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

Abstract

We used clustered regularly interspaced short palindromic repeats/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 Bruton tyrosine kinase inhibition by ibrutinib, and the activated B-cell (ABC) subtype.Replacing antigen-bindingBCRregions had no effect onBCR signaling inGCB-DLBCLlines, reflecting this subtype's exclusive use of tonicBCRsignaling. Conversely, Y188Fmutation in the immunoreceptor tyrosine-based activationmotif ofCD79A inhibited tonic BCR signaling in GCB-DLBCL lines but did not affect their calcium flux after BCR cross-linking 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 (KO) of the BCR or 2 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 KO, was highly variable; in contrast, pan-AKTKOwas uniformly toxic. This discrepancywas explained by finding thatBCRKO-induced changes inAKT activity (measured by gene expression,CXCR4 level, and a fluorescent reporter) correlated with changes in proliferation andwith baseline BCR surface density. PTEN protein expression and BCR surface density may influence clinical response to therapeutic inhibition of tonic BCR signaling in DLBCL.