Essential role for cyclic-AMP responsive element binding protein 1 (CREB) in the survival of acute lymphoblastic leukemia

Abstract

Acute lymphoblastic leukemia (ALL) relapse remains a leading cause of cancer related death in children, therefore, new therapeutic options are needed. Recently, we showed that a peptide derived from Cyclic-AMP Responsive Element Binding Protein (CREB) was highly phosphorylated in pediatric leukemias. In this study, we determined CREB phosphorylation and mRNA levels showing that CREB expression was significantly higher in ALL compared to normal bone marrow (phosphorylation: P < 0.0001, mRNA: P = 0.004). High CREB and phospho-CREB expression was correlated with a lower median overall survival in a cohort of 140 adult ALL patients. ShRNA mediated knockdown of CREB in ALL cell lines blocked leukemic cell growth by inducing cell cycle arrest and apoptosis. Gene expression array analysis showed downregulation of CREB target genes regulating cell proliferation and glucose metabolism and upregulation of apoptosis inducing genes. Similar to CREB knockdown, the CREB inhibitor KG-501 decreased leukemic cell viability and induced apoptosis in ALL cell lines, as well as primary T-ALL samples, with cases showing high phospho-CREB levels being more sensitive than those with lower phospho-CREB levels. Together, these in vitro findings support an important role for CREB in the survival of ALL cells and identify this transcription factor as a potential target for treatment.

Keywords: CREB; acute lymphoblastic leukemia; cyclic-AMP responsive element binding protein; targeted therapy.

Figure 1. Normalized CREB expression profiles in pediatric ALL and normal bone marrow (NBM) mononuclear cells

CREB mRNA expression levels for normal bone marrow and ALL patients, ALL patients are displayed as a group and distinguished based on immunophenotype A. CREB_S133 phosphorylation determined using phospho-kinase arrays for normal bone marrow and ALL patients. ALL patients are again shown as one group and distinguished based on immunophenotype B. Asterisks (*) indicate significant differences (P < 0.05) of normal bone marrow vs ALL or BCP-ALL vs T-ALL as determined by the Mann-Whitney U test. Horizontal bars indicate median values.

Figure 2. Relationship between CREB phosphorylation and event-free survival in pediatric ALL

Kaplan-Meier plot of event-free survival time as function of CREB phosphorylation. The estimated event-free survival time appeared to be lower for patients with high CREB phosphorylation intensities (P = 0.073).

Figure 3. High CREB expression and phosphorylation is associated with reduced overall survival in adult ALL

Box-plots of CREB A. and phospho-CREB B. protein levels of 140 Ph-negative adult ALL patients distinguished based on immunophenotype. Expression levels were normalized using normal CD34⁺ cells as a comparison because of their known high CREB expression. To determine the overall survival, patients were divided into three groups based on CREB C. and phospho-CREB D. expression levels. The proportion of surviving patients was lower for patients with high CREB or phospho-CREB expression levels compared to patients with an expression at normal CD34⁺ levels (P = 0.002 and P = 0.001, respectively) or below normal CD34⁺ levels (P = 0.015 and P < 0.001, respectively)

Figure 4. CREB is essential for leukemic cell growth and viability

CREB is successfully downregulated in 4 ALL cell lines using three different CREB shRNAs. Downregulation was confirmed by Western blot analysis as shown in de left panels. Growth curve analysis shows that CREB downregulation results in a decrease in cell growth (right panels). Growth was measured in duplicate by absolute cell counts every 48 hours using a Coulter Counter. The results are shown as mean ± standard deviation.

Figure 5. CREB downregulation induces cell cycle arrest and apoptosis

Cell cycle analysis shows a reduction in the percentage of cells in S and G₂/M phases with a concomitant increase of cells in sub-G₀ and G₀/G₁ phases in comparison with shControl A. Annexin-V staining shows an increase in apoptotic cell numbers for CREB downregulated cells as compared to control transduced cells B. The results are shown as mean ± standard deviation. Asteriks (*) indicate significant differences (P < 0.05) by Student t-test.

Figure 6. Effect of CREB knockdown on glucose metabolism

Schematic overview of metabolic enzymes involved in glucose metabolism affected by shRNA mediated CREB knockdown. Downregulated genes, as determined by gene expression profiling, and their role in glucose metabolism are shown in red.

Figure 7. Inhibition of CREB induces cell death in a dose-dependent manner

CREB and phospho-CREB expression was determined by Western blot analysis for BCP-ALL and T-ALL cell lines and primary samples A. Effects of the CREB inhibitor KG-501 were determined after 24 or 48 hours of incubation for primary samples and cell lines, respectively. CREB inhibition results in a dose-dependent decrease in cell viability as determined by WST-1 conversion. Cell viability percentages were plotted against concentration of KG-501 (μM) using four ALL cell lines B., four primary BCP-ALL samples C., and four primary T-ALL samples D.