Analysis of the PI3/Akt/Survivin Pathway in Acute Myelogenous Leukemia.

The PI3/Akt pathway has been implicated in the pathogenesis of a wide variety of cancers including Acute Myeloid Leukemia (AML). Activated Akt is known to function as an essential survival factor by inhibiting apoptosis through its ability to phosphorylate several targets including Bad, FoxO transcription factors, Raf-1, caspase-9 and inhibitor of apoptosis protein family (IAPs). Survivin is a member of IAP regulating both apoptosis and cell cycle progression. Survivin binds to several structural components of the mitotic apparatus and can block apoptosis by inhibiting caspases 9, 3 and 7. Recently the importance of PI3/Akt/survivin pathway in solid neoplasias such as breast or prostate cancer has been highlighted but its role in AML remains unknown. In this work we analyzed the PI3/Akt/survivin pathway in human AML.

Bone marrow samples obtained at diagnosis of 68 AML consecutive patients and K562, MV4-11 and HL-60 cell lines were included. Patients Median age was 62 years (range 8–89). Median leukocyte count was 10.3×10⁹/L (range 0.8–285). FAB subtypes were: M0=8, M1=20, M2=13, M3=8, M4=11, M5=7 and M6=1 with the following cytogenetic findings: t(15;17)=8, t(8;21)=2, complex karyotype=8, 11q23=2, normal karyotype=36 and others=12. There were 11.3% patients with FLT3-ITD and 16.9% with NPM1 mutation. Cytoplasmic and nuclear Proteins were harvested with Q-proteome cell compartment (Qiaqen) and protein concentration assayed using Protein Assay Kit (Bio-Rad). Total Akt, Akt-pSer473 and survivin proteins were detected by Western Blot and were visualized by enhanced chemiluminescence (ECL-Plus, GE Healthcare) in Chemigenius-2 and quantified using Gene-Tools software. Cell cycle analysis was assessed by double Hoechst 33342- Pyronin Y staining and flow cytometry in FACSvantage. Inhibition experiments were done using Ly294002 at 25 μM, and Wortmaninn at 250 nM for 12 hours.

In our series p-Ser473Akt was detected in 56% of AML marrow samples (with high levels expression in 27%) and in all cell lines. In cytoplasmic protein extracts, Survivin WT and 2B isoform were detected in 45% and 45.2% of patients respectively. All three leukemic cell lines showed only Survivin 2B expression. Interestingly, there was strong statistical correlation between the levels of p-Ser473Akt with cytoplasmic Survivin (P=.01). Inhibitors of PI3K/Akt pathway LY294002 and Wortmaninn both decreased in vitro p-Ser473Akt expression but only the irreversible action of Wortmaninn caused a marked dowregulation of cytoplasmic survivin. Meaningfully, lack of cytoplasmic Survivin was associated with an increased proportion of cells in Go cell cycle phase (11.1 % vs. 3.6%, P=.04). Moreover, cytoplasmic Survivin WT localization and high p-Ser473 Akt levels, were both significantly correlated with less unfavourable FAB leukemia subtype and cytogenetic risk (P<.01), more CR achievement with one induction cycle (P<.01), less relapse rate (P=.01) and less mortality rate (P=.03).

Survivin cytoplasmic expression is regulated by PI3-kinase/Akt pathway in AML. The activation of PI3/Akt/survivin pathway is associated with an increased proliferative status and our series suggest that this finding could be associated with a more favorable outcome.

Financial support: This study was supported by a grant of Conserjeria de Salud, Junta de Andalucia 2006/0355. J. Serrano López is a post-doc fellow from Fundación Española de Hematologíıa y Hemoterapia

No relevant conflicts of interest to declare.