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-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 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-AKT KO was uniformly toxic. This discrepancy was explained by finding that BCR KO-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.

The B‐cell receptor in control of tumor B‐cell fitness: Biology and clinical relevance

“…The involvement of self‐antigens driving the proliferation of a subset of ABC‐type DLBCL cell lines through chronic BCR engagement has been also documented . Gene editing based on CRISPR/Cas9 technology has recently been employed to further identify genetic determinants required for DLBCL and BL growth . In these studies, components of the BCR signaling complex (CD79A and CD79B), as well as several proximal regulators/effectors of BCR signaling including CD19, Lyn, Blk, Syk, Csk, Blnk, Plcγ2, Btk, and Ptprc were shown to be required for optimal growth of the malignant B cells of most lymphoma cell lines tested in vitro .…”

“…Gene editing based on CRISPR/Cas9 technology has recently been employed to further identify genetic determinants required for DLBCL and BL growth . In these studies, components of the BCR signaling complex (CD79A and CD79B), as well as several proximal regulators/effectors of BCR signaling including CD19, Lyn, Blk, Syk, Csk, Blnk, Plcγ2, Btk, and Ptprc were shown to be required for optimal growth of the malignant B cells of most lymphoma cell lines tested in vitro . Notably, the dependence on particular BCR signaling effectors differed between lymphoma lines representing, respectively, the ABC‐ and GC‐type DLBCL .…”

“…Notably, the dependence on particular BCR signaling effectors differed between lymphoma lines representing, respectively, the ABC‐ and GC‐type DLBCL . Specifically, whereas ABC‐type DLBCL tumor lines were dependent on a BCR signaling axis centered around the activation of the PI3K, mTOR, and NF‐κB signaling pathways, the GC‐type DLBCL tumors (as well as most BL lines) relied on PI3K‐AKT and mTOR signaling, while they resisted to NF‐κB inhibition . The dependence of the malignant B cells from different types of B‐LPD on selected BCR signaling pathways likely reflects the different state of activation and mode of action of the BCR in the cell‐of‐origin of the individual tumors …”

“…Interference with BCR expression may alter the response of malignant B cells to the chemokine CXCL12 . In particular, in tumors arising from GC B cells, the interference with BCR expression/function could increase CXCR4 expression, and thereby, possibly its signaling upon binding to CXCL12 released by neighboring stromal cells. This behavior could resemble that of dark zone GC B cells where substantial downregulation of sIg expression correlates with high CXCR4 expression and heightened responsiveness to CXCL12 .…”

“…In this context, the choice of which BCR inhibitor is employed in the therapy can be of crucial relevance, in an effort to concomitantly eliminate both BCR‐proficient and BCR‐negative tumor B cells. Indeed, whereas Ig‐negative lymphoma cells may more easily resist to inhibition of proximal effectors of BCR signaling, such as SYK and BTK, they may continue to depend on downstream effectors including specific PI3K isoforms, which could get activated in these cells through alternative pathways initiated by growth factors and/or other stimuli received from the tumor microenvironment, or through the selection of specific genetic alterations including PTEN mutations …”