SUMMARY B-cell receptor (BCR) signaling pathway components represent promising treatment targets in diffuse large B-cell lymphoma (DLBCL) and additional B-cell tumors. BCR signaling activates spleen tyrosine kinase (SYK) and downstream pathways including PI3K/AKT and NF-κB. In previous studies, chemical SYK blockade selectively decreased BCR signaling and induced apoptosis of BCR-dependent DLBCLs. Herein, we characterize distinct SYK/PI3K-dependent survival pathways in DLBCLs with high or low baseline NF-κB activity including selective repression of the pro-apoptotic HRK protein in NF-κB-low tumors. We also define SYK/PI3K-dependent cholesterol biosynthesis as a feed-forward mechanism of maintaining the integrity of BCRs in lipid rafts in DLBCLs with low or high NF-κB. In addition, SYK amplification and PTEN deletion are identified as selective genetic alterations in primary “BCR”-type DLBCLs.

Serum‐ and glucocorticoid‐induced protein kinase‐1 (SGK‐1) plays a critical role in regulation of the epithelial sodium channel, ENaC. SGK‐1 also shares significant catalytic domain homology with protein kinase B (PKB/AKT‐1) and is a downstream effector of antiapoptotic phosphoinositide 3‐kinase signaling. Steady‐state levels of an active SGK‐1 are tightly regulated by rapid transcriptional activation and post‐translational modification including phosphorylation. We show here that endogenous SGK‐1 protein is polyubiquitinated and rapidly degraded by the 26S proteasome. In contrast to other rapidly degraded kinases, neither the catalytic activity of SGK‐1 nor activation site phosphorylation was required for its ubiquitin modification and degradation. Instead, SGK‐1 degradation required a lysine‐less six‐amino‐acid (amino acids 19–24) hydrophobic motif (GMVAIL) within the N‐terminal domain. Deletion of amino acids 19–24 significantly increased the half‐life of SGK1 and prevented its ubiquitin modification. Interestingly, this minimal region was also required for the association of SGK‐1 with the endoplasmic reticulum. Ubiquitin modification and degradation of SGK‐1 were increasingly inhibited by the progressive mutation of six N‐terminal lysine residues surrounding the GMVAIL motif. Mutation of all six lysines to arginine did not disrupt the subcellular localization of SGK‐1 despite a significant decrease in ubiquitination, implying that this modification per se was not required for targeting to the endoplasmic reticulum. These results suggest that constitutive ubiquitin‐mediated degradation of SGK‐1 is an important mechanism regulating its biological activity.

Purpose B cell receptor (BCR) mediated signaling is important in the pathogenesis of a subset of diffuse large B cell lymphomas (DLBCL) and the BCR-associated kinases SYK and BTK have recently emerged as potential therapeutic targets. We sought to identify a signature of activated BCR signaling in DLBCL to aid the identification of tumors that may be most likely to respond to BCR-pathway inhibition. Experimental Design We applied quantitative immunofluorescence (qIF) using antibodies to phosphorylated forms of proximal BCR signaling kinases LYN, SYK and BTK and antibody to BCR-associated transcription factor FOXO1 on BCR-crosslinked formalin-fixed paraffin-embedded (FFPE) DLBCL cell lines as a model system and on two clinical cohorts of FFPE DLBCL specimens (n=154). Results A robust signature of active BCR signaling was identified and validated in BCR-crosslinked DLBCL cell lines and in 71/154 (46%) of the primary DLBCL patient specimens. Further analysis of the primary biopsy samples revealed increased nuclear exclusion of FOXO1 among DLBCL with qIF evidence of active BCR signaling compared to those without (p = 0.004). Nuclear exclusion of FOXO1 was also detected in a subset of DLBCL without evidence of proximal BCR signaling suggesting that alternative mechanisms for PI3K/AKT activation may mediate FOXO1 subcellular localization in these cases. Conclusion This study establishes the feasibility of detecting BCR activation in primary FFPE biopsy specimens of DLBCL. It lays a foundation for future dissection of signal transduction networks in DLBCL and provides a potential platform for evaluating individual tumors in patients receiving novel therapies targeting the BCR pathway.