Role of Sprouty2 in the Pathogenesis of Chronic Lymphocytic Leukemia

INTRODUCTION: Chronic Lymphocytic Leukemia (CLL) is the most common leukemia in adults representing about 35% of all adult leukemias in the United States. CLL is a B-cell neoplasm which is characterized by accumulation of long lived mature B-cells in peripheral blood (PB), bone marrow (BM) and lymph nodes (LN). We and others have reported that CLL cells in tissue microenvironments have constitutively activated MAPK-Erk and AKT signaling pathways, which promotes their proliferation and survival. However, the molecular mechanisms and the gene(s) responsible for the activation of these pathways in CLL are not clearly understood. We performed gene expression profiling of CLL cells from PB, BM and LN and transcriptome analyses of CLL cells from PB from good and poor prognosis patients. Our studies identified Sprouty 2 (Spry2), a negative feedback regulator as one of the key genes involved in the regulation of MAPK-Erk and Akt pathways in CLL. We observed that Spry2 levels were down-regulated in poor prognosis CLL cells. Interestingly, Spry2 is epigenetically silenced in most human and mouse lymphomas and ectopic expression of Spry2 induces apoptosis in mouse B cells. We categorized CLL patients into good and poor prognosis to further evaluate Spry2 in CLL biology. Poor prognosis patients were defined by unmutated immunoglobulin variable heavy chain (IgVH), 11q22 deletion, 17p deletion, trisomy 12 or high CD38 and Zap70 expression. Good prognosis patients were defined as having mutated IgVH, 13q14 deletions or low CD38 and Zap70 expression.

RESULTS: We found that Spry2 expression was significantly decreased by 3.2 log2-fold, (p=0.0001) at transcript and protein levels, respectively, in CLL cells from patients with poor prognosis compared to patients with good prognosis. Spry2 is a negative feedback regulator which attenuates cell proliferation, migration and survival. To determine the effect of Spry2 on CLL cells we transfected Mec-1 cells (stable human CLL cell line) and primary human CLL cells with a Spry2 cDNA containing vector. Ectopic expression of Spry2 induced spontaneous apoptosis in Mec-1 cells and primary CLL cells from poor prognosis patients. These results indicate that Spry2 negatively regulates the survival of human CLL cells. CLL cells have been shown to have active B-cell receptor (BCR) signaling promoting proliferation in vivo. To study the effect of BCR stimulation on Spry2 we stimulated BCR on Mec-1 and primary CLL cells by BCR cross-linking. Unexpectedly, the rate of proliferation of Mec-1 cells decreased upon BCR stimulation. We were able to demonstrate a transient increase in Spry2 levels that peaked at 12 hours post BCR stimulation, which we suspected was mediating the decrease in proliferation observed. We therefore repeated the BCR stimulation experiment using siRNA to inhibit the transient increase in Spry2 in primary CLL cells from seven good prognosis patients and in the Mec-1 cells. As predicted, we observed a significant increase in proliferation in all primary CLL samples and in the Mec-1 cells upon Spry2 knock down following BCR stimulation. Spry2 attenuates ligand induced MAPK-Erk and AKT signaling leading to suppression of proliferation and survival. We also examined the effect of Spry2 on MAPK-Erk and Akt signaling in CLL cells. Upon induction of exogenous Spry2 expression in Mec-1 cells there was a significant decrease in p-Akt and p-Erk levels. Conversely, Spry2 knock down resulted in an increase in p-Erk and p-Akt and enhanced cell proliferation.

CONCLUSIONS: Spry2 is significantly down-regulated in the B-cells from patients with poor prognosis CLL. Down-regulation of Spry2 in CLL cells leads to hyperactivation of MAPK-Erk and Akt signaling and exogenous and endogenous expression of Spry2 leads to spontaneous apoptosis in human CLL cells. These results identify Spry2 as a negative regulator of CLL cell survival and proliferation and provide a potential molecular target for therapeutic intervention.