scholarly journals Conditional Knockout of Sfpi1 in Post GC B and Plasma Cells Induces B Cell Lymphoma and Plasma Cell Neoplasm

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 29-29
Author(s):  
Shinya Endo ◽  
Hiromichi Yuki ◽  
Yoshihiro Komohara ◽  
Shikiko Ueno ◽  
Nao Nishimura ◽  
...  
Keyword(s):  
B Cell ◽  
Cell Lines ◽  
Germinal Center ◽  
Plasma Cells ◽  
Cell Lymphoma ◽  
B Cell Lymphoma ◽  
Myeloma Cell ◽  
Conditional Knockout ◽  
Down Regulation ◽  
Maturation Stages

Abstract PU.1 is an Ets family transcription factor, which is essential for differentiation of both myeloid and lymphoid lineages. It was previously reported that conditional knockout of the upstream enhancer region (URE) located in 14 kb 5’ of the murine PU.1 gene resulted in down-regulation of PU.1 expression in granulocytes and B lymphocytes by 80% compared to that of wild type and induced acute myeloid leukemia and CLL-like diseases in mice. Therefore, down-regulation of PU.1 in myeloid and B cell lineages results in hematological malignancies. We previously reported that PU.1 is down-regulated in 5 out of 7 myeloma cell lines as well as primary myeloma cells from a subset of myeloma patients; that the promoter and the URE located in 17 kb 5’ of the human PU.1 gene that is homologous to that in 14 kb 5’ of murine PU.1 gene are highly methylated in these cell lines; and that conditionally expressed PU.1 with tet-off system induces cell growth arrest and apoptosis in myeloma cell lines, U266 and KMS12PE, suggesting that the down-regulation of PU.1 is necessary for myeloma cell growth. Here, to evaluate tumor suppressor activity of PU.1 in mature B and plasma cells in vivo, we generated Cγ1-Cre PU.1 knockout mice by crossing Cγ1-Cre and PU.1-loxP mice. We confirmed that PU.1 alleles were both conditionally deleted in the maturation stages of B cells from post germinal center B to plasma cells. By 18-24 months of age, about 77.7% (10 of 13) of the knockout mice had developed serum M proteins. To induce B cell differentiation to plasma cells, those mice were immunized with NP-CGG and 76.9% (20 of 26) of the mice developed serum M protein. ELISA of sera from those mice revealed that IgG was not elevated compared to those from the PU.1-loxP mice, which was thought because Cγ1-Cre locus fails to produce IgG1. Instead, a small number (5 of 20) of the mice showed relatively large amounts of IgM and/or IgA. When 11 such mice were sacrificed, 7 had developed splenomegaly and/or intestinal B cell lymphoma. Immunostaining revealed that B220+ cells had infiltrated into the tumors and various organs including the spleen, liver, and bone marrow. Those cells were monoclonal for κ chain and partly CD138 positive. When we transplanted those tumor cells into Rag2-/- Jak3-/- immunedeficient mice, all the mice died within 3 weeks. Thus, PU.1 apparently functions as a tumor suppressor in mature B cells and its deletion in late B cell maturation stages produces B cell lymphoma with M proteinemia. The remaining 4 mice developed high titer IgM and/or IgA levels and flow cytometry of bone marrow cells and splenocytes revealed that those cells were monoclonal for κ chain and positive for B220 and IgM and/or IgA, suggesting that those mice suffered from multiple myeloma or monoclonal gammopathy with undetermined sighnificance (MGUS). These data strongly suggest that conditional knockout of PU.1 in post germinal center B and plasma cells results in B cell lymphoma and plasma cell neoplasms related to multiple myeloma. Disclosures No relevant conflicts of interest to declare.

IL-4 Affects Proliferation, Chemosensitivity-and Rituximab Sensitivity of Germinal Center B-Cell like (GCB) and Activated B-Cell like (ABC) Diffuse Large B-Cell Lymphoma Differently.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 242-242 ◽  
Author(s):  
Hovav Nechushtan ◽  
Joseph D. Rosenblatt ◽  
Izidore S. Lossos
Keyword(s):  
Cell Death ◽  
B Cell ◽  
Cell Lines ◽  
Germinal Center ◽  
Cell Lymphoma ◽  
B Cell Lymphoma ◽  
Cell Lysis ◽  
Large B Cell Lymphoma ◽  
Expression Of Genes ◽  
Large B Cell

Abstract Diffuse Large B-cell Lymphoma (DLBCL) represent a diverse group of lymphoid neoplasms with heterogeneous clinical, histological, immunophenotypic, cytogenetic and molecular genetic features. Approximately 50% of DLBCL patients are not cured by the standard combination chemotherapy regimens. DLBCL can be subclassified into GCB-like DLBCL which are characterized by expression of genes normally expressed in germinal center B cells, and having a significantly better overall survival (OS) than the ABC-like DLBCL, which are characterized by expression of genes induced during in vitro activation of normal B cells. At least two markers of the GCB-phenotype - BCL6 and HGAL - are IL-4 target genes, increased expression of which independently predicts better OS. These observations suggest that endogenous or exogenously administered IL-4 may influence behavior of DLBCL. IL-4 mRNA was detected at low levels in 5 of 7 GCB-like and in all 4 ABC-like DLBCL tumor specimens. Two of 7 GCB-like tumors showed high expression levels of IL-4 as determined by real-time RT-PCR. Examination of the effects of IL-4 on proliferation of GCB-like (SUDHL6, SUDHL4 and OCILY19) and ABC-like (OCILY10 and OCILY3) DLBCL cell lines showed that IL-4 mildly increased DNA synthesis, as assessed by thymidine incorporation, in all the GCB-like DLBCL. Conversely, IL-4 markedly decreased proliferation in the ABC-like DLBCL cell lines by inducing G1 arrest. IL-4 also differently affected the sensitivity of GCB-like and ABC-like DLBCL to doxorubicin. IL-4 reduced doxorubicin-induced cell death of ABC-like cell lines (20–50% reduction) while it markedly increased the killing of the GCB-like cells (40–80% induction). IL-4 also prevented serum starvation-induced cell death of the ABC-like DLBCL, but it increased cell death of the GCB-like DLBCL cell lines. Recently, Rituximab was shown to improve survival of DLBCL patients when added to the CHOP regimen. The precise mechanisms of its action are unknown; however present data suggest that it may affect lymphoma cells either by activation of complement lysis or by mediating ADCC. IL-4 reduced the complement mediated Rituximab cell lysis of the ABC-like cell lines, while it increased the complement mediated Rituximab cell lysis of the GCB-like DLBCL cell lines. Expression levels of surface markers that modulate complement cell lysis (CD46, CD55 and CD59) were not affected by IL-4 exposure. In contrast, IL-4 did not affect killing of GCB-like and ABC-like cells by ADCC. These observations suggest that DLBCL subtypes may respond differently to the in vivo cytokine milieu of the tumor. Different responsiveness to IL-4 may modulate tumor sensitivity to the current therapeutic modalities and can potentially be explored to augment response to chemotherapy and Rituximab.

Acadesine Inhibits Proliferation and Induces Apoptosis In Multiple Myeloma Cells, and Synergizes with Standard of Care Antimyeloma Agents

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 5023-5023
Author(s):  
Susana Hernández-García ◽  
Mercè de Frias ◽  
Clara Campàs ◽  
Bruno Paiva ◽  
Enrique M. Ocio ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
B Cell ◽  
Cell Lines ◽  
Plasma Cells ◽  
Cell Lymphoma ◽  
Hematologic Malignancy ◽  
B Cell Lymphoma ◽  
In Vivo Studies ◽  
Mutational Status

Abstract Abstract 5023 Multiple myeloma (MM) is a malignancy characterized by the accumulation of plasma cells. The disease represents the second most common hematologic malignancy and remains incurable, despite recent advances in its treatment. Therefore, studies to develop new therapies are still necessary, particularly in patients with bad prognostic factors, such as 17p deleted/p53 mutated patients. In this study we describe the preclinical activity of 5-Aminoimidazole-4-carboxamide-1–4-ribofuranoside (AICAR or acadesine) in multiple myeloma. Acadesine is an analog of AMP that is widely used as an activator of AMP-kinase (AMPK), a protein that regulates the responses of the cell to energy changes. Acadesine induces apoptosis in different cell types including CLL, mantle cell lymphoma (MCL) and splenic marginal zone B-cell lymphoma (SMZL) cells and tumor cell lines, without affecting primary T lymphocytes. Thus, acadesine is a promising drug for the treatment of B-cell neoplasms. A clinical phase I/II study of acadesine is currently being performed in CLL patients. We studied the effects of acadesine on the MTT metabolization of several multiple myeloma cell lines (MM1S, MM1R, RPMI-8266, RPMI-LR5, U266, U266-LR7, U266 Dox4, MM144, MGG, SJR, OPM-2, NCIH-929). Acadesine inhibited MM cell growth and induced apoptosis, with IC50 values in the micromolar range, and independently of the p53 mutational status. Cancer treatment, including myeloma, is generally based on combinations of drugs with different mechanisms of action. Thus, we studied the effect of acadesine in double combinations with drugs used in myeloma therapy, such as dexamethasone, melphalan, doxorubicin, bortezomib, and lenalidomide. Analyses of these data using the Chou and Talalay method indicated that acadesine was synergistic with dexamethasone (CI values of 0.60), and particularly with lenalidomide (CI values of 0.42). These promising results with double combinations promoted the investigation of triple combinations in the MM1S cell line. Triple combination of acadesine plus dexamethasone plus lenalidomide or bortezomib notably improved the efficacy of the respective double combinations, being the combination of acadesine plus lenalidomide plus dexamethasone especially efficient. Further studies to determinate the mechanism of action, and in vivo studies in MM1S xenograph are ongoing. Disclosures: de Frias: Advancell: Employment. Campàs:Advancell: Employment.

SYK and STAT3 Are Active in Diffuse Large B-Cell Lymphoma: Activity of Cerdulatinib, a Dual SYK/JAK Inhibitor

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 926-926
Author(s):  
Y. Lynn Wang ◽  
Jiao Ma ◽  
Wei Xing ◽  
Pin Lu ◽  
Karen Dresser ◽  
...  
Keyword(s):  
B Cell ◽  
Cell Lines ◽  
Cell Lymphoma ◽  
Staining Pattern ◽  
B Cell Lymphoma ◽  
Down Regulation ◽  
Dual Inhibitor ◽  
Large B Cell Lymphoma ◽  
Bcr Signaling ◽  
Large B Cell

Abstract Non-Hodgkin Lymphoma (NHL) represents about 5 percent of all cancers diagnosed in the United States. While incidence of NHL has increased slightly over the past decade, death rates have been declining steadily. These declines in mortality can be attributed to improvements in treatment that are based on an increased understanding of the biology of the disease. Diffuse large B-cell lymphoma (DLBCL) accounts for ~30% of NHLs and greater than 80% of aggressive NHLs. Recent studies including large-scale genetic analyses have demonstrated the critical roles of the B-cell receptor’s (BCR) and JAK/STAT pathways in DLBCL. Herein, we investigated the anti-lymphoma activity of cerdulatinib (aka PRT062070), a novel compound that dually targets both SYK and JAK/STAT signaling pathways. To determine whether targeting both SYK and JAK/STAT is relevant in DLBCL, we examined the expression of p-SYK (pY525/526) and p-STAT3 (pY705) on a tissue microarray of 62 DLBCL primary tumors, including 41 GCB and 21 non-GCB cases. p-SYK expression was detected in 29 (47%) cases with a characteristic peri-membrane staining pattern. Of those 29 p-SYK positive cases, 17 were GCB type (17/41, 41%) and 12 were non-GCB type (12/21, 57%). p-STAT3 exhibits a characteristic nuclear staining pattern in DLBCL cases. A total of 26 (42%) stained positive for p-STAT3; 16 were GCB type (16/41, 39%) and 10 were non-GCB type (10/21, 48%). Interestingly, there are 19 cases (31%) with reactivity for both p-SYK and p-STAT3, among which, 11 were GCB type (27%) and 8 were non-GCB type (38%). SYK and STAT3 are also phosphorylated in a panel of nine DLBCL cell lines. Immunoblotting analyses showed that ABC and GCB subtypes of DLBCL cells appear to exhibit different JAK/STAT and BCR signaling profiles. For instance, p-AKT was highly expressed in GCB cells, whereas p-STAT3 was more strongly expressed in ABC cells. Overall, the DLBCL cells are more sensitive to the dual inhibitor than to the SYK-specific inhibitor alone. In both GCB and ABC cell lines, cerdulatinib induced apoptosis via down-regulation of MCL1 protein and PARP cleavage. The compound also blocked G1/S transition and caused cell cycle arrest through inhibition of RB phosphorylation and down-regulation of cyclin E. Further analyses of the cell signaling activities showed that STAT3 phosphorylation was sensitive to inhibition by cerdulatinib in ABC cell lines while phosphorylation of SYK, PLCg2, AKT and ERK was sensitive to inhibition by cerdulatinib in GCB cell lines. Importantly, JAK/STAT and BCR signaling can be blocked by cerdulatinib in GCB and non-GCB primary human DLBCL cells, which led to cell death of these cells. Our work provided mechanistic insights into the actions of SYK/JAK dual inhibitor cerdulatinib, suggesting that the drug may be a potent treatment of DLBCL with a broader anti-tumor activity in both ABC and GCB subtypes of the lymphoma. Disclosures Pandey: Portola Pharmaceuticals: Employment. Conley:Portola Pharmaceuticals: Employment. Coffey:Portola Pharmaceuticals: Employment.

scholarly journals A Novel FBXO45-Gef-H1 Axis Controls Oncogenic Signaling in B-Cell Lymphoma

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 711-711
Author(s):  
Anagh Anant Sahasrabuddhe ◽  
Xiaofei Chen ◽  
Kaiyu Ma ◽  
Rui Wu ◽  
Richa Kapoor ◽  
...  
Keyword(s):  
B Cell ◽  
Cell Lines ◽  
Germinal Center ◽  
De Novo ◽  
Cell Lymphoma ◽  
B Cell Lymphoma ◽  
Lymphoma Cell ◽  
In Vivo Studies

Abstract Introduction: Diffuse large B cell lymphoma (DLBCL) is the most common form of malignant lymphoma and may arise de novo, or through transformation from a pre-existing low-grade B cell lymphoma such as follicular lymphoma (FL). However, the post-translational mechanisms and deregulated pathways underlying the pathogenesis of disease evolution are not fully understood. Methods: We employed integrated functional and structural genomics and mass spectrometry (MS)-driven proteomics which implicated a possible novel tumor suppressor role for a conserved E3 ubiquitin ligase FBXO45 in DLBCL pathogenesis. We generated conditional knockout mice targeting loss of Fbxo45 in germinal center (GC) B-cells using the Cg1-Cre-loxP system and an assortment of CRISPR-mediated knockouts of FBXO45 in B cell lymphoma cells (FL518, BJAB, U2932). We engineered B cell lines (BJAB, U2932) to inducibly express FLAG-tagged FBXO45 to identify candidate substrates of FBXO45 using liquid chromatography-tandem MS. In vitro biochemical and in vivo studies using a variety of genetically-modified lines in xenograft studies in immunodeficient mice were performed to validate observations from proteogenomic studies. Whole genome sequencing (WGS) and genomic copy number studies were interrogated to investigate structural alterations targeting FBXO45 in primary human lymphoma samples. Results: Conditional targeting of Fbxo45 in GCB-cells in transgenic mice resulted in abnormal germinal center formation with increased number and size of germinal centers. Strikingly, targeted deletion of Fbxo45 in GCB-cells resulted in spontaneous B cell lymphomas with (22/22);100%) penetrance and none of the wild-type (WT) littermates (0/20; 0%) developed lymphoma at 24 months. Macroscopic examination revealed large tumor masses, splenomegaly, and lymphadenopathy at different anatomic locations including ileocecal junction, mesenteric, retroperitoneal and cervical lymph nodes and thymus. Next generation sequencing of immunoglobulin heavy chain genes revealed monoclonal or oligoclonal B cell populations. Using proteomic analysis of affinity-purified FBXO45-immunocomplexes and differential whole proteome analysis from GCB-cells of Fbxo45 wt/wt vs Fbxo45 fl/fl mice, we discovered that FBXO45 targets the RHO guanine exchange factor GEF-H1 for ubiquitin-mediated proteasomal degradation. FBXO45 exclusively interacts with GEF H1 among 8 F-box proteins investigated and silencing of FBXO45 using three independent shRNA and CRISPR-Cas9-mediated knockouts in B-cell lymphoma cell lines promotes RHOA and MAPK activation, B cell growth and enhances proliferation. GEF-H1 is stabilized by FBXO45 depletion and GEF-H1 ubiquitination by FBXO45 requires phosphorylation of GEF-H1. Importantly, FBXO45 depletion and expression of a GEF-H1 mutant that is unable to bind FBXO45 results in GEF-H1 stabilization, promotes hyperactivated RHO and MAPK signaling and B-cell oncogenicity in vitro and in vivo. Notably, this phenotype is reverted by co-silencing of GEF-H1. Inducible ectopic expression of FBXO45 triggers accelerated turnover of GEF H1 and decreased RHOA signaling. Genomic analyses revealed recurrent loss targeting FBXO45 in transformed DLBCL (25%), de novo DLBCL (6.6%) and FL (2.3%). In keeping with our observation of prolonged hyperactivation of pERK1/2 consequent to FBXO45 ablation, in vitro and in vivo studies using B-cell lymphoma cell lines and xenografts demonstrated increased sensitivity to pharmacologic blockade with the MAP2K1/2 (ERK1/2) inhibitor Trametinib. Conclusions: Our findings define a novel FBXO45-GEF-H1-MAPK signalling axis, which plays an important role in DLBCL pathogenesis. Our studies carry implications for potential exploitation of this pathway for targeted therapies. Disclosures Siebert: AstraZeneca: Speakers Bureau. Lim: EUSA Pharma: Honoraria.

Potent targeting of B cell lymphoma and plasma cell tumors by a tetravalent, Fc-engineered antibody directed against the glycoantigen CD75s.

2019 ◽  
Vol 37 (15_suppl) ◽  
pp. e14004-e14004
Author(s):  
Katja Klausz ◽  
Amir Karimzadeh-Tabrizi ◽  
Malena Buck ◽  
Anna Otte ◽  
Steffen Krohn ◽  
...  
Keyword(s):  
B Cell ◽  
Plasma Cells ◽  
Cell Lymphoma ◽  
B Cell Lymphoma ◽  
Burkitt’S Lymphoma ◽  
Myeloma Cell ◽  
Burkitt's Lymphoma ◽  
The Novel ◽  
Effector Cells ◽  
Effector Functions

e14004 Background: Monoclonal antibodies are established treatment options for B cell-derived malignancies, but relapse is still the major challenge. Novel target structures may open alternative avenues to develop effective antibody therapies. Here, we characterized the novel tetravalent antibody ‘EBU-141 Tetra’ and identified the glycoantigen CD75s (α-2,6-sialylated lactosamines) as suitable target structure for antibody-based therapy. CD75s was detected on most B cell lymphomas, including Burkitt’s lymphoma, FL, DLBCL, MCL, CLL, and plasma cell tumors. Classical Hodgkin lymphomas were consistently negative while reactivity on individual cases of peripheral T cell lymphoma was seen. To evaluate CD75s as a target for antibody therapy, we generated a tetravalent, Fc-engineered chEBU-141 IgG1 antibody with enhanced avidity for CD75s and potent effector functions. Methods: ‘EBU-141 Tetra’ was produced by transient transfection and purified by affinity chromatography. Direct anti-tumor effects and Fc-mediated effector functions were investigated in cell proliferation assays, by fluorescence microscopy and in 51Cr release experiments using lymphoma and myeloma cell lines and patient-derived tumor cells. Peripheral blood mononuclear cells and monocyte-derived macrophages of healthy donors were used as human effector cells in the experiments. Results: ‘EBU-141 Tetra’ showed improved binding to CD75s on cell surface of mature B cell lymphoma as well as myeloma plasma cells compared to the conventional chimeric antibody chEBU-141 IgG1. The higher avidity for CD75s resulted in markedly improved ADCC activity of ‘EBU-141 Tetra’ against Daudi Burkitt’s lymphoma, U266 plasma cells and CLL patient-derived tumor cells with EC50 values in the low nanomolar range. In addition, ‘EBU-141 Tetra’ demonstrated efficient phagocytosis of Burkitt’s lymphoma and myeloma cell lines. Thus, the novel tetravalent, chimeric, Fc-engineered antibody ‘EBU-141 Tetra’ efficiently recruits immune effector cells for tumor cell lysis. Conclusions: Our findings further demonstrate that highly potent IgG-like antibodies against glycan-structures can be generated from mouse IgM antibodies and may open a new therapeutic window for therapy of patients with mature B cell lymphomas and multiple myeloma.

MicroRNA Expression Profiling of Diffuse Large B-Cell Lymphoma Reveals Differences between Germinal Center-Like and Activated B Cell-Like Subtypes.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 3002-3002
Author(s):  
Charles H. Lawrie ◽  
Shamit Soneji ◽  
Christopher D. Cooper ◽  
Chris Hatton
Keyword(s):  
Gene Expression ◽  
B Cell ◽  
Cell Lines ◽  
Expression Profile ◽  
Germinal Center ◽  
Cell Lymphoma ◽  
B Cell Lymphoma ◽  
Large B Cell Lymphoma ◽  
Large B Cell ◽  
Clinical Cases

Abstract MicroRNAs (miRNA) are a recently discovered class of short non-coding RNA molecules that negatively regulate gene expression. They have been shown to play a critical role in many biological functions. In humans about 320 miRNAs have been identified, some of which are expressed in a cell-specific and developmental stage-specific manner. Recently it has been shown that the expression profile of miRNAs can be used to subtype clinical cases (and cell lines) according to diagnosis with a greater degree of accuracy than traditional gene expression analysis. The identity of miRNAs associated with different lymphoma types however remains poorly defined. Previous expression studies have revealed the presence of at least two subtypes of diffuse large B-cell lymphoma (DLBCL) representing the postulated cell of origin; those that are germinal center B cell derived (GCB-type) and those that are activated B-cell derived (ABC-type). The latter subtype has been linked with poor prognostic outcome. It is not known whether these subtypes are also defined at the miRNA level. Therefore we examined the miRNA expression profile of DLBCL cell lines of defined subtypes as well as sub-populations of B-lymphocytes by microarray analysis. Consistent with recent publications, we found that mir-19a, 19b and 17-5p (part of mir-17-92 cluster) were up-regulated in cell lines but not in normal lymphocyte populations. Furthermore, cluster analysis showed that GCB-type cell lines (SUD-HL4, SUD-HL6 & SUD-HL10) have a distinct miRNA profile from ABC-type cell lines (OCI-Ly3 & OCI-Ly10). Most notably, high levels of expression of mir-155, mir-181b and mir-325 were found in ABC-type cell lines whilst high levels of mir-181a were found in GCB-type cell lines. We looked at expression of mir-155, 181a, 143, 145, 378 and 16 in these cell lines as well as clinical cases of DLBCL by RNase-protection assay. Consistent with the microarray data, we found that mir-155 was expressed in ABC-type cell lines but not GCB-type cell lines whilst the converse was true for mir-181a. Clinical cases showed similar patterns of expression but have still to be sub-typed according to immunohistochemical markers. Although still preliminary, our data suggests that miRNA profiling may be a useful tool in predicting the subtype of DLBCL cases and hence clinical outcome.

Recurrent 11q24.3 Gain Contributes to the Pathogenesis of Diffuse Large B Cell Lymphoma (DLBCL) by Deregulating ETS1 and FLI1

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 1386-1386
Author(s):  
Paola Bonetti ◽  
Monica Testoni ◽  
Marta Scandurra ◽  
Maurilio Ponzoni ◽  
Roberto Piva ◽  
...  
Keyword(s):  
Gene Expression ◽  
Transcription Factors ◽  
B Cell ◽  
Germinal Center ◽  
Conflicts Of Interest ◽  
Cell Lymphoma ◽  
Functional Characterization ◽  
B Cell Lymphoma ◽  
Transcriptional Program ◽  
Down Regulation

Abstract Abstract 1386 DLBCL represents the most common form of B-cell non-Hodgkin lymphoma (B-NHL). It is an aggressive and heterogeneous disease, comprising at least three distinct subtypes based on gene expression profile analysis: germinal center B cell-like DLBCL (GCB), activated B cell-like DLBCL (ABC) and primary mediastinal B-cell lymphoma (PMBL). These subtypes are supposed to derive from B cells at different stages of differentiation. Normal germinal center (GC) B-cell differentiation requires a complex transcriptional program and alterations of genes involved in this process are relevant for DLBCL pathogenesis. Identification and functional characterization of new genetic lesions would provide critical information to better understand the pathogenesis of DLBCL. With this aim, we studied the genomic profiles of 166 DLBCL patients, identified and characterized a recurrent gain mapping to chromosome 11q24.3. Methods. Genomic profiles were obtained from 166 Affymetrix 250K SNP arrays and integrated with gene expression data (GeneChip U133 plus 2.0) in 54 cases. Data were validated by PCR and immunohistochemistry. Gene silencing experiments were done with shRNA. Results. A minimal common region 11q24.3 gain was present in 26% of DLBCL samples and it encompassed six genes (ETS1, FLI1, KCNJ1, KCNJ5, P53AIP1, RICS). Samples with the 11q24.3 gain were significantly associated with high expression of the transcription factors ETS1 and FLI1. Data were confirmed by real-time PCR and by immunohistochemical analysis. Gene expression analysis showed 228 transcripts with a significantly different expression between cases with or without the lesion (p<0.01, >2-fold change): 215 genes were up-regulated in the patients with the gain and 13 were down-regulated, suggesting that this lesion has an impact on the transcriptional program of the tumor cells. To study the biological meaning of the lesion, ETS1 and FLI1 expression was down-regulated in a DLBCL cell line bearing the same lesion observed in clinical specimens (OCI-Ly7). Results showed that ETS1 and FLI1 down-regulation caused a reduced proliferation rate and activation of apoptosis leading to cell death. Concomitant ETS1 and FLI1 down-regulation resulted in a more severe phenotype. Only FLI1 was confirmed to be essential for cell viability in other DLBCL cell lines (SUDHL4, VAL, U2932), whereas ETS1 did not, suggesting a distinct role of the two transcription factors in different DLBCL samples. Preliminary results showed that down-regulation of ETS1 affected the transcriptional program of GC B-cell terminal differentiation causing an up-regulation of BLIMP1, the master regulator of plasma cells differentiation. Conclusions. In DLBCL, a recurrent gain at 11q24.3 determines the over-expression of the transcription factors ETS1 and FLI1. Functional experiments showed that the lesion might sustain DLBCL proliferation and viability, and contribute to a differentiation blockade of the GC B-cell towards a plasma cell lineage by negatively regulating BLIMP1. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Targeting the Glyco-Antigen CD75s with the Tetravalent, Fc-Engineered Antibody 'Ebu-141 Tetra' Induces Potent Killing of B Cell Lymphoma and Plasma Cell Tumors

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 4178-4178
Author(s):  
Katja Klausz ◽  
Amir Karimzadeh-Tabrizi ◽  
Malena Buck ◽  
Steffen Krohn ◽  
Anna-Kathrin Otte ◽  
...  
Keyword(s):  
B Cell ◽  
Research Funding ◽  
Plasma Cells ◽  
Cell Lymphoma ◽  
B Cell Lymphoma ◽  
Burkitt’S Lymphoma ◽  
Myeloma Cell ◽  
Burkitt's Lymphoma ◽  
Effector Cells ◽  
Binding Avidity

Abstract While monoclonal antibodies (MoAb) are already well established for the treatment of B cell-derived malignancies and usually show a good safety profile, not all patients benefit and relapses may be a problem. In order to identify novel surface structures suitable for antibody-based therapies and to improve killing mechanisms, 'EBU-141 Tetra' was developed. The parental MoAb EBU-141 is of mouse IgMk isotype, was generated in our laboratory and recognizes the glyco-antigen CD75s (previously CDw75), which are α-2,6-sialylated lactosamines on cell surface glycoproteins and glycolipids. CD75s was found on normal mature B cells and subsets of T cells, but is also expressed on certain lymphatic and solid tumors, e.g. mature B cell lymphoma, pancreatic and prostate cancer cells. EBU-141 specifically binds to CD75s on most B cell lymphoma, including Burkitt's lymphoma, FL, DLBCL, MCL, CLL, and interestingly plasma cell tumors. In addition, EBU-141 showed reactivity on a few cases of peripheral T-cell lymphoma, whereas classical Hodgkin lymphomas were consistently negative. Previously a chimeric IgG1k antibody, chEBU-141, was derived from EBU-141. Compared to the parental IgM antibody, chEBU-141 showed strongly reduced binding avidity, but was moderately effective in triggering antibody-dependent cell-mediated cytotoxicity (ADCC) of mature B cell lymphoma and malignant plasma cells via recruitment of NK cells. However, chEBU-141 lacked the potent complement-dependent cytotoxicity (CDC) observed with the parental EBU-141 antibody. The aim of this study was to generate a tetravalent binding, Fc-engineered chEBU-141 IgG1 antibody with enhanced binding avidity for CD75s and potent effector functions for antibody-based therapy of mature B cell lymphomas and multiple myeloma. Using the variable regions of EBU-141, the chimeric IgG1κ antibody with a protein-engineered Fc and tetravalent binding properties, named 'EBU-141 Tetra', was generated. This MoAb and relevant controls were produced by transient transfection of 293T cells and purified from cell culture supernatants by affinity chromatography. Direct anti-tumor effects and Fc-mediated modes of action were investigated in cell proliferation assays and chromium release experiments using lymphoma and myeloma cell lines. Peripheral blood mononuclear cells and serum of healthy donors were used as source of human effector cells and complement in the cytotoxicity experiments. The 'EBU-141 Tetra' showed improved binding to CD75s on cell surface of mature B cell lymphoma as well as myeloma plasma cells compared to the bivalent binding chEBU-141 IgG1. The higher avidity for CD75s resulted in markedly improved ADCC activity of the 'EBU-141 Tetra' against Daudi Burkitt's lymphoma and U266 plasma cells with EC50 values in the picomolar range and higher maximum lysis rates. In addition, the 'EBU-141 Tetra' regained CDC activity of the parental EBU-141 and demonstrated efficient killing of Burkitt's lymphoma and myeloma cell lines with human serum as complement source. Thus, recruitment of immune effector cells and activation of the complement system are the main modes of action of the novel, tetravalent, chimeric, Fc-engineered antibody 'EBU-141 Tetra' antibody. Our findings further demonstrate that highly potent IgG-like antibodies against glycan-structures can be generated from mouse IgM antibodies and may open a new therapeutic window for therapy of patients with mature B cell lymphomas and multiple myeloma. Disclosures Klausz: Affimed: Research Funding. Otte:Affimed: Research Funding. Klapper:HTG Molecular Diagnostics, Inc.: Research Funding; Amgen: Honoraria, Research Funding; F.Hoffman-La Roche: Honoraria, Research Funding; Takeda: Honoraria, Research Funding; Regeneron: Honoraria, Research Funding. Peipp:Affimed: Research Funding. Gramatzki:Affimed: Research Funding.

scholarly journals Deletion of Murine Rhoh induces More Aggressive Diffuse Large B Cell Lymphoma (DLBCL) Via Interaction with Kaiso and Regulation of BCL-6 Expression

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 1574-1574
Author(s):  
Hiroto Horiguchi ◽  
Marioara Felicia Ciuculescu ◽  
Anja Troeger ◽  
Haiming Xu ◽  
Christian Brendel ◽  
...  
Keyword(s):  
T Cell ◽  
B Cell ◽  
Cell Lines ◽  
Nuclear Localization ◽  
Germinal Center ◽  
Cell Lymphoma ◽  
B Cell Lymphoma ◽  
Lymphoma Cell ◽  
Germinal Center Formation

Abstract RHOH encodes a GTPase-deficient, hematopoietic-specific small GTPase first identified as a hypermutable gene in DLBCL (Pasqualucci et al. 2001). RhoH is critical for T cell receptor signaling and Rhoh-deficient (RhohKO) mice have T cell lymphopenia (Gu et al., 2006) and loss of function mutations of RHOH are associated with Epidermodysplasia Verruciformis (Crequer et al., 2012). However, the role of RhoH in the biology of DLBCL is still unknown and its role in B lymphoid development is incompletely studied. We investigated the role of RhoH in normal germinal center formation and in a murine model of DLBCL by crossing RhohKO mice with Iµ-HABcl-6 transgenic (Bcl-6Tg) mice (Cattoretti G, et al., 2005). In young RhohKOmice, deficient development of CXCR5+ follicular T helper (Tfh) cells results in defective germinal center (GC) formation and impaired immunoglobulin switching in vivo. In spite of this defect in GC formation, RhohKO; Bcl-6Tg (KOTg) mouse demonstrated accelerated lymphoma progression associated with larger spleens and significantly earlier death (Log-rank test p<0.01, Figure 1). Immunohistochemistry data suggested increased expression of IRF-4 and enhanced expression of BCL-6 in KOTg mice, findings confirmed by immunoblot and consistent with an activated B-cell (ABC)-DLBCL phenotype. To analyze the mechanism underlying these results, B cell lymphoma cell lines from KOTg lymphoma mice were established. Multiple attempts to establish RhohWT lymphoma cell lines failed, although we also successfully established a lymphoma cell line from RhohKO; Bcl-6(ntg) (KONtg) mice. Re-expression of RhoH in these lines via retrovirus mediated gene transfer led to significantly decreased proliferation (5.9x106±9.6x105 cells vs 8.6x106±9.6x105 cells after 5-days culture; KOTg vs KOTg-RhoH, mean±SEM, p<0.05) that was associated with clear reduction in BCL-6 expression. These data suggest that BCL-6 is a direct or an indirect transcriptional target of RhoH. Our laboratory previously reported that KAISO, a dual-specific, Broad complex, Trantrak, Bric-a-brac/Pox virus, Zinc finger (POZ-ZF) transcription factor interacts and colocalizes with RhoH in the nucleus, whereas knockdown of RhoH inhibits the nuclear localization of KAISO in Jurkat cells (Mino A, et al., 2016). In addition, Kaiso has been shown to be a key regulator of spleen germinal center formation by repressing Bcl-6 expression in splenocytes (Koh D, et al., 2013). We hypothesized that the deletion of Rhoh may lead to the decreased nuclear localization of KAISO and result in increased the expression of Bcl-6. We first confirmed that RhoH bound KAISO in RhoH-transduced KO lymphoma cells by co-immunoprecipitation. Further immunoblot analysis and quantitative PCR (qPCR) demonstrated decreased BCL-6 expression in lymphoma cells in which RhoH was re-expressed (KOTg-RhoH and KONtg-RhoH) compared with empty vector-transduced lymphoma cell lines. Interestingly, p53 a BCL-6 target was increased in RhoH-transduced lymphoma cell lines. These data indicate that RhoH affects BCL-6 expression in B cell lymphoma cell lines and suggest that RhoH may be involved in DLBCL development by co-regulating BCL-6 expression affecting downstream targets via interaction with KAISO. Figure. Figure. Disclosures Williams: Bluebird Bio: Research Funding.

Therapeutic Targeting of the Bcl-6: p53 Axis with Histone Deacetylase Inhibitors in Diffuse Large B-Cell Lymphoma,

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 3733-3733 ◽  
Author(s):  
Jennifer E Amengual ◽  
Matko Kalac ◽  
Luigi Scotto ◽  
Patrick A Sleckman ◽  
Enrica Marchi ◽  
...  
Keyword(s):  
Tumor Suppressor ◽  
B Cell ◽  
Cell Lines ◽  
Germinal Center ◽  
Research Funding ◽  
Cell Lymphoma ◽  
Hdac Inhibitors ◽  
B Cell Lymphoma ◽  
Large B Cell Lymphoma ◽  
Large B Cell

Abstract Abstract 3733 Diffuse large B-cell lymphoma (DLBCL) is the most common non-Hodgkin's Lymphoma. Despite advances in treatment, 1/3 of patients die from their disease. Gene expression profiling has delineated three subtypes with different genetic features known to be prognostic: the Activated B-cell (ABC), Germinal Center (GC), and grey zone types. For example, ABC DLBCL is addicted to NFkB over-expression. The oncogene, BCL6, encodes a transcription factor that functions as a transcriptional repressor within normal germinal center B-cells. Constitutive activation of Bcl-6 leads to GC-type DLBCL by turning off genes expressing cell cycle dependent kinase inhibitors, and essential tumor suppressor genes, like p53. There is a critical inverse relationship between Bcl-6 and p53, the functional status of which is linked to each transcription factor's degree of acetylation. Deacetylation of Bcl-6 is required for maintaining its effects as a transcriptional repressor. Conversely, acetylation of p53 is activating when class III histone deacetylases (HDAC), also known as sirtuins, are inhibited by drugs such as niacinamide. HDAC inhibitors are presently approved for T-cell lymphoma and may require the targeting of additional pathways to be effective in B-cell lymphomas. Trichostatin A and niacinamide modulate Bcl-6 in lymphoma cell lines. One therapeutic strategy that could favorably shift the relationship between oncogenes and tumor suppressors is the pharmacologic modification of Bcl-6 and p53 using HDAC inhibitors. Eight DLBCL cell lines were screened (4 ABC: Su-DHL2, HBL-1, OCI-Ly10, RIVA; 4 GC:OCI-Ly1, OCI-Ly7, Su-DHL6, Su-DHL4) with four class I/II HDAC inhibitors (romidepsin, vorinostat, panobinostat and belinostat) in combination with niacinamide (sirtuin inhibitor) at two dose levels each at three time points. Cell growth inhibition was measured by luminescence cell viability and apoptosis flow cytometry assays. Synergy was measured by the relative risk ratio (RRR) calculation where values <1 represent synergy. Synergy was achieved in significantly greater number and intensity in the GC versus ABC cell lines. Specifically, romidepsin in combination with niacinamide achieved the greatest synergy. To analyze mechanism of action, DLBCL cell lines were treated with combinations of class I/II HDAC inhibitors and niacinamide. Cells of both GC and ABC subtypes treated with the combination resulted in increased acetylation of p53, and increased p21 and BLIMP-1 content compared to controls. These results did not correlate with cytotoxicity as the ABC cell lines did not achieve the same synergy as the GC cells. GC cells treated with the same combinations resulted in acetylation of Bcl-6 compared with controls as measured by immunoprecipitation and Western blotting assays; ABC cells do not express Bcl-6. This finding correlated with cytotoxicity implying that a rational second pathway must be targeted to shift the balance between oncogene and tumor suppressor activity to achieve effective cell kill. p300 content was also increased suggesting that treatment with HDAC inhibitors recruit or upregulate its production and activity leading to increased acetylation. Using a novel double transgenic mouse model of aggressive spontaneous B-cell lymphoma (l-myc overexpressing crossed with CD19-tagged mCherry luciferase), in vivo effects of the drug combination were studied. These mice express equal basal amounts of Bcl-6 and p53 as GC cell lines. Mice treated with niacinamide 20 mg/kg and romidepsin 2.3mg/kg IP for 5 hours achieved increased acetylation of Bcl-6 and p53, and accumulation of p21 and BLIMP1 compared with controls. Importantly, mice treated with the combination of niacinamide 40 mg/kg and romidepsin 2.3 mg/kg IP achieved decreased tumor burden as measured by bioluminescence signal intensity compared to mice treated with each drug alone and controls. Presently, we are translating these concepts and observations in a proof-of-principle phase I trial evaluating the safety of vorinostat plus niacinamide in lymphoid malignancies. By targeting the specific pathogenetic features of DLBCL, it may be possible to tailor future treatment platforms for discrete subtypes of DLBCL. Disclosures: Off Label Use: The drugs evaluated are not approved for use in DLBCL. O'Connor:Celgene: Consultancy, Research Funding; Merck: Research Funding; Novartis: Research Funding; Spectrum: Research Funding.

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