Overexpression of the Autoimmunity-Associated Tyrosine Phosphatase PTPN22 in CLL Cells Enhances Antiapoptotic B-Cell Receptor Signals Through Selective Activation of the AKT Pathway

Abstract 619

The phosphatase PTPN22 is an important negative regulator of T cell receptor (TCR) signaling. A gain of function polymorphism in this phosphatase is strongly linked with increased risk for the development of several common autoimmune diseases, including rheumatoid arthritis, insulin-dependent diabetes mellitus and systemic lupus erythematosus. The exact mechanism how the more active PTPN22 variant contributes to the development of these autoimmune diseases is still unclear, but it is has been postulated that increased activity of this enzyme may prevent negative selection of autoreactive T cells by blocking proapoptotic TCR signals induced by autoantigen.

We now report that PTPN22 is significantly overexpressed in CLL B cells. Of the 91 investigated CLL samples, in 66 (73%) the levels of PTPN22 were at least 2 fold higher and often exceeded by 10 fold the levels in normal tonsilar B cells (CLL, mean PTPN22 levels 5.7, SD +/−4.3; normal tonsilar B cells, mean PTPN22 levels 0.9, SD +/−0.1). PTPN22 levels were somewhat higher in CD38−positive than CD38−negative cases (P=0.031), whereas no association was observed with IGHV mutation status, ZAP-70 expression and time to treatment.

To determine the functional consequences of PTPN22 overexpression, we performed RNA interference experiments in primary CLL B cells with high PTPN22. We evaluated the effects of PTPN22 silencing in unstimulated CLL cells as well as CLL cells stimulated through the B cell receptor (BCR), given the important role of the BCR signaling pathway in the pathophysiology of CLL. Both soluble and immobilized anti-IgM antibodies were used for BCR stimulation, as these two BCR crosslinking agents have opposing effects on leukemic cell survival.

Silencing of PTPN22 had no effect on the viability of unstimulated CLL cells (n=11, viable CLL cells: siControl 49%, siPTPN22 48%, P=n.s.), but significantly inhibited the antiapoptotic effect of immobilized anti-IgM (viable CLL cells: siControl 55%, siPTPN22 48%, P=0.001) and enhanced the proapoptotic effect of soluble anti-IgM (viable CLL cells: siControl 44%, siPTPN22 31%, P<0.001). Consistent with these data, overexpression of PTPN22 in the lymphoma B cell line B104 significantly blocked apoptosis induced by soluble anti-IgM (control 22%, PTPN22 35%) without affecting the survival of unstimulated cells (control 52%, PTPN22 51%).

The previous experiments suggested that PTPN22 functions as a molecular switch that enhances antiapoptotic and inhibits proapoptotic BCR signals. To establish the molecular mechanisms underlying these different activities, we studied BCR signal transduction in primary CLL cells transfected with control or PTPN22-specific siRNA. Downregulation of PTPN22 increased anti-IgM-induced activation of several BCR signaling molecules, including LYN, SYK, ERK, JNK and the proapoptotic p38MAPK, confirming that PTPN22 is primarily a negative regulator of BCR signaling. However, activation of the antiapoptotic AKT kinase was substantially reduced by silencing of PTPN22, as evidenced by decreased phosphorylation of AKT and its direct targets GSK3b and FOXO3a. Lack of PTPN22 expression was further associated with increased activity of the phosphatase SHIP, a key negative regulator of the AKT kinase. Opposite effects on the activity of these signaling molecules were observed when PTPN22 was overexpressed in B104 cells, thus validating the data obtained by PTPN22 knockdown in primary CLL cells.

In summary, this study shows that the phosphatase PTPN22, which has been implicated in the pathogenesis of several common autoimmune diseases, is significantly overexpressed in CLL B-cells. Overexpression of PTPN22 inhibits activation of molecules that propagate the proapoptotic BCR signal, such as p38MAPK, but at the same time enhances the antiapoptotic signal delivered through the AKT kinase. The mechanism how PTPN22 enhances activation of AKT in response to BCR engagement is by preventing Lyn-mediated activation of the negative regulator SHIP. Collectively, these data show that PTPN22 is an important regulatory molecule in CLL and a potential therapeutic target. Inhibitors of this phosphatase, which are currently being developed for the treatment of autoimmune diseases associated with the gain of function PTPN22 variant, would be expected to convert the BCR survival signal into a death signal and may thus provide means for selective targeting of the malignant clone.