Defective Inhibitory Signaling in CLL B Cells and Increased Recruitment of PI3K by c-Cbl in Zap-70+ CLL.

An inhibitory signaling pathway involving sialic acid 9-O-acetyl esterase (SIAE), sialic acid binding lectins (Siglecs) particularly Siglec-2/CD22, the Lyn tyrosine kinase, and the SH2 domain containing tyrosine phosphatase, SHP-1, attenuates B cell receptor signaling and sets a threshold for B cell activation. A key step in the process is the requirement that SIAE access N-glycans on Siglec ligands and remove 9-O-acetyl groups from terminal αa2,6 linked sialic acid moieties. Siglec-ligand interaction is followed by phosphorylation of ITIM tyrosines on CD22 by Lyn, and the recruitment of SHP-1 by CD22 resulting in signal attenuation (Cariappa et al., J.Exp.Med.2009, 206, 125). While Lyn has both positive and negative signaling functions, knockout mice studies suggest that inhibitory functions are dominant. Previous studies have shown that CLL cells overexpress active Lyn at the protein level and that Lyn is localized to sites beyond the plasma membrane. Although cell surface expression of CD22 is reduced in CLL, it is not known if CD22 can be accessed in CLL cells by promiscuously active Lyn. We sought to ask if cancer progression in CLL involves the evolution of mechanisms to evade inhibitory signaling, thus tipping the balance towards positive, pro-proliferative signaling by Lyn.

CLL B cells from patient and control subjects were isolated. Immunoprecipitation and Western blot approaches were used to quantitate the total cellular levels of Lyn and CD22αa and β proteins at the protein level, the ratio of CD22 phosphorylated on an inhibitory tyrosine to total CD22, recruitment of SHP-1 by CD22, the activation of Syk, the expression of c-Cbl and the recruitment of PI3K by c-Cbl in CLL and control B cells.

A modest decrease in total CD22αa and β proteins was observed in CLL but a dramatic reduction in the proportion of ITIM-phosphorylated CD22, and a reduction in the recruitment of SHP-1 by CD22 in CLL B cells. Decreased inhibitory signaling in CLL correlates with an increase in active Syk. An increase in c-Cbl protein levels was observed and an increased recruitment of p85PI3K was observed specifically in Zap-70 positive CLL.

Defective inhibitory signaling may contribute to disease progression in CLL. This defect probably results from the inability of CD22 to access 9-O-deacetylated ligands even in the presence of active Lyn. Enhanced constitutive BCR signaling prevails in all CLL patients but in Zap70+ CLL patients p85PI3K is more readily recruited by c-Cbl.

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