DEREGULATION OF PI3K/AKT SIGNALING IN BONE MARROW MESENCHIMAL STROMAL CELLS FROM PATIENTS WITH DE NOVO AND THERAPY-RELATED ACUTE MYELOID LEUKEMIA

Background: In addition to neoplastic transformation of hematopoietic progenitors, a damage of bone marrow microenvironment can contribute to leukemia development and maintenance. Several functional and morphological abnormalities of bone marrow mesenchimal stromal cells (BM-MSCs) have been described in myeloid neoplasms. Nevertheless molecular bases of differences between MSCs from normal and leukemic bone marrows are still unknown. PI3K/AKT signaling pathway is involved in several MSC functions and deregulation of genes belonging to these pathways have been described in MSCs from different type of cancers. Aims: To study the expression profile of genes belonging to PI3K/AKT signaling pathway in MSCs from patients with de novo and therapy related Acute Myeloid Leukemia (t-AML), using as normal counterpart BM-MSCs isolated from patients with limited stage lymphoma without bone marrow involvement. Methods: Study population included 5 patients with limited stage diffuse large B cell lymphoma (DLBCL) without bone marrow involvement, as normal control, and 10 patients with AML, including 5 de novo and 5 therapy-related cases. Bone marrow mononuclear cells were obtained by Ficoll-gradient centrifugation of bone marrow samples and cultured in Complete Human MesenCultR Medium (Stem Cell Technologies) in 25 cm2 flask at 37°C. After 24 hours nonadherent cells were removed and adherent cells were cultured up to 70% confluence, then trypsinized and passed to a new flask. Cells at 2nd passage were collected by trypsinization, RNA was extracted using RNase mini kit (Qiagen) and cDNA was synthesized by QuantiTect Reverse Transcription kit (Qiagen). The Human PI3K-AKT PCR array (RT2ProfilerTM PCR Array; SABioscience) was used to analyze mRNA levels of 84 key genes involved in PI3K-AKT Signaling Pathway, in a 96-well plate in the CFX96 thermocycler (Bio-Rad). Relative changes in gene expression were calculated using the ΔΔCt method. An average Ct value of five housekeeping genes (GAPDH, βactin, β2-microglobulin, HPRT1 and RPLPO) was used to normalize the gene expression between sample groups. Fold change (FC) variations ≥1.5 in association to statistically significant T-test (P-value≤0.05) were used for the statistical analysis. Results: Comparison of MSCs from AML samples versus normal controls identified three genes significantly down-regulated in leukemic samples, including GSK3B (P=0.0002, FC=-1.56), MTCP1 (P=0.019, FC=-1.62) and RASA1 (P=0.013, FC=-1.58). Stratifying the analysis according to AML subtypes, three genes were significantly down-regulated in t-AML versus normal bone marrow, including GSK3B (P=0.0009 and FC=-1.63), PTEN (P=0.004 and FC=-1.50) and SOS1 (P=0.004 and FC=-1.50). Similarly when comparing MSC from de novo AML versus normal bone marrow, GSK3B, MTCP1 and RASA1 resulted still down-regulated in MSC from leukemic samples (P=0.005 and FC=-1.51; P=0.018 and FC=-1.96; P=0.007 and FC=-1.81, respectively). No differences were found in the expression levels of studied genes between de novo and therapy- related AML. Summary and Conclusions: Deregulation of genes belonging to PI3K/AKT signaling pathway may contribute to MSC dysfunction described in leukemic bone marrows and can affect their ability to interact with leukemic blasts and normal hematopoietic cells, eventually contributing to bone marrow failure and leukemia development. GSK3B was the most significantly and commonly down-regulated gene in MSCs from leukemic samples and codifies for the serine/ threonine protein kinase Gsk3β which is also involved in additional signaling pathways, such as Raf/Mek/Erk and Wnt/β-catenin.