Cytoplasmic localization of wild-type survivin is associated with constitutive activation of the PI3K/Akt signaling pathway and represents a favorable prognostic factor in patients with acute myeloid leukemia

Abstract

Survivin is over-expressed in most hematologic malignancies but the prognostic significance of the subcompartmental distribution of wild-type or splicing variants in acute myeloid leukemia has not been addressed yet. Using western blotting, we assessed the expression of wild-type survivin and survivin splice variants 2B and Delta-Ex3 in nuclear and cytoplasmic protein extracts in samples taken from 105 patients at the time of their diagnosis of acute myeloid leukemia. Given that survivin is a downstream effector of the PI3K/Akt signaling pathway, survivin expression was also correlated with pSer473-Akt. Wild-type survivin and the 2B splice variant were positive in 76.3% and 78.0% of samples in the nucleus, cytoplasm or both, whereas the Delta-Ex3 isoform was only positive in the nucleus in 37.7% of samples. Cytoplasmic localization of wild-type survivin was significantly associated with the presence of high levels of pSer473-Akt (P<0.001). Inhibition of the PI3K/Akt pathway with wortmannin and Ly294002 caused a significant reduction in the expression of cytoplasmic wild-type survivin. The presence of cytoplasmic wild-type survivin and pSer473-Akt was associated with a lower fraction of quiescent leukemia stem cells (P=0.02). The presence of cytoplasmic wild-type survivin and pSer473-Akt were favorable independent prognostic factors. Moreover, the activation of the PI3K/Akt pathway with expression of cytoplasmic wild-type survivin identified a subgroup of acute myeloid leukemia patients with an excellent outcome (overall survival rate of 60.0±21.9% and relapse-free survival of 63.0±13.5%). Our findings suggest that cytoplasmic wild-type survivin is a critical downstream effector of the PI3K/Akt pathway leading to more chemosensitive cells and a more favorable outcome in acute myeloid leukemia.

Figure 1.

Sub-compartmental localization of WT survivin and isoforms in AML. Y axis represents percentages of cases over total AML samples. White bars: expression in both cytoplasm and nucleus; gray bars: cytoplasmic expression only; gray-shaded bars: nuclear expression only, and black bars: no survivin expression.

Figure 2.

Western blot analysis of total Akt, pSer473-Akt, WT survivin and survivin isoforms in cytoplasmic and nuclear protein extracts of AML samples. Bone marrow aspirates from five representative AML patients at diagnosis (P1 to P5) and a control (C) from a healthy bone marrow donor. Cells were starved and maintained in a serum-free medium without cytokines for 4–5 h before protein extraction. Splicing variants of nuclear survivin are identified by different molecular weights: 2B 18 KDa, and Delta-Ex3 14 KDa and confirmed with blocking peptide experiments. Laminin-A and actin were used as controls for nuclear and cytoplasmic extracts, respectively. CytSurWT: WT cytoplasmic survivin; nSurWT: WT nuclear survivin; nSur2B: nuclear survivin 2B and nSurDEx3: nuclear survivin Delta-Ex3.

Figure 3.

Inhibition of the PI3K/Akt pathway using Ly294002 (Ly) and wortmannin (wort) in AML cell lines. Western blot analysis of pSer473-Akt and CytSurWT after culture for 24 h with different inhibitors using MV4-11 (A) and HL-60 (B) cell lines. (C) Mean plus SD of raw volumes of WT survivin of quadruplicate experiments in MV4-11 and HL-60; control, white bars; Ly, gray bars; wortmannin, gray shaded bars; and black bars, Ly+wortmannin. (D) Percentage of DiOC₆ loss in the same experiments in MV4-11 (white bars) and HL-60 (gray bars). Ly: Ly294002, Wort: wortmannin, Rap: rapamycin.

Figure 4.

Cell cycle analysis of leukemic cells at diagnosis. In each dot plot, R3 displays G0 cells that are Hoechst 3342 positive but low/negative for the RNA-binding dye pyronin Y and R4 displays S/G2/M cells. Events were gated on forward and side scatter signals (R1) and CD34-CD33/CD38 expression (R2). To set up regions lymphocytes from chronic lymphoid leukemia were used. (A) A representative patient expressing cytoplasmic survivin WT and high levels of pSer473-Akt. (B) A representative patient lacking cytoplasmic WT survivin and negative for pSer473-Akt.

Figure 5.

Outcomes of AML patients according to subcellular WT survivin localization. Overall survival was calculated with Kaplan-Meier curves for all 66 intensively treated patients and relapse-free survival was calculated for intensively treated patients achieving complete remission. (A) Kaplan-Meier curves for overall survival according to subcompartmental WT survivin expression. (B) Kaplan-Meier curves for relapse-free survival according subcompartmental WT survivin expression.

Figure 6.

Outcomes of AML patients according to normalized values of pSer473-Akt. Overall survival was calculated with Kaplan-Meier curves for all 66 intensively treated patients and relapse-free survival was calculated for intensively treated patients achieving complete remission. (A) Kaplan-Meier curves for overall survival according to pSer473-Akt normalized values. (B) Kaplan-Meier curves for relapse-free survival according to pSer473-Akt normalized values.

Figure 7.

Outcomes of AML patients according to the PI3K/Akt/CytSurWT activation pathway. Overall survival was calculated with Kaplan-Meier curves for all 66 intensively treated patients and relapse-free survival was calculated for intensively treated patients achieving complete remission. (A) Kaplan-Meier curves for overall survival according to PI3K/Akt/CytSurWT activation pathway status. (B) Kaplan-Meier curves for relapse-free survival according to PI3K/Akt/CytSurWT activation pathway status.