No abstract

4977 Introduction PI3K/AKT signalling pathway is involved in cell growth, proliferation and apoptosis. PI3K/AKT constitutive activation is observed in several solid tumors and leukemic cells. Inhibition of PI3K/AKT activity using specific inhibitor, LY294002, results in apoptosis. Deguelin is a natural product isolated from the leguminous, Mundulea sericea, with antitumorigenic effect in vitro and in vivo. The inhibition effect of Deguelin in PI3K/AKT signalling pathway in leukaemia cells was also observed in vitro. Aims We evaluated PI3K/AKT activation using p-AKT expression by flow cytometry in P39 cell line and CD34+ hematopoietic progenitors. PI3K/AKT activity inhibition mediated by deguelin and its proapoptotic effect was tested in P39 cell line. Material and Methods The high-risk MDS cell line P39 and leukaemia cell lines, Jurkat and HL60, were maintained in RPMI1640 and FBS 10%. Jurkat and HL60 were used as positive and negative control for p-AKT expression, respectively. Cell lines and CD34+ cells from bone marrow healthy donors (n=5) were assessed for p-AKT expression. Flow cytometry detection of intracellular Ser 473 p-AKT was performed after permeabilization and fixation with a saponin based solution with Tween20 and FACSlysing solution (Becton Dickinson-BD). An alexa-fluor 488-conjugated rabbit antibody to Ser 473 p-AKT (Cell Signalling Technology) was employed. A double immunostaining procedure using CD45-PerCP and CD34+PE was performed to analyse p-AKT expression in CD34+ cells from bone marrow healthy donors. After incubation, cells were analysed on a FACSCalibur (BD). The p-AKT activity was determined using Kolmogorov-Smirnov test (D). Difference between groups was analyzed using the Mann Whitney test. For treatment and determination of apoptosis, cells were exposed to deguelin (at concentrations of 10-300nM) and LY294002 (15-50uM) for 24-48h. To determine apoptotic changes, cells were stained withy Annexin-V/FITC and propidium iodide and examined on a FACScalibur (BD). Results Jurkat cells showed constitutive PI3K/AKT activation with a mean D value for p-AKT expression of D=0,84 ± 0,02. P39 cells also showed a constitutive PI3K/AKT activation with p-AKT expression as high as in Jurkat cells (mean D= 0,76±0,07). P-AKT expression was significant lower in CD34+cells from health donors (D=0,38±0,06) than in Jurkat and P39, respectively (p=0,0043 and p=0,015). Inhibition of p-AKT was observed using different concentrations of LY294002 (15-50uM) in Jurkat cells and apoptosis was observed after 24 and 48h when cells were treated with 50uM. For P39, although apoptosis was observed, no p-AKT inhibition was detected after LY294002 treatment. Treatment with deguelin induced apoptosis in Jurkat via p-AKT inhibition. No effect on apoptosis or p-AKT expression was observed in P39 cell line after deguelin treatment. Conclusions PI3/AKT constitutive activation was observed in P39 cell line and suggests that PI3/AKT can play a role in MDS progression. Deguelin effect in PI3K/AKT pathway can be cell specific, as observed in Jurkat but not in P39 cell line. Thus, deguelin in combination with other agents should be tested as a potential therapeutic option for MDS via PI3K/AKT inhibition. Disclosures No relevant conflicts of interest to declare.

1871 Introduction: PI3K/AKT pathway is involved in cell growth, proliferation and apoptosis. A key downstream effector is the phosphorylated serine-threonine Akt (p-AKT). Constitutive activation of PI3K/AKT has been observed in solid tumours and leukemic cells. Inhibition of PI3K/AKT activity, results in apoptosis in cell lines (CL) after treatment with different compounds, e.g. deguelin, a natural product from the leguminous Mundulea sericea, with antitumour effects. Aims: To evaluate PI3K/AKT activation in MDS patients and its therapeutic potential in MDS. Methods: PI3K/AKT activation was evaluated by flow cytometry (FC) using an alexa-fluor 488-antibody Ser 473 p-AKT (Cell Signalling Technology). A triple immunostaining procedure using CD45-PerCP and CD34-PE was used for p-AKT expression in CD34+ primary samples. The p-AKT activity was determined using Kolmogorov-Smirnov test (D). CD34+ cells from healthy donors and Jurkat cells were used as negative and positive controls respectively. Apoptosis (determined by Annexin V and PI/7AAD) and cell cycle arrest (using RNAse and PI) were determined following treatments with LY294002 (50uM), and deguelin (100-500nM) in P-39 myeloid leukemia cell line, with constitutive PI3K/AKT activation. Apoptosis was determined in bone marrow mononuclear cells and CD34+ cells from MDS patients with the same treatments. To evaluate in vivo activity of deguelin, we used a xenotransplant model. Briefly, NODSCID mice were injected intrafemurally with P-39 CL and 12 days post transplant a three week-course of treatment, every other day, was started (deguelin 4mg/Kg, n=3 vs vehicle, n=3). Results: P-39 CL showed constitutive PI3K/AKT activation with levels significantly higher than in CD34+cells from controls (median±SD= 0.73. Disclosures: No relevant conflicts of interest to declare.