B-cell receptor dependent phagocytosis and presentation of particulate antigen by chronic lymphocytic leukemia cells.

Aim: T-helper cells could play an important role in the pathogenesis of chronic lymphocytic leukemia (CLL), a common B-cell neoplasm. Although CLL cells can present soluble antigens targeted from the B-cell receptor to T-helper cells via major histocompatibility complex (MHC) class II, antigens recognized by some CLL cells may be encountered in a particulate form. Here the ability of CLL cells to internalize and present anti-immunoglobulin M (IgM) beads as a model for the interaction of CLL cells with particulate antigens was investigated. Methods: The effect of anti-IgM beads on antigen presentation pathways was analyzed using RNA-seq and internalization of anti-IgM beads by primary CLL cells was investigated using confocal microscopy and flow cytometry. Antigen presentation was investigated by analyzing activation of a T-cell line expressing a T-cell receptor specific for a peptide derived from mouse κ light chains after incubating CLL cells with a mouse κ light chain-containing anti-IgM monoclonal antibody. Kinase inhibitors were used to characterize the pathways mediating internalization and antigen presentation. Results: Stimulation of surface IgM of CLL cells increased expression of the antigen presentation machinery and CLL cells were able to phagocytose anti-IgM beads. Internalization of anti-IgM beads was associated with MHC class II-restricted activation of cognate T-helper cells. Antigen presentation by CLL cells was dependent on activity of spleen tyrosine kinase (SYK) and phosphatidylinositol 3-kinase delta (PI3Kδ) but was unaffected by inhibitors of Bruton's tyrosine kinase (BTK). Conclusions: CLL cells can internalize and present antigen from anti-IgM beads. This capacity of CLL cells may be particularly important for recruitment of T-cell help in vivo in response to particulate antigens.

B-cell receptor signaling induces proteasomal degradation of PDCD4 via MEK1/2 and mTORC1 in malignant B cells.

B-cell receptor (BCR) signaling plays a major role in the pathogenesis of B-cell malignancies and is an established target for therapy, including in chronic lymphocytic leukemia cells (CLL), the most common B-cell malignancy. We previously demonstrated that activation of BCR signaling in primary CLL cells downregulated expression of PDCD4, an inhibitor of the translational initiation factor eIF4A and a potential tumor suppressor in lymphoma. Regulation of the PDCD4/eIF4A axis appeared to be important for expression of the MYC oncoprotein as MYC mRNA translation was increased following BCR stimulation and MYC protein induction was repressed by pharmacological inhibition of eIF4A. Here we show that MYC expression is also associated with PDCD4 down-regulation in CLL cells in vivo and characterize the signaling pathways that mediate BCR-induced PDCD4 down-regulation in CLL and lymphoma cells. PDCD4 downregulation was mediated by proteasomal degradation as it was inhibited by proteasome inhibitors in both primary CLL cells and B-lymphoma cell lines. In lymphoma cells, PDCD4 degradation was predominantly dependent on signaling via the AKT pathway. By contrast, in CLL cells, both ERK and AKT pathways contributed to PDCD4 down-regulation and dual inhibition using ibrutinib with either MEK1/2 or mTORC1 inhibition was required to fully reverse PDCD4 down-regulation. Consistent with this, dual inhibition of BTK with MEK1/2 or mTORC1 resulted in the strongest inhibition of BCR-induced MYC expression. This study provides important new insight into the regulation of mRNA translation in B-cell malignancies and a rationale for combinations of kinase inhibitors to target translation control and MYC expression.