Inhibition of autoantigen-induced B-cell receptor (BCR) internalization as a therapeutic strategy in diffuse large B...

BCR signal dependency is a hallmark of diffuse large B-cell lymphoma (DLBCL) and other B-cell lymphoid malignancies originating from germinal centers. Chronic-active BCR signaling, typical for the more aggressive activated B-cell subtype (ABC) of DLBCLs, is often attributed to activating mutations within the BCR signaling cascade and continuous stimulation of the BCR by autoantigens. In certain ABC-DLBCLs, the BCR forms an intracellular multiprotein supercomplex with TLR9 and MYD88, which generates signals from endolysosomes. However, it is not clear whether the internalization of BCR is required for sustained signaling, nor have the mechanisms responsible for BCR trafficking been defined. To address these questions, we developed DLBCL cell models with modified ovalbumin (OVA)-specific hypervariable regions (HVRs) in the BCRs using CRISPR-Cas9 technology. Modified BCRs were incapable of binding self-antigens, while still responding in a controlled fashion to stimulation with ovalbumin. Using these genetic models, we demonstrated that autoantigens drive a complex BCR-dependent signaling program and facilitate the assembly of the intracellular BCR-TLR9-IκB complex, promoting NFκB pathway activation. Furthermore, we showed that the binding of autoantigens to the BCR leads to the internalization of the BCR-autoantigen complex via clathrin-mediated endocytosis (CME). Using genetic models with inducible inhibition of this endocytic pathway, we found that BCR internalization is essential for the oncogenic activation of the BCR-dependent signaling pathways and the formation of the BCR-TLR9-IκB complex in autoantigen-dependent ABC-DLBCL cells. Finally, CME inhibition with dynamin-2 antagonists, such as phenothiazine derivatives, reduces BCR signaling, cell viability, and synergizes with SYK and PI3Kδ inhibitors. Since phenothiazines have well-defined safety and pharmacokinetic profiles, our data provide a framework for the rational design of clinical trials employing these drugs in the autoantigen-dependent subset of DLBCL.