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Background: Follicular lymphoma (FL) shows marked variation in clinical course including spontaneous regression and histologic transformation (HT). Watchful waiting (W&W) is routinely applied to pts with newly diagnosed FL, but monitoring strategies are not standardized. Pts with progression within 1-2y of diagnosis have worse outcomes, but the biologic basis is unclear and biologic-based classifiers are not routinely applied at diagnosis. Circulating tumor DNA (ctDNA) is a highly tumor-specific biomarker that is prognostic in aggressive B-cell lymphomas, but its ability to serially monitor FL remains undefined. We applied a next-generation sequencing assay to identify tumor clonotypes for serial monitoring of peripheral blood in pts with untreated FL as part of an ongoing prospective clinical trial [NCT03190928]. Methods: Pts with grade I-II or 3A FL are eligible if evaluable disease on CT or FDG-PET, age ≥18, ECOG ≤2, no evidence of HT, and no prior systemic therapy. Pts undergo W&W until they meet uniform protocol-defined treatment criteria and remain on study until second-line therapy. Baseline testing includes labs, peripheral blood flow cytometry, BM biopsy/aspirate, CT and FDG-PET scans, and research biopsy. Pt have clinic visits every 4m for 2y, every 6m in years 3-5, then annually. CT scans are every 8m for 2y, then annually. FDG-PET scans are at baseline, at 2y, and any time of suspected progression. Peripheral blood samples including Streck tubes (plasma) and PBMCs are drawn at each clinic visit and stored. For ctDNA analysis, tumor DNA was amplified from FFPE using locus-specific primer sets for the Ig heavy-chain and light-chain loci along with BCL1/BCL2 translocations (Adaptive Biotechnologies). Amplified products were sequenced and tumor clonotypes were identified in plasma and PBMCs. Serial tracking of ctDNA was done in plasma and blinded to clinical outcomes. Results: 77 pts enrolled between July 2017 and July 2021. Median age was 57 (range 24-83) including 14 (18%) low-risk, 29 (38%) intermediate-risk, and 34 (44%) high-risk by FLIPI. Fourteen (18%) pts had stage I-II disease. Forty-three (56%) pts had monoclonal B-cells on peripheral blood flow cytometry. Twenty-nine (38%) pts progressed requiring frontline therapy including 7 (9%) pts with HT. Twenty-five (32%) pts were monitored ≥2y with no progression including 10 (13%) pts with evidence of at least some spontaneous regression by CT. Twenty (26%) pts were on study for Conclusions: ctDNA quantified from plasma in FL mirrors TMTV. Serial monitoring of ctDNA in patients without therapy demonstrated various patterns of fluctuation, including some patients in which ctDNA became undetectable coincident with spontaneous clinical regressions. ctDNA thus provides a non-invasive platform to monitor the natural history of FL, enabling future studies of tumor immune surveillance in this disease. Figure 1 Figure 1. Disclosures Jacob: Adaptive Biotechnologies: Current Employment, Current equity holder in publicly-traded company. Bagaev: BostonGene Corp.: Current Employment, Current holder of stock options in a privately-held company, Patents & Royalties: BostonGene. Meerson: BostonGene: Current Employment, Current holder of stock options in a privately-held company, Patents & Royalties: BostonGene. Postovalova: BostonGene Corp.: Current Employment, Current holder of stock options in a privately-held company, Patents & Royalties: BostonGene. Kudryashova: BostonGene: Current Employment, Current holder of stock options in a privately-held company, Patents & Royalties: BostonGene. Kotlov: BostonGene Corp: Current Employment, Current holder of stock options in a privately-held company, Patents & Royalties. Fowler: BostonGene: Current Employment, Current holder of stock options in a privately-held company.

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.

Kelly et al. report the development of two highly selective and bioavailable small molecule IRAK4 inhibitors and show for the first time their therapeutic efficacy in autoimmune disorders and in a specific subset of diffuse large B cell lymphomas in mice., Pathological activation of the Toll-like receptor signaling adaptor protein MYD88 underlies many autoimmune and inflammatory disease states. In the activated B cell–like (ABC) subtype of diffuse large B cell lymphoma (DLBCL), the oncogenic MYD88 L265P mutation occurs in 29% of cases, making it the most prevalent activating mutation in this malignancy. IRAK4 kinase accounts for almost all of the biological functions of MYD88, highlighting IRAK4 as a therapeutic target for diseases driven by aberrant MYD88 signaling. Using innovative structure-based drug design methodologies, we report the development of highly selective and bioavailable small molecule IRAK4 inhibitors, ND-2158 and ND-2110. These small molecules suppressed LPS-induced TNF production, alleviated collagen-induced arthritis, and blocked gout formation in mouse models. IRAK4 inhibition promoted killing of ABC DLBCL lines harboring MYD88 L265P, by down-modulating survival signals, including NF-κB and autocrine IL-6/IL-10 engagement of the JAK–STAT3 pathway. In ABC DLBCL xenograft models, IRAK4 inhibition suppressed tumor growth as a single agent, and in combination with the Bruton’s tyrosine kinase (BTK) inhibitor ibrutinib or the Bcl-2 inhibitor ABT-199. Our findings support pharmacological inhibition of IRAK4 as a therapeutic strategy in autoimmune disorders, in a genetically defined population of ABC DLBCL, and possibly other malignancies dependent on aberrant MYD88 signaling.

The two major subtypes of diffuse large B cell lymphoma (DLBCL)—activated B cell–like (ABC) and germinal center B cell–like (GCB)—arise by distinct mechanisms, with ABC selectively acquiring mutations that target the B cell receptor (BCR), fostering chronic active BCR signaling(1). The ABC subtype has a ∼40% cure rate with currently available therapies, which is worse than the rate for GCB DLBCL, and highlights the need for ABC subtype-specific treatment strategies(2). We hypothesized that ABC, but not GCB, DLBCL tumors would respond to ibrutinib, an inhibitor of BCR signaling. In a phase 1/2 clinical trial that involved 80 subjects with relapsed or refractory DLBCL, ibrutinib produced complete or partial responses in 37% (14/38) of those with ABC DLBCL, but in only 5% (1/20) of subjects with GCB DLBCL (P = 0.0106). ABC tumors with BCR mutations responded to ibrutinib frequently (5/9; 55.5%), especially those with concomitant myeloid differentiation primary response 88 (MYD88) mutations (4/5; 80%), a result that is consistent with in vitro cooperation between the BCR and MYD88 pathways. However, the highest number of responses occurred in ABC tumors that lacked BCR mutations (9/29; 31%), suggesting that oncogenic BCR signaling in ABC does not require BCR mutations and might be initiated by non-genetic mechanisms. These results support the selective development of ibrutinib for the treatment of ABC DLBCL.

The importance of understanding the genetic and biochemical basis of B cell receptor (BCR) survival signaling in diffuse large B cell lymphoma (DLBCL) is underscored by the recent clinical success of agents that target the BCR pathway. DLBCL is composed of multiple distinct molecular subtypes with divergent clinical outcomes. The activated B cell-like (ABC) subtype is the most aggressive form of DLBCL and is often resistant to standard chemotherapies. ABC DLBCL expresses numerous genes found in antigen-activated B cells, and genetic and pharmacologic studies have demonstrated that ABC DLBCL tumors are addicted to NF-κB activity. The origins of this NF-κB activity remained obscure until RNA interference screens established that the majority of ABC DLBCL cell lines rely on expression of BCR components and downstream signaling effectors for NF-κB activation. Pharmacological inhibition with ibrutinib of Bruton’s tyrosine kinase (Btk), a kinase that is required for BCR signaling to engage NF-κB, is selectively toxic for ABC DLBCL tumors; a finding that has now been translated to the clinic. These novel targets not only offer a promising new therapy options for ABC DLBCL, but also demonstrate the value of a deep molecular understanding of oncogenic signaling pathways.

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.

Diffuse large B-cell lymphoma (DLBCL) is an aggressive cancer of aberrant B-lymphocytes. Although a portion of DLBCL is curable with standard immunochemotherapy, patients who fail this treatment have a poor prognosis. Recently, cancer genomics has paved the way for better understanding of the genetic basis of lymphoma pathogenesis. Characterization of point mutations and structural alterations has uncovered novel molecular targets for lymphoma therapy and provided a comprehensive view of lymphoma development. By performing multiplatform genomic analysis of DLBCL biopsy samples, we have identified KLHL14 as a recurrent target of somatic mutations in activated B-cell-like (ABC) DLBCL biopsies (10.8% of patients). KLHL14 contains a BTB (broad complex, tramtrack, and bric a brac) domain that can potentially mediate dimerization and binding to Cullin3 (CUL3)-a essential scaffold component of the Cullin-RING-based E3 ubiquitin ligase complexes. KLHL14 also contains kelch repeats that can form a B-propeller tertiary structure that can serve as a substrate-binding domain. KLHL14 is highly expressed in B-cells but is found at low levels in non-immune tissues. Deficiency of KLHL14 in mice leads to embryonic lethality while KLHL14 heterozygous mice show reduction of B-1a cells, suggesting a role for KLHL14 in B-cell homeostasis. Importantly, KLHL14 mutations are highly enriched in tumors belonging to the recently defined MCD (MYD88L265P/CD79B mutation) genetic subtype of DLBCL, the subset of ABC DLBCLs. Somatic mutations primarily localize to the N-terminus of the protein in the BTB domain and BACK (BTB and C-terminal Kelch) domain. However, the impact of these mutations as well as the molecular function of KLHL14 is largely unknown. To investigate the biological effect of KLHL14 loss of function, we used an inducible CRISPR/Cas9 system to delete KLHL14 in ABC DLBCL cell lines and monitored cell growth. Ablation of KLHL14 resulted in an increase in cell proliferation and survival, supporting a role for KLHL14 as a tumor suppressor. Next, we performed a multiplatform -omic analysis (proteomics, phosphoproteomics, ubiquitinomics, high-throughput sequencing) to explore the signaling networks and interactome of KLHL14. Whereas ectopic expression of wild-type KLHL14 altered the dynamics of tyrosine phosphorylation and ubiquitylation events in ABC DLBCL lines, KLHL14 lymphoma-associated mutant alleles had little if any effect, suggesting that they are loss-of-function variants. Gene expression profiling by RNA-sequencing revealed that KLHL14-inactivated cells have a higher NF-kB target gene expression than wild-type cells. Thus, tumor-associated inactivating mutations of KLHL14 depend on a subset of essential NF-kB-related oncoproteins for their survival and this might contribute to the proliferative advantage of DLBCL. In summary, we have uncovered a tumor suppressive function of KLHL14 and found that KLHL14 mutants promote ABC DLBCL survival by increasing NF-kB activity. These findings suggest that tumors with KLHL14 inactivating mutations may serve as a marker of resistance to anti-NF-kB treatment and provide the basis for treating MCD subtype patients with downstream NF-kB pathway inhibitors in the clinical settings. Disclosures Staudt: Nanostring: Patents & Royalties.

Blastic plasmacytoid dendritic cell neoplasm (BPDCN) is an aggressive and largely incurable hematologic malignancy originating from plasmacytoid dendritic cells (pDCs). Using RNA interference screening, we identified the E-box transcription factor TCF4 as a master regulator of the BPDCN oncogenic program. TCF4 served as a faithful diagnostic marker of BPDCN, and its downregulation caused the loss of the BPDCN-specific gene expression program and apoptosis. High-throughput drug screening revealed that bromodomain and extra-terminal domain inhibitors (BETi’s) induced BPDCN apoptosis, which was attributable to disruption of a BPDCN-specific transcriptional network controlled by TCF4-dependent super-enhancers. BETi’s retarded the growth of BPDCN xenografts, supporting their clinical evaluation in this recalcitrant malignancy.

Dysregulation of cyclin-dependent kinase 4 (CDK4) and CDK6 by gain of function or loss of inhibition is common in human cancer, including multiple myeloma, but success in targeting CDK with broad-spectrum inhibitors has been modest. By selective and reversible inhibition of CDK4/CDK6, we have developed a strategy to both inhibit proliferation and enhance cytotoxic killing of cancer cells. We show that induction of prolonged early-G1 arrest (pG1) by CDK4/CDK6 inhibition halts gene expression in early-G1 and prevents expression of genes programmed for other cell-cycle phases. Removal of the early-G1 block leads to S-phase synchronization (pG1-S) but fails to completely restore scheduled gene expression. Consequently, the IRF4 protein required to protect myeloma cells from apoptosis is markedly reduced in pG1 and further in pG1-S in response to cytotoxic agents, such as the proteasome inhibitor bortezomib. The coordinated loss of IRF4 and gain of Bim sensitize myeloma tumor cells to bortezomib-induced apoptosis in pG1 in the absence of Noxa and more profoundly in pG1-S in cooperation with Noxa in vitro. Induction of pG1 and pG1-S by reversible CDK4/CDK6 inhibition further augments tumor-specific bortezomib killing in myeloma xenografts. Reversible inhibition of CDK4/CDK6 in sequential combination therapy thus represents a novel mechanism-based cancer therapy.

SummaryT helper 17 (Th17) cells play an important role in mucosal host defense through production of the signature cytokines IL-17 and IL-22. Prostaglandin E2 (PGE2) has been shown to enhance IL-17 production by mature Th17 cells. However, when present during Th17 cell differentiation, we found that PGE2 inhibited the transcription factor IRF4 and suppressed production of IL-17 but not IL-22. We show that IRF4 was required for IL-17 expression but inhibited IL-22 expression, highlighting the potential for discordant regulation of these two cytokines in Th17 cells. The pathogenic fungus Cryptococcus neoformans produces PGE2, and we found that it uses PGE2- and IRF4-dependent mechanisms to specifically inhibit induction of IL-17 during Th17 cell differentiation. Blockade of host PGE2 during infection led to increased IL-17 production from CD4+ T cells and increased survival of mice. These findings suggest that host- or pathogen-derived PGE2 can act directly on Th17 cells during differentiation to inhibit IL-17-dependent antimicrobial responses.

Modulating aberrant transcription of oncogenes is a relatively unexplored opportunity in cancer therapeutics. In approximately 10% of multiple myelomas, the initiating oncogenic event is translocation of musculoaponeurotic fibrosarcoma oncogene homolog (MAF), a transcriptional activator of key target genes, including cyclinD2. Our prior work showed that MAF is up-regulated in an additional 30% of multiple myeloma cases. The present study describes a common mechanism inducing MAF transcription in both instances. The second mode of MAF transcription occurred in myelomas with multiple myeloma SET domain (MMSET) translocation. MMSET knockdown decreased MAF transcription and cell viability. A small-molecule screen found an inhibitor of mitogen-activated protein kinase kinase (MEK), which activates extracellular signal-regulated kinase (ERK)-MAP kinases, reduced MAF mRNA in cells representing MMSET or MAF subgroups. ERK activates transcription of FOS, part of the AP-1 transcription factor. By chromatin immunoprecipitation, FOS bound the MAF promoter, and MEK inhibition decreased this interaction. MEK inhibition selectively induced apoptosis in MAF-expressing myelomas, and FOS inactivation was similarly toxic. Reexpression of MAF rescued cells from death induced by MMSET depletion, MEK inhibition, or FOS inactivation. The data presented herein demonstrate that the MEK-ERK pathway regulates MAF transcription, providing molecular rationale for clinical evaluation of MEK inhibitors in MAF-expressing myeloma.

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.

A fundamental mystery of the biology of germinal center B cells is why it seems that these rapidly dividing B cells lack expression of c-Myc, a transcriptional regulator intimately linked to cell metabolism and proliferation. This mystery has now been resolved.

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.

T follicular helper (Tfh) cells are specialized CD4 helper T cells that signal B cells to produce high affinity antibodies and provide long-term humoral immunity. Identifying genes that govern Tfh differentiation and function could provide targets for improving vaccines and therapies for autoimmunity. However, Tfh differentiation is only partially understood, and not fully recapitulated in vitro. To discover novel Tfh-regulating genes, we developed a CRISPR-based functional genetic system to mutate genes in primary mouse T cells, and measured the effects on Tfh cells in vivo. Transgenic Cas9-expressing mouse CD4 T cells were transduced in vitro with an optimized retroviral vector encoding an sgRNA and GFP. With a guide targeting Tcf7 (encoding a Tfh-essential transcription factor Tcf1), >90% cells could be transduced; up to >80% of GFP+ cells were Tcf1neg by flow and mutational analyses. To evaluate the functional consequence of CRISPR editing, we adoptively transferred virus-specific CD4 T cells that were transduced with either a control or Tcf7 sgRNA construct into wild-type hosts. After viral challenge, control T cells differentiated into both Tfh and non-Tfh cells, while very few Tcf7-edited T cells became Tfh cells. Similarly, guides against the Tfh master transcription factor Bcl6 abrogated Tfh differentiation in vivo. Next, we constructed sgRNA libraries of up to 400 guides, targeting up to 80 genes, including ones implicated in primary immunodeficiencies and druggable targets. NGS analysis of T cells post-viral challenge revealed depletion of multiple guides targeting known Tfh-required genes in the Tfh vs non-Tfh populations, validating this approach. We are currently following up on potential hits from these screens.

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.

Bortezomib induces remissions in 30%-50% of patients with relapsed mantle cell lymphoma (MCL). Conversely, more than half of patients' tumors are intrinsically resistant to bortezomib. The molecular mechanism of resistance has not been defined. We generated a model of bortezomib-adapted subclones of the MCL cell lines JEKO and HBL2 that were 40- to 80-fold less sensitive to bortezomib than the parental cells. Acquisition of bortezomib resistance was gradual and reversible. Bortezomib-adapted subclones showed increased proteasome activity and tolerated lower proteasome capacity than the parental lines. Using gene expression profiling, we discovered that bortezomib resistance was associated with plasmacytic differentiation, including up-regulation of IRF4 and CD38 and expression of CD138. In contrast to plasma cells, plasmacytic MCL cells did not increase immunoglobulin secretion. Intrinsically bortezomib-resistant MCL cell lines and primary tumor cells from MCL patients with inferior clinical response to bortezomib also expressed plasmacytic features. Knockdown of IRF4 was toxic for the subset of MCL cells with plasmacytic differentiation, but only slightly sensitized cells to bortezomib. We conclude that plasmacytic differentiation in the absence of an increased secretory load can enable cells to withstand the stress of proteasome inhibition. Expression of CD38 and IRF4 could serve as markers of bortezomib resistance in MCL. This study has been registered at http://clinicaltrials.gov as NCT00131976.

The transcription factor IRF4 (interferon regulatory factor 4) is required during an immune response for lymphocyte activation and the generation of immunoglobulin-secreting plasma cells. Multiple myeloma, a malignancy of plasma cells, has a complex molecular aetiology with several subgroups defined by gene expression profiling and recurrent chromosomal translocations. Moreover, the malignant clone can sustain multiple oncogenic lesions, accumulating genetic damage as the disease progresses. Current therapies for myeloma can extend survival but are not curative. Hence, new therapeutic strategies are needed that target molecular pathways shared by all subtypes of myeloma. Here we show, using a loss-of-function, RNA-interference-based genetic screen, that IRF4 inhibition is toxic to myeloma cell lines, regardless of transforming oncogenic mechanism. Gene expression profiling and genome-wide chromatin immunoprecipitation analysis uncovered an extensive network of IRF4 target genes and identified MYC as a direct target of IRF4 in activated B cells and myeloma. Unexpectedly, IRF4 was itself a direct target of MYC transactivation, generating an autoregulatory circuit in myeloma cells. Although IRF4 is not genetically altered in most myelomas, they are nonetheless addicted to an aberrant IRF4 regulatory network that fuses the gene expression programmes of normal plasma cells and activated B cells.

Summary: Genomics has provided a lever to pry open lymphoid cells and examine their regulatory biology. The large body of available gene expression data has also allowed us to define the of coordinately expressed genes, termed gene expression signatures, which characterize the states of cellular physiology that reflect cellular differentiation, activation of signaling pathways, and the action of transcription factors. Gene expression signatures that reflect the action of individual transcription factors can be defined by perturbing transcription factor function using RNA interference (RNAi), small-molecule inhibition, and dominantnegative approaches. We have used this methodology to define gene expression signatures of various transcription factors controlling B-cell differentiation and activation, including BCL-6, B lymphocyte-induced maturation protein-1 (Blimp-1), X-box binding protein-1 (XBP1), nuclear factor-kB (NF-kB), and c-myc. We have also curated a wide variety of gene expression signatures from the literature and assembled these into a signature database. Statistical methods can define whether any signature in this database is differentially expressed in independent biological samples, an approach we have used to gain mechanistic insights into the origin and clinical behavior of B-cell lymphomas. We also discuss the use of genomic-scale RNAi libraries to identify genes and pathways that may serve as therapeutic targets in B-cell malignancies.

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.

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.

The immunoglobulin kappa light chain locus has two enhancer elements: the intronic enhancer, which lies between the Jkappa cluster and the Ckappa exon, and the 3'kappa enhancer, which is located downstream of Ckappa. To address the contribution of these elements to the developmentally regulated activation of germline kappa locus transcription and rearrangement, we purified primary pro- and pre-B cells and determined by in vivo footprinting the sites within each enhancer that were occupied. We found that the kappa intronic enhancer NF-kappaB site is occupied in both pro- and pre-B cells, while CRE, BSAP, and PU.1/pip sites within the 3'kappa enhancer undergo changes in occupancy as cells progress from the pro-B to the pre-B cell stage. These findings suggest that regulation of the kappa locus in primary pre-B cells may be mediated by factors that bind the 3'kappa enhancer.

Polymerase chain reaction (PCR) primers were designed to specifically amplify exon 2 of the DRw52-associated DRB1 alleles for subsequent typing by sequence-specific oligonucleotide probe (SSOP) hybridization and chemiluminescent detection. The DRw52 DRB1 group, encoding 22 of the 44 W.H.O. designated DRB1 allelic products, was divided by differential PCR with two polymorphism-directed forward primers. Based on a polymorphism at codon 13, these forward primers separate the DRw52-associated alleles into subsets; one comprised of the alleles of DR3/DRw11/DRw6 and the other of DRw8/DRw12/DRB1*1404. The DRB1 alleles in the latter subset were then defined by SSOP hybridization to the amplified DNA. The preferential amplification also resulted in SSOP definition of 15 alleles in the DR3/DRw11/DRw6 subset but some DRw11/DRw13 heterozygous allelic combinations were still unresolved. Two reverse PCR primers specific for the polymorphism at codon 86 were used to obtain amplified material to which SSOP reactivity provided definitive identification of the ambiguous heterozygotes.

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.

The differentiation of B cells into immunoglobulin-secreting plasma cells is controlled by two transcription factors, Blimp-1 and XBP1. By gene expression profiling, we defined a set of genes whose induction during mouse plasmacytic differentiation is dependent on Blimp-1 and/or XBP1. Blimp-1-deficient B cells failed to upregulate most plasma cell-specific genes, including xbp1. Differentiating xbp1-deficient B cells induced Blimp-1 normally but failed to upregulate genes encoding many secretory pathway components. Conversely, ectopic expression of XBP1 induced a wide spectrum of secretory pathway genes and physically expanded the endoplasmic reticulum. In addition, XBP1 increased cell size, lysosome content, mitochondrial mass and function, ribosome numbers, and total protein synthesis. Thus, XBP1 coordinates diverse changes in cellular structure and function resulting in the characteristic phenotype of professional secretory cells.

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.