Phosphorylation of GSK3α/β correlates with activation of AKT and is prognostic for poor overall survival in acute myeloid leukemia patients

Abstract

Background: Acute myeloid leukemia (AML) patients with highly active AKT tend to do poorly. Cell cycle arrest and apoptosis are tightly regulated by AKT via phosphorylation of GSK3α and β isoforms which inactivates these kinases. In the current study we examine the prognostic role of AKT mediated GSK3 phosphorylation in AML.

Methods: We analyzed GSK3α/β phosphorylation by reverse phase protein analysis (RPPA) in a cohort of 511 acute myeloid leukemia (AML) patients. Levels of phosphorylated GSK3 were correlated with patient characteristics including survival and with expression of other proteins important in AML cell survival.

Results: High levels of p-GSK3α/β correlated with adverse overall survival and a lower incidence of complete remission duration in patients with intermediate cytogenetics, but not in those with unfavorable cytogenetics. Intermediate cytogenetic patients with FLT3 mutation also fared better respectively when p-GSK3α/β levels were lower. Phosphorylated GSK3α/β expression was compared and contrasted with that of 229 related cell cycle arrest and/or apoptosis proteins. Consistent with p-GSK3α/β as an indicator of AKT activation, RPPA revealed that p-GSK3α/β positively correlated with phosphorylation of AKT, BAD, and P70S6K, and negatively correlated with β-catenin and FOXO3A. PKCδ also positively correlated with p-GSK3α/β expression, suggesting crosstalk between the AKT and PKC signaling pathways in AML cells.

Conclusions: These findings suggest that AKT-mediated phosphorylation of GSK3α/β may be beneficial to AML cell survival, and hence detrimental to the overall survival of AML patients. Intrinsically, p-GSK3α/β may serve as an important adverse prognostic factor for a subset of AML patients.

Keywords: AKT; GSK3; Leukemia; PKC delta; RPPA; Signal transduction.

Supplemental Fig. 1

GSK3 α/β total and serine 21/9 phosphorylated protein expression in AML blast cells by FAB and cytogenetics. RPPA was performed as described in the “Materials and methods” section. Expression of phosphorylated and total GSK3 α/Β was compared by FAB category (A and C for phosphorylated and total, respectively) and cytogenetic category (B and D for phosphorylated and total, respectively) from AML patients versus CD34 + bone marrow cells from 11 normal donors.

Supplemental Fig. 2

Proteins correlated with total GSK3 α/β expression in AML patients. Negative and positive correlation from a list of 229 proteins also assayed compared to total GSK3 α/β expression using the same reverse phase proteomics analysis on the same samples; Pearson correlation coefficient R > 0.25, p < 0.001.

Fig. 1

GSK3 α/β total and serine 21/9 phosphorylated protein expression in AML blast cells compared to normal counterpart CD34 + cells. RPPA was performed as described in the “Materials and methods” section. Expression of total (A) and phosphorylated (B) GSK3 α/Β was compared in blast cells from 511 AML patients versus CD34 + bone marrow cells from 11 normal donors.

Fig. 2

Correlation of overall survival with serine 21/9 GSK 3α/β phosphorylation levels in AML patient cytogenetic subsets. The Kaplan–Meier curve for overall survival (OS) for the total AML patient set (A; N = 415), intermediate cytogenetic patient set (B; N = 187), and unfavorable cytogenetic patient set (C; N = 195) based on expression of phosphorylated GSK3 α/β.

Fig. 3

Correlation of remission duration with serine 21/9 GSK 3α/β phosphorylation levels in AML patient cytogenetic subsets. The Kaplan–Meier curve for remission duration for the total treated AML patient set (A; N = 231), treated intermediate cytogenetic patient set (B; N = 120), and treated intermediate cytogenetic with the FLT3 mutant patient set (C; N = 35) based on expression of phosphorylated GSK3α/β.

Fig. 4

Proteins correlated with phosphorylated GSK3α/β expression in AML patients. Negative and positive correlation from a list of 229 proteins also assayed compared to phosphorylated GSK3α/β expression using the same reverse phase proteomics analysis on the same samples; Pearson correlation coefficient R > 0.25, p < 0.001.

Fig. 5

Proteins correlated with phosphorylated GSK3α/β expression in the intermediate cytogenetic group AML patients. Negative and positive correlation from a list of 229 proteins also assayed compared to phosphorylated GSK3α/β expression using the same reverse phase proteomics analysis on the same samples; Pearson correlation coefficient R > 0.25, p < 0.001.

Fig. 6

Role of GSK3α/β on AKT suppression of FOXO3A and PKCδ in AML cell lines. (A) Protein lysate (200,000 cell equivalents) from OCI-AML3 cells containing control (NS) lentiviral plasmid or cells containing FOXO3A shRNA using 2 different lentiviral plasmids, and THP-1 cells or HL60 cells containing control (NS) lentiviral plasmid or cells containing PKCδ shRNA lentiviral plasmid were subject to SDS/PAGE and Western analysis performed for GSK3α/β, phosphorylated GSK3α/β, PKC d, FOXO3A, MYC, and Tubulin. Ratios were determined by densitometric analysis of the bands depicted in the figure and normalized to each NS control. (B) Protein lysate (200,000 cell equivalents) from OCI-AML3 cells treated with vehicle (-/-; 0.1 % DMS) or Calbiochem GSK3 inhibitor XVI (5 μM) or MK-2206 AKT inhibitor (1 μM) for 6 h were subject to SDS/PAGE and Western analysis performed for GSK3α/β, phosphorylated GSK3α/β, PKCδ, phosphorylated PKCδ, and Tubulin. Ratios were determined by densitometric analysis of the bands depicted in the figure and normalized to vehicle treated control.

Fig. 7

Model of AKT and GSK3α/β-mediated pathways in AML cells. Extracellular survival signals activate AKT which suppresses GSK3α/β. When AKT is suppressed, FOXO3A and PKCδ are inhibited independent of GSK3α/β. GSK3α/β when active will suppress MYC, MCL-1, and β-catenin.