Chronic active B-cell-receptor signalling in diffuse large B-cell lymphoma

Abstract

A role for B-cell-receptor (BCR) signalling in lymphomagenesis has been inferred by studying immunoglobulin genes in human lymphomas and by engineering mouse models, but genetic and functional evidence for its oncogenic role in human lymphomas is needed. Here we describe a form of 'chronic active' BCR signalling that is required for cell survival in the activated B-cell-like (ABC) subtype of diffuse large B-cell lymphoma (DLBCL). The signalling adaptor CARD11 is required for constitutive NF-kappaB pathway activity and survival in ABC DLBCL. Roughly 10% of ABC DLBCLs have mutant CARD11 isoforms that activate NF-kappaB, but the mechanism that engages wild-type CARD11 in other ABC DLBCLs was unknown. An RNA interference genetic screen revealed that a BCR signalling component, Bruton's tyrosine kinase, is essential for the survival of ABC DLBCLs with wild-type CARD11. In addition, knockdown of proximal BCR subunits (IgM, Ig-kappa, CD79A and CD79B) killed ABC DLBCLs with wild-type CARD11 but not other lymphomas. The BCRs in these ABC DLBCLs formed prominent clusters in the plasma membrane with low diffusion, similarly to BCRs in antigen-stimulated normal B cells. Somatic mutations affecting the immunoreceptor tyrosine-based activation motif (ITAM) signalling modules of CD79B and CD79A were detected frequently in ABC DLBCL biopsy samples but rarely in other DLBCLs and never in Burkitt's lymphoma or mucosa-associated lymphoid tissue lymphoma. In 18% of ABC DLBCLs, one functionally critical residue of CD79B, the first ITAM tyrosine, was mutated. These mutations increased surface BCR expression and attenuated Lyn kinase, a feedback inhibitor of BCR signalling. These findings establish chronic active BCR signalling as a new pathogenetic mechanism in ABC DLBCL, suggesting several therapeutic strategies.

Figures

Figure 1. BTK is a critical kinase for survival of ABC DLBCL cells

A. RNA interference screen in lymphoma and multiple myeloma cell lines. An shRNA library targeting 442 kinases was screened in the indicated cell lines as described. Shown is the selective toxicity of two BTK shRNAs after 3 weeks in culture. Bar values are mean +/− s.d. of four independent transductions. B. Selective toxicity of a BTK shRNA for ABC DLBCLs with wild type CARD11. DLBCL cell lines were infected with a retrovirus that expresses BTK shRNA #1 together with GFP. Shown is the fraction of GFP+ cells relative to the GFP+ fraction on day 2. C. BTK kinase activity is required for survival of ABC DLBCL cells. OCI-Ly10 cells were transduced with cDNAs encoding wild type or mutant BTK (kinase-dead allele or analog-sensitive kinase allele (ASKA)29). Wild type but not kinase-dead BTK rescued cells with endogenous BTK knockdown. The ASKA isoform-specific kinase inhibitor 1-NM-PP1 (2 mM) killed cells bearing the BTK ASKA allele.

Figure 2. Chronic active BCR signaling in ABC DLBCL lines

A. Survival of DLBCL cell lines following shRNA-mediated knockdown of BCR signaling components CD79A, SYK, and CARD11. B. Knockdown of immunoglobulin heavy or light chain is toxic for ABC DLBCLs with chronic active BCR signaling. C. Phosphoproteins in multiple signaling pathways depend upon chronic active BCR signaling. Indicated ABC DLBCL cell lines were transduced with an shRNA targeting CD79A and phosphorylated or total proteins were assessed by Western blotting before and after 48-hour shRNA induction. D. Clustering of IgM in the plasma membrane was observed only in ABC DLBCL lines with chronic active BCR signaling, using TIRF microscopy. Plasma membrane density was revealed by membrane dye R18. E. Reduced diffusion of surface IgM in ABC DLBCL lines with chronic active BCR signaling as compared to the GCB DLBCL line, as quantified by TIRF microscopy. F. Immobile BCR clusters are characteristic of lines representing ABC DLBCL but not other lymphoma types.

Figure 3. CD79A and CD79B ITAM mutations in ABC DLBCL

A. CD79B and CD79A ITAM mutations in DLBCL biopsies and lines (case number in parenthesis). B. CD79B ITAM mutation frequencies in lymphoma biopsies. C. Mutant CD79A and CD79B isoforms increase surface IgM. The GCB DLBCL line BJAB was reconstituted with either wild type or mutant CD79A/B proteins. Surface IgM is depicted relative to CD79 RNA levels, estimated using bicistronic expression of CD8. “Synthetic” mutants were not observed in patient samples. D. CD79B mutations prevent down-modulation of surface BCR by BCR signaling. The ABC DLBCL line HBL-1 was reconstituted with wild type or Y196H mutant CD79B and treated for 24 hours with DMSO or dasatinib, a BCR signaling inhibitor. Surface IgM (mean fluorescence intensity; M.F.I.) is depicted relative to the levels in cells with wild type CD79B treated with DMSO. Error bars depict +/− s.e.m.; 2 experiments. E. CD79B mutations inhibit LYN kinase activity in ABC DLBCLs. The indicated ABC DLBCL lines were reconstituted with wild type or Y196F mutant CD79B. LYN kinase activity in immunoprecipitates was estimated by densitometric analysis of Western blots as phospho-LYN (using anti-phospho-tyrosine antibody 4G10) relative to total LYN.

Figure 4. Therapeutic strategies to target chronic active BCR signaling

A. Viability of DLBCL lines assessed by MTT assay after 4 days of treatment with varying doses of dasatinib, the BTK inhibitor PCI-32765 (compound 13 in ref.25), or an IKKβ inhibitor. B. Effect of dasatinib on phospho-protein levels in ABC DLBCL cells. Three ABC DLBCL lines were treated with dasatinib (50 nM) for the indicated times and analyzed by Western blotting.