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BACKGROUND: Follicular lymphoma (FL) is an indolent malignancy of germinal center B-cell origin. FL patients experience remarkable heterogeneity in their disease trajectory, with many patients slowly progressing over several years, and a subset of patients experiencing an aggressive clinical course. Uncovering the cell-intrinsic and -extrinsic factors that govern differential progression and outcome in FL patients is thus essential. Beyond the genetic and epigenetic aberrations that contribute to FL oncogenesis, the tumor microenvironment (TME) plays an integral role in supporting the proliferation and survival of malignant cells. In a process described as "re-education", FL tumor cells may actively subvert the normal functions of non-malignant cells present in the TME, including T cells, follicular dendritic cells (FDCs), macrophages, dendritic cells, and stromal cells to support their survival and growth. METHODS: To address the importance of the TME in FL, we molecularly profiled excisional lymph node biopies from untreated FL patients using multiple platforms: bulk RNA sequencing (RNAseq), single-cell RNA sequencing (scRS), and a unique high content imaging method, Iterative Bleaching Extends MultipleXity (IBEX), which utilizes chemical bleaching to image 40+ proteins in the same tissue section by antibody staining. In combination with advanced computational tools for the quantitative analysis of cell types and distribution in tissues, we have used this approach to evaluate the TME:FL interaction within 8 FL samples, with 4 normal lymph nodes as controls. RESULTS: Both FL and TME components reconstructed from bulk RNA-seq were similar to the cellular composition revealed by scRS and IBEX analyses. Moreover, the bulk RNAseq and scRS identified the expression of genes involved in tumorigenesis and oncogenic signaling, often unique to each case. However, RNAseq-based approaches often miss important cellular and acellular components not readily extracted from dense tissues. For example, IBEX imaging can trace the pattern of blood vessels within a section, which cannot be achieved with non-imaging methods. In one case, our multi-parameter imaging studies revealed the close spatial interaction between clonal FL B cells, expressing a B-cell receptor (BCR) possessing a de novo N-linked glycosylation site introduced by somatic hypermutation, and cells expressing dendritic cell-specific intercellular adhesion molecule-3-grabbing non-integrin (DC-SIGN). This contact may activate pro-survival signaling in the malignant B cells. CONCLUSIONS: Integration of bulk RNAseq, scRS, clonotype analysis, and IBEX reveals both shared and unique aspects of different FL tumors. These data highlight the importance of integrating direct tissue analysis by high-content imaging with methods examining aspects of isolated cells. This approach may provide a more complete understanding of tumor biology, which in turn will identify patients at risk for developing aggressive disease and rationally improve treatment strategies for FL. This research was supported in part by the Intramural Research Program of the NIH, NIAID and NCI Figure Disclosures Bagaev: BostonGene: Current Employment, Current equity holder in private company, Patents & Royalties. Plotnikova:BostonGene: Current Employment, Current equity holder in private company, Patents & Royalties. Galkin:BostonGene: Current Employment, Patents & Royalties. Postovalova:BostonGene: Current Employment, Current equity holder in private company. Svekolkin:BostonGene: Current Employment, Current equity holder in private company, Patents & Royalties. Isaev:BostonGene: Current Employment, Current equity holder in private company, Patents & Royalties. Lozinsky:BostonGene: Current Employment, Current equity holder in private company, Patents & Royalties. Meerson:BostonGene: Current Employment. Varlamova:BostonGene: Current Employment, Current equity holder in private company, Patents & Royalties. Ovcharov:BostonGene: Current Employment, Current equity holder in private company, Patents & Royalties. Polyakova:BostonGene: Current Employment, Current equity holder in private company, Patents & Royalties. Nomie:BostonGene: Current Employment, Current equity holder in private company. Kotlov:BostonGene: Current Employment, Current equity holder in private company, Patents & Royalties. Tsiper:BostonGene: Current Employment, Current equity holder in private company, Patents & Royalties. Frenkel:BostonGene: Current Employment, Current equity holder in private company, Patents & Royalties. Attaulakhanov:BostonGene: Current Employment, Current equity holder in private company, Patents & Royalties. Fowler:BostonGene: Current Employment, Current equity holder in private company, Patents & Royalties.

Recombination activating gene 1 (RAG1) and RAG2 are critical enzymes for initiating variable-diversity-joining [V(D)J] segment recombination, an essential process for antigen receptor expression and lymphocyte development. The BCL11A transcription factor is required for B cell and plasmacytoid dendritic cell (pDC) development, but its molecular function(s) in early B cell fate specification and commitment is unknown. We show here that the major B cell isoform, BCL11A-XL, binds directly to the RAG1 promoter as well as directly to regulatory regions of transcription factors previously implicated in both B cell and pDC development to activate RAG1 and RAG2 gene transcription in pro- and pre-B cells. We employed BCL11A overexpression with recombination substrates to demonstrate direct consequences of BCL11A/RAG modulation on V(D)J recombination. We conclude that BCL11A is a critical component of a transcriptional network that regulates B cell fate by controlling V(D)J recombination.

B cell receptor (BCR) signalling has emerged as a therapeutic target in B cell lymphomas, but inhibiting this pathway in diffuse large B cell lymphoma (DLBCL) has benefited only a subset of patients1. Gene expression profiling identified two major subtypes of DLBCL, known as germinal centre B cell-like and activated B cell-like (ABC)2,3, that show poor outcomes after immunochemotherapy in ABC. Autoantigens drive BCR-dependent activation of NF-κB in ABC DLBCL through a kinase signalling cascade of SYK, BTK and PKCβ to promote the assembly of the CARD11-BCL10-MALT1 adaptor complex, which recruits and activates IκB kinase4-6. Genome sequencing revealed gain-of-function mutations that target the CD79A and CD79B BCR subunits and the Toll-like receptor signalling adaptor MYD885,7, with MYD88(L265P) being the most prevalent isoform. In a clinical trial, the BTK inhibitor ibrutinib produced responses in 37% of cases of ABC1. The most striking response rate (80%) was observed in tumours with both CD79B and MYD88(L265P) mutations, but how these mutations cooperate to promote dependence on BCR signalling remains unclear. Here we used genome-wide CRISPR-Cas9 screening and functional proteomics to determine the molecular basis of exceptional clinical responses to ibrutinib. We discovered a new mode of oncogenic BCR signalling in ibrutinib-responsive cell lines and biopsies, coordinated by a multiprotein supercomplex formed by MYD88, TLR9 and the BCR (hereafter termed the My-T-BCR supercomplex). The My-T-BCR supercomplex co-localizes with mTOR on endolysosomes, where it drives pro-survival NF-κB and mTOR signalling. Inhibitors of BCR and mTOR signalling cooperatively decreased the formation and function of the My-T-BCR supercomplex, providing mechanistic insight into their synergistic toxicity for My-T-BCR+ DLBCL cells. My-T-BCR supercomplexes characterized ibrutinib-responsive malignancies and distinguished ibrutinib responders from non-responders. Our data provide a framework for the rational design of oncogenic signalling inhibitors in molecularly defined subsets of DLBCL.

Endosomal TLRs play an important role in systemic autoimmune diseases, such as systemic erythematosus lupus, in which DNA- and RNA-associated autoantigens activate autoreactive B cells through TLR9- and TLR7-dependent pathways. Nevertheless, TLR9-deficient autoimmune-prone mice develop more severe clinical disease, whereas TLR7-deficient and TLR7/9–double deficient autoimmune-prone mice develop less severe disease. To determine whether the regulatory activity of TLR9 is B cell intrinsic, we directly compared the functional properties of autoantigen-activated wild-type, TLR9-deficient, and TLR7-deficient B cells in an experimental system in which proliferation depends on BCR/TLR coengagement. In vitro, TLR9-deficient cells are less dependent on survival factors for a sustained proliferative response than are either wild-type or TLR7-deficient cells. The TLR9-deficient cells also preferentially differentiate toward the plasma cell lineage, as indicated by expression of CD138, sustained expression of IRF4, and other molecular markers of plasma cells. In vivo, autoantigen-activated TLR9-deficient cells give rise to greater numbers of autoantibody-producing cells. Our results identify distinct roles for TLR7 and TLR9 in the differentiation of autoreactive B cells that explain the capacity of TLR9 to limit, as well as TLR7 to promote, the clinical features of systemic erythematosus lupus.

We have shown previously that priming of respiratory mucosa with live Lactobacillus species promotes robust and prolonged survival from an otherwise lethal infection with pneumonia virus of mice, a property known as heterologous immunity. Lactobacillus priming results in a moderate reduction in virus recovery and a dramatic reduction in virus-induced proinflammatory cytokine production; the precise mechanisms underlying these findings remain to be elucidated. Because B cells have been shown to promote heterologous immunity against respiratory virus pathogens under similar conditions, in this study we explore the role of B cells in Lactobacillus-mediated protection against acute pneumovirus infection. We found that Lactobacillus-primed mice feature elevated levels of airway Igs IgG, IgA, and IgM and lung tissues with dense, B cell (B220+)–enriched peribronchial and perivascular infiltrates with germinal centers consistent with descriptions of BALT. No B cells were detected in lung tissue of Lactobacillus-primed B cell deficient μMT mice or Jh mice, and Lactobacillus-primed μMT mice had no characteristic infiltrates or airway Igs. Nonetheless, we observed diminished virus recovery and profound suppression of virus-induced proinflammatory cytokines CCL2, IFN-γ, and CXCL10 in both wild-type and Lactobacillus-primed μMT mice. Furthermore, Lactobacillus plantarum–primed, B cell–deficient μMT and Jh mice were fully protected from an otherwise lethal pneumonia virus of mice infection, as were their respective wild-types. We conclude that B cells are dispensable for Lactobacillus-mediated heterologous immunity and were not crucial for promoting survival in response to an otherwise lethal pneumovirus infection.

The requirement for the B-cell transcription factor OCT2 (octamer-binding protein 2, encoded by Pou2f2) in germinal center B cells has proved controversial. Here, we report that germinal center B cells are formed normally after depletion of OCT2 in a conditional knockout mouse, but their proliferation is reduced and in vivo differentiation to antibody-secreting plasma cells is blocked. This finding led us to examine the role of OCT2 in germinal center-derived lymphomas. shRNA knockdown showed that almost all diffuse large B-cell lymphoma (DLBCL) cell lines are addicted to the expression of OCT2 and its coactivator OCA-B. Genome-wide chromatin immunoprecipitation (ChIP) analysis and gene-expression profiling revealed the broad transcriptional program regulated by OCT2 that includes the expression of STAT3, IL-10, ELL2, XBP1, MYC, TERT, and ADA. Importantly, genetic alteration of OCT2 is not a requirement for cellular addiction in DLBCL. However, we detected amplifications of the POU2F2 locus in DLBCL tumor biopsies and a recurrent mutation of threonine 223 in the DNA-binding domain of OCT2. This neomorphic mutation subtly alters the DNA-binding preference of OCT2, leading to the transactivation of noncanonical target genes including HIF1a and FCRL3 Finally, by introducing mutations designed to disrupt the OCT2-OCA-B interface, we reveal a requirement for this protein-protein interface that ultimately might be exploited therapeutically. Our findings, combined with the predominantly B-cell-restricted expression of OCT2 and the absence of a systemic phenotype in our knockout mice, suggest that an OCT2-targeted therapeutic strategy would be efficacious in both major subtypes of DLBCL while avoiding systemic toxicity.

High expression of the forkhead box P1 (FOXP1) transcription factor distinguishes the aggressive activated B cell (ABC) diffuse large B-cell lymphoma (DLBCL) subtype from the better prognosis germinal center B-cell (GCB)-DLBCL subtype and is highly correlated with poor outcomes. A genetic or functional role for FOXP1 in lymphomagenesis, however, remains unknown. Here, we report that sustained FOXP1 expression is vital for ABC-DLBCL cell-line survival. Genome-wide analyses revealed direct and indirect FOXP1 transcriptional enforcement of ABC-DLBCL hallmarks, including the classical NF-κB and MYD88 (myeloid differentiation primary response gene 88) pathways. FOXP1 promoted gene expression underlying transition of the GCB cell to the plasmablast--the transient B-cell stage targeted in ABC-DLBCL transformation--by antagonizing pathways distinctive of GCB-DLBCL, including that of the GCB "master regulator," BCL6 (B-cell lymphoma 6). Cell-line derived FOXP1 target genes that were highly correlated with FOXP1 expression in primary DLBCL accurately segregated the corresponding clinical subtypes of a large cohort of primary DLBCL isolates and identified conserved pathways associated with ABC-DLBCL pathology.

After neonatal HTLV-I infection through breast feeding, approximately 5% of HTLV-I carriers eventually develop Adult T-Cell Leukemia/Lymphoma (ATLL) with a latency of ~50 years, suggesting that acquired genetic and epigenetic changes in cellular genes act in concert with HTLV-I to initiate and maintain oncogenic transformation. We and others have recently utilized next generation sequencing technology to identify mutated genes that could be pivotal in the pathogenesis of ATLL. However, due to the complexity of genomic/epigenetic alteration in the ATLL genome, the identification of indispensable genes for proliferation and/or survival of ATLL cells remains a formidable challenge. To discover essential regulatory networks that are required for the proliferation and survival of ATLL cells, we performed a pooled shRNA screen in 8 ATLL cell lines using a library enriched for shRNAs targeting lymphoid regulatory factors and discovered that two BATF3 shRNAs and one IRF4 shRNA were highly toxic for all ATLL lines, but had little if any effect in other T cell and B cell lines. It is recently shown that a transcriptional complex of Irf4 and Batf binds to AP1-IRF composite (AICE) DNA motifs and plays key roles in the differentiation and function of certain mouse helper T cell subsets. A close paralogue of Batf, Batf3, is an indispensable transcription factor in a mouse dendritic cell subset, but also appears to play a redundant role with Batf in the differentiation of TH2 cells and can substitute for Batf in Batf knockout T cells. Our observations from shRNA screening suggested that IRF4 and BATF3 may cooperate to drive a transcriptional program that is essential for ATLL viability. We next used genome-wide chromatin precipitation (ChIP-seq) to identify the loci that are bound by BATF3 and IRF4. The set of binding peaks and the associated genes in IRF4 and BATF3 ChIP-seq intersected significantly. By integrating the ChIP-seq and gene expression profiling data of shBATF3- and shIRF4-ATLL cells, we defined a set of 68 BATF3-IRF4 direct target genes. Gene set enrichment analysis using gene expression profiling data from primary T cell lymphomas demonstrated that BATF3-IRF4 direct target genes were significantly enriched among genes that are more highly expressed in ATLL than in peripheral T-cell lymphoma, not otherwise specified (PTCL-NOS), suggesting that the BATF3 and IRF4 cooperatively regulate transcription in primary ATLL cells. HBZ is unique among HTLV-I viral proteins in being maintained in expression in all ATLL cases, suggesting that it may help maintain the malignant phenotype. Given that BATF3 and IRF4 are essential regulators in ATLL, we hypothesized possible relationship between HBZ and BATF3-IRF4 complex. We defined HBZ direct target genes by integrating the ChIP-seq and gene expression profiling data of HBZ-knockout ATLL cell lines by CRISPR/Cas9. Notably we discovered that BATF3 was among these. BATF3 mRNA and protein expression decreased following HBZ inactivation. The above considerations suggested that pharmacologic inhibition of the BATF3-IRF4 regulatory network might be a means to attack the HBZ oncogenic program therapeutically. ChIP-seq analysis of two enhancer marks, H3K27ac and BRD4, identified super-enhancers at the BATF3 locus in two ATLL cell lines. The small molecule JQ1 prevents the BET-protein BRD4 from interacting with chromatin, which is required for the function of super-enhancers. JQ1 treatment reduced BATF3 mRNA and protein levels in all ATLL lines tested, correlating with the eviction of BRD4 from the BATF3 super-enhancer. MYC mRNA and protein expression was also broadly downmodulated by JQ1. JQ1 treatment was consistently toxic for all ATLL cell lines tested at dose ranges that killed cell line models of T-ALL and DLBCL, which are known to rely on BET-proteins. In a dose-dependent manner, JQ1 also reduced the viability of primary ATLL samples and downregulated their expression BATF3 and MYC mRNA. Finally, we treated mouse xenograft models of ATLL with the BET-protein inhibitor CPI-203, a JQ1 analog with superior bioavailability in mice. In two different xenograft models, we observed significant tumor regression or growth inhibition, without evidence of systemic toxicity. Our study demonstrates that the HTLV-I virus exploits a regulatory module that can potentially be attacked therapeutically with BET protein inhibitors. Disclosures Yu: Celgene Corporation: Employment.

IRF4, a member of the Interferon Regulatory Factor (IRF) family of transcription factors, is expressed in cells of the immune system, where it transduces signals from various receptors to activate or repress gene expression. IRF4 expression is a key regulator of several steps in lymphoid-, myeloid-, and dendritic-cell differentiation, including the differentiation of mature B cells into antibody-secreting plasma cells. IRF4 expression is also associated with many lymphoid malignancies, with recent evidence pointing to an essential role in multiple myeloma, a malignancy of plasma cells. Interference with IRF4 expression is lethal to multiple myeloma cells, irrespective of their genetic etiology, making IRF4 an “Achilles' heel” that may be exploited therapeutically.

Memory B cells provide rapid protection to previously encountered antigens; however, how these cells develop from germinal center B cells is not well understood. A previously described in vitro culture system using human tonsillar germinal center B cells was used to study the transcriptional changes that occur during differentiation of human memory B cells. Kinetic studies monitoring the expression levels of several known late B cell transcription factors revealed that BCL-6 is not expressed in memory B cells generated in vitro, and gene expression profiling studies confirmed that BCL-6 is not expressed in these memory B cells. Furthermore, ectopic expression of BCL-6 in human B cell cultures resulted in formation of fewer memory B cells. In addition, the expression profile of in vitro memory B cells showed a unique pattern that includes expression of genes encoding multiple costimulatory molecules and cytokine receptors, antiapoptotic proteins, T cell chemokines, and transcription factors. These studies establish new molecular criteria for defining the memory B cell stage in human B cells.

The serine-threonine kinase ataxia-telangiectasia mutated (ATM) plays a central role in maintaining genomic integrity. In mice, ATM deficiency is exclusively associated with T-cell lymphoma development, whereas B-cell tumors predominate in human ataxia-telangiectasia patients. We demonstrate in this study that when T cells are removed as targets for lymphomagenesis and as mediators of immune surveillance, ATM-deficient mice exclusively develop early-onset immunoglobulin M(+) B-cell lymphomas that do not transplant to immunocompetent mice and that histologically and genetically resemble the activated B cell-like (ABC) subset of human diffuse large B-cell lymphoma (DLBCL). These B-cell lymphomas show considerable chromosomal instability and a recurrent genomic amplification of a 4.48-Mb region on chromosome 18 that contains Malt1 and is orthologous to a region similarly amplified in human ABC DLBCL. Of importance, amplification of Malt1 in these lymphomas correlates with their dependence on nuclear factor (NF)-κB, MALT1, and B-cell receptor (BCR) signaling for survival, paralleling human ABC DLBCL. Further, like some human ABC DLBCLs, these mouse B-cell lymphomas also exhibit constitutive BCR-dependent NF-κB activation. This study reveals that ATM protects against development of B-cell lymphomas that model human ABC DLBCL and identifies a potential role for T cells in preventing the emergence of these tumors.

We have identified two intronic regions of mouse prdm1, the gene encoding B lymphocyte-induced maturation protein-1 (Blimp-1), which confer transcriptional repression in response to Bcl-6. The Bcl-6 response element in intron 5, which is conserved between mice and humans, was studied in detail. It binds Bcl-6 in vitro and was shown by chromatin immunoprecipitation to be occupied by Bcl-6 in vivo. Neither Bcl-6 response element functions as a STAT3-response element, showing that STAT3 does not compete with Bcl-6 at these sites. Bcl-6−/− mice confirm the biological importance of Bcl-6-dependent repression of prdm1. These mice have elevated Ab response, increased Ig-secreting cells, and increased Blimp-1+ cells in spleen following immunization and their splenic B cells show accelerated plasmacytic development in vitro.

When the regulation of B-cell differentiation and activation is disrupted, lymphomas and leukaemias can occur. The processes that normally create immunoglobulin diversity might be misdirected, resulting in oncogenic chromosomal translocations that block differentiation, prevent apoptosis and/or promote proliferation. Prolonged or unregulated antigenic stimulation might contribute further to the development and progression of some malignancies. Lymphoid malignancies often resemble normal stages of B-cell differentiation, as shown by molecular techniques such as gene-expression profiling. The similarities and differences between malignant and normal B cells indicate strategies for the treatment of these cancers.

Recombination activating gene 1 (RAG1) and RAG2 are critical enzymes for initiating variable-diversity-joining [V(D)J] segment recombination, an essential process for antigen receptor expression and lymphocyte development. The BCL11A transcription factor is required for B cell and plasmacytoid dendritic cell (pDC) development, but its molecular function(s) in early B cell fate specification and commitment is unknown. We show here that the major B cell isoform, BCL11A-XL, binds directly to the RAG1 promoter as well as directly to regulatory regions of transcription factors previously implicated in both B cell and pDC development to activate RAG1 and RAG2 gene transcription in pro- and pre-B cells. We employed BCL11A overexpression with recombination substrates to demonstrate direct consequences of BCL11A/RAG modulation on V(D)J recombination. We conclude that BCL11A is a critical component of a transcriptional network that regulates B cell fate by controlling V(D)J recombination.

Burkitt's lymphoma (BL) can often be cured by intensive chemotherapy, but the toxicity of such therapy precludes its use in the elderly and in patients with endemic BL in developing countries, necessitating new strategies(1). The normal germinal centre B cell is the presumed cell of origin for both BL and diffuse large B-cell lymphoma (DLBCL), yet gene expression analysis suggests that these malignancies may use different oncogenic pathways(2). BL is subdivided into a sporadic subtype that is diagnosed in developed countries, the Epstein-Barr-virus-associated endemic subtype, and an HIV-associated subtype, but it is unclear whether these subtypes use similar or divergent oncogenic mechanisms. Here we used high-throughput RNA sequencing and RNA interference screening to discover essential regulatory pathways in BL that cooperate with MYC, the defining oncogene of this cancer. In 70% of sporadic BL cases, mutations affecting the transcription factor TCF3 (E2A) or its negative regulator ID3 fostered TCF3 dependency. TCF3 activated the pro-survival phosphatidylinositol-3-OH kinase pathway in BL, in part by augmenting tonic B-cell receptor signalling. In 38% of sporadic BL cases, oncogenic CCND3 mutations produced highly stable cyclin D3 isoforms that drive cell cycle progression. These findings suggest opportunities to improve therapy for patients with BL.

SummaryWe performed a loss-of-function RNA interference screen to define therapeutic targets in multiple myeloma, a genetically diverse plasma cell malignancy. Unexpectedly, we discovered that all myeloma lines require caspase-10 for survival irrespective of their genetic abnormalities. The transcription factor IRF4 induces both caspase-10 and its associated protein cFLIPL in myeloma, generating a protease that does not induce apoptosis but rather blocks an autophagy-dependent cell death pathway. Caspase-10 inhibits autophagy by cleaving the BCL2-interacting protein BCLAF1, itself a strong inducer of autophagy that acts by displacing beclin-1 from BCL2. While myeloma cells require a basal level of autophagy for survival, caspase-10 tempers this response to avoid cell death. Drugs that disrupt this vital balance may have therapeutic potential in myeloma.

The mechanisms that drive normal B cell differentiation and activation are frequently subverted by B cell lymphomas for their unlimited growth and survival. B cells are particularly prone to malignant transformation because the machinery used for antibody diversification can cause chromosomal translocations and oncogenic mutations. The advent of functional and structural genomics has greatly accelerated our understanding of oncogenic mechanisms in lymphomagenesis. The signaling pathways that normal B cells utilize to sense antigens are frequently derailed in B cell malignancies, leading to constitutive activation of prosurvival pathways. These malignancies co-opt transcriptional regulatory systems that characterize their normal B cell counterparts and frequently alter epigenetic regulators of chromatin structure and gene expression. These mechanistic insights are ushering in an era of targeted therapies for these cancers based on the principles of pathogenesis.

BackgroundGene-targeted iMycEμmice that carry a His6-tagged mouseMyc(c-myc)cDNA,MycHis, just 5' of the immunoglobulin heavy-chain enhancer, Eμ, are prone to B cell and plasma cell neoplasms, such as lymphoblastic B-cell lymphoma (LBL) and plasmacytoma (PCT). Cell lines derived from Myc-induced neoplasms of this sort may provide a good model system for the design and testing of new approaches to prevent and treat MYC-driven B cell and plasma cell neoplasms in human beings. To test this hypothesis, we used the LBL-derived cell line, iMycEμ-1, and the newly established PCT-derived cell line, iMycEμ-2, to evaluate the growth inhibitory and death inducing potency of the cancer drug candidate, CDDO-imidazolide (CDDO-Im).MethodsMorphological features and surface marker expression of iMycEμ-2 cells were evaluated using cytological methods and FACS, respectively. mRNA expression levels of the insertedMycHisand normalMycgenes were determined by allele-specific RT-PCR and qPCR. Myc protein was detected by immunoblotting. Cell cycle progression and apoptosis were analyzed by FACS. The expression of 384 "pathway" genes was assessed with the help of Superarray©cDNA macroarrays and verified, in part, by RT-PCR.ResultsSub-micromolar concentrations of CDDO-Im caused growth arrest and apoptosis in iMycEμ-1 and iMycEμ-2 cells. CDDO-Im-dependent growth inhibition and apoptosis were associated in both cell lines with the up-regulation of 30 genes involved in apoptosis, cell cycling, NFκB signaling, and stress and toxicity responses. Strongly induced (≥10 fold) were genes encoding caspase 14, heme oxygenase 1 (Hmox1), flavin-containing monooxygenase 4 (Fmo4), and three members of the cytochrome P450 subfamily 2 of mixed-function oxygenases (Cyp2a4, Cyp2b9, Cyp2c29). CDDO-Im-dependent gene induction coincided with a decrease in Myc protein.ConclusionGrowth arrest and killing of neoplastic mouse B cells and plasma cells by CDDO-Im, a closely related derivative of the synthetic triterpenoid 2-cyano-3,12-dioxooleana-1,9-dien-28-oic acid, appears to be caused, in part, by drug-induced stress responses and reduction of Myc.

There is evidence that the insulin-like growth factor-I (IGF-I) receptor is required for transformation by a variety of viral and cellular oncogenes in a mouse embryo fibroblast model. To further investigate the IGF-I receptor signaling pathways that are required for the permissive effect of the receptor on transformation by SV40 T antigen, we established three independent fibroblast cell lines each from wild-type and IGF-I receptor null embryos (R−). We transfected the wild-type and R− cell lines with an SV40 T antigen plasmid and selected three clones from each cell line that expressed T antigen. As in previous reports, none of the cloned R− cell lines expressing T antigen were transformed as measured by the ability to form large colonies in soft agar. However, with further passage, all three T antigen–expressing clones from one of the R− cell lines (R−3) formed large colonies in soft agar and the transformation of these T antigen–expressing clones was confirmed by tumorigenesis experiments in immunodeficient mice. DNA microarray analysis comparing gene expression between early passage and late passage R−3/T antigen clones showed, among other changes, an increase in the expression of ErbB-3 mRNA in the late passage clones. Also, the expression of ErbB-3 protein was dramatically increased in the late passage R−3/T antigen clones. We conclude that late passage IGF-I receptor null mouse embryo fibroblasts can be transformed by SV40 T antigen, and that ErbB-3 may play a role in permitting transformation by T antigen. (Cancer Res 2006; 66(8): 4233-9)

Summary Molecular mechanisms underlying the coordination of isotype switching with plasma cell differentiation are poorly understood. We show that interferon regulatory factor-4 (IRF-4) regulates both processes by controlling the expression of the Aicda and Prdm1 genes, which encode AID and Blimp-1, respectively. Genome-wide analysis demonstrated that Irf4 −/− B cells failed to induce the entire Blimp-1-dependent plasma cell program. Restoration of AID or Blimp-1 expression in Irf4 −/− B cells promoted isotype switching or secretion, respectively. IRF-4 was expressed in a graded manner in differentiating B cells and targeted Prdm1 . Higher concentration of IRF-4 induced Prdm1 and consequently the transition from a germinal center gene expression program to that of a plasma cell. We propose a gene-regulatory network in which graded expression of IRF-4 developmentally coordinates isotype switching with plasma cell differentiation.

Gene-targeted mice that contain a His6-tagged mouse c-Myc cDNA, MycHis, inserted head to head into different sites of the mouse immunoglobulin heavy-chain locus, Igh, mimic the chromosomal T(12;15)(Igh-Myc) translocation that results in the activation of Myc in the great majority of mouse plasmacytomas. Mice carrying MycHis just 5′ of the intronic heavy-chain enhancer Eμ (strain iMycEμ) provide a specific model of the type of T(12;15) found in a subset (∼20%) of plasmacytomas that develop “spontaneously” in the gut-associated lymphoid tissue (GALT) of interleukin-6 transgenic BALB/c (C) mice. Here we show that the transfer of the iMycEμ transgene from a mixed genetic background of segregating C57BL/6 × 129/SvJ alleles to the background of C increased the incidence of GALT plasmacytomas by a factor of 2.5 in first-generation backcross mice (C.iMycEμ N1). Third-generation backcross mice (C.iMycEμ N3, ∼94% C alleles) were hypersusceptible to inflammation-induced peritoneal plasmacytomas (tumor incidence, 100%; mean tumor onset, 86 ± 28 days) compared with inbred C mice (tumor incidence, 5% on day 150 after tumor induction). Peritoneal plasmacytomas of C.iMycEμ N3 mice overexpressed MycHis, produced monoclonal immunoglobulin, and exhibited a unique plasma cell signature upon gene expression profiling on mouse Lymphochip cDNA microarrays. These findings indicated that the iMycEμ transgene accelerates plasmacytoma development by collaborating with tumor susceptibility alleles of strain C and circumventing the requirement for tumor precursors to acquire deregulated Myc by chromosomal translocation.

Background Myc-induced lymphoblastic B-cell lymphoma (LBL) in iMycEμ mice may provide a model system for the study of the mechanism by which human MYC facilitates the initiation and progression of B cell and plasma cell neoplasms in human beings. We have recently shown that gene-targeted iMycEμ mice that carry a His6-tagged mouse Myc cDNA, Myc His, just 5' of the immunoglobulin heavy-chain enhancer, Eμ, are prone to B cell and plasma cell tumors. The predominant tumor (~50%) that arose in the iMycEμ mice on the mixed genetic background of segregating C57BL/6 and 129/SvJ alleles was LBL. The purpose of this study was to establish and characterize a cell line, designated iMycEμ-1, for the in-depth evaluation of LBL in vitro. Methods The morphological features and the surface marker expression profile of the iMycEμ-1 cells were evaluated using cytological methods and FACS, respectively. The cytogenetic make-up of the iMycEμ-1 cells was assessed by spectral karyotyping (SKY). The expression of the inserted Myc His gene was determined using RT-PCR and qPCR. Clonotypic immunoglobulin gene arrangements were detected by Southern blotting. The global gene expression program of the iMycEμ-1 cells and the expression of 768 "pathway" genes were determined with the help of the Mouse Lymphochip© and Superarray© cDNA micro- and macroarrays, respectively. Array results were verified, in part, by RT-PCR and qPCR. Results Consistent with their derivation from LBL, the iMycEμ-1 cells were found to be neoplastic IgMhighIgDlow lymphoblasts that expressed typical B-cell surface markers including CD40, CD54 (ICAM-1), CD80 (B7-1) and CD86 (B7-2). The iMycEμ-1 cells harbored a reciprocal T(9;11) and three non-reciprocal chromosomal translocations, over-expressed Myc His at the expense of normal Myc, and exhibited gene expression changes on Mouse Lymphochip© microarrays that were consistent with Myc His-driven B-cell neoplasia. Upon comparison to normal B cells using eight different Superarray© cDNA macroarrays, the iMycEμ-1 cells showed the highest number of changes on the NFκB array. Conclusion The iMycEμ-1 cells may provide a uniquely useful model system to study the growth and survival requirements of Myc-driven mouse LBL in vitro.

BCL-6, a transcriptional repressor frequently translocated in lymphomas, regulates germinal center B cell differentiation and inflammation. DNA microarray screening identified genes repressed by BCL-6, including many lymphocyte activation genes, suggesting that BCL-6 modulates B cell receptor signals. BCL-6 repression of two chemokine genes, MIP-1α and IP-10, may also attenuate inflammatory responses. Blimp-1, another BCL-6 target, is important for plasmacytic differentiation. Since BCL-6 expression is silenced in plasma cells, repression of blimp-1 by BCL-6 may control plasmacytic differentiation. Indeed, inhibition of BCL-6 function initiated changes indicative of plasmacytic differentiation, including decreased expression of c-Myc and increased expression of the cell cycle inhibitor p27kip1. These data suggest that malignant transformation by BCL-6 involves inhibition of differentiation and enhanced proliferation.

Genetic alterations of the BCL-6 gene in mice and man have established BCL-6 as a pivotal regulator of normal differentiation of B and T lymphocytes as well as one of the most frequently translocated oncogenes in human B cell lymphomas. As an oncogene, BCL-6 has not been easy to place into existing paradigms of cellular transformation. Rather, it is likely that the function of BCL-6 as a regulator of lymphocyte differentiation is subverted in BCL-6-induced lymphomas. The lymphomas in which BCL-6 is translocated are all suspected to arise from the germinal center B lymphocyte. Given the selective expression of BCL-6 protein in normal germinal center B lymphocytes and the requirement for BCL-6 in germinal center development, the functions of BCL-6 in normal and malignant B cells are probably intertwined. The BCL-6 protein is a potent transcriptional repressor which presumably controls lymphocyte differentiation and induces lymphomas by regulating the expression of key downstream target genes.

Abstract 287 Mantle cell lymphoma (MCL) is a lymphoproliferative disorder of mature B-cells with an aggressive course and short survival. The proteasome inhibitor bortezomib (BZM) induces clinical responses in up to 50% of patients. Conversely, in half of the cases the lymphoma cells are intrinsically resistant or rapidly develop resistance to BZM. To investigate the mechanisms of BZM resistance, we generated HBL2 and JEKO bortezomib resistant (HBL2-BR, JEKO-BR) derivative lines by continuous culture in sub-lethal concentrations of BZM. After several months, clones of HBL2-BR and JEKO-BR were obtained showing BZM IC50 at 48h of 41.6 and 44.6 nM, compared to 6 and 4.9 nM for the respective parental lines. Acquired resistance to BZM remained stable over months but gradually decreased with extended passages in the absence of BZM, suggesting adaptive changes rather than a single gene mutation as the basis of BZM resistance. BR cells exhibited higher proteasome activity, which was dose-dependently inhibited by higher concentrations of BZM. However, BR cells were able to survive with lower proteasome activity than the parental cells, indicating that BR cells had acquired additional changes. To investigate these changes, we use gene expression profiling (GEP) on Affymetrix U133A plus 2 arrays to compared HBL2-BR (in triplicate) and JEKO-BR (in duplicate) subclones to the corresponding parental lines. Unexpectedly, Gene Set Enrichment Analysis (GSEA) of microarray data revealed reduced expression of the mature B-cell gene signature (including genes for CD19, BLNK, SPIB, SYK) and increased expression of plasma cell differentiation signatures (including genes for CD38, IRF4, BLIMP, CD138) in both HBL-2 BR and JEKO-BR. BR lines also expressed higher protein levels of the master plasma cell regulators BLIMP and IRF4, but did not show enhanced expression of the secretory program controlled by XBP1. Flow cytometry analysis confirmed that BR cells had dramatically reduced expression of B-cell surface markers, including CD19, CD24 and CD52, and expressed plasma cell markers, such as CD38 and CD138. Consistent with a partial plasmacytoid phenotype, BR cells tended to be somewhat larger and more granular than parental cells. Loss of BZM resistance over months of culture in the absence of BZM was paralleled by the recovery of CD19 and CD24 expression and down-regulation of CD38, supporting a mechanistic link between the acquisition of a plasmacytoid phenotype and BZM resistance. We have previously shown that the MCL cell lines Mino and REC-1 are less sensitive to BZM than HBL-2, JEKO and most other MCL cell lines. Here we found that these constitutively resistant cells also showed plasmacytoid features including CD38 and CD138 surface expression, increased granularity and size, and an enlarged endoplasmic reticulum (ER). Combined these changes may enhance the ability of the cells to deal with an increased protein load due to bortezomib inhibition. In addition, we also observed higher expression of IRF4 and its target genes in the constitutively resistant cells, as well as higher IRF4 and CD38 expression in primary tumor cells of patients with poor response to BZM. Given the important role of IRF4 as a survival factor in multiple myeloma, we tested whether BZM treatment could decrease IRF4 expression in MCL cells. Indeed, within 24 hours BZM dose-dependently decreased IRF4 expression and the degree of downregulation of IRF4 correlated with the induction of apoptosis. Knockdown of IRF4 expression by shRNA has been shown to be toxic to myeloma cells (Shaffer et al, Nature 2008). Surprisingly, we found a similar toxic effect of IRF4 knockdown using the same inducible shRNA system in the MCL cell lines HBL2, JEKO and REC, which was more prominent in the latter BZM resistant cell line. These results identify loss of IRF4 expression as an additional mechanism by which BZM may induce cell death. However, overexpression of IRF4 in MCL cells is not sufficient to induce bortezomib resistance, indicating that several components of the plasma cell program cooperate to protect cells from BZM induced apoptosis. Furthermore, we have identified markers of BZM resistance that may be clinically relevant predictors of outcome. Disclosures: No relevant conflicts of interest to declare.

Blimp-1, a transcriptional repressor, drives the terminal differentiation of B cells to plasma cells. Using DNA microarrays, we found that introduction of Blimp-1 into B cells blocked expression of a remarkably large set of genes, while a much smaller number was induced. Blimp-1 initiated this cascade of gene expression changes by directly repressing genes encoding several transcription factors, including Spi-B and Id3, that regulate signaling by the B cell receptor. Blimp-1 also inhibited immunoglobulin class switching by blocking expression of AID, Ku70, Ku86, DNA-PKcs, and STAT6. These findings suggest that Blimp-1 promotes plasmacytic differentiation by extinguishing gene expression important for B cell receptor signaling, germinal center B cell function, and proliferation while allowing expression of important plasma cell genes such as XBP-1.

A compendium of global gene expression measurements from DNA microarray analysis of immune cells identifies gene expression signatures defining various lineages, differentiation stages, and signaling pathways. Germinal center (GC) B cells represent a discrete stage of differentiation with a unique gene expression signature. This includes genes involved in proliferation, as evidenced by high expression of G2/M phase regulators and low expression of ribosomal and metabolic genes that are transcriptional targets of c- myc . GC B cells also lack expression of the NF-κB signature genes, which may favor apoptosis. Finally, the transcriptional repression signature of BCL-6 reveals how this factor can prevent terminal differentiation of B cells and cause B cell lymphomas.

The oncogene c-maf is translocated in ∼5%–10% of multiple myelomas. Unexpectedly, we observed c-maf expression in myeloma cell lines lacking c-maf translocations and in 50% of multiple myeloma bone marrow samples. By gene expression profiling, we identified three c-maf target genes: cyclin D2 , integrin β7 , and CCR1 . c-maf transactivated the cyclin D2 promoter and enhanced myeloma proliferation, whereas dominant inhibition of c-maf blocked tumor formation in immunodeficient mice. c-maf-driven expression of integrin β7 enhanced myeloma adhesion to bone marrow stroma and increased production of VEGF. We propose that c-maf transforms plasma cells by stimulating cell cycle progression and by altering bone marrow stromal interactions. The frequent overexpression of c-maf in myeloma makes it an attractive target for therapeutic intervention.