3240 Introduction: Treatment of adult acute lymphoblastic leukemia (ALL) has shown only modest improvements over the last 2 decades, with overall survival of 15% to 40%. The mitogen-activated protein kinase (MAPK) signaling cascade and the phosphoinosytol-3 phosphate/AKT (PI3K/AKT) pathways are involved in proliferation and differentiation of hematopoietic cells. It has been reported that those pathways are frequently activated in solid tumors and acute myeloid leukemia. However, their role in adult ALL is still uncertain. Better understanding of such pathways is necessary for development of novel therapeutic strategies. Aims: To evaluate the phopho-ERK and phospho-AKT protein expression in ALL at diagnosis and to correlate with biological and clinical parameters. Material and Methods: Twenty eight patients (median age 33y, 14–69y) with ALL at diagnosis were studied. Bone marrow and/or peripheral blood mononuclear cells (PBMC) (10 fresh and 18 cryopreserved cells at diagnosis) were stained using phospho-ERK and phospho-AKT/alexafluor 488 monoclonal antibodies (Cell Signaling Technology, Beverly, MA) and their expression was evaluated by flow cytometry (FACScalibur cytometer and CELL Quest program - BDB, San Jose, CA). The monoclonal antibodies CD19, CD10, CD3, CD45, IgM, CD34, CD7, CD2 were used for leukemic cells characterization by four-colour staining. Healthy donor PBMC and Jurkat cell line were used as controls: normal T lymphocytes are negative for p-ERK and Jurkat cell line express p-ERK and p-AKT in low levels. Samples were analyzed for constitutive expression of p-ERK and p-AKT and also after cell activation by phorbol-myristate-acetate (PMA). The expression of these proteins was evaluated by Kolmogorov-Smirnov test using fluorescence ratio between control isotype and phospho-protein (D). p-ERK and p-AKT expression was also evaluated in fresh and frozen samples of the same patients (2 cases) and similar results were obtained. In addition, patients were evaluated for multidrug resistance (MDR) through p-glycoprotein (PGP) expression and Rhodamine (Rh) efflux test and minimal residual disease (MRD) detection at the end of induction by flow cytometry. Results: Twenty cases were B-ALL (EGIL B-I 3, B-II 9, B-III 8, B-IV 5) and 3 T-ALL. Median WBC count was 25.3×109/L (2.3-373×109/L). The expression of p-ERK and p-AKT varied and the median value of p-ERK expression was D = 0.16 (0.01-0.80) and p-AKT median D = 0.08 (0.00-0.63). Considering these values as cutoff there was no difference regarding the patients` age and WBC count at the diagnosis between the positive and negative groups. In regards to EGIL subtypes, p-ERK expression was higher in T-ALL [median 0.50 (0.18-0.54)] than in B-precursor ALL [median 0.14 (0.01-0.80)] (p=0.03). Conversely p-AKT expression was similar in all cases, although high levels were observed among BIII cases. The frequency of Rh efflux was 88% in pERK negative cases and 66% in positive group but there was no difference on PGP expression. On the contrary, PGP expression and Rh efflux were more frequently seen in p-AKT positive cases (88% and 100%, respectively) than p-AKT negative ones (63% and 60%). MRD analysis was performed in seven patients. Two cases presented detectable MRD (>0.01%) and both were p-ERK positive and p-AKT negative. Interestingly all five MRD (-) cases were p-ERK negative and p-AKT positive. In addition, PMA test showed p-ERK activation of normal T lymphocytes and the expression was increased in 52% of ALL cases upon treatment. Conclusion: MAPK and PI3/AKT activation varied among ALL patients. MAPK pathway showed to be more activated in T-ALL than in precursor-B ALL. The functional analysis of these pathways can address the role in ALL pathogenesis. Both pathways may be potential therapeutic targets for novel therapies. (Support: FAPESP proc.09/51002-8). Disclosures: No relevant conflicts of interest to declare.

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.