Tonic B-cell receptor signaling in diffuse large B-cell lymphoma

Ondrej Havranek, Jingda Xu, Stefan Köhrer, Zhiqiang Wang, Lisa Becker, Justin M Comer, Jared Henderson, Wencai Ma, John Man Chun Ma, Jason R Westin, Dipanjan Ghosh, Nicholas Shinners, Luhong Sun, Allen F Yi, Anusha R Karri, Jan A Burger, Tomasz Zal, R Eric Davis

We used CRISPR/Cas9-mediated genomic modification to investigate B-cell receptor (BCR) signaling in cell lines of diffuse large B-cell lymphoma (DLBCL). Three manipulations that altered BCR genes without affecting surface BCR levels showed that BCR signaling differs between the germinal center B-cell (GCB) subtype, which is insensitive to BTK inhibition by ibrutinib, and the activated B-cell (ABC) subtype. Replacing antigen-binding BCR regions had no effect on BCR signaling in GCB-DLBCL lines, reflecting this subtype's exclusive use of tonic BCR signaling. Conversely, Y188F mutation in the immunoreceptor tyrosine-based activation motif of CD79A inhibited tonic BCR signaling in GCB-DLBCL lines but did not affect their calcium flux after BCR crosslinking, or the proliferation of otherwise-unmodified ABC-DLBCL lines. CD79A-GFP fusion showed BCR clustering or diffuse distribution, respectively, in lines of ABC and GCB subtypes. Tonic BCR signaling acts principally to activate AKT, and forced activation of AKT rescued GCB-DLBCL lines from knockout of the BCR or two mediators of tonic BCR signaling, SYK and CD19. The magnitude and importance of tonic BCR signaling to proliferation and size of GCB-DLBCL lines, shown by the effect of BCR knockout, was highly variable; in contrast, pan-AKT knockout was uniformly toxic. This discrepancy was explained by finding that BCR knockout-induced changes in AKT activity (measured by gene expression, CXCR4 level, and a fluorescent reporter) correlated with changes in proliferation, and with baseline BCR surface density. PTEN protein expression and BCR surface density may influence clinical response to therapeutic inhibition of tonic BCR signaling in DLBCL.