DAP Kinase-Related Apoptosis-Inducing Protein Kinase 2 (DRAK2) Is a Key Regulator and Molecular Marker in Chronic Lymphocytic Leukemia

Abstract

Chronic lymphocytic leukemia (CLL) is the most common adult leukemia in the Western World and it is characterized by a marked degree of clinical heterogeneity. An impaired balance between pro- and anti-apoptotic stimuli determines chemorefractoriness and outcome. The low proliferation rate of CLL cells indicates that one of the primary mechanisms involved in disease development may be an apoptotic failure. Here, we study the clinical and functional significance of DRAK2, a novel stress response kinase that plays a critical role in apoptosis, T-cell biology, and B-cell activation in CLL. We have analyzed CLL patient samples and showed that low expression levels of DRAK2 were significantly associated with unfavorable outcome in our CLL cohort. DRAK2 expression levels showed a positive correlation with the expression of DAPK1, and TGFBR1. Consistent with clinical data, the downregulation of DRAK2 in MEC-1 CLL cells strongly increased cell viability and proliferation. Further, our transcriptome data from MEC-1 cells highlighted MAPK, NF-κB, and Akt and as critical signaling hubs upon DRAK2 knockdown. Taken together, our results indicate DRAK2 as a novel marker of CLL survival that plays key regulatory roles in CLL prognosis.

Keywords: CLL; DAPK1; DRAK2; STK17B; prognostic indicator.

Figure 1

DRAK2 expression distribution and clinical impact in CLL. Domain architecture of DAPK1 and DRAK2 (Upper Panels (A,B)) and scatter plot showing the distribution of (lower panel (A)) DAPK1 expression level (lower panel (B)) and DRAK2 expression level in a cohort of heterogeneously selected CLL cases. (C) Kaplan-Meier overall survival curve for patients with a high and low level of DRAK2 expression (D) Kaplan-Meier overall survival curves for the 13q deleted patient subgroup with a high and low level of DRAK2 expression.

Figure 2

Correlation between DRAK2 and key targets of survival cellular pathways. Pearson’s correlation analysis showing strong, positive relationship between (A) DAPK1 and DRAK2 RNA expression and (B) TGFBR1 and DRAK2 RNA expression.

Figure 3

Immunofluorescence analyses of DRAK2 cellular localization in MEC-1 cells. MEC-1 cells were stained with different fluorophores to visualize cellular compartments: cytoplasm/actin filaments (visualized with phalloidin) and nucleus (visualized with DAPI). Cells were analyzed using a ZEISS Axio Imager microscope. Z1 to generate 2D view (A) using ZEISS Apotome that allows the collection of confocal-like “Z” stacks to project images, as shown in 3D (B,C).

Figure 4

DRAK2 impact on the cellular viability (A,B) and proliferation (C). Cellular viability was measured using the CellTiter-Glo^® Luminescent Cell Viability Assay (A), trypan blue staining (B), and the cell proliferation endpoint was evaluated by cell counting following EdU (5ethynyl-2′-deoxiuridine) (C). All experiments were repeated 3 times, and error bars represent the standard error of the mean of 5 experiments.

Figure 5

Microarray analysis reveals that MEC1 cells produce a differential gene expression signature upon DRAK2 siRNA knockdown. (A) DRAK2 siRNA knockdown in MEC1 cells strongly reduces DRAK2 RNA and (B) protein levels. (C) Volcano plot in which gene expression is calculated as a linear fold change relative to MEC1 cells treated with siRNA control. p-value is calculated by one-way between-subject ANOVA for unpaired samples. Red data points represent genes expressed > 1.2-fold and blue data points represent genes expressed < 1.2-fold in MEC1-siRNA-DRAK2 versus MEC1-siRNA-control, where p < 0.05. (D). Ingenuity’s pathway analysis of DRAK2 mediated putative genes, and biological pathways are depicted using a color-coded heatmap. The color intensity of the squares in the heatmaps reflects the strength of the absolute z-score for predictions (orange = positive, blue = negative). The size of the squares reflects the z-score values. The top five molecular and cellular functions deregulated in MEC1 cells upon DRAK2 knockdown are given in the lower panel.