Chronic Lymphocytic Leukemia B-Cells Lack or Express Markedly Reduced Levels of the Phosphatase PHLPP, Which Regulates B-Cell Receptor Induced AKT Activation.

Abstract

The PI3K/AKT pathway plays a central role in regulating cellular growth and survival. This pathway is activated by signals derived from various receptors and is tightly regulated through the action of several phosphatases, including SHIP and PTEN, which hydrolyze the PI3K product PIP3, and the recently identified PHLPP, which directly dephosphorylates AKT. Hyperactivation of the PI3K/AKT pathway has been implicated in the pathogenesis of many types of cancer, including chronic lymphocytic leukemia (CLL) and B-cell lymphoma. In addition, gene expression profiling and real-time RT/PCR analysis have recently shown differential expression of PHLPP mRNA in CLL subsets classified according to the presence of the 13q14 abnormality, with many CLL cases demonstrating absent PHLPP expression altogether. These findings prompted us to compare the levels of PHLPP expression in primary CLL B-cells (n=17) with normal tonsillar B-cells (n=4) and various lymphoma cell lines, including the diffuse large B-cell lymphomas (DLBCL) DHL-4, DHL-6, DHL-8, DHL-10, WSU, Toledo, Ly1, Ly3, Ly7 and Ly18, the Burkitt's lymphoma BJAB and the prolymphocytic leukemia MEC1. Immunoblotting analysis revealed abundant and uniform expression of PHLPP in normal B-cells and in 7 out of 12 investigated lymphoma cell lines. Higher levels were observed in the BJAB, Ly1 and Ly18 cell lines, whereas PHLPP was undetectable in the DLBCL cell lines WSU and Toledo. Remarkably, PHLPP was either not expressed or was expressed at markedly reduced levels in all of the investigated CLL samples, with levels of expression ranging from 0 to 10% of the levels in normal B-cells. In contrast, the levels of expression of the phosphatase SHIP were relatively similar between CLL and normal B-cells. To determine what are the consequences of reduced PHLPP expression on signaling through AKT in malignant B-lymphocytes, we downregulated PHLPP in BJAB and DHL-4 cells by RNA interference. A significant reduction in the levels of PHLPP was achieved in both cell lines, which amounted to 20–40% of the levels in cells transfected with the control siRNA. Immunoblotting analysis of protein extracts from cells transfected with PHLPP and control siRNA did not show a difference in AKT phosphorylation on Ser473 and Thr308, indicating that a reduction in PHLPP expression is not sufficient to augment basal AKT activity. To determine the effects of PHLPP downregulation on agonist-induced AKT activation, we investigated phosphorylation on Ser473 and Thr308 in BJAB and DHL-4 cells stimulated through the B-cell receptor. In both cell lines downregulation of PHLPP resulted in more than a 50% increase in BCR-induced AKT phosphorylation. In contrast, phosphorylation of other signaling molecules that are also activated by BCR crosslinking, such as PLCγ2 and ERK, appeared unaffected by PHLPP downregulation. These data confirm the functional relevance of PHLPP in AKT regulation in B-lymphoid cells and implicate reduced or absent PHLPP expression in CLL B-cells as a potential determinant of BCR-induced AKT signaling in CLL.