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Early growth response gene (EGR1) is a transcription factor known to be a downstream effector of B-cell receptor signaling and Janus kinase 1 (JAK1) signaling in diffuse large B-cell lymphoma (DLBCL). While EGR1 is characterized as a tumor suppressor in leukemia and multiple myeloma, the role of EGR1 in lymphoma is unknown. Here we demonstrate that EGR1 is a potential oncogene that promotes cell proliferation in DLBCL. IHC analysis revealed that EGR1 expression is elevated in DLBCL compared with normal lymphoid tissues and the level of EGR1 expression is higher in activated B cell–like subtype (ABC) than germinal center B cell–like subtype (GCB). EGR1 expression is required for the survival and proliferation of DLBCL cells. Genomic analyses demonstrated that EGR1 upregulates expression of MYC and E2F pathway genes through the CBP/p300/H3K27ac/BRD4 axis while repressing expression of the type I IFN pathway genes by interaction with the corepressor NAB2. Genetic and pharmacologic inhibition of EGR1 synergizes with the BRD4 inhibitor JQ1 or the type I IFN inducer lenalidomide in growth inhibition of ABC DLBCL both in cell cultures and xenograft mouse models. Therefore, targeting oncogenic EGR1 signaling represents a potential new targeted therapeutic strategy in DLBCL, especially for the more aggressive ABC DLBCL.Implications:The study characterizes EGR1 as a potential oncogene that promotes cell proliferation and defines EGR1 as a new molecular target in DLBCL, the most common non-Hodgkin lymphoma.

Supplementary Table S1 including analyzed RNA-seq and ChIP-seq data

Supplementary Methods, Supplementary Figures and Figure Legends. Figure S1. EGR1 is a downstream target of BCR and JAK1 signaling (related to Figure 1). Figure S2. Sensitivity of MCL cell lines to EGR1 shRNAs and statistical analysis (related to Figure 2). Figure S3. EGR1 motif analysis and gene set enrichment analysis of RNA-seq data (related to Figure 3). Figure S4. Enrichment of E2F pathway genes in ABC DLBCL after EGR1 knockdown (related to Figure 4). Figure S5. EGR1 regulates expression of MYC and MYC targets and OCI-Ly10 xenografts (related to Figure 4). Figure S6. Enrichment of EGR1 on EGR1 own promoter and EP300 regulatory (5'UTR and enhancer) regions (related to Figure 4). Figure S7. Inhibition of EGR1 transcription and tumor growth by mithramycin (related to Figure 4). Figure S8. Synergism of mithramycin and lenalidomide in ABC DLBCL (related to Figure 6). Figure S9. Expression of MYC and MYC target genes is higher in ABC DLBCL than GCB DLBCL patient samples (related to the Discussion). Figure S10. No correlation between EGR1 and MYC expression in DLBCL cell lines and patient samples (related to the Discussion).

Subunits of the chromatin remodeler SWI/SNF are the most frequently disrupted genes in cancer. However, how post-translational modifications (PTM) of SWI/SNF subunits elicit epigenetic dysfunction remains unknown. Arginine-methylation of BAF155 by coactivator-associated arginine methyltransferase 1 (CARM1) promotes triple-negative breast cancer (TNBC) metastasis. Herein, we discovered the dual roles of methylated-BAF155 (me-BAF155) in promoting tumor metastasis: activation of super-enhancer-addicted oncogenes by recruiting BRD4, and repression of interferon α/γ pathway genes to suppress host immune response. Pharmacological inhibition of CARM1 and BAF155 methylation not only abrogated the expression of an array of oncogenes, but also boosted host immune responses by enhancing the activity and tumor infiltration of cytotoxic T cells. Moreover, strong me-BAF155 staining was detected in circulating tumor cells from metastatic cancer patients. Despite low cytotoxicity, CARM1 inhibitors strongly inhibited TNBC cell migration in vitro, and growth and metastasis in vivo. These findings illustrate a unique mechanism of arginine methylation of a SWI/SNF subunit that drives epigenetic dysregulation, and establishes me-BAF155 as a therapeutic target to enhance immunotherapy efficacy.

Relapsed/refractory T cell acute lymphoblastic leukemia (T-ALL) is difficult to salvage especially in heavily pretreated patients, thus novel targeted agents are sorely needed. Hyperactivated JAK/STAT and BCL2 overexpression promote increased T-ALL proliferation and survival, and targeting these pathways with ruxolitinib and venetoclax may provide an alternative approach to achieve clinical remissions. Ruxolitinib and venetoclax show a dose-dependent effect individually, but combination treatment synergistically reduces survival and proliferation of Jurkat and Loucy cells in vitro. Using a xenograft CXCR4+ Jurkat model, the combination treatment fails to improve survival, with death from hind limb paralysis. Despite on-target inhibition by the drugs, histopathology demonstrates increased leukemic infiltration into the central nervous system (CNS), which expresses CXCL12, as compared to liver or bone marrow. Liquid chromatography-tandem mass spectroscopy shows that neither ruxolitinib nor venetoclax can effectively cross the blood-brain barrier, limiting efficacy against CNS T-ALL. Deletion of CXCR4 on Jurkat cells by CRISPR/Cas9 results in prolonged survival and a reduction in overall and neurologic clinical scores. While combination therapy with ruxolitinib and venetoclax shows promise for treating T-ALL, additional inhibition of the CXCR4-CXCL12 axis will be needed to eliminate both systemic and CNS T-ALL burden and maximize the possibility of complete remission.

DNA methyltransferases (DNMTs) are an evolutionarily conserved family of DNA methylases, transferring a methyl group onto the fifth carbon of a cytosine residue. The mammalian DNMT family includes three major members that have functional methylation activities, termed DNMT1, DNMT3A, and DNMT3B. DNMT3A and DNMT3B are responsible for methylation establishment, whereas DNMT1 maintains methylation during DNA replication. Accumulating evidence demonstrates that regulation of DNA methylation by DNMTs is critical for normal hematopoiesis. Aberrant DNA methylation due to DNMT dysregulation and mutations is known as an important molecular event of hematological malignancies, such as DNMT3A mutations in acute myeloid leukemia. In this review, we first describe the basic methylation mechanisms of DNMTs and their functions in lymphocyte maturation and differentiation. We then discuss the current understanding of DNA methylation heterogeneity in leukemia and lymphoma to highlight the importance of studying DNA methylation targets. We also discuss DNMT mutations and pathogenic roles in human leukemia and lymphoma. We summarize the recent understanding of how DNMTs interact with transcription factors or cofactors to repress the expression of tumor suppressor genes. Finally, we highlight current clinical studies using DNMT inhibitors for the treatment of these hematological malignancies.

Arginine methylation is a common posttranslational modification that governs important cellular processes and impacts development, cell growth, proliferation, and differentiation. Arginine methylation is catalyzed by protein arginine methyltransferases (PRMTs), which are classified as type I and type II enzymes responsible for the formation of asymmetric and symmetric dimethylarginine, respectively. PRMT5 is the main type II enzyme that catalyzes symmetric dimethylarginine of histone proteins to induce gene silencing by generating repressive histone marks, including H2AR3me2s, H3R8me2s, and H4R3me2s. PRMT5 can also methylate nonhistone proteins such as the transcription factors p53, E2F1 and p65. Modifications of these proteins by PRMT5 are involved in diverse cellular processes, including transcription, translation, DNA repair, RNA processing, and metabolism. A growing literature demonstrates that PRMT5 expression is upregulated in hematologic malignancies, including leukemia and lymphoma, where PRMT5 regulates gene expression to promote cancer cell proliferation. Targeting PRMT5 by specific inhibitors has emerged as a potential therapeutic strategy to treat these diseases.

Early growth response gene (EGR1) is a transcription factor known to be a downstream effector of B-cell receptor signaling and Janus kinase 1 (JAK1) signaling in diffuse large B-cell lymphoma (DLBCL). While EGR1 is characterized as a tumor suppressor in leukemia and multiple myeloma, the role of EGR1 in lymphoma is unknown. Here we demonstrate that EGR1 is a potential oncogene that promotes cell proliferation in DLBCL. IHC analysis revealed that EGR1 expression is elevated in DLBCL compared with normal lymphoid tissues and the level of EGR1 expression is higher in activated B cell–like subtype (ABC) than germinal center B cell–like subtype (GCB). EGR1 expression is required for the survival and proliferation of DLBCL cells. Genomic analyses demonstrated that EGR1 upregulates expression of MYC and E2F pathway genes through the CBP/p300/H3K27ac/BRD4 axis while repressing expression of the type I IFN pathway genes by interaction with the corepressor NAB2. Genetic and pharmacologic inhibition of EGR1 synergizes with the BRD4 inhibitor JQ1 or the type I IFN inducer lenalidomide in growth inhibition of ABC DLBCL both in cell cultures and xenograft mouse models. Therefore, targeting oncogenic EGR1 signaling represents a potential new targeted therapeutic strategy in DLBCL, especially for the more aggressive ABC DLBCL. Implications: The study characterizes EGR1 as a potential oncogene that promotes cell proliferation and defines EGR1 as a new molecular target in DLBCL, the most common non-Hodgkin lymphoma.

Regulation of the anti-apoptotic BCL2 protein determines cell survival and is frequently abnormal in B cell lymphomas. An evolutionarily conserved post-translational mechanism for over-expression of BCL2 in human B cell lymphomas and the BCL2 paralogue CED-9 in Caenorhabditis elegans results from loss-of-function mutations in human FBXO10 and its C.elegans paralogue DRE-1, a BCL2/CED-9-binding subunit of the SKP-CULLIN-FBOX (SCF) ubiquitin ligase. Here, we tested the role of FBXO10 in BCL2 regulation by producing mice with two different CRISPR/Cas9-engineered Fbxo10 mutations: an Asp54Lys (E54K) missense mutation in the FBOX domain and a Cys55SerfsTer55 frameshift (fs) truncating mutation. Mice homozygous for either mutant allele were born at the expected Mendelian frequency and appeared normal in body weight and appearance as adults. Spleen B cells from homozygous mutant mice did not have increased BCL2 protein, nor were the numbers of mature B cells or germinal centre B cells increased as would be expected if BCL2 was increased. Other lymphocyte subsets that are also regulated by BCL2 levels also displayed no difference in frequency in homozygous Fbxo10 mutant mice. These results support one of two conclusions: either FBXO10 does not regulate BCL2 in mice, or it does so redundantly with other ubiquitin ligase complexes. Possible candidates for the latter include FBXO11 or ARTS-XIAP. The difference between the role of FBXO10 in regulating BCL2 protein levels in C. elegans and in human DLBCL, relative to single-gene deficient mouse leukocytes, should be further investigated.

Regulation of the anti-apoptotic BCL2 protein determines cell survival and is frequently abnormal in B cell lymphomas. An evolutionarily conserved post-translational mechanism for over-expression of BCL2 in human B cell lymphomas and the BCL2 paralogue CED-9 in Caenorhabditis elegans results from loss-of-function mutations in human FBXO10 and its C.elegans paralogue DRE-1, a BCL2/CED-9-binding subunit of the SKP-CULLIN-FBOX (SCF) ubiquitin ligase. Here, we tested the role of FBXO10 in BCL2 regulation by producing mice with two different CRISPR/Cas9-engineered Fbxo10 mutations: an Asp54Lys (E54K) missense mutation in the FBOX domain and a Cys55SerfsTer55 frameshift (fs) truncating mutation. Mice homozygous for either mutant allele were born at the expected Mendelian frequency and appeared normal in body weight and appearance as adults. Spleen B cells from homozygous mutant mice did not have increased BCL2 protein, nor were the numbers of mature B cells or germinal centre B cells increased as would be expected if BCL2 was increased. Other lymphocyte subsets that are also regulated by BCL2 levels also displayed no difference in frequency in homozygous Fbxo10 mutant mice. These results support one of two conclusions: either FBXO10 does not regulate BCL2 in mice, or it does so redundantly with other ubiquitin ligase complexes. Possible candidates for the latter include FBXO11 or ARTS-XIAP. The difference between the role of FBXO10 in regulating BCL2 protein levels in C. elegans and in human DLBCL, relative to single-gene deficient mouse leukocytes, should be further investigated.

Regulating B-cell receptor (BCR) signaling after antigenic stimulation is essential to properly control immune responses. Currently known mechanisms of inhibiting BCR signaling are via co-receptor stimulation and downstream immunoreceptor tyrosine-based inhibition motif (ITIM) phosphorylation. Herein we demonstrate that BCR stimulation induces rapid and reversible palmitoylation of the SCF-FBXO10 ubiquitin E3 ligase. This results in FBXO10 relocation to the cell membrane, where it targets the human germinal center-associated lymphoma (HGAL) protein for ubiquitylation and degradation, leading to decreases in both BCR-induced calcium influx and phosphorylation of proximal BCR effectors. Importantly, FBXO10 recognition and degradation of HGAL is phosphorylation independent and instead relies on a single evolutionarily conserved HGAL amino acid residue (H91) and FBXO10 relocalization to the cytoplasmic membrane. Together our findings demonstrate the first evidence of negative BCR signaling regulation from direct BCR stimulation and define the temporospatial functions of the FBXO10-HGAL axis. FBXO10 is infrequently mutated in DLBCL but some of these mutations deregulate BCR signaling. These observations may have important implications on lymphomagenesis and other immune processes.

Nuclear factor-κB (NF-κB) is a family of transcription factors that play a key role in cell survival and proliferation in many hematological malignancies, including multiple myeloma (MM). Bortezomib, a proteasome inhibitor used in the management of MM, can inhibit both canonical and noncanonical activation of NF-κB in MM cells. However, we previously reported that a significant fraction of freshly isolated MM cells harbor bortezomib-resistant NF-κB activity. Here, we report that hyaluronan and proteoglycan link protein 1 (HAPLN1) is produced in bone marrow stromal cells from MM patients, is detected in patients' bone marrow plasma, and can activate an atypical bortezomib-resistant NF-κB pathway in MM cells. We found that this pathway involves bortezomib-resistant degradation of the inhibitor of NF-κB (IκBα), despite efficient bortezomib-mediated inhibition of proteasome activity. Moreover, HAPLN1 can also confer bortezomib-resistant survival of MM cells. We propose that HAPLN1 is a novel pathogenic factor in MM that induces an atypical NF-κB activation and thereby promotes bortezomib resistance in MM cells.

Treatment of relapsed/refractory B cell acute lymphoblastic leukemia (B-ALL) remains a challenge particularly in patients who do not respond to chemotherapy or immunotherapy. While a subset of these patients can be cured with allogeneic bone marrow transplant, success of the transplant is contingent upon achieving a minimal residual disease negative complete remission. The objective of this study was to assess the efficacy of fedratinib, a semi selective JAK2 inhibitor and venetoclax, a selective BCL-2 inhibitor, on the B-ALL cell lines RS4;11, NALM-6, and SUP-B15. While select studies have examined the efficacy of these drugs on B-ALL as single agents, data is lacking on whether these drugs work optimally as single agents or in combination. Cell lines were treated with either vehicle (DMSO), single agent fedratinib or venetoclax, or the combination of both inhibitors for 48 and 72 hours. Fedratinib doses were tested from 150 nM to 1 uM and venetoclax doses were tested from 2.5 nM to 500 nM. Live/dead discrimination was performed using flow cytometry and a 72-hour MTS assay to assess proliferation. Drug synergy was calculated using CompuSyn software to generate combination index (CI) scores, with a CI score less than 1 reflecting synergy. RNA was isolated from treated leukemia cells and processed on a Nanostring nCounter platform to assess potential changes in gene expression. Results were then analyzed using Rosalind data processing software. In RS4;11, the combination of 5 nM venetoclax and 525 nM fedratinib significantly increased cell death compared to single agent venetoclax (p=0.0451) and fedratinib (p=0.0077). Furthermore, the combination significantly decreased leukemia cell proliferation compared to single agent fedratinib (p Disclosures Capitini: Novartis: Honoraria; Nektar Therapeutics: Honoraria.

PRMT5, which regulates gene expression by symmetric dimethylation of histones and non-histone target proteins, is overexpressed and plays a pathogenic role in many cancers. In diffuse large B cell lymphoma (DLBCL), the mechanisms of PRMT5 dysregulation and its role in lymphomagenesis remain largely unknown. Here we demonstrate that B cell receptor (BCR) signaling regulates PRMT5 expression in DLBCL cells. Immunohistochemical analysis reveals elevated levels of PRMT5 expression in DLBCL cases and in germinal center (GC) B cells when compared to naive B cells. PRMT5 can be induced in naive B cells by BCR stimulation. We discovered that BTK-NF-κB signaling induces PRMT5 transcription in activated B cell-like (ABC) DLBCL cells while BCR downstream PI3K-AKT-MYC signaling upregulates PRMT5 expression in both ABC and GCB DLBCL cells. PRMT5 inhibition inhibits the growth of DLBCL cells in vitro and patient derived xenografts. Genomic and biochemical analysis demonstrate that PRMT5 promotes cell cycle progression and activates PI3K-AKT signaling, suggesting a feedback regulatory mechanism to enhance cell survival and proliferation. Co-targeting PRMT5 and AKT by their specific inhibitors is lethal to DLBCL cell lines and primary cancer cells. Therefore, this study provides a mechanistic rationale for clinical trials to evaluate PRMT5 and AKT inhibitors for DLBCL.

Lymphoma is a complex disease that arises from cells of the immune system with an intricate pathology. While lymphoma may be classified as Hodgkin or non-Hodgkin, each type of tumor is genetically and phenotypically different and highly invasive tissue biopsies are the only method to investigate these differences. Noninvasive imaging strategies, such as immunoPET, can provide a vital insight into disease staging, monitoring treatment response in patients, and dose planning in radioimmunotherapy. ImmunoPET imaging with radiolabeled antibody-based tracers may also assist physicians in optimizing treatment strategies and enhancing patient stratification. Currently, there are two common biomarkers for molecular imaging of lymphoma, CD20 and CD30, both of which have been considered for investigation in preclinical imaging studies. In this review, we examine the current status of both preclinical and clinical imaging of lymphoma using radiolabeled antibodies. Additionally, we briefly investigate the role of radiolabeled antibodies in lymphoma therapy. As radiolabeled antibodies play critical roles in both imaging and therapy of lymphoma, the development of novel antibodies and the discovery of new biomarkers may greatly affect lymphoma imaging and therapy in the future.

Targeting Bruton tyrosine kinase (BTK) by ibrutinib is an effective treatment for patients with relapsed/refractory mantle cell lymphoma (MCL). However, both primary and acquired resistance to ibrutinib have developed in a significant number of these patients. A combinatory strategy targeting multiple oncogenic pathways is critical to enhance the efficacy of ibrutinib. Here, we focus on the BCL2 anti-apoptotic pathway. In a tissue microarray of 62 MCL samples, BCL2 expression positively correlated with BTK expression. Increased levels of BCL2 were shown to be due to a defect in protein degradation because of no or little expression of the E3 ubiquitin ligase FBXO10, as well as transcriptional upregulation through BTK-mediated canonical nuclear factor-κB activation. RNA-seq analysis confirmed that a set of anti-apoptotic genes (for example, BCL2, BCL-XL and DAD1) was downregulated by BTK short hairpin RNA. The downregulated genes also included those that are critical for B-cell growth and proliferation, such as BCL6, MYC, PIK3CA and BAFF-R. Targeting BCL2 by the specific inhibitor ABT-199 synergized with ibrutinib in inhibiting growth of both ibrutinib-sensitive and -resistant cancer cells in vitro and in vivo. These results suggest co-targeting of BTK and BCL2 as a new therapeutic strategy in MCL, especially for patients with primary resistance to ibrutinib.

Signal transducer and activator of transcription 3 (STAT3) is an important and the most studied transcription factor in the Janus kinase (JAK)/STAT signaling pathway. STAT3 mediates the expression of various genes that play a critical role in many cellular and biological processes, such as cell proliferation, survival, differentiation, migration, angiogenesis, and inflammation. STAT3 and associated JAKs are activated and tightly regulated by a variety of cytokines and growth factors and their receptors in normal immune responses. However, abnormal expression of STAT3 leads to its constitutive activation, which promotes malignant transformation and tumor progression through oncogenic gene expression in numerous human cancers. Human lymphoma is a heterogeneous malignancy of T and B lymphocytes. Constitutive signaling by STAT3 is an oncogenic driver in several types of B-cell lymphoma and most of T-cell lymphomas. Aberrant STAT3 activation can also induce inappropriate expression of genes involved in tumor immune evasion such as PD-L1. In this review, we focus on the oncogenic role of STAT3 in human lymphoma and highlight potential therapeutic intervention by targeting JAK/STAT3 signaling.

STAT3 is constitutively activated in many cancers and regulates gene expression to promote cancer cell survival, proliferation, invasion, and migration. In diffuse large B cell lymphoma (DLBCL), activation of STAT3 and its kinase JAK1 is caused by autocrine production of IL-6 and IL-10 in the activated B cell–like subtype (ABC). However, the gene regulatory mechanisms underlying the pathogenesis of this aggressive lymphoma by STAT3 are not well characterized. Here we performed genome-wide analysis and identified 2,251 STAT3 direct target genes, which involve B cell activation, survival, proliferation, differentiation, and migration. Whole-transcriptome profiling revealed that STAT3 acts as both a transcriptional activator and a suppressor, with a comparable number of up- and down-regulated genes. STAT3 regulates multiple oncogenic signaling pathways, including NF-κB, a cell-cycle checkpoint, PI3K/AKT/mTORC1, and STAT3 itself. In addition, STAT3 negatively regulates the lethal type I IFN signaling pathway by inhibiting expression of IRF7, IRF9, STAT1, and STAT2. Inhibition of STAT3 activity by ruxolitinib synergizes with the type I IFN inducer lenalidomide in growth inhibition of ABC DLBCL cells in vitro and in a xenograft mouse model. Therefore, this study provides a mechanistic rationale for clinical trials to evaluate ruxolitinib or a specific JAK1 inhibitor combined with lenalidomide in ABC DLBCL.

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 Janus kinase (JAK)/signal transducer and activator of transcription (STAT) pathway is central to signaling by receptors of diverse cytokines, growth factors, and other related molecules. Many of these receptors transmit anti-apoptosis, proliferation, and differentiation signals that are critical for normal hematopoiesis and immune response. However, the JAK/STAT signaling pathway is deregulated in many hematologic malignancies, and as such is co-opted by malignant cells to promote their survival and proliferation. It has recently come to light that an alternative mechanism, wherein nuclear JAKs epigenetically modify the chromatin to increase gene expression independent of STATs, also plays an important role in the pathogenesis of many hematologic malignancies. In this review, we will focus on common genetic alterations of the JAK family members in leukemia and lymphoma, and provide examples in which JAKs regulate gene expression by targeting the cancer epigenome.

Epstein-Barr virus (EBV) latently infects normal B cells and contributes to the development of certain human lymphomas. Newly infected B cells support a highly transforming form (type III) of viral latency; however, long-term EBV infection in immunocompetent hosts is limited to B cells with a more restricted form of latency (type I) in which most viral gene expression is silenced by promoter DNA methylation. How EBV converts latency type is unclear, although it is known that type I latency is associated with a germinal center (GC) B cell phenotype, and type III latency with an activated B cell (ABC) phenotype. In this study, we have examined whether expression of TET2, a cellular enzyme that initiates DNA demethylation by converting 5-methylcytosine (5mC) into 5-hydroxymethylcytosine (5hmC), regulates EBV latency type in B cells. We found that TET2 expression is inhibited in normal GC cells and GC type lymphomas. In contrast, TET2 is expressed in normal naive B cells and ABC type lymphomas. We also demonstrate that GC type cell lines have increased 5mC levels and reduced 5hmC levels in comparison to those of ABC type lines. Finally, we show that TET2 promotes the ability of the EBV transcription factor EBNA2 to convert EBV-infected cells from type I to type III latency. These findings demonstrate that TET2 expression is repressed in GC cells independent of EBV infection and suggest that TET2 promotes type III EBV latency in B cells with an ABC or naive phenotype by enhancing EBNA2 activation of methylated EBV promoters. IMPORTANCE EBV establishes several different types of viral latency in B cells. However, cellular factors that determine whether EBV enters the highly transforming type III latency, versus the more restricted type I latency, have not been well characterized. Here we show that TET2, a cellular enzyme that initiates DNA demethylation by converting 5-methylcytosine (5mC) into 5-hydroxymethylcytosine (5hmC), regulates EBV latency type in B cells by enhancing the ability of the viral transcription factor EBNA2 to activate methylated viral promoters that are expressed in type III (but not type I) latency. Furthermore, we demonstrate that (independent of EBV) TET2 is turned off in normal and malignant germinal center (GC) B cells but expressed in other B cell types. Thus, restricted TET2 expression in GC cells may promote type I EBV latency.

JAKs are non-receptor tyrosine kinases that are generally found in association with cytokine receptors. In the canonical pathway, roles of JAKs have well been established in activating STATs in response to cytokine stimulation to modulate gene transcription. In contrast, a noncanonical role of JAK2 has recently been discovered, in which JAK2 in the nucleus imparts the epigenetic regulation of gene transcription through phosphorylation of tyrosine 41 on the histone protein H3. Recent work further demonstrated that this noncanonical mechanism is conserved with JAK1, which is activated by the autocrine cytokines IL6 and IL10 in activated B-cell–like diffuse large B-cell lymphoma (ABC DLBCL), a cancer type that is particularly difficult to treat and has poor prognosis. However, how JAK1 gains access to the nucleus to enable epigenetic regulation remains undefined. Here, we investigated this question and revealed that JAK1 has a classical nuclear localization signal toward the N-terminal region, which can be recognized by multiple importin α isoforms. Moreover, the nuclear import of JAK1 is independent of its kinase activity but is required for the optimal expansion of ABC DLBCL cells in vitro. Implications: This study demonstrates that the nuclear import of JAK1 is essential for the optimal fitness of ABC DLBCL cells, and targeting JAK1 nuclear localization is a potential therapeutic strategy for ABC DLBCL. Mol Cancer Res; 15(3); 348–57. ©2016 AACR.

Background: Mantle cell lymphoma (MCL), which accounts for around 7% of non-Hodgkin lymphomas, is currently incurable. MCL's key survival pathway is the B-cell receptor pathway, and Bruton's tyrosine kinase (BTK), the critical component of this pathway, is fundamentally activated in MCL. Ibrutinib, an agent that inhibits BTK, produced a 68% clinical response rate in patients with relapsed/refractory MCL, leading to its FDA approval. However, if MCL patients experience relapse following ibrutinib therapy or do not respond to ibrutinib, the 1-year survival rate is only 22%. Therefore, determining the mechanisms underlying ibrutinib resistance and identifying alternative therapeutic options are urgent unmet needs. Our group and others have shown that the BTK mutation associated with ibrutinib resistance is rare in MCL, and targeting alternative survival pathways to overcome ibrutinib resistance has not shown promising results. Methods: To explore novel biomarkers or therapeutic targets, we performed whole exome and transcriptome sequencing and RNA-seq of ibrutinib-treated clinical specimens. Patient primary cells were isolated from MCL patients treated with ibrutinib either prior to treatment or at treatment discontinuation. Whole exome sequencing (WES) was performed to determine the mutational landscape of ibrutinib resistance. RNA-seq was employed to compare the gene expression profiles between ibrutinib-sensitive and -resistant patient samples. Gene set enrichment analysis was utilized to identify dysregulated molecular pathways associated with the resistant phenotype. The RNA-seq data were then validated with western blots. Functional studies targeting this molecular pathway were conducted, and in vivo efficacy was investigated in the ibrutinib-resistant MCL patient-derived xenograft (PDX) mouse model. Results: Co-deletion of CDKN2A and MTAP was frequently detected in ibrutinib-resistant tumors but not in ibrutinib-sensitive tumors. We also found that metabolic reprogramming towards oxidative phosphorylation (OXPHOS) potentially drives ibrutinib resistance. IACS-010759, a small molecule developed by MD Anderson Cancer Center, inhibits OXPHOS and overcomes ibrutinib resistance in vitro and in PDX models. Nevertheless, the potential drivers of this observed metabolic reprogramming towards OXPHOS remain unclear. MTAP encodes 5-methylthioadenosine phosphorylase, a critical enzyme in the methionine salvage pathway, and is frequently lost in cancer cells, resulting in accumulation of metabolite methylthioadenosine and overexpression of protein arginine methyltransferase 5 (PRMT5). Thus, PRMT5 is a potential druggable therapeutic target to overcome ibrutinib resistance in MCL. We found that PRMT5 is highly expressed in ibrutinib-resistant MCL but not ibrutinib-sensitive MCL (7 ibrutinib-resistant vs. 15 ibrutinib-sensitive patient samples, p = 0.0051), especially in MTAP-deleted MCL specimens. Furthermore, patients with high PRMT5 levels had poor clinical outcomes on ibrutinib treatment. PRMT5 regulates tumor survival pathways by the methylation of histones H3R8, H4R3, and post-translational methylation of non-histone proteins, including p53, E2F1, BCL6, p65, and others. A novel PRMT5 inhibitor, GSK3226595, significantly inhibited tumor growth in an ibrutinib-resistant MCL PDX model (n = 5; vehicle vs. GSK3226595, p = 0.000048) and had no significant mouse body weight change (GSK3226595 vs. vehicle, p = 0.08). Conclusion: This study may pave the way to develop a PRMT5/OXPHOS-based therapeutic strategy to overcome ibrutinib resistance in MCL. Disclosures Wang: Juno Therapeutics: Research Funding; Aviara: Research Funding; Dava Oncology: Honoraria; Celgene: Honoraria, Research Funding; Loxo Oncology: Research Funding; VelosBio: Research Funding; Janssen: Consultancy, Honoraria, Research Funding, Speakers Bureau; Pharmacyclics: Honoraria, Research Funding; AstraZeneca: Consultancy, Honoraria, Research Funding, Speakers Bureau; MoreHealth: Consultancy, Equity Ownership; Acerta Pharma: Consultancy, Research Funding; Kite Pharma: Consultancy, Research Funding; Guidepoint Global: Consultancy; BioInvent: Consultancy, Research Funding.

Diffuse large B cell lymphoma (DLBCL) grows rapidly and represents 30% to 40% of newly diagnosed lymphomas, and comprises two main molecular subtypes: activated B cell-like (ABC) and germinal center B cell-like (GCB). ABC DLBCL is more aggressive with a current cure rate of only ~40%. The B cell antigen receptor (BCR) signaling pathway is known for its critical role in the survival and proliferation of ABC DLBCL cells. Our recent studies revealed that IL6- and IL10-mediated JAK1/STAT3 activation enhances the survival and proliferation of ABC DLBCL cells. Furthermore, we discovered that EGR1, a transcription factor known to be a downstream effector of BCR signaling, is also a downstream target of JAK1 signaling in DLBCL. While EGR1 has been characterized as a tumor suppressor in leukemia, the role of EGR1 in human lymphoma remains unclear. Initial immunohistochemical analysis of 102 DLBCL cases revealed that not only is the level of EGR1 elevated in DLBCL as compared to normal human tonsils, lymph nodes and mantle cell lymphomas, but its level is higher in ABC DLBCL (non-GCB) than GCB DLBCL, indicating functional significance of EGR1 in DLBCL. In addition, we found that 7 out of 8 ABC DLBCL cell lines are sensitive to EGR1 knockdown, whereas only half of 8 GCB DLBCL cell lines are affected. Our genomics studies discovered dual functions of EGR1 in regulating three signaling pathways. EGR1 mediates transcriptional activation associated with two hallmark oncogenic pathways, JAK1/STAT3 and BCR signaling. Our EGR1 ChIP-seq results revealed a large overlap of EGR1 genomic occupancy with that of BRD4, a highly expressed bromo and extraterminal (BET) protein in DLBCL known to induce MYC expression and activation of MYC target genes. This agrees with the RNA-seq results showing that multiple oncogenic pathways including MYC are activated by EGR1 in ABC DLBCL. The synergistic inhibition of cell growth was observed between EGR1 shRNA and JQ1, an inhibitor of BRD4. Surprisingly, the RNA-seq data and pathway analyses also revealed EGR1-mediated suppression of the type I interferon pathway, which otherwise causes cancer cell death. The majority of the interferon pathway genes are up-regulated upon EGR1 knockdown. EGR1-modulated suppression of the interferon pathway appears to be functionally significant as EGR1 knockdown by shRNA synergizes with the type I interferon inducer lenalidomide in growth inhibition of ABC DLBCL cells in vitro and in a xenograft mouse model. EGR1 is known to bind the promoter of p300 and activate p300 expression. P300 catalyzes H3K27 acetylation (H3K27ac), an active histone mark that recruits BRD4 for transcription of MYC and MYC pathway genes, among others. Indeed, immunoblot analysis revealed that EGR1 knockdown reduces H3K27ac expression in ABC DLBCL cells, leading to reduced expression of MYC and MYC target genes based on H3K27ac ChIP qPCR analysis. The results provide a molecular basis for oncogenic cooperation between EGR1 and BRD4 in ABC DLBCL. In summary, our study demonstrates that EGR1 is an oncogenic driver orchestrating multiple important signaling pathways. Notably, EGR1 creates therapeutic vulnerability in ABC DLBCL because both EGR1-mediated transcriptional activation and repression provide growth advantage to the cancer cells. Disclosures No relevant conflicts of interest to declare.

T-cell acute lymphoblastic leukemia (T-ALL) is a hematologic malignancy that accounts for 25% of adult and 15% of pediatric acute lymphoblastic leukemia (ALL) cases. Relapsed or refractory T-ALL is difficult to salvage with chemotherapy, which causes long-term toxicity, and is often fatal. The JAK/STAT and BCL-2 pathways are upregulated in T-ALL and promote increased T-ALL proliferation and survival. Currently, targeted therapies of the JAK/STAT and BCL-2 pathways have not been investigated in combination. I propose that dual inhibition of the JAK/STAT and BCL-2 pathways, with ruxolitinib and venetoclax respectively, will lead to maximal T-ALL cell death. Jurkat cells were treated with single doses of ruxolitinib (0.156µM - 5µM) or venetoclax (1.56nM - 50nM) in vitro, and analyzed by trypan blue exclusion, MTT and flow cytometry at 24, 48 and 72h post-treatment. Results demonstrate decreased proliferation by MTT, decreased viability by trypan blue exclusion, and increased apoptosis by flow cytometry for the three highest doses of ruxolitinib (1.25µM, 2.5µM and 5µM) and venetoclax (12.5nM, 25nM and 50nM). A synergistic effect was achieved for all three assays at 48 and 72h when cells were treated with a combination dose of ruxolitinib (1.25µM) and venetoclax (25nM; CI Citation Format: Kirsti L. Walker, Sabrina A. Kabakov, Fen Zhu, Sydney L. Olson, Lixin Rui, Christian M. Capitini. CXCR4 blockade of T cell acute lymphoblastic leukemia causes systemic disease in an NSG model allowing ruxolitinib and venetoclax to synergistically treat cancer burden [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2975.

Protein arginine methyltransferase PRMT5, which regulates gene expression by symmetric dimethylation of histones and non-histone target proteins, is overexpressed and plays a pathogenic role in many cancers. A growing literature demonstrates a critical role of PRMT5 in tumorigenesis. PRMT5 expression is upregulated in various cancers, including diffuse large B cell lymphoma (DLBCL), the most common, aggressive form of non-Hodgkin lymphoma. PRMT5 upregulation is associated with Epstein-Barr virus (EBV) infection. Given that less than 10% of DLBCL is EBV-positive, the molecular mechanisms of PRMT5 overexpression in DLBCL are still largely unknown. Here, we demonstrated increased expression of PRMT5 in DLBCL cell lines and in a tissue microarray of 104 DLBCL cases when compared with normal naïve B cells. Notably, the level of PRMT5 expression was also elevated in germinal center B cells, which experience antigen stimulation and give rise to DLBCL. In addition, PRMT5 expression in these DLBCL cases correlated with BTK expression, a key component of the B cell antigen receptor (BCR) signaling pathway and an effective drug target in DLBCL. We dissected the mechanisms of differential BCR signaling in regulating PRMT5 expression. Using the CRISPR/Cas9 genome editing technology or by specific pharmacological inhibitors, we discovered that two main BCR downstream signaling pathways, NF-kB and PI3K/AKT, contribute to PRMT5 overexpression. Specifically, the NF-kB member p65 and MYC, a downstream target of the PI3K/AKT signaling pathway, induced PRMT5 transcription. More importantly, inhibition of PRMT5 by its sgRNA or its specific inhibitor was lethal to all DLBCL cell lines tested but not to normal B cells. The antitumor effect of PRMT5 sgRNA or its inhibitor was further revealed in two DLBCL xenograft mouse models as well as in patient derived xenografts (PDX). Further RNA-Seq and biochemical analysis demonstrated that PRMT5 promotes cell cycle progression and activates PI3K-AKT signaling, suggesting a positive feedback regulatory mechanism to enhance cell survival and proliferation. To intervene in the positive feedback regulatory mechanism, we used the PRMT5 inhibitor GSK3326595 and the AKT inhibitor AZD5363, both of which are currently used in clinical trials for solid cancers. Indeed, our in vitro analysis in DLBCL cell lines demonstrated their synergistic cytotoxicity. We will further test the synergism of these two inhibitors in xenograft mouse models as well as in primary cancer cells. Taken together, our study elucidates the mechanisms of PRMT5 upregulation and also demonstrates an oncogenic role for PRMT5 in DLBCL, suggesting targeting PRMT5 by a specific inhibitor or co-targeting with an AKT inhibitor as a potential therapeutic strategy for DLBCL patients. Citation Format: Lixin Rui. PRMT5 upregulation and its oncogenic cooperation with PI3K/AKT signaling in diffuse large B cell lymphoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3436.

Cell death is a common metazoan cell fate, and its inactivation is central to human malignancy. In Caenorhabditis elegans, apoptotic cell death occurs via the activation of the caspase CED-3 following binding of the EGL-1/BH3-only protein to the antiapoptotic CED-9/ BCL2 protein. Here we report a major alternative mechanism for caspase activation in vivo involving the F-box protein DRE-1. DRE-1 functions in parallel to EGL-1, requires CED-9 for activity, and binds to CED-9, suggesting that DRE-1 promotes apoptosis by inactivating CED-9. FBXO10, a human protein related to DRE-1, binds BCL2 and promotes its degradation, thereby initiating cell death. Moreover, some human diffuse large B-cell lymphomas have inactivating mutations in FBXO10 or express FBXO10 at low levels. Our results suggest that DRE-1/FBXO10 is a conserved regulator of apoptosis. Fil: Chiorazzi, Michael. The Rockefeller University. Laboratory of Developmental Genetics; Estados Unidos de América; Fil: Rui, Lixin. National Cancer Institute. Center for Cancer Research. Metabolism Branch; Estados Unidos de América; Fil: Yang, Yandan. National Cancer Institute. Center for Cancer Research. Metabolism Branch; Estados Unidos de América; Fil: Ceribelli, Michelle. National Cancer Institute. Center for Cancer Research. Metabolism Branch; Estados Unidos de América; Fil: Tishbi, Nima. The Rockefeller University. Laboratory of Developmental Genetics; Estados Unidos de América; Fil: Maurer. Carine W.. The Rockefeller University. Laboratory of Developmental Genetics; Estados Unidos de América; Fil: Ranuncolo, Stella Maris. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Oncología; Argentina; National Cancer Institute. Center for Cancer Research. Metabolism Branch; Estados Unidos de América; Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay; Argentina; Fil: Zhao, Hong. National Cancer Institute. Center for Cancer Research. Metabolism Branch; Estados Unidos de América; Fil: Xu, Weihong. National Cancer Institute. Center for Cancer Research. Metabolism Branch; Estados Unidos de América; Fil: Chan, Wing-Chung C.. University of Nebraska. Department of Pathology and Microbiology; Estados Unidos de América; Fil: Jaffe, Elaine S.. National Cancer Institute. Center for Cancer Research. Laboratory of Pathology; Estados Unidos de América; Fil: Gascoyne, Randy D.. British Columbia Cancer Agency; Canadá; Fil: Campo, Elias. Universidad de Barcelona. Hospital Clínico; España; Fil: Rossenwald, Andreas. University of Wurzburg. Department of Pathology; Alemania; Fil: Ott, German. Robert-Bosch-Krankenhaus and Dr. Margarete Fischer-Bosch Institute for Clinical Pharmacology. Department of Clinical Pathology; Alemania; Fil: Delabie, Jan. Oslo University Hospital. Pathology Clinic; Noruega; Fil: Rimsza, Lisa M.. University of Arizona. Department of Pathology; Estados Unidos de América; Southwest Oncology Group; Estados Unidos de América; Fil: Shaham, Shai. The Rockefeller University. Laboratory of Developmental Genetics; Estados Unidos de América; Fil: Staudt, Louis M.. National Cancer Institute. Center for Cancer Research. Metabolism Branch; Estados Unidos de América

Janus kinases (JAKs) classically signal by activating STAT transcription factors but can also regulate gene expression by epigenetically phosphorylating histone H3 on tyrosine 41 (H3Y41-P). In diffuse large B-cell lymphomas (DLBCLs), JAK signaling is a feature of the activated B-cell (ABC) subtype and is triggered by autocrine production of IL-6 and IL-10. Whether this signaling involves STAT activation, epigenetic modification of chromatin, or both mechanisms is unknown. Here we use genetic and pharmacological inhibition to show that JAK1 signaling sustains the survival of ABC DLBCL cells. Whereas STAT3 contributed to the survival of ABC DLBCL cell lines, forced STAT3 activity could not protect these cells from death following JAK1 inhibition, suggesting epigenetic JAK1 action. JAK1 regulated the expression of nearly 3,000 genes in ABC DLBCL cells, and the chromatin surrounding many of these genes was modified by H3Y41-P marks that were diminished by JAK1 inhibition. These JAK1 epigenetic target genes encode important regulators of ABC DLBCL proliferation and survival, including IRF4, MYD88, and MYC. A small molecule JAK1 inhibitor cooperated with the BTK inhibitor ibrutinib in reducing IRF4 levels and acted synergistically to kill ABC DLBCL cells, suggesting that this combination should be evaluated in clinical trials.

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.

SummaryChromosome band 9p24 is frequently amplified in primary mediastinal B cell lymphoma (PMBL) and Hodgkin lymphoma (HL). To identify oncogenes in this amplicon, we screened an RNA interference library targeting amplicon genes and thereby identified JAK2 and the histone demethylase JMJD2C as essential genes in these lymphomas. Inhibition of JAK2 and JMJD2C cooperated in killing these lymphomas by decreasing tyrosine 41 phosphorylation and increasing lysine 9 trimethylation of histone H3, promoting heterochromatin formation. MYC, a major target of JAK2-mediated histone phosphorylation, was silenced after JAK2 and JMJD2C inhibition, with a corresponding increase in repressive chromatin. Hence, JAK2 and JMJD2C cooperatively remodel the PMBL and HL epigenome, offering a mechanistic rationale for the development of JAK2 and JMJD2C inhibitors in these diseases.

Triple-negative breast cancers (TNBC) lack the expression of estrogen receptor (ER), progesterone receptor (PR) and human epidermal growth factor receptor 2 (HER2). TNBC is the most aggressive breast cancer subtype and is often associated with distant metastasis, poor prognosis, and lack of targeted treatment options. The failure to identify a few genes driving metastasis through sequencing of paired tumors at primary and metastatic sites implies that epigenetic mechanisms may play important roles in TNBC metastasis. Recently, we found that an epigenetic enzyme coactivator associated arginine methyltransferase 1 (CARM1) is highly expressed in TNBC and its high expression correlates with poor prognosis. Subsequently we identified BRG1-Associated Factor 155 (BAF155), a component of SWI/SNF chromatin remodeling complex, as a novel CARM1 substrate. Transcriptome analyses of BAF155 methylation competent and defective cell lines identified many genes involved in regulating cancer metastasis are methyl-BAF155 targets. Methylation of BAF155 was found to promote metastasis in the TNBC xenografted mouse models. We generated a methyl-BAF155 antibody that could specifically detect methylated BAF155 (me-BAF155) by IHC and chromatin immunoprecipitation followed by sequencing (ChIP-seq). We mapped global me-BAF155 genomic association sites using ChIP-seq and discovered that me-BAF155 was highly enriched in super-enhancers (SEs), regulatory elements on chromatin that are known to drive high transcription of many oncogenes. SEs are known to recruit BRD4, a BET family bromodomain protein. Indeed, global me-BAF155 binding peaks greatly overlap with those of BRD4 binding sites. Treatment of TNBC cells with either BRD4 inhibitor JQ1 or CARM1 inhibitor TP-064 significantly reduced the expression of me-BAF155 target genes. Together our data suggest that me-BAF155 is an important epigenetic target for treatment of cancer metastasis in TNBC cells. Citation Format: Eui-Jun Kim, Yidan Wang, Li Lu, Lixin Rui, Xuehua Zhong, Kari B. Wisinski, Wei Xu. Methylated BAF155 promotes cancer metastasis by cooperating with BRD4 to potentiate genes addicted to super-enhancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4313.

It is now widely accepted that lymphomagenesis is a multistep transformation process. A number of genetic changes and environmental and infectious factors contributing to the development and malignant progression of B-cell lymphoproliferative disorders are well documented. Reciprocal chromosomal translocations involving the immunoglobulin loci are a hallmark of most mature B cell lymphomas and lead to dysregulated expression of proto-oncogenes (c-myc) important for cell proliferation or genes involved in cell cycle progression (cyclin D1), differentiation block (bcl-6, PAX5) and cell survival (bcl-2, NF-kappaB). In addition, genetic alterations that inactivate tumor suppressor genes (p53, p16) have been frequently detected in some lymphoma tissues. Many of these genes are normally regulated by signals from the B cell antigen receptor. The high prevalence of bacterial and viral infection in lymphoma patients supports the hypothesis that infectious agents may play a contributory role in the development and evolution of B cell lymphoproliferative disorders by either directly inducing polyclonal B cell hyperactivation (EBV, HCV), or providing a chronic antigenic stimulus (EBV, HCV, HBV, H. pylori), or mimicking B cell antigen receptor signaling (EBV, HCV, HHV8), although whether these are causative factors or they are secondary to genetic changes in lymphomagenesis remains to be defined. Stimulatory signals from reactive T cells, local cytokines and growth factors can also contribute, to some extent, to the progression of transformation. Modulation of B cell antigen receptor signaling therefore emerges as a potentially powerful strategy for controlling the growth of certain B cell lymphomas.

The NF-κB family of transcription factors regulates numerous cellular processes, including cell proliferation and survival responses. The constitutive activation of NF-κB has also emerged as an important oncogenic driver in many malignancies, such as activated B-cell like diffuse large B cell lymphoma, among others. In this study, we investigated the impact and mechanisms of action of Withaferin A, a naturally produced steroidal lactone, against both signal-inducible as well as constitutive NF-κB activities. We found that Withaferin A is a robust inhibitor of canonical and constitutive NF-κB activities, leading to apoptosis of certain lymphoma lines. In the canonical pathway induced by TNF, Withaferin A did not disrupt RIP1 polyubiquitination or NEMO-IKKβ interaction and was a poor direct IKKβ inhibitor, but prevented the formation of TNF-induced NEMO foci which colocalized with TNF ligand. While GFP-NEMO efficiently formed TNF-induced foci, a GFP-NEMO(Y308S) mutant that is defective in binding to polyubiquitin chains did not form foci. Our study reveals that Withaferin A is a novel type of IKK inhibitor which acts by disrupting NEMO reorganization into ubiquitin-based signaling structures in vivo.

Janus kinases (JAKs) are non-receptor tyrosine kinases that are generally found bound to cytokine receptors. JAKs have well established roles in activating signal transducers and activators of transcription (STATs) in response to cytokine stimulation in the cytoplasm. However, the epigenetic regulation of gene transcription by nuclear JAK2 through phosphorylation of tyrosine 41 on the histone protein H3 has recently been discovered. Our very recent work demonstrated that this histone targeting mechanism applies to another member of the JAK family, JAK1, which is activated by the autocrine cytokines IL6 and IL10 in activated B-cell-like diffuse large B cell lymphoma (ABC DLBCL). Genome-wide mapping of the H3Y41p histone mark revealed 2956 potential JAK1 target genes, 91% of which do not have a STAT motif in their promoter region. Here we investigated the JAK1 nuclear transfer mechanism in ABC DLBCL. Using cell fractionation and immunofluorescence assays, we detected a nuclear pool of JAK1 in both HEK 293T cells and ABC DLBCL cell lines. In searching for a putative nuclear localization signal (NLS) of JAK1, we identified a basic amino acid enriched sequence (KRKK…KHKK, starting from amino acid 342) that was reminiscent of a classical bipartite NLS. Immunofluorescence with GFP-tagged JAK1 truncations and full length JAK1 with mutations in putative NLS revealed that the first group of amino acids (KRKK) was the NLS that was responsible for the nuclear transport of JAK1. Our results, however, indicate that the active transport of JAK1 into the nucleus is independent of its activation status, since nucleocytoplasmic distribution did not change either in HEK293T cells when constitutively activated JAK1 was expressed or in ABC DLBCL cells with IL-6 stimulation or in GCB DLBCL cells that lack cytokine signaling. To identify potential importins that recognize the NLS of JAK1 and subsequently transport JAK1 into the nucleus, we used immunoprecipitation and GST pull down assays. We found that importin a4, a5 and a7 (encoded by KPNA3, KPNA1, and KPNA6 respectively) physically interacted with the NLS of JAK1 and these interactions were abolished when the 4 amino acids in the NLS were mutated to alanine, suggesting these alpha importins recognize the NLS of JAK1 (KRKK) and mediate the nuclear transport of JAK1. To test whether the NLS-mediated nuclear transfer that allows JAK1 to function in the nucleus is important for the fitness of ABC DLBCL cells, we performed JAK1 knockdown and rescue experiments in TMD8 and OCI-Ly10 cells, in which endogenous JAK1 was knocked down by an shRNA and an shRNA-resistant cDNA of either wild type or NLS-defective JAK1 was constantly expressed. The results revealed that knockdown of JAK1 led to reduced viable cells in the culture, suggesting that JAK1 expression is required for cancer cell survival. As expected, overexpression of wild type JAK1 completely reversed the toxicity due to knockdown of endogenous JAK1. However, overexpression of the NLS-defective JAK1 did not rescue the toxic effects of JAK1 shRNA. Thus, the findings suggest that nuclear transport of JAK1 is an essential process to maintain the survival of ABC DLBCL, and targeting the JAK1 NLS might be a potential targeted therapeutic strategy. Disclosures No relevant conflicts of interest to declare.

Activated B-cell-like diffuse large B-cell lymphoma (ABC DLBCL) is characterized by increased expression and activator of signal transducer and activator of transcription 3 (STAT3). ABC DLBCL cells require STAT3 for growth in culture. In ABC DLBCL cells, eosinophils and perhaps all cells, four variant STAT3 mRNAs (Sα, ΔSα, Sβ and ΔSβ) are present as a result of two alternative splicing events, one that results in the inclusion of a 55-residue C-terminal transactivation domain (α) or a truncated C-terminal domain with 7 unique residues (β) and a second that includes (S) or excludes (ΔS) the codon for Ser-701 in the linker between the SH2 and C-terminal domains. A substantial literature indicates that both α and β variants are required for optimal STAT3 function, but nothing is known about functions of ΔS variants. We used a knockdown/re-expression strategy to explore whether survival of ABC DLBCL cells requires that the four variants be in an appropriate ratio. No single variant rescued survival as well as STAT3Sα-C, Sα with activating mutations (A661C and N663C) in the SH2 domain. Better rescue was achieved when all four variants were re-expressed or Sα and ΔSα or Sβ and ΔSβ were re-expressed in pairs. Rescue correlated with expression of STAT3-sensitive genes NFKBIA and NFKBIZ. We consider a variety of explanations why a mix of S and ΔS variants of STAT3 should enable survival of ABC DLBCL cells.

Mantle cell lymphoma (MCL), a B-cell non-Hodgkin lymphoma, remains incurable with current treatment modalities. Bruton tyrosine kinase (BTK), a key component of the early BCR signaling pathway, has emerged as a promising therapeutic target. Ibrutinib, a specific inhibitor that binds covalently to the active site of BTK at cysteine 481, has been approved for the treatment of MCL. In a recent Phase II study, treatment for relapsed or refractory MCL with Ibrutinib alone achieved a response rate of 68%. Although this response rate is striking, approximately one third of patients show primary resistance, and acquired resistance with a C481S mutation in BTK can also develop. To overcome primary and acquired resistance to BTK inhibition, a combinatory strategy that targets multiple pathways is needed. Here, we focused on the BCL2 anti-apoptotic pathway. Our previous study established FBXO10 as the E3 ubiquitin ligase that targets BCL2 for proteasomal degradation and revealed reduced levels of FBXO10 expression in MCL cell lines. To examine BCL2 and FBXO10 expression in patient samples, we performed immunohistochemical (IHC) analysis in a tissue microarray (TMA) that contained 62 MCL cases. Our data showed high levels of BCL2 expression but no or low FBXO10 expression in MCL cases. Based on this initial expression analysis, we hypothesized that a defect in FBXO10-mediated proteasomal degradation contributes to high BCL2 expression in MCL. To test this hypothesis, we genetically manipulated FBXO10 expression in MCL cell lines. Flow cytometric analysis revealed that overexpression of FBXO10 resulted in reduced BCL2 expression. Inversely, we silenced endogenous FBXO10 by shRNA. Indeed, after blocking protein synthesis by cycloheximide, the amount of BCL2 protein was sustained in these FBXO10 shRNA-expressing cells. These results indicate that BCL2 is the predominantly expressed anti-apoptotic protein in MCL, due to a specific defect in its proteasomal degradation. Inhibition of BCL2 by the specific inhibitor ABT-199 killed cancer cells in culture and also inhibited xenografted tumor growth. BCL2 is a target gene of the BTK/canonical NF-κB signaling pathway. We next analyzed BTK expression in the above TMA and found that BTK was highly expressed and positively correlated with BCL2 expression in these cases. BTK inhibition reduced BCL2 expression as shown in the BCR-dependent cell lines Jeko and Mino. RNA-seq analysis confirmed that a set of anti-apoptotic genes (e.g. BCL2, BCL-XL and DAD1) was downregulated by BTK shRNA. The downregulated genes also included those that are critical for B cell growth and proliferation, such as BCL6, MYC, PIK3CA and BAFF-R. Elevated BCL2, however, can also result from other mechanisms, such as amplification. Indeed, this genomic alteration is present in some MCL cell lines, including Granta-519 and Z138 used for this study. Since these cell lines are insensitive or less sensitive to Ibrutinib, we hypothesized that BCL2 upregulation is a mechanism underlying primary resistance to Ibrutinib, and thus targeting BCL2 by ABT-199 increases or restores sensitivity of these cells to Ibrutinib. The results supported this hypothesis and demonstrated a synergistic effect of ABT-199 and Ibrutinib on growth inhibition of Granta-519 and Z138 cells both in vitro and in vivo. In summary, this study elucidated mechanisms of BCL2 overexpression and association with the BCR/BTK signaling pathway in MCL. Our data provided a mechanistic rationale for co-targeting of these two oncogenic pathways by ABT-199 and Ibrutinib as a new therapeutic strategy in a pre-clinical setting. Most importantly, the findings indicate that this combination has a synergistic effect on the killing of both BCR-dependent and independent MCL cells. Since these two signaling pathways are deregulated in DLBCL and follicular lymphoma, co-targeting by Ibrutinib and ABT-199 may provide an attractive therapeutic strategy for these additional lymphoma patients as well. Disclosures Kahl: Roche/Genentech: Consultancy; Seattle Genetics: Consultancy; Millennium: Consultancy; Cell Therapeutics: Consultancy; Celgene: Consultancy; Infinity: Consultancy; Pharmacyclics: Consultancy; Juno: Consultancy.

Expression of the Human Germinal center Associated Lymphoma (HGAL) gene is restricted to germinal center (GC) B-lymphocytes and GC-derived lymphomas. HGAL expression identifies lymphomas characterized by a better prognosis. We previously showed that HGAL is a unique adaptor protein that regulates both cell motility and B-cell receptor (BCR) signaling, processes that are central for successful completion of the GC reaction. In our previous studies we demonstrated that upon BCR activation HGAL binds to and increases Syk kinase activity, resulting in enhanced BCR signaling. Syk induces HGAL phosphorylation, leading to its redistribution from lipid rafts to the bulk membrane and cytoplasm, followed by its rapid degradation. The mechanism of HGAL degradation is currently unknown. To elucidate the mechanism of HGAL degradation, we hypothesized that HGAL is degraded by a SKP1-CUL1-F-box protein (SCF) ubiquitin ligase complex. Consequently, we examined the ability of HGAL to be recruited to the SCF by screening a panel of F-box proteins transfected into HEK293T cells by reciprocal coimmunoprecipitations (Co-IPs). These studies revealed that HGAL specifically bound only FBXO10. Concordantly, HGAL Co-IPed with endogenous SKP1 and CUL1 in the presence of FBXO10. Furthermore, a dose dependent reduction of HGAL levels was observed in Raji cells transfected with increasing quantities of FBXO10 expressing plasmid. In contrast, none of the other FBXO11 tested F-box proteins affected HGAL levels. The reduction in HGAL protein level by FBXO10 was due to enhanced proteolysis, as demonstrated by the decrease in HGAL protein half-life and the rescue of its levels by either addition of proteasome inhibitor MG132 or expression of a dominant-negative CUL1 mutant (CUL1 (1-242)). Furthermore, depletion of FBXO10 by two short hairpin RNA (shRNA) constructs in Raji cells increased HGAL stability and half-life. SCF complexes mediates protein degradation by polyubiquitination. We therefore examined if HGAL protein can be ubiquinated. To this end we cotransfected 293T cells with HGAL-V5, FBXO10-FLAG, HA-ubiquitin with or without CUL1 (1-242) and blotted immunoprecipitated HGAL for ubiquitin. FBXO10 expression markedly increased HGAL ubiquitination and this was significantly decreased in cells coexpressing CUL1 (1-242). Similarly, HGAL ubiquitination was markedly decreased in cells expressing FBXO10 (FBXO10- ΔF-box), in which the F-box motif, necessary for linking the target protein to the SCF ubiquitin ligase moieties, is deleted. These results support the function of FBXO10 in binding HGAL and facilitating its ubiquitination by the SCF complex prior to its degradation. We next analyzed the functional significance of HGAL degradation by FBXO10 on BCR activation, as measured by intracellular and transmembrane Ca2+ mobilization. Overexpression of FBXO10 in Raji cells expressing endogenous HGAL led to decreased levels of HGAL and in Ca2+ mobilization. In contrast, FBXO10 did not affect BCR-induced Ca2+ mobilization in Raji cells in which HGAL was deleted by crispr/Cas9 targeting. These results suggest that FBXO10 affects BCR signaling via regulating HGAL levels. While FBXO10 mediates HGAL degradation post BCR-induced HGAL phosphorylation, FBXO10 also bound HGAL in the absence of BCR activation. The binding of FBXO10 to HGAL was unaffected in Raji cells incubated with anti-IgM antibodies. Treatment of cellular lysates with l-phosphatase did not affect FBXO10 binding to HGAL. These findings indicate that FBXO10-dependent degradation of HGAL is phosphorylation independent. In contrast, HGAL degradation by FBXO10 was dependent on HGAL lipid raft localization, since HGAL myristoylation and palmitoylation mutants (G2A, C43A/C45A, and G2A/C43A/C45A) exhibited decreased Co-IP with the FBXO10 protein. To further map FBXO10 binding motifs of HGAL, we generated HGAL deletion and mutant constructs and examined their ability to Co-IP with FBXO10. These studies revealed that HGAL amino acids 91-95(HRVLC) mediate HGAL binding to FBXO10. In summary, our results demonstrate that SCFFBXO10 is the ubiquitin ligase for HGAL and regulates BCR signaling via controlling HGAL expression. Some diffuse large cell lymphomas expressing HGAL harbor inactivating mutations or express low levels of FBXO10, thus predisposing to enhanced BCR signaling- a phenomenon implicated in lymphomagenesis. Disclosures No relevant conflicts of interest to declare.

At great human cost, cancer is the largest genetic experiment ever conducted. This review highlights how lymphoid malignancies have genetically perverted normal immune signaling and regulatory mechanisms for their selfish oncogenic goals of unlimited proliferation, perpetual survival and evasion of the immune response.

Chromosomal translocations are critically involved in the molecular pathogenesis of B-cell lymphomas, and highly recurrent and specific rearrangements have defined distinct molecular subtypes linked to unique clinicopathological features(1,2). In contrast, several well-characterized lymphoma entities still lack disease-defining translocation events. To identify novel fusion transcripts resulting from translocations, we investigated two Hodgkin lymphoma cell lines by whole-transcriptome paired-end sequencing (RNA-seq). Here we show a highly expressed gene fusion involving the major histocompatibility complex (MHC) class II transactivator CIITA (MHC2TA) in KM-H2 cells. In a subsequent evaluation of 263 B-cell lymphomas, we also demonstrate that genomic CIITA breaks are highly recurrent in primary mediastinal B-cell lymphoma (38%) and classical Hodgkin lymphoma (cHL) (15%). Furthermore, we find that CIITA is a promiscuous partner of various in-frame gene fusions, and we report that CIITA gene alterations impact survival in primary mediastinal B-cell lymphoma (PMBCL). As functional consequences of CIITA gene fusions, we identify downregulation of surface HLA class II expression and overexpression of ligands of the receptor molecule programmed cell death 1 (CD274/PDL1 and CD273/PDL2). These receptor-ligand interactions have been shown to impact anti-tumour immune responses in several cancers(3), whereas decreased MHC class II expression has been linked to reduced tumour cell immunogenicity(4). Thus, our findings suggest that recurrent rearrangements of CIITA may represent a novel genetic mechanism underlying tumour-microenvironment interactions across a spectrum of lymphoid cancers.

Condensins are ubiquitously expressed multiprotein complexes that are important for chromosome condensation and epigenetic regulation of gene transcription, but whose specific roles in vertebrates are poorly understood. We describe a mouse strain, nessy, isolated during an ethylnitrosourea screen for recessive immunological mutations. The nessy mouse has a defect in T lymphocyte development that decreases circulating T cell numbers, increases their expression of the activation/memory marker CD44, and dramatically decreases the numbers of CD4 + CD8 + thymocytes and their immediate DN4 precursors. A missense mutation in an unusual alternatively spliced first exon of the kleisin β gene, a member of the condensin II complex, was shown to be responsible and act in a T cell-autonomous manner. Despite the ubiquitous expression and role of condensins, kleisin β nes/nes mice were viable, fertile, and showed no defects even in the parallel pathway of B cell lymphocyte differentiation. These data define a unique lineage-specific requirement for kleisin β in mammalian T cell differentiation.

Immunological tolerance has historically been considered as arising primarily by elimination or inactivation of immature lymphocytes with self-reactive antigen receptors, during the early phase of repertoire formation in the thymus or bone marrow. Indeed, the clinical importance of thymic repertoire formation has now been established by two devastating genetic disorders autoimmune polyendocrine syndrome 1 (APS1 or APECED) and immunodeficiency polyendocrinopathy enteropathy X-linked syndrome (IPEX) and their mouse counterparts. In APS1 there is a failure to delete organ-specific T cells in the thymus caused by mutation of the AIRE gene1,2, while defects in the FOXP3 gene in IPEX render the thymus unable to form a specific class of suppressor CD4 T cells3,4, in both cases resulting in inexorable and severe autoimmunity. Tolerance mechanisms that act later, on mature lymphocytes in the peripheral lymphoid organs and circulation, have garnered less attention – not because they are less important but because they have proved much less experimentally accessible. Two mechanisms that stop B and T cells from proliferating and clonally expanding anergy and activation-induced cell death have been studied in some detail 5,6. Defects in one form of activation-induced cell death cause the severe autoimmune lymphoproliferative syndrome (ALPS) when the FAS gene is defective in man or mouse7,8. Arguably the two most critical steps for preventing autoantibody secretion, however, are the penultimate processes of affinity maturation in germinal centres and terminal differentiation into plasma cells. Tolerance mechanisms acting at these steps are nevertheless poorly characterized. Here we summarize recent work fromour groupproviding insights into these late-stage tolerancemechanisms.