Key Points• Complete genome sequence analysis of 40 DLBCL tumors and 13 cell lines reveals novel somatic point mutations, rearrangements, and fusions. • Recurrence of mutations in genes involved in B-cell homing were identified in germinal center B-cell DLBCLs.Diffuse large B-cell lymphoma (DLBCL) is a genetically heterogeneous cancer composed of at least 2 molecular subtypes that differ in gene expression and distribution of mutations. Recently, application of genome/exome sequencing and RNAseq to DLBCL has revealed numerous genes that are recurrent targets of somatic point mutation in this disease. Here we provide a whole-genome-sequencing-based perspective of DLBCL mutational complexity by characterizing 40 de novo DLBCL cases and 13 DLBCL cell lines and combining these data with DNA copy number analysis and RNA-seq from an extended cohort of 96 cases. Our analysis identified widespread genomic rearrangements including evidence for chromothripsis as well as the presence of known and novel fusion transcripts. We uncovered new gene targets of recurrent somatic point mutations and genes that are targeted by focal somatic deletions in this disease. We highlight the recurrence of germinal center B-cell-restricted mutations affecting genes that encode the S1P receptor and 2 small GTPases (GNA13 and GNAI2) that together converge on regulation of B-cell homing. We further analyzed our data to approximate the relative temporal order in which some recurrent mutations were acquired and demonstrate that ongoing acquisition of mutations and intratumoral clonal heterogeneity are common features of DLBCL. This study further improves our understanding of the processes and pathways involved in lymphomagenesis, and some of the pathways mutated here may indicate new avenues for therapeutic intervention. (Blood. 2013;122(7):1256-1265 Introduction Diffuse large B-cell lymphoma (DLBCL) is an aggressive nonHodgkin lymphoma (NHL) with at least 2 molecular subtypes that demonstrate distinct clinical outcomes and gene expression profiles. Because these cancers derive from mature B cells, the mutations that arise in DLBCLs can result from somatic hypermutation that targets a small number of genes, 1 as well as structural rearrangements that arise from double-strand breaks that can be initiated by the B-cell recombination apparatus. In recent years, multiple groups have used massively parallel sequencing (genome/ exome sequencing and RNA-seq) to ascertain the full set of genes targeted by somatic single-nucleotide variants (SNVs) in this disease.2-5 On the basis of these and earlier studies, 6 it is now known that the 2 molecular subtypes also harbor distinct repertoires of somatic copy number alterations (CNAs) and SNVs. In particular, mutations affecting genes involved in B-cell receptor signaling and nuclear factor kB are common in the activated B-cell variety, 7 whereas those affecting certain genes with roles in histone modification may be more common in the germinal center B-cell (GCB) subtype. 2,8,9 These studies have confirmed t...

Antigen receptor signalling activates the canonical NF-κB pathway via the CARD11/BCL10/MALT1 (CBM) signalosome involving key, yet ill-defined roles for linear ubiquitination. The paracaspase MALT1 cleaves and removes negative checkpoint proteins, amplifying lymphocyte responses in NF-κB activation and in B-cell lymphoma subtypes. To identify new human MALT1 substrates, we compare B cells from the only known living MALT1mut/mut patient with healthy MALT1+/mut family members using 10-plex Tandem Mass Tag TAILS N-terminal peptide proteomics. We identify HOIL1 of the linear ubiquitin chain assembly complex as a novel MALT1 substrate. We show linear ubiquitination at B-cell receptor microclusters and signalosomes. Late in the NF-κB activation cycle HOIL1 cleavage transiently reduces linear ubiquitination, including of NEMO and RIP1, dampening NF-κB activation and preventing reactivation. By regulating linear ubiquitination, MALT1 is both a positive and negative pleiotropic regulator of the human canonical NF-κB pathway—first promoting activation via the CBM—then triggering HOIL1-dependent negative-feedback termination, preventing reactivation.

Somatic hypermutation (SHM) in the variable region of immunoglobulin genes (IGV) naturally occurs in a narrow window of B cell development to provide high-affinity antibodies. However, SHM can also aberrantly target proto-oncogenes and cause genome instability. The role of aberrant SHM (aSHM) has been widely studied in various non-Hodgkin's lymphoma particularly in diffuse large B-cell lymphoma (DLBCL). Although, it has been speculated that aSHM targets a wide range of genome loci so far only twelve genes have been identified as targets of aSHM through the targeted sequencing of selected genes. A genome-wide study aiming at identifying a comprehensive set of aSHM targets recurrently occurring in DLBCL has not been previously undertaken. Here, we present a comprehensive assessment of the somatic hypermutated genes in DLBCL identified through an analysis of genomic and transcriptome data derived from 40 DLBCL patients. Our analysis verifies that there are indeed many genes that are recurrently affected by aSHM. In particular, we have identified 32 novel targets that show same or higher level of aSHM activity than genes previously reported. Amongst these novel targets, 22 genes showed a significant correlation between mRNA abundance and aSHM.

When B cells encounter antigens on the surface of an antigen-presenting cell (APC), B cell receptors (BCRs) are gathered into microclusters that recruit signaling enzymes. These microclusters then move centripetally and coalesce into the central supramolecular activation cluster of an immune synapse. The mechanisms controlling BCR organization during immune synapse formation, and how this impacts BCR signaling, are not fully understood. We show that this coalescence of BCR microclusters depends on the actin-related protein 2/3 (Arp2/3) complex, which nucleates branched actin networks. Moreover, in murine B cells, this dynamic spatial reorganization of BCR microclusters amplifies proximal BCR signaling reactions and enhances the ability of membrane-associated antigens to induce transcriptional responses and proliferation. Our finding that Arp2/3 complex activity is important for B cell responses to spatially restricted membrane-bound antigens, but not for soluble antigens, highlights a critical role for Arp2/3 complex-dependent actin remodeling in B cell responses to APC-bound antigens.

In vivo, B cells are often activated by antigens that are displayed on the surface of antigen-presenting cells (APCs). Binding of membrane-associated antigens to the B cell receptor (BCR) causes rapid cytoskeleton-dependent changes in the spatial organization of the BCR and other B cell membrane proteins, leading to the formation of an immune synapse. This process has been modeled using antigens attached to artificial planar lipid bilayers or to plasma membrane sheets. As a more physiological system for studying B cell-APC interactions, we have expressed model antigens in easily transfected adherent cell lines such as Cos-7 cells. The model antigens that we have used are a transmembrane form of a single-chain anti-Igκ antibody and a transmembrane form of hen egg lysozyme that is fused to a fluorescent protein. This has allowed us to study multiple aspects of B cell immune synapse formation including cytoskeletal reorganization, BCR microcluster coalescence, BCR-mediated antigen gathering, and BCR signaling. Here, we provide protocols for expressing these model antigens on the surface of Cos-7 cells, transfecting B cells with siRNAs or with plasmids encoding fluorescent proteins, using fixed cell and live cell fluorescence microscopy to image B cell-APC interactions, and quantifying APC-induced changes in BCR spatial organization and signaling.