in MesenCult Medium. Cells at 2nd passage were used for all experiments. The Human PI3K-AKT PCR array (SABioscience) was used to analyze mRNA levels of 84 key genes involved in PI3K-AKT signaling pathway, comparing 5 de novo AML, 5 de novo MDS and 5 t-MN vs 5 normal bone marrows. Relative changes in gene expression were calculated using the DDCt method. Fold change variations ≥1.5 in association to statistically significant T-test (p-value≤ 0.05) were used for the statistical analysis. Genes resulted significantly deregulated were validated by quantitative real time RT-PCR, in 10 de novo AML samples, 10 de novo MDS, 10 t-MN and 10 normal controls. Total GSK3b protein level and its Ser-9 phosphorylated isoform were measured by Western blot in a subgroup of MDS patients and controls. PI3K-AKT PCR arrays revealed a significantly down-regulation of GSK3b, MTCP1, RASA1 and SOS1 genes in MSC from all leukemic and myelodysplastic bone marrows compared to control group. After validation, all genes were confirmed as significantly down-regulated in de novo MDS samples respect to normal controls: GSK3b (p=0.0056, FC=-1.94), RASA1 (p =0.0044, FC=-2.19), SOS1 (p=0.0047, FC=-1.9) and MTCP1 (p=0.0026, FC=-1.93). No significant differences were found in the expression levels of studied genes in the validation group among de novo AML, t-MN and controls. Western blot analysis confirmed the down-regulation of both GSK3b total protein and of its Ser-9 phosphorylated isoform in MDS MSC respect to controls. Deregulation of genes belonging to PI3K/AKT signaling pathway may contribute to MSC dysfunction described in MDS bone marrows and can affect their ability to interact with normal hematopoietic cells, participating to bone marrow failure and myelodysplastic development. GSK3b, a crucial regulatory kinase interacting with multiple signaling pathways, is one of the most significantly down-regulated genes in MSC from myelodysplastic bone marrows and its functional significance is under investigation.