Cell Lymphomagenesis Research Articles

Untangling the Epigenetic Imbalance in B cell Lymphoma

Structural and functional genomics of B cell lymphoma identified several recurrent lesions in epigenetic machinery that drive disease pathogenesis. However, the functional relationship between these lesions and disease phenotype has not been well studied. This review is crafted with the aim to delineate pathologic derangements in the epigenetic system that underlies the imbalance and to identify opportunities for strategic design of novel therapeutic interventions by restoring the balance in epigenetic signaling of B cell lymphoma. Selective gain or loss of epigenetic machinery components at the genetic or proteomic level through various regulatory cues including genetic lesions and transcriptional or post-translational regulations often bring regulatory system functional imbalance. This also impinges upon our understanding of consequences when epigenetic regulation goes awry leading to loss of cellular identity and unchecked cellular proliferation, a hallmark of cancer. Such deregulations prompted by epigenetic imbalance are prone to spuriously activate proto-oncogene and/or cripple the tumor suppressor function to gain growth-promoting advantages, particularly in B cell lymphomagenesis. Often, the restoration of epigenetic balance through pharmacologic approaches caused synthetic lethality to neoplastic cells. Therefore, a detailed understanding needed to utilize these phenomena as novel therapeutic interventions targeting B cell lymphoma. This review illustrates the previously underrepresented importance of epigenetic imbalance caused by genetic lesions and restoration of functional epigenetic balance as a novel treatment strategy for B cell lymphoma.

MYC-regulated lncRNA NEAT1 promotes B cell proliferation and lymphomagenesis via the miR-34b-5p-GLI1 pathway in diffuse large B-cell lymphoma

BackgroundLncRNA NEAT1 has been identified as a tumour driver in many human cancers. However, the underlying mechanism of lncRNA NEAT1 in diffuse large B-cell lymphoma (DLBCL) progression is unclear.MethodsThe expression levels of NEAT1, GLI1 and miR-34b-5p were detected by RT-qPCR and Western blotting in DLBCL tissues and cell lines. MTT and colony formation assays were performed to examine cell proliferation, while annexin-V staining and TUNEL assays were performed to measure cell apoptosis. The effect of NEAT1, GLI1 and miR-34b-5p on cell cycle-associated proteins was evaluated by Western blotting. Dual-luciferase reporter and RNA immunoprecipitation (RIP) assays were employed to investigate the interaction between NEAT1 and miR-34b-5p or GLI1 and miR-34b-5p. Moreover, chromatin immunoprecipitation (ChIP) was performed to demonstrate the interaction between MYC and NEAT1.ResultsNEAT1 and GLI1 were upregulated while miR-34b-5p was downregulated in DLBCL tissues and cell lines compared to normal controls. Knockdown of NEAT1 or overexpression of miR-34b-5p inhibited cell proliferation but promoted cell apoptosis. Overexpression of NEAT1 reversed GLI1-knockdown induced attenuation of cell proliferation. In other words, NEAT1 acted as a competing endogenous RNA (ceRNA), regulating the miR-34b-5p-GLI1 axis, further affecting the proliferation of DLBCL. Moreover, MYC modulated NEAT1 transcription by directly binding to the NEAT1 promoter.ConclusionWe revealed that MYC-regulated NEAT1 promoted DLBCL proliferation via the miR-34b-5p-GLI1 pathway, which could provide a novel therapeutic target for DLBCL.

Epigenetic Therapy as a Putative Molecular Target to Modulate B Cell Biology and Behavior in the Context of Immunological Disorders.

Histone Deacetylase- (HDAC-) dependent epigenetic mechanisms have been widely explored in the last decade in different types of malignancies in preclinical studies. This effort led to the discovery and development of a range of new HDAC inhibitors (iHDAC) with different chemical properties and selective abilities. In fact, hematological malignancies were the first ones to have new iHDACs approved for clinical use, such as Vorinostat and Romidepsin for cutaneous T cell lymphoma and panobinostat for multiple myeloma. Besides these promising already approved iHDACs, we highlight a range of studies focusing on the HDAC-dependent epigenetic control of B cell development, behavior, and/or function. Here, we highlight 21 iHDACs which have been studied in the literature in the context of B cell development and/or dysfunction mostly focused on B cell lymphomagenesis. Regardless, we have identified 55 clinical trials using 6 out of 21 iHDACs to approach their putative roles on B cell malignancies; none of them focuses on peritoneal B cell populations. Since cells belonging to this peculiar body compartment, named B1 cells, may contribute to the development of autoimmune pathologies, such as lupus, a better understanding of the HDAC-dependent epigenetic mechanisms that control its biology and behavior might shed light on iHDAC use to manage these immunological dysfunctions. In this sense, iHDACs might emerge as a promising new approach for translational studies in this field. In this review, we discuss a putative role of iHDACs in the modulation of peritoneal B cell subpopulation's balance as well as their role as therapeutic agents in the context of chronic diseases mediated by peritoneal B cells.

IBTK Haploinsufficiency Affects the Tumor Microenvironment of Myc-Driven Lymphoma in E-myc Mice

The tumor microenvironment is a dynamic and interactive supporting network of various components, including blood vessels, cytokines, chemokines, and immune cells, which sustain the tumor cell’s survival and growth. Murine models of lymphoma are useful to study tumor biology, the microenvironment, and mechanisms of response to therapy. Lymphomas are heterogeneous hematologic malignancies, and the complex microenvironment from which they arise and their multifaceted genetic basis represents a challenge for the generation and use of an appropriate murine model. So, it is important to choose the correct methodology. Recently, we supported the first evidence on the pro-oncogenic action of IBTK in Myc-driven B cell lymphomagenesis in mice, inhibiting apoptosis in the pre-cancerous stage. We used the transgenic Eμ-myc mouse model of non-Hodgkin’s lymphoma and Ibtk hemizygous mice to evaluate the tumor development of Myc-driven lymphoma. Here, we report that the allelic loss of Ibtk alters the immunophenotype of Myc-driven B cell lymphomas, increasing the rate of pre-B cells and affecting the tumor microenvironment in Eμ-myc mice. In particular, we observed enhanced tumor angiogenesis, increasing pro-angiogenic and lymphangiogenic factors, such as VEGF, MMP-9, CCL2, and VEGFD, and a significant recruitment of tumor-associated macrophages in lymphomas of Ibtk+/- Eμ-myc compared to Ibtk+/+ Eμ-myc mice. In summary, these results indicate that IBTK haploinsufficiency promotes Myc tumor development by modifying the tumor microenvironment.

High-Dimensional Heterogeneity of Waldenström Macroglobulinemia within Its Immune Tumor Microenvironment

Waldenström macroglobulinemia (WM), a malignant B-cell lymphoplasmacytic lymphoma, is a rare subtype of non-Hodgkin lymphoma representing about 1% of all cases. To better understand the WM pathogenesis, we performed large-scale data-driven proteomic profile of WM tumor cells associated with tumor-driven immune changes in the tumor microenvironment of 66 bone marrow (BM) samples from WM patients compared to 10 age-matched healthy donors (HD) by time-of-flight mass cytometry (CyTOF) technology. Our workflow has been designed based on extensive 3 CyTOF antibody panels to evaluate WM tumor within B cell lymphopoiesis concurrently with immune landscape of the tumor microenvironment in WM by state-of-art technology CyTOF. To map B cell lymphomagenesis in WM, we defined whole spectrum of maturation of B cell development, from hematopoietic stem cells and B cell precursors through immature B cells, transitional B cells, and naïve B cells together with memory un-switched and switched B cells, plasmablasts and plasma cells in BM samples of WM patients by positive and negative co-expression of 13 B cell-stage specific markers. Various immunophenotyping aberrancies within WM B lymphomagenesis were associated with WM clones characterized by significant increase of 11 B subset clusters from un-switched and switched memory B cells to plasma cells. Interestingly, WM clusters differ in intra-clonal expression of activation surface molecules (CD23, CD24, CD25, CD81, CD329, CD200, and CD319); transcriptional factors and regulators controlling B cell development (MYD88, Bcl-6, IRF-4, sXBP-1, and FGFR-3) and stemness-related markers (Oct3/4, Nanog, Sox-2, c-Myc, and Notch-1) in WM supporting the idea of sub-clonal heterogeneity insight of WM tumor. Moreover, decrease in cell frequency of B cell precursors (pro-B and pre-BI), naive B cells, and plasmablasts were observed in WM patients versus HD. To generate a comprehensive view of the tumor microenvironment, we observed significant upregulation of g/dT cells, CD4+ and CD8+ T effector cells, CD8+ T effector memory cells, monocytes, and neutrophils immune subsets and downregulation of immature T cells, CD8+ T naïve cells, plasmacytoid dendritic cells, myelo/mono progenitor clusters. Ibrutinib (IBRU) treatment has been effective in relapsed/refractory WM patients; therefore highest numbers of WM patients were receiving IBRU therapy in our cohort. IBRU treated WM patients had decreased frequency of naive B, CD4+ T naive cells and specific clusters of un-switched and switched memory B cells. Moreover, responder versus non-responders to IBRU therapy revealed increase of CD8+T effector memory cells. In sum, correspondence analysis reflecting data of each patient and immune subsets revealed stratification of WM patients with reflection on their clinical outcome, therefore providing the rational for prediction of WM patient status. This study was supported by APVV-16-0484 and VEGA 2/0076/17. Disclosures Hunter: Janssen: Consultancy. Richardson:Karyopharm: Membership on an entity's Board of Directors or advisory committees; Janssen: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees, Research Funding; Takeda: Membership on an entity's Board of Directors or advisory committees, Research Funding; Bristol-Myers Squibb: Research Funding; Amgen: Membership on an entity's Board of Directors or advisory committees; Oncopeptides: Membership on an entity's Board of Directors or advisory committees, Research Funding; Sanofi: Membership on an entity's Board of Directors or advisory committees. Kastritis:Amgen: Honoraria, Research Funding; Janssen: Honoraria, Research Funding; Takeda: Honoraria; Pfizer: Honoraria; Prothena: Honoraria; Genesis: Honoraria. Treon:BMS: Research Funding; Janssen: Consultancy; Pharmacyclics: Research Funding. Anderson:Celgene: Consultancy, Speakers Bureau; Takeda: Consultancy, Speakers Bureau; Sanofi-Aventis: Other: Advisory Board; Bristol-Myers Squibb: Other: Scientific Founder; Oncopep: Other: Scientific Founder; Amgen: Consultancy, Speakers Bureau; Janssen: Consultancy, Speakers Bureau.

Role of Ubiquitin E3 Ligase UBR5 in B-Cell Development and Lymphoma

Mantle Cell Lymphoma (MCL) is a non-Hodgkin's lymphoma (NHL) that typically affects older adults. In MCL, there is uncontrolled growth of B cells within the mantle zone of lymph node and spleen germinal centers. The vast majority of MCL cases have a translocation of chromosome 11 and 14 which juxtaposes the cyclin D1 gene, CCND1, with the immunoglobin heavy chain gene promoter (Eµ) and leads to the overexpression of CCND1. This translocation of CCND1 is not enough to cause MCL by itself and additional mutations are needed for MCL to develop. Although MCL only represents ~5% of NHL patients, it has the poorest survival rate among NHL sub-types due to a lack of successful therapeutic treatments. As such, it is important to identify potential targets for treatment. Recently published data shows that in 18% of all MCL patients have a mutation in the gene encoding the E3 ubiquitin ligase, UBR5. UBR5 is part of the ubiquitin proteasome system, a degradation/recycling pathway in which proteins are tagged with a ubiquitin protein and degraded by the proteasome. Of the identified UBR5 mutations, over 60% of the mutations are truncations at the carboxy terminus that cut off the cysteine residue linked to ubiquitin transfer in exon 59 suggesting a catalytic dead mutant protein is produced. Interestingly, UBR5HECT mutations are specific to MCL and are not found in other sub-types of NHL. By studying UBR5, we can determine the role of UBR5 mutations in MCL, elucidate molecular mechanism of UBR5 in B cell development, and identify potential therapeutic targets. In order to study the role of UBR5 in B cell development and MCL, we generated a conditional mouse model targeting exon 58 similar to mutations in MCL patients and crossed the mice with Mb1CRE/WT mice to delete exon 58 specifically in B cells at the pro-pre B cell stage of B cell development. Mb1CRE/WT; Ubr5fl/fl mice shows that mice lacking the carboxy terminus of UBR5 have a block in B cell differentiation at the mature B cell IgM+ IgD+ stage within the spleen. Mb1CRE/WT; Ubr5fl/fl mice showed a marked decrease of mature IgM+ IgD+ B cells in the BM and spleen. Specifically, within the spleen, Mb1CRE/WT; Ubr5fl/fl mice produce abnormal follicular B cells (higher IgM and CD23 expression and lower IgD and CD22 expression) and significant reductions in marginal zone B cells, plasma cells, size of germinal centers, and number of germinal centers. Mass spectrometry comparing mouse B220+ splenocytes in Mb1CRE/WT; Ubr5fl/fl compared to Mb1CRE/WT; Ubr5+/+ mice showed that UBR5 in B cells in Mb1CRE/WT; Ubr5fl/fl mice has over two-fold more expression. Additionally, IgD was the most downregulated protein, B cell specific proteins were downregulated, and proteins involved with mRNA splicing were upregulated within the mass spectrometry. The loss of the UBR5 HECT domain leads to increased UBR5 half-life and upregulation of spliceosome proteins. Coupled together, this suggests that the loss of the carboxy terminus of UBR5 impedes B cell maturation by disrupting IgD expression, potentially by interfering with mRNA splicing. Finally, we have crossed and are currently aging our Mb1CRE/WT; Ubr5fl/fl mice with an EµCCND1 mouse model to determine if Ubr5 mutations lead to tumorigenesis. These studies aim to identify the role of UBR5 in the context of normal B cell development and lymphomagenesis with the goal of identifying therapeutic targets for drug discovery. Disclosures No relevant conflicts of interest to declare.

B Cell Restricted Expression of Mutated IKZF3 modulates BCR Signaling and Homing Pathways in a Mouse Model of CLL

Mutation in IKZF3 (AIOLOS), a lymphoid transcription factor with a key role in B cell development and function, has been identified as a putative driver of chronic lymphocytic leukemia (CLL) through large-scale WES studies of CLL patients. Prevalent in ~3% CLLs and associated with fludarabine resistance, mutated IKZF3 has been detected uniquely as a hotspot mutation (L162R), localized within the DNA binding domain. The functional effects exerted by this mutation on lymphomagenesis remain unexplored, and were the subject of the current study. We generated a B-cell restricted knock-in mouse line by crossing mice with the Ikzf3 mutant floxed allele with Cd19-Cre animals (i.e. Cre recombinase expression under the Cd19 promoter), and established cohorts carrying either heterozygous mutant-Ikzf3 (Ikzf3Het), homozygous mutant-Ikzf3 (Ikzf3Homo) or wild-type Ikzf3 (Ikzf3WT). We thereafter monitored the animals for leukemia development by serial flow cytometry analyses of peripheral blood. Notably, we detected appearance of ~10-50% clonal B220+CD5+Igk+in 8 of 30 (27%) of the Ikzf3Het mice by 20 months of age, but not in age-matched Ikzf3WTmice (n=30). The cohort of Ikzf3Homo mice has been monitored thus far for 18 months, with identification already of one of 30 (3%) with leukemia with a ~15 months onset. Across animals with CLL-like disease, the pattern of disease localization was consistently observed as detected in the spleen but not in the bone marrow, suggesting preferential homing of Ikzf3-mutant cells to the splenic microenvironment. To dissect the mechanisms underlying Ikzf3-mediated leukemogenesis, we asked whether presence of mutated-Ikzf3 was associated with changes in B cell transcriptional programs in young mice without disease. We performed RNA-sequencing using splenic B cells from Ikzf3Het, Ikzf3Homo or Ikzf3wt animals (3 mice/group, 3-month old). The most striking changes were identified between Ikzf3Homo to Ikzf3wt mice, with ~1400 differentially expressed genes in Ikzf3Homocells compared to Ikzf3wt (>2 fold, adjusted p<0.05). Within this Ikzf3Homo-specific signature, we identified genes regulating B cell positioning within the light zone of germinal centers (GCs, p<0.0001) and BCR signaling pathway members (p<0.05), by gene set enrichment analysis (GSEA). The enhanced activation of the BCR pathway was further confirmed by western blot analysis of splenic B cells stimulated with anti-IgM, with Ikzf3Homo and Ikzf3het mice showing upregulation of phosphorylation in SYK, AKT and ERK after BCR engagement, as compared to Ikzf3WT. Allo-immunization with the T-cell dependent antigen sheep red blood cells (SRBC) for 10 days elicited a higher rate of germinal centers (GC) formation in Ikzf3Homo mice than Ikzf3WT (p<0.01, ANOVA with Tukey's correction), further indicating an enhanced response to BCR stimulation in presence of the mutation. Overall, phenotypic changes of Ikzf3het appeared less pronounced than Ikzf3Homo, in young mice. To further define the functional effects of mutant-Ikzf3 in transcriptional regulation, we performed chromatin immunoprecipitation sequencing (ChIP-seq) using anti-AIOLOS antibody in Ikzf3WT and Ikzf3Homo B cells. While we observed no significant changes in the conserved DNA binding motif or the genomic localization of AIOLOS binding sites, stronger AIOLOS binding at the transcription start sites (TSS) was detected in Ikzf3Homo versus Ikzf3WTcells (p=1.3747x10-5, Student's t-test). These results indicate an enhanced binding of mutated AIOLOS to DNA which may in turn dysregulate gene expression. As an example, upregulation of the chemokine receptor Cxcr4 in Ikzf3Homo B cells was associated with an enrichment of mutant AIOLOS at its promoter region. Functionally, we confirmed that such upregulation was associated with increased migration of Ikzf3Homo cells to SDF-1 in vitro, in a transwell-based chemotaxis assay (p<0.01, ANOVA with Tukey's correction). Overall, this novel model provides evidence of an oncogenic role of IKZF3 mutation in CLL. Ikzf3 mutation induces broad transcriptional and functional changes associated with dysregulation of BCR signaling and homing mechanisms, which may favor disease initiation and progression in vivo. Comparative analyses between Ikzf3-mutant murine CLLs and IKZF3-mutant primary samples are ongoing, to further refine the relevance of Ikzf3 in B cell function and lymphomagenesis. Disclosures Neuberg: Pharmacyclics: Research Funding; Madrigal Pharmaceuticals: Equity Ownership; Celgene: Research Funding. Wu:Neon Therapeutics: Other: Member, Advisory Board; Pharmacyclics: Research Funding.

STAT3 Dysregulation in Mature T and NK Cell Lymphomas.

T cell lymphomas comprise a distinct class of non-Hodgkin’s lymphomas, which include mature T and natural killer (NK) cell neoplasms. While each malignancy within this group is characterized by unique clinicopathologic features, dysregulation in the Janus tyrosine family of kinases/Signal transducer and activator of transcription (JAK/STAT) signaling pathway, specifically aberrant STAT3 activation, is a common feature among these lymphomas. The mechanisms driving dysregulation vary among T cell lymphoma subtypes and include activating mutations in upstream kinases or STAT3 itself, formation of oncogenic kinases which drive STAT3 activation, loss of negative regulators of STAT3, and the induction of a pro-tumorigenic inflammatory microenvironment. Constitutive STAT3 activation has been associated with the expression of targets able to increase pro-survival signals and provide malignant fitness. Patients with dysregulated STAT3 signaling tend to have inferior clinical outcomes, which underscores the importance of STAT3 signaling in malignant progression. Targeting of STAT3 has shown promising results in pre-clinical studies in T cell lymphoma lines, ex-vivo primary malignant patient cells, and in mouse models of disease. However, targeting this pleotropic pathway in patients has proven difficult. Here we review the recent contributions to our understanding of the role of STAT3 in T cell lymphomagenesis, mechanisms driving STAT3 activation in T cell lymphomas, and current efforts at targeting STAT3 signaling in T cell malignancies.

Germinal Centre B Cell Functions and Lymphomagenesis: Circuits Involving MYC and MicroRNAs.

Background: The transcription factor MYC regulates several biological cellular processes, and its target gene network comprises approximately 15% of all human genes, including microRNAs (miRNAs), that also contribute to MYC regulatory activity. Although miRNAs are emerging as key regulators of immune functions, the specific roles of miRNAs in the regulation/dysregulation of germinal centre B-cells and B-cell lymphomas are still being uncovered. The regulatory network that integrates MYC, target genes and miRNAs is a field of intense study, highlighting potential pathways to be explored in the context of future clinical approaches. Methods: The scientific literature that is indexed in PUBMED was consulted for publications involving MYC and miRNAs with validated bioinformatics analyses or experimental protocols. Additionally, seminal studies on germinal centre B-cell functions and lymphomagenesis were reported. Conclusions: This review summarizes the interactions between MYC and miRNAs through regulatory loops and circuits involving target genes in germinal centre B-cell lymphomas with MYC alterations. Moreover, we provide an overview of the understanding of the regulatory networks between MYC and miRNAs, highlighting the potential implication of this approach for the comprehension of germinal centre B-cell lymphoma pathogenesis. Therefore, circuits involving MYC, target genes and miRNAs provide novel insight into lymphomagenesis that could be useful for new improved therapeutic strategies.

Abstract SY35-02: Bone marrow remodeling in response to distant tumor starts early in transformation

Abstract Bone marrow (BM) stromal cell are sensors of immunological stress signals from peripheral tissue. We showed that chronicity of such signals induces persistent changes in the stromal architecture providing favorable conditions for the establishment and progression of a myeloproliferative spur (Blood 2012, 120: 3541). A key regulator of bone marrow stromal changes is the secreted protein acidic and rich in cysteine (SPARC) a matricellular protein with both structural and regulatory functions on extracellular matrix (ECM) organization. Up- and downregulation of SPARC has been indeed associated with different hematologic neoplasms including myeloid and lymphoid malignancies (Cancer Res. 2017, 77: 3685; Cancer Discovery 2014, 4: 110). We identified SPARC as a common stromal trait of the BM osteoblastic niche and SLO germinal center (GC) microenvironments, where it regulates physiological B lymphopoiesis and B cell development, as well as B cell lymphomagenesis (Oncoimmunology 2014). Like infections and autoimmunity, also the presence of solid tumors involves the establishment of a chronic state of inflammation that can act locally but also be sensed systemically, particularly by the BM. The last responds with an expansion of accessory cells further fostering tumor growth and dissemination.Others and we have indeed demonstrated in tumor mouse models the expansion in the BM of the myeloid compartment that parallels tumor progression. In this context we contributed to identify some of the key molecules involved in the BM-tumor cross talk, such as VEGF, MMP-9 (Cancer Res. 2007, 67:11438). Tumor-expanded myeloid cells are generally endowed with immune suppressive features, which actively contribute to cancer-associated systemic immune suppression.We have recently described two additional mechanisms that contribute to the shaping of the myeloid cell phenotype and suppressive activity in mammary tumors and involve SPARC (Cell Report 2016, 17: 233) and another matricellular protein, i.e. osteopontin (Cancer Res. 2014, 74: 4706). New data indicate that tumor-induced myeloid cell expansion in the BM is associated with profound changes not only in the hematopoietic compartment, but also in the stromal niche organization. In a spontaneous model of mammary carcinogenesis we show significant alterations in the density and localization of Nestin+ mesenchymal stromal elements and in the expression of the prototypical stroma-derived chemotactic factor CXCL12, which were both increased in the hematopoietic interstitium of mammary tumor bearing mice. How early during transformation these hematopoietic and stromal changes are detectable, and the identification of soluble factors involved in these BM modification are the focus of our present research and will be presented in this symposium.Understanding the molecular determinants of cancer-driven hematopoietic adaptation would offer a new prospect on cancer early detection and prognostication, eventually prompting novel strategies to interfere with such detrimental changes besides the specific targeting of functional hematopoietic subsets. Citation Format: Mario P. Colombo. Bone marrow remodeling in response to distant tumor starts early in transformation [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 SY35-02.

The HVEM-BTLA Axis Restrains T Cell Help to Germinal Center B Cells and Functions as a Cell-Extrinsic Suppressor in Lymphomagenesis

SummaryThe tumor necrosis factor receptor superfamily member HVEM is one of the most frequently mutated surface proteins in germinal center (GC)-derived B cell lymphomas. We found that HVEM deficiency increased B cell competitiveness during pre-GC and GC responses. The immunoglobulin (Ig) superfamily protein BTLA regulated HVEM-expressing B cell responses independently of B-cell-intrinsic signaling via HVEM or BTLA. BTLA signaling into T cells through the phosphatase SHP1 reduced T cell receptor (TCR) signaling and preformed CD40 ligand mobilization to the immunological synapse, thus diminishing the help delivered to B cells. Moreover, T cell deficiency in BTLA cooperated with B cell Bcl-2 overexpression, leading to GC B cell outgrowth. These results establish that HVEM restrains the T helper signals delivered to B cells to influence GC selection outcomes, and they suggest that BTLA functions as a cell-extrinsic suppressor of GC B cell lymphomagenesis.

Non-oncogene Addiction to SIRT3 Plays a Critical Role in Lymphomagenesis

Diffuse large B cell lymphomas (DLBCLs) are genetically heterogeneous and highly proliferative neoplasms derived from germinal center (GC) B cells. Here, we show that DLBCLs are dependent on mitochondrial lysine deacetylase SIRT3 for proliferation, survival, self-renewal, and tumor growth invivo regardless of disease subtype and genetics. SIRT3 knockout attenuated B cell lymphomagenesis in VavP-Bcl2 mice without affecting normal GC formation. Mechanistically, SIRT3 depletion impaired glutamine flux to the TCA cycle via glutamate dehydrogenase and reduction in acetyl-CoA pools, which in turn induce autophagy and cell death. We developed a mitochondrial-targeted class I sirtuin inhibitor, YC8-02, which phenocopied the effects of SIRT3 depletion and killed DLBCL cells. SIRT3 is thus a metabolic non-oncogene addiction and therapeutic target for DLBCLs.

165 Contribution of TOX1 and STAT3 pathways to the development of cutaneous T-cell lymphoma (CTCL)

Thymocyte selection associated high mobility group box 1 (Tox1), a transcription factor that is essential to establish the CD4+ lineage, is overexpressed in malignant cells from patients with CTCL. Knockdown of Tox1 leads to decreased malignant cell viability, while treatment with HDAC inhibitors results in normalization of Tox1expression. Another gene which is consistently overexpressed in patient samples is signal transducers and activators of transcription3 (Stat3), a transcription factor essential for the differentiation of Th17 and follicular helper T cells. Treating CTCL cell lines with a STAT3 inhibitor results in decreased cell number and increased apoptosis, highlighting the importance of this pathway for malignant cell survival. Importantly, the Koralov lab has developed a mouse model which constitutively expresses a hyperactive STAT3 allele, STAT3C, that recapitulates several key features of MF. To examine the contribution of TOX1overexpression to CTCL pathogenesis, we have introduced Tox1 cDNA downstream of a floxed stop cassette into the ubiquitously expressed Rosa26 locus of C57Bl/6J embryonic stem (ES) cells. We have screened the ES clones to validate the presence of the correctly targeted allele, and we have treated ES clones with TAT-Cre to delete the floxed stop cassette and drive subsequent expression of Tox1 cDNA. We have now generated R26Tox1stopflmice using tetraploid complementation to generate 100% ES cell derived animals. These animals will be crossed to CD4Cre and CD4Cre STAT3Cstopflstrains, thus enabling us to study the contribution of Tox1 overexpression to T cell lymphomagenesis and allowing us to examine synergy between Tox1 overexpression and hyperactive STAT3 signaling in CTCL pathogenesis. We hope that these mice will pave the way to a better understanding of this enigmatic malignancy and allow us to develop a relevant small animal model of this disease.

Microbial Agents as Putative Inducers of B Cell Lymphoma in Sjögren's Syndrome through an Impaired Epigenetic Control: The State-of-The-Art.

Introduction Understanding the mechanisms underlying the pathogenesis of Sjögren's syndrome (SS) is crucially important in order to be able to discriminate the steps that lead to B cell transformation and promptly identify the patients at risk of lymphomagenesis. The aim of this narrative review is to describe the evidence concerning the role that infections or dysbiosis plays in the epigenetic control of gene expression in SS patients and their possible involvement in B cell lymphomagenesis. Materials and Methods We searched the PubMed and Google Scholar databases and selected a total of 92 articles published during the last 25 years that describe experimental and clinical studies of the potential associations of microbiota and epigenetic aberrations with the risk of B cell lymphoma in SS patients. Results and Discussion The genetic background of SS patients is characterized by the hyperexpression of genes that are mainly involved in regulating the innate and adaptive immune responses and oncogenesis. In addition, salivary gland epithelial cells and lymphocytes both have an altered epigenetic background that enhances the activation of proinflammatory and survival pathways. Dysbiosis or chronic latent infections may tune the immune response and modify the cell epigenetic machinery in such a way as to give B lymphocytes an activated or transformed phenotype. It is also worth noting that transposable integrated retroelements may participate in the pathogenesis of SS and B cell lymphomagenesis by inducing DNA breaks, modulating cell gene expression, or generating aberrant transcripts that chronically stimulate the immune system. Conclusions Microorganisms may epigenetically modify target cells and induce their transcriptome to generate an activated or transformed phenotype. The occurrence of lymphoma in more than 15% of SS patients may be the end result of a combination of genetics, epigenetics, and dysbiosis or latent infections.

Reciprocal negative regulation between the tumor suppressor protein p53 and B cell CLL/lymphoma 6 (BCL6) via control of caspase-1 expression

Even in the face of physiological DNA damage or expression of the tumor suppressor protein p53, B cell CLL/lymphoma 6 (BCL6) increases proliferation and antagonizes apoptotic responses in B cells. BCL6 represses TP53 transcription and also appears to inactivate p53 at the protein level, and additional findings have suggested negative mutual regulation between BCL6 and p53. Here, using Bcl6-/- knockout mice, HEK293A and HCT116 p53-/- cells, and site-directed mutagenesis, we found that BCL6 interacts with p53 and thereby inhibits acetylation of Lys-132 in p53 by E1A-binding protein p300 (p300), a modification that normally occurs upon DNA damage-induced cellular stress and whose abrogation by BCL6 diminished transcriptional activation of p53 target genes, including that encoding caspase-1. Conversely, we also found that BCL6 protein is degraded via p53-induced, caspase-mediated proteolytic cleavage, and the formation of a BCL6-p53-caspase-1 complex. Our results suggest that p53 may block oncogenic transformation by decreasing BCL6 stability via caspase-1 up-regulation, whereas aberrant BCL6 expression inactivates transactivation of p53 target genes, either by inhibiting p53 acetylation by p300 or repressing TP53 gene transcription. These findings have implications for B cell development and lymphomagenesis.

The non-canonical NF-kappaB Signaling Pathway Contributes to the Expansion and Lymphomagenesis of CD40-activated B Cells

The non-canonical NF-κB pathway is highly conserved, can be activated by TNF-receptors e.g. CD40 and is dysregulated in several lymphomas (Krappmann and Vincendeau 2016). In human DLBCL hyper-activation of the non-canonical NF-kB signaling pathway could be detected in about 15% (Zhang et al. 2015). Upon activation of the non-canonical NF-κB pathway RelB is translocated to the nucleus and acts as transcription factor. RelB is important to maintain viability of a Hodgkin Lymphoma cell line (Ranuncolo et al. 2012), but interestingly B cell specific inactivation of RelB in mature B cells of transgenic mice have only a minor effect on B cell maintenance and activation (De Silva et al. 2016). Therefore it is still elusive, whether RelB-regulated genes contribute to lymphoma development.To study the contribution of RelB-regulated genes to lymphoma development we used a mouse model expressing a constitutively active CD40 receptor in B cells (LMP1/CD40stopflmice). LMP1/CD40 is a fusion protein comprising the signaling domain of human CD40 and the transmembrane domain of LMP1 and promotes a persistently activation of CD40 signaling. In LMP1/CD40stopfl//CD19-Cre (LMP1/CD40) mice the constitutive CD40 activation leads to the selective activation of the non-canonical NF-kB pathway and the MAP kinase pathways JNK and ERK. LMP1/CD40-expression positively affects cell survival and proliferation of B cells resulting in a B cell expansion and accumulation of B cells in follicles of young mice. Mice older than 12 months develop lymphomas with a high incidence. In most cases the lymphoma was monoclonal and had the phenotype B220low, CD21- and CD23- (Hojer et al. 2014; Hömig-Hölzel et al. 2008). Recently, we observed that this aberrant population appears at around 6 months of age and increases continuously during the aging process.To unveil the contribution of the non-canonical NF-κB pathway to the phenotype and lymphoma development of constitutively CD40-activated B cells we crossed conditional RelBfl/fl mice to LMP1/CD40stopfl//CD19-Cre mice (RelBKO//LMP1/CD40 mice). In young mice we were able to observe that LMP1/CD40 B cells without functional RelB were less expanded in the spleen and lymph nodes, but had elevated B cell numbers in the blood and a higher number of recirculating B cells in the bone marrow. This indicates that RelB-regulated genes are important for the retention of CD40-activated B cells in B cell follicles of secondary lymphoid organs. In addition, the survival of ex vivo isolated LMP1/CD40-expressing RelB-deficient B cells was diminished in comparison to LMP1/CD40-expressing RelB-proficent B cells, but RelBKO//LMP1/CD40 B cells still survived better than control B cells. Furthermore, we found that RelBKO//LMP1/CD40 B cells were less activated than LMP1/CD40 B cells, but had a higher expression of the tested activation markers than control B cells.In order to characterize the influence of RelB-regulated genes on lymphoma development in LMP1/CD40 mice we analyzed LMP1/CD40-expressing mice at different time points. The aberrant population, arising in LMP1/CD40 mice, was also detectable in aged RelBKO//LMP1/CD40 mice, though showed a delayed progression and always had a lower expansion than in age-matched LMP1/CD40 mice. Both transgenic mice developed monoclonal lymphomas. However, the double mutant mice had a lower tumor incidence than LMP1/CD40 mice with functional RelB. Nevertheless, the risk of lymphoma development in RelBKO//LMP1/CD40 was still higher than in wildtype mice.In the last part of this study we performed a RNA-Seq. analysis to see which genes are RelB-dependent in chronically CD40-activated B cells. We identified several RelB-dependent genes and 21 of these had been correlated with Non-Hodgkin Lymphoma. Some of these candidates could be promising new therapy targets and are under further investigation.In conclusion we provide evidence that RelB-regulated genes contribute to the survival and activation of B cells, which are chronically activated by CD40 signaling. Furthermore, we found that CD40 signaling enhances the retention of B cells in B cell follicles through activation of the non-canonical NF-kB pathway. Moreover, we demonstrated that RelB-regulated genes contribute to B cell lymphomagenesis and identified several RelB-regulated genes that may contribute to the expansion and lymphomagenesis of B cells receiving a chronic CD40 signal. DisclosuresWeigert:Roche: Research Funding; Novartis: Research Funding.

MYC Functions As a Master Switch for Natural Killer Cell-Mediated Immune Surveillance of Lymphoid Malignancies

Background: The MYC oncogene drives T and B cell lymphoid malignancies, including Burkitt's lymphoma (BL) and Acute Lymphoblastic Leukemia (ALL). Such lymphomas are said to be “oncogene-addicted” to MYC. In order to develop targeted therapies for MYC-driven cancers, it is vital to understand how MYC regulates both cell autonomous and non-cell-autonomous processes, including host immunity.Approach: We have used a particularly tractable approach for studying the role of oncogenic MYC on the host immune system during lymphomagenesis through a tetracycline (tet)-system regulated transgenic mouse model of MYC-driven T cell lymphoma (SRα-tTA/Tet-O-MYC mice; Felsher and Bishop, Molecular Cell, 1999). By delineating the global immunological changes during primary MYC-driven T cell lymphomagenesis in SRα-tTA/Tet-O-MYC mice using mass cytometry (CyTOF) and CIBERSORT, we identified anti-tumor immune subsets that can be developed as therapies to treat MYC-driven lymphomas.Results: Amongst the immune subsets evaluated, our results demonstrated a specific systemic suppression of natural killer (NK) cell-mediated surveillance in SRα-tTA/Tet-O-MYC mice bearing overt MYC-driven T cell lymphomas. Inactivation of lymphoma-intrinsic MYC restores NK cell-mediated immune surveillance, suggesting that the regulation of NK cell-mediated surveillance may be integral to MYC-induced lymphomagenesis. We observed that lymphoma-specific MYC transcriptionally represses STAT1/2 and secretion of Type I Interferons (IFNs) required for normal NK cell homeostasis and NK cell-mediated immune surveillance in the tumor microenvironment. Concordantly, treating T cell lymphoma-bearing SRα-tTA/Tet-O-MYC mice with Type I IFN improves survival by rescuing NK cell production, and in part overriding MYC-mediated suppression of NK surveillance. We showed that human lymphomas with both high levels of MYC and low levels of STAT1/2 have lower NK surveillance, and are associated with poor prognosis. Finally, we established the therapeutic implications of our findings by showing that adoptive transfer of NK cells significantly delays lymphoma initiation, and recurrence after MYC inactivation; suggesting that NK cell-based therapy may be effective against MYC-driven lymphomas.Conclusion: Subversion of NK cell surveillance is integral to MYC-induced lymphomagenesis. Our studies provide a rationale for further developing the NK subset as a cell-based immunotherapy to effectively treat MYC-driven lymphomas in the future. DisclosuresNo relevant conflicts of interest to declare.

Selective HDAC3 Inhibition Induces Apoptosis in B Cell Lymphoma through Protein Acetylation

Acetylation is a reversible process under the control of histone acetyltransferases (HATs) and histone deacetylases (HDACs). Acetyl proteome analysis revealed that acetylation regulates various cellular processes through both histone and non-histone proteins (Choudhary et al., Science, 2008). Subsets of diffuse large B cell lymphoma (DLBCL) and follicular lymphoma (FL) have inactivating mutations of HATs, CBP and p300 (Pasqualucci et al., Nature, 2011). In addition, p300 mutation is a poor prognostic factor in FL patients (Pastore et al., Lancet Oncol, 2015). In a mouse model, CBP deficiency promotes B cell lymphomagenesis through H3K27 deacetylation of enhancer mediated by the HDAC3, which forms repressor complex with Bcl-6, SMRT and NCoR (Jiang et al., Cancer Discov, 2016).Human HDACs consist of 18 isoenzymes that are classified into 4 classes (I-IV). Among the 4 classes, class I includes 4 HDACs (HDAC1, 2, 3 and 8) that are ubiquitous. Four HDAC inhibitors are FDA approved for the treatment of cutaneous T cell lymphoma (romidepsin, vorinostat), peripheral T cell lymphoma (belinostat) and multiple myeloma (panobinostat). These inhibitors mainly target class I and class II HDACs, each with a different specificity. This non-specific nature of current HDAC inhibitors limits their efficacy and causes adverse effects, therefore, several selective HDAC inhibitors has been developed. Selective HDAC3 inhibition restricts myeloma cell growth in vitro and in vivo more efficiently than selective HDAC1 or HDAC2 inhibition, allowing for DNMT1 acetylation, accelerating its degradation by the proteasome (Harada et al., Leukemia, 2017). However, the efficacy of selective HDAC3 inhibition against B cell lymphoma remains unclear.We first knocked down HDAC3 by shRNA in a B-cell lymphoma cell line (KIS1). We found that selective HDAC3 knock down suppressed cell growth and induced apoptosis. We next tested selective HDAC3 inhibitors (RGFP966 and AA-1) using five B cell lymphoma cell lines (Raji, Ramos, KIS1, SUDHL-6 and Granta519). We confirmed that these inhibitors reduced cell viability in all treated cell lines, significantly increased the number of apoptotic cells in Ramos, KIS1 and SUDHL-6 and induced cell cycle arrest in Raji and Granta519, rather than apoptosis. Lentiviral shRNA against HDAC3 or the inhibitors also led to the cleavage and subsequent activation of caspase9 and caspase3. These results show that HDAC3 inhibition induces apoptosis by activating the intrinsic apoptotic pathway.To clarify how HDAC3 inhibitors affect global protein acetylation and how they induce apoptosis in B cell lymphoma, we conducted acetyl proteome analysis using LC-MS/MS. Briefly, we immunoprecipitated acetyl lysine peptides from whole cell lysates of SUDHL-6 with or without RGFP966 treatment, and analyzed them by mass spectrometry. We identified 673 and 1,328 acetylation sites in RGFP966 treated and untreated samples, respectively, and 1,425 acetylation sites in total. Among these 1,425 sites, 153, including histones and HATs, were more than two fold upregulated in the RGFP966 treated sample. To identify which cellular processes are affected by RGFP966 treatment, we performed Gene Ontology (GO) enrichment analysis of the proteins with upregulated acetylation. GO enrichment analysis revealed that bromodomain, glycolysis and unfolded protein binding were significantly enriched terms upon RGFP966 treatment.Our data show that selective HDAC3 inhibition is also effective against B cell lymphoma and that protein folding and metabolic processes as well as epigenetic mechanisms might be involved in activation of the intrinsic apoptosis pathway upon HDAC3 inhibition. We discuss how acetylation of these proteins leads to cell growth inhibition and apoptosis in B cell lymphoma. DisclosuresTakaori-Kondo:Bristol-Myers Squibb: Honoraria; Pfizer: Honoraria; Celgene: Honoraria, Research Funding; Novartis: Honoraria; Janssen Pharmaceuticals: Honoraria.

MEF2B Instructs Germinal Center Development and Acts as an Oncogene in B Cell Lymphomagenesis

The gene encoding the MEF2B transcription factor is mutated in germinal center (GC)-derived B cell lymphomas, but its role in GC development and lymphomagenesis is unknown. We demonstrate that Mef2b deletion reduces GC formation in mice and identify MEF2B transcriptional targets in GC, with roles in cell proliferation, apoptosis, GC confinement, and differentiation. The most common lymphoma-associated MEF2B mutant (MEF2BD83V) ishypomorphic, yet escapes binding and negative regulation by components of the HUCA complex and classIIa HDACs. Mef2bD83V expression in mice leads to GC enlargement and lymphoma development, a phenotype that becomes fully penetrant in combination with BCL2 de-regulation, an event associated with human MEF2B mutations. These results identify MEF2B as a critical GC regulator and a driver oncogene in lymphomagenesis.

Co-Expression of the Epstein-Barr Virus-Encoded Latent Membrane Proteins and the Pathogenesis of Classic Hodgkin Lymphoma.

The Epstein-Barr virus (EBV) is present in the tumour cells of a subset of patients with classic Hodgkin lymphoma (cHL), yet the contribution of the virus to the pathogenesis of these tumours remains only poorly understood. The EBV genome in virus-associated cHL expresses a limited subset of genes, restricted to the non-coding Epstein-Barr virus-encoded RNAs (EBERs) and viral miRNA, as well as only three virus proteins; the Epstein-Barr virus nuclear antigen-1 (EBNA1), and the two latent membrane proteins, known as LMP1 and LMP2, the latter of which has two isoforms, LMP2A and LMP2B. LMP1 and LMP2A are of particular interest because they are co-expressed in tumour cells and can activate cellular signalling pathways, driving aberrant cellular transcription in infected B cells to promote lymphomagenesis. This article seeks to bring together the results of recent studies of the latent membrane proteins in different B cell systems, including experiments in animal models as well as a re-analysis of our own transcriptional data. In doing so, we summarise the potentially co-operative and antagonistic effects of the LMPs that are relevant to B cell lymphomagenesis.