The Level of AKT Phosphorylation on Threonine 308 but Not on Serine 473 Is Associated with High-Risk Cytogenetics and Predicts Poor Overall Survival in Acute Myeloid Leukemia.

The PI3K/Akt pathway is an important signaling pathway governing cell survival, proliferation and drug resistance in AML. However, the prognosis impact of Akt phosphorylation in AML has yielded some controversies as far as the phosphorylation on Ser473 is concerned. Because full activation of Akt requires phosphorylation on both Thr308 and Ser473, the assessment of these two sites would allow a more complete analysis of Akt activation in AML cells. We have studied the level of phosphorylation on both sites in primary AML samples by flow cytometry. We retrospectively assessed samples from 58 AML patients treated between 12/2000 and 02/2005. The clinical characteristics were: median age, 46.5 y (15–61); sex ratio (M/F) 1.24; FAB, 1 M0, 29 M1/M2, 25 M4/M5, 1 M6, 2 unclassified; median WBC, 33.4 G/L (1.2–225); Hb, 9.2 g/dL (5–13.5); platelets, 48 G/L (6–328); % marrow blasts, 78 (20–97); cytogenetic risk group (favorable, 28%; intermediate, 31%; unfavorable, 19%); FLT3-ITD, 10/54 (19%). The complete response rate after induction chemotherapy (anthracyclines + ara-C) was 83%. Patients in CR1 were referred to consolidation chemotherapy, autologous or allogeneic stem-cell transplantion according to the cytogenetic risk. The global overall survival (OS), event-free survival (EFS) and relapse-free survival (RFS) were of 22.1, 14.1, and 15.6 months, respectively. The levels of Akt phosphorylation (Thr308 and Ser473) were evaluated on pretreatment marrow samples and expressed by the ratio between mean fluorescence intensity of the stained AML sample and non-relevant IgG control (rMFI). The values of both Thr308 and Ser473 represented a continuum ranging from 0.3 to 5.6 and 0.4 to 2.87, respectively. The median level of phosphorylation was 2.3 (±0.89) and 1.3 (±0.27) for Thr308 and Ser473, respectively. There were no significant correlations between age, gender, FAB, WBC, FLT3-ITD and Akt phosphorylation. However, the level of phosphorylation on Thr308 but not on Ser473 was significantly correlated with the cytogenetic risk group. Indeed, the median rMFIs on Thr308 were 1.95 (± 0.37); 2.2 (±1) and 3.1 (±0.49) for favorable, intermediate and unfavorable karyotype, respectively (p=0.0069). In univariate analysis, unfavorable karyotype, FLT3-ITD and Thr308^high correlated with poor OS, EFS, and RFS. Thr308^high patients (> median value) had significantly shorter OS (11 vs 47 months; p=0.01), EFS (9 vs 26 months; p=0.005) and RFS (10 months vs not reached; p=0.02) than Thr308^low patients. In the Cox model for multivariate analysis, only the cytogenetic risk independently predicted worse RFS, EFS and OS. Neither mutation screening for AKT1 E17K in an independant series of 148 AML cases nor changes in the level of PTEN expression and phosphorylation could explain the increased phosphorylation of Thr308 in high-risk cytogenetics AML cells. However, we found a statistically significant correlation between protein phosphatase 2A (PP2A) activity and complex karyotypes. Indeed, PP2A activity was markedly reduced by 40% in complex karyotypes compared with normal karyotypes. To specifically assess the role of Akt in the survival and proliferation of complex karyotype AML cells, we used the allosteric Akt kinase inhibitor targeting Akt1 and Akt2 (Akt-i). Akt-i inhibited the phosphorylation of Akt on both sites in these cells. Akt-i inhibited the clonogenic activity of AML cells from normal and complex karyotype samples in a dose-dependent manner, but this effect was significantly more potent in complex than in normal karyotype samples. After 24 h incubation with 10 μM Akt-i, features of apoptosis were detected by Annexin V staining (25% ±3,38 in treated vs 14% in untreated cells). Moreover, Akt-i enhanced the toxicity of daunorubicin (37% ±2,9, Akt-i+dnr vs 25% ±3,5, dnr). We also detected Akt phosphorylation in the immature leukemic compartment (CD34⁺ CD38⁻ CD123⁺). Accordingly, Akt-i also induced apoptosis and enhanced dnr activity in CD34⁺ CD38⁻ CD123⁺ cells from complex karyotype samples. This study shows that AML cells with complex karyotype display high level of phosphorylation of AktThr308 and suggests that this anti-apoptotic pathway may represent a valuable therapeutic target. Thus, it will be important to determine in clinical trials if this aberrant activation of Akt sensitizes AML cells to PI3K/Akt inhibitors specifically in this subgroup of very high-risk patients.