An Activating Mutation (Ser525Pro) within the Transactivation Domain of REL in Two Patients with Human B-Cell Lymphomas Enhances REL’s In Vitro Transforming Activity.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 2617-2617
Author(s):  
Heiko Trautmann ◽  
Daniel T. Starczynowski ◽  
Christiane Pott ◽  
Lana Harder ◽  
Norbert Arnold ◽  
...  
Keyword(s):  
B Cell ◽  
Cell Lymphoma ◽  
B Cell Lymphoma ◽  
Transactivation Domain ◽  
Wild Type ◽  
Spleen Cells ◽  
B Cell Lymphomas ◽  
Chicken Spleen ◽  
Human B Cell

Abstract REL/NF-κB transcription factors are implicated in the control of apoptosis and cell growth particular in hematopoetic lineages. The REL locus at chromosomal region 2p13–16 is frequently amplified in B-cell lymphomas including diffuse-large B-cell lymphoma (DLBCL) and may play a role in lymphomagenesis. Overexpression of wild-type REL can transform chicken lymphoid cells in culture, and several experimentally-generated mutations within the REL C-terminal transactivation domain (TAD) have been previously shown to enhance REL’s transforming ability. We analysed 83 B-cell lymphomas included in the ‘Deutsche Krebshilfe’ funded network „Molecular Mechanisms in Malignant Lymphoma“ for the presence of activating mutations in the coding region of REL. We performed a systematic dHPLC screening for mutation discovery and identified an identical point mutation in two human B-cell lymphomas (a t(14;18)-positive follicular lymphoma and a mediastinal B-cell lymphoma) that changes Ser525 to Pro within the REL TAD. In the mediastinal B-cell lymphoma, the mutation in REL was proven to be of germline origin. FISH showed an amplification of the REL locus in the tumor cells of this case. Quantitative allelic discrimination of S525P indicates that the mutant REL gene was over-represented in both cases. By in vitro experiments we could show that the S525P mutation enhances the in vitro transforming ability of REL in chicken spleen cells. In addition, REL-S525P differs from wild-type REL in its ability to activate certain κB site-containing reporter plasmids in transient transfection assays. In particular, REL-S525P has a reduced ability to activate the human manganese superoxide dismutase (MnSOD) promoter in A293 cells; however, the MnSOD protein is over-expressed in REL-S525P-transformed chicken spleen cells as compared to wild-type REL-transformed cells. Ser525 of REL falls within a sequence that is similar to other known phosphorylation sites of the IκB kinase, and REL-S525P shows a reduced ability to be phosphorylated by IKKα in vitro. The S525P mutation reduces IKKα- and TNFα-stimulated transactivation by REL, as measured in GAL4 reporter assays. Furthermore, REL-S525P-transformed chicken spleen cells are more resistant to TNFα-induced cell death than cells transformed by wild-type REL. These results represent the first identification of a tumor-derived activating mutation in the REL proto-oncogene, and they suggest that the S525P mutation contributes to the development of human B-cell lymphomas by altering REL’s ability to induce target gene expression by affecting an IKKα-regulated transactivation activity.

Growth inhibition and apoptosis of human B-cell lymphoma in vitro and in vivo by Bcl-2 short hairpin RNA

2012 ◽  
Vol 29 (1) ◽  
pp. 244-252
Author(s):  
YUCHUN LIU ◽  
YONGMEI SHEN ◽  
CHENHAO QIN ◽  
YIZHEN SHI ◽  
GUANG RONG ◽  
...  
Keyword(s):  
Growth Inhibition ◽  
B Cell ◽  
Cell Lymphoma ◽  
B Cell Lymphoma ◽  
Short Hairpin Rna ◽  
Hairpin Rna ◽  
Short Hairpin ◽  
Human B Cell

Targeting Bcl-2 family members with the BH3 mimetic AT-101 markedly enhances the therapeutic effects of chemotherapeutic agents in in vitro and in vivo models of B-cell lymphoma

Blood ◽  
2008 ◽  
Vol 111 (11) ◽  
pp. 5350-5358 ◽  
Author(s):  
Luca Paoluzzi ◽  
Mithat Gonen ◽  
Jeffrey R. Gardner ◽  
Jill Mastrella ◽  
Dajun Yang ◽  
...  
Keyword(s):  
Drug Resistance ◽  
B Cell ◽  
Cell Lymphoma ◽  
Family Members ◽  
B Cell Lymphoma ◽  
B Cell Lymphomas ◽  
In Vivo Models ◽  
Acquired Drug Resistance ◽  
Bh3 Mimetic

Abstract Overexpression of antiapoptotic members of the Bcl-2 family are observed in approximately 80% of B-cell lymphomas, contributing to intrinsic and acquired drug resistance. Nullifying antiapoptotic function can potentially overcome this in-trinsic and acquired drug resistance. AT-101 is a BH3 mimetic known to be a potent inhibitor of antiapoptotic Bcl-2 family members including Bcl-2, Bcl-XL, and Mcl-1. In vitro, AT-101 exhibits concentration- and time-dependent cytotoxicity against lymphoma and multiple myeloma cell lines, enhancing the activity of cytotoxic agents. The IC50 for AT-101 is between 1 and 10 μM for a diverse panel of B-cell lymphomas. AT-101 was synergistic with carfilzomib (C), etoposide (E), doxorubicin (D), and 4-hydroxycyclophosphamide (4-HC) in mantle cell lymphoma (MCL) lines. In a transformed large B-cell lymphoma line (RL), AT-101 was synergistic when sequentially combined with 4-HC, but not when both drugs were added simultaneously. AT-101 also induced potent mitochondrial membrane depolarization (ΔΨm) and apoptosis when combined with carfilzomib, but not with bortezomib in MCL. In severe combined immunodeficient (SCID) beige mouse models of drug-resistant B-cell lymphoma, 35 mg/kg per day of AT-101 was safe and efficacious. The addition of AT-101 to cyclophosphamide (Cy) and rituximab (R) in a schedule-dependent manner enhanced the efficacy of the conventional therapy.

scholarly journals Identification of Potent Paracaspase MALT1 Inhibitors for Hematological Malignancies

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 30-30
Author(s):  
Wu Yin ◽  
Nie Zhe ◽  
Andrew Placzek ◽  
Michael Trzoss ◽  
Goran Krilov ◽  
...  
Keyword(s):  
B Cell ◽  
Cell Lymphoma ◽  
Synergistic Effects ◽  
Xenograft Model ◽  
B Cell Lymphoma ◽  
B Cell Lymphomas ◽  
Publicly Traded ◽  
Ongoing Work

Introduction: MALT1 (mucosa-associated lymphoid tissue lymphoma translocation protein 1), was identified as a translocation protein fused with cIAP2 in mucosa-associated lymphoid tissue (MALT) B cell lymphomas. MALT1, a key mediator of NF-κB signaling and the main driver of a subset of B-cell lymphomas, functions via formation of a complex with CARMA1 and BCL10 to mediate antigen receptor-induced lymphocyte activation. MALT1 has been considered as a potential therapeutic target for several non-Hodgkin B cell lymphomas as well as chronic lymphocytic leukemia (CLL). Here, we describe the discovery of novel, potent MALT1 inhibitors that result in antiproliferative effects in non-Hodgkin B-cell lymphoma cells. Results: We have identified novel small molecule MALT1 inhibitors using our proprietary physics-based Free Energy Perturbation (FEP+) modeling technology. Our compounds show potent (sub nM) inhibition of MALT1 enzymatic activity, as well as high binding affinity (sub nM) to MALT1 protein measured by Surface Plasmon Resonance (SPR). BCL10 is a binding partner of MALT1 that is cleaved by MALT1 at the C-terminus. Our inhibitors were efficacious in a target engagement assay showing prevention of BCL10 cleavage in Activated B-cell (ABC) subtype of diffuse large B cell lymphoma (DLBCL) cell lines OCI-LY3 and OCI-LY10, which are Bruton tyrosine kinase (BTK) inhibitor ibrutinib-resistant and -responsive respectively. Our compounds are potent inhibitors of IL10 secretion in both OCI-LY3 and OCI-LY10 cells, which is consistent with the inhibition of NF-κB signaling. We also examined the effect of our MALT1 inhibitors on ABC-DLBCL cell proliferation. Our inhibitors demonstrated potent anti-proliferative effects in both OCI-LY3 and OCI-LY10 cell lines, as well as synergistic effects with ibrutinib in a BTKi sensitive ABC-DLBCL cell panel. Examinations of a protease panel and off-target safety screening panel, as well as in vivo high dose tolerability study showed our compound had excellent selectivity and significant safety margin. Plasma IL10 and tumor BCL10 have been identified as robust PD markers in PK/PD studies in both OCI-LY3 and OCI-LY10 tumor bearing mice. Dose-dependent tumor growth inhibition was observed after 3 weeks of treatment in OCI-LY3 xenograft model, with efficacy also observed in combination with venetoclax. Ongoing work: We are continuing to explore the synergistic effects of our compounds with BTK inhibitors in B-cell lymphoma mouse models. Preliminary data showed potent inhibition of IL-2 secretion in Jurkat cells from our compound treatment. Additional studies are ongoing to elucidate the role of MALT1 inhibition in Treg as well as Teffector cells in vitro and in vivo. Refinement of the current inhibitor series, using co-crystal structures, is in progress in preparation for further development of optimized molecules. Conclusion and Future Plans: We have identified novel potent MALT1 protease small molecule inhibitors that are efficacious in the in vitro B-cell lymphoma cell proliferation assays and in the in vivo B-cell lymphoma xenograft model. Our data suggest that targeting MALT1 may expand therapy options for patients with selected B-cell lymphomas, such as ABC-DLBCL. Our work provided insight into the anti-tumor efficacy of our inhibitors in B-cell lymphomas as single agent, and ongoing work will continue to assess the potential combination with BTKi to overcome drug-induced resistance in patients with relapsed/refractory B-cell lymphoma. Disclosures Yin: Schrodinger: Current Employment, Current equity holder in publicly-traded company. Zhe:Schrodinger: Current Employment, Current equity holder in publicly-traded company. Placzek:Schrodinger: Current Employment, Current equity holder in publicly-traded company. Trzoss:Schrodinger: Current Employment, Current equity holder in publicly-traded company. Krilov:Schrodinger: Current Employment, Current equity holder in publicly-traded company. Feng:Schrodinger: Current Employment, Current equity holder in publicly-traded company. Lawrenz:Schrodinger: Current Employment, Current equity holder in publicly-traded company. Pelletier:Schrodinger: Current Employment, Current equity holder in publicly-traded company. Lai:Triplet Therapeutics: Current Employment, Current equity holder in private company. Bell:Schrodinger: Current Employment, Current equity holder in publicly-traded company. Calkins:Schrodinger: Current Employment, Current equity holder in publicly-traded company. Grimes:Schrodinger: Current Employment, Current equity holder in publicly-traded company. Tang:Schrodinger: Current Employment, Current equity holder in publicly-traded company. McRobb:Schrodinger: Current Employment, Current equity holder in publicly-traded company. Gerasyuto:Schrodinger: Current Employment, Current equity holder in publicly-traded company. Feher:Schrodinger: Current Employment, Current equity holder in publicly-traded company. Mondal:Schrodinger: Current Employment, Current equity holder in publicly-traded company. Jensen:Schrodinger: Current Employment, Current equity holder in publicly-traded company. Wright:Schrodinger: Current Employment, Current equity holder in publicly-traded company. Akinsanya:Schrodinger: Current Employment, Current equity holder in publicly-traded company.

Direct Growth Inhibition of Both Mouse and Human B Cell Lymphomas by CpG Oligodeoxynucleotides

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 2854-2854
Author(s):  
Reiko E Yamada ◽  
David J Betting ◽  
Michael Ahdoot ◽  
Kristopher K Steward ◽  
John M Timmerman
Keyword(s):  
B Cells ◽  
B Cell ◽  
Cell Lines ◽  
B Cell Lymphoma ◽  
B Cell Lymphomas ◽  
Cpg Oligodeoxynucleotides ◽  
Mantle Cell ◽  
Class B ◽  
Human B Cell

Abstract Abstract 2854 Immunostimulatory CpG oligodeoxynucleotides (ODN) are potent activators of T cell immunity and antibody-dependent cellular cytotoxicity (ADCC), and under study as immunotherapeutic agents for a variety of cancers, including B cell lymphomas. Recently, anti-CD20 antibody-CpG conjugates have been shown to eradicate rituximab-resistant B cell lymphoma in a syngeneic murine lymphoma model (D. Betting et al, ASH 2009). CpG is known to strongly stimulate the proliferation of normal B cells. Paradoxically, CpG has been reported to markedly inhibit the in vitro growth of the murine B cell lymphoma A20 (J. Li et al, J. Immunol. 2007), thereby prompting us to investigate the direct effects of CpGs on the growth of human B cell lymphomas. We first demonstrated that CpGs, especially those of the B class, potently inhibited proliferation of the A20 mouse B cell line in vitro by up to 81.5% (class A 58.7% and class C 52.7%). Moreover, in non-tumor bearing mice intratumoral injections of CpG activated normal B cells, while mice bearing subcutaneous A20 tumors showed suppressed tumor growth after CpG injections. Similarly, in humans, CpGs strongly stimulated the proliferation of normal peripheral blood B cells (stimulation index for class B 27.5 at 5 μg/ml). A panel of 12 human lymphoma cell lines (DLBCL, Burkitt's, mantle cell) were cultured in the presence or absence of varying concentrations of CpGs of A, B, or C classes (50, 10, or 2 μg/ml) or control ODN. Proliferation was measured by [3H]-thymidine incorporation in quadruplicate 72 hour cultures, and apoptosis measured by Annexin-V and PI flow cytometry. In contrast to the stimulation observed with normal human B cells, the proliferation of all 12 lymphoma lines were inhibited by CpGs. The strongest inhibitory effects were seen with CpG 7909, a class B CpG under clinical development for cancer therapy (Pfizer, PF-3512676). Raji cells were inhibited by 77.9%, 40.7%, and 8.8% at CpG concentrations of 50, 10, and 2 μg/ml, respectively (p≤0.01 for all comparisons vs. media alone). Among the 12 tested cell lines, the percentage growth inhibition using 50 μg/ml CpG 7909 was 61.2–80.4% for germinal center-type DLBCL (SUDHL-4, SUDHL-6, OCI-Ly19), 50–59.5% for activated B cell-type DLBCL (SUDHL-2, OCI-Ly3, OCI-Ly10), 56.4–79.3% for Burkitt's lymphomas (Raji, Ramos, Daudi, BJAB), and 69.6–69.9% for mantle cell lymphomas (Jeko-1, Granta-519). Interestingly, although all of the human cell lines expressed TLR9 by semi-quantitative RT-PCR, inhibition in the proliferation levels did not correlate with TLR9 expression levels. CpG 7909 also induced significant levels of apoptosis in Raji and Jeko-1 cells, 10.1% and 27.6% respectively at 50 μg/ml. In conclusion, we have demonstrated that CpGs have divergent effects on normal versus malignant B cells in both mouse and human systems. Delivery of CpG to mouse lymphoma cells inhibited their growth in vivo, while normal mouse B cells were activated. Furthermore, CpGs directly inhibit the proliferation of a large panel of human B cell lymphomas representing the majority of aggressive histologies. These results provide a novel mechanism of action for CpGs as therapeutic agents for B cell lymphomas. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Somatic mutations at EZH2 Y641 act dominantly through a mechanism of selectively altered PRC2 catalytic activity, to increase H3K27 trimethylation

Blood ◽  
2011 ◽  
Vol 117 (8) ◽  
pp. 2451-2459 ◽  
Author(s):  
Damian B. Yap ◽  
Justin Chu ◽  
Tobias Berg ◽  
Matthieu Schapira ◽  
S.-W. Grace Cheng ◽  
...  
Keyword(s):  
B Cell ◽  
Mode Of Action ◽  
Cell Lymphoma ◽  
B Cell Lymphoma ◽  
Histone Methyltransferase ◽  
Dominant Mode ◽  
Wild Type ◽  
Allele Specific

Abstract Next-generation sequencing of follicular lymphoma and diffuse-large B-cell lymphoma has revealed frequent somatic, heterozygous Y641 mutations in the histone methyltransferase EZH2. Heterozygosity and the presence of equal quantities of both mutant and wild-type mRNA and expressed protein suggest a dominant mode of action. Surprisingly, B-cell lymphoma cell lines and lymphoma samples harboring heterozygous EZH2Y641 mutations have increased levels of histone H3 Lys-27–specific trimethylation (H3K27me3). Expression of EZH2Y641F/N mutants in cells with EZH2WT resulted in an increase of H3K27me3 levels in vivo. Structural modeling of EZH2Y641 mutants suggests a “Tyr/Phe switch” model whereby structurally neutral, nontyrosine residues at position 641 would decrease affinity for unmethylated and monomethylated H3K27 substrates and potentially favor trimethylation. We demonstrate, using in vitro enzyme assays of reconstituted PRC2 complexes, that Y641 mutations result in a decrease in monomethylation and an increase in trimethylation activity of the enzyme relative to the wild-type enzyme. This represents the first example of a disease-associated gain-of-function mutation in a histone methyltransferase, whereby somatic EZH2 Y641 mutations in lymphoma act dominantly to increase, rather than decrease, histone methylation. The dominant mode of action suggests that allele-specific EZH2 inhibitors should be a future therapeutic strategy for this disease.

The Acquirement of Rituximab Resistance Is Associated with a Global Downregulation of CD54 in B-Cell Lymphoma Cells: The Potential Role of Adhesion Molecules in Rituximab Anti-Tumor Activity

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 2607-2607
Author(s):  
Ping-Chiao Tsai ◽  
Naveen Bangia ◽  
Scott Olejniczak ◽  
Francisco J. Hernandez-Ilizaliturri ◽  
Myron Czuczman
Keyword(s):  
Flow Cytometry ◽  
B Cell ◽  
Adhesion Molecules ◽  
Cell Lymphoma ◽  
B Cell Lymphoma ◽  
Cell Aggregation ◽  
B Cell Lymphomas ◽  
Cell Clustering

Abstract Cell adhesion plays an important role in the cell-cell communication and provides important signals for cell survival, migration, aggregation, or other cell functions. Preclinical studies have been conducted to investigate the expression profiles of different adhesion molecules on the surface of malignant B-cells in an attempt to explain differences in the clinical behavior and patterns of spread between non-Hodgkin’s lymphoma (NHL) and chronic lymphocytic leukemia (CLL). Of interest, CLL cells have lower levels of both adhesion molecules and CD20 when compared to follicular lymphomas (FL). Recently, knockout studies had demonstrated that CD26, an adhesion molecule, modified responses to chemotherapy in B-cell lymphomas. It is unclear if the expression of adhesion molecules affects rituximab activity. To this end, we studied the patterns of cell aggregation and expression of adhesion molecules in a panel of rituximab-sensitive (RSCL) and rituximab-chemotherapy lymphoma cell lines (RRCL) that had been extensively characterized by our group (Czuczman S. et al. Clin Cancer Res.2008; 14:1561–70). Homotypic adhesion of B-cells is known to, due to the interaction of ICAM-1(CD54) and LFA-1(CD11a). Expression of CD54 and its ligand CD11a was studied by flow cytometry analysis and polymerase chain reaction (PCR, CD54 only). Patterns of cell aggregation in RSCL and RRCL in resting conditions were studied by inverted light microscopy. To define further the role of CD54 in B-cell aggregation and rituximab activity, RSCL (Raji and RL cells) were exposed to RPMI, rituximab (10mg/ml), isotype (10mg/ml) with or without a blocking anti-CD54 monoclonal antibody (0.25mg/ml) and patterns of cell aggregation were evaluated by inverted light microscopy, and photographs were captured at different time intervals. Experiments were conducted with or without the potent pan-caspase inhibitor Q-VD-OPh and performed in triplicates. Cell death was detected by propidium iodine staining and quantified by flow cytometry. Differences in the expression levels of CD54 were observed in the NHL cells tested. RRCL were found to have lower levels of CD54 at the surface protein and gene level. No differences in the CD11a were observed. RSCL aggregate and form clusters under culture conditions whereas RRCL do not aggregate in vitro. In vitro exposures to rituximab lead to a rapid cell clustering in RSCL. Blocking CD54 using mAbs prevented spontaneous and rituximab induced cell clustering, resulting in a phenotype similar to the RRCL. Of interest, in vitro exposure to anti-CD54 mAb and to a lesser degree rituximab resulted in apoptosis of RSCL, suggesting that cell adhesion is important for survival in B-cell lymphomas. The decrease in cell aggregation following CD54 blocking was not reduced by inhibition of caspase activation suggesting that cell death was not the dominant factor in preventing cell clustering in RSCL. In summary, our data suggests that CD54 is important for B-cell lymphoma cell aggregation and survival. Furthermore, blocking of CD54 appears to abolish the clustering effects of rituximab in vitro. Loss of CD54 is observed in rituximab-chemotherapy cell lines and may disrupt signaling events that control cell proliferation (i.e. pro- or anti-apoptotic proteins) rendering these cells resistant to rituximab and chemotherapy drugs. Ongoing studies in lymphoma severe combined immunodeficiency mice (SCID) are underway to further define the role of CD54 in the progression of B-cell lymphomas and responses to rituximab activity in vivo.

scholarly journals Targeted Therapy of B Cell Lymphoma with a Direct Inhibitor of the NF-κB Subunit c-Rel

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 507-507
Author(s):  
Yusuke Shono ◽  
Andrea Z. Tuckett ◽  
Hsiou-Chi Liou ◽  
Samedy Ouk ◽  
Ekaterina Doubrovina ◽  
...  
Keyword(s):  
B Cell ◽  
Cell Lines ◽  
Cell Lymphoma ◽  
B Cell Lymphoma ◽  
Tnf Α ◽  
Wide Range ◽  
In Vitro Treatment ◽  
Human B Cell

Abstract NF-kB plays important roles in immunity and oncogenesis, indicating that therapeutic targeting of this pathway could be beneficial in various clinical settings; however,an NF-kB-specific inhibitor does not exist in clinical practice to date. One approach toward development of such a compound is small-molecule-mediated direct inhibition of one or several members of the NF-kB family of transcription factors, a network that comprises five structurally related proteins including p50, p52, RelA, RelB and c-Rel. After screening of a library of 15,000 small molecules with a biochemical assay, we identified two scaffolds with inhibitory activity specific for the NF-kB subunit c-Rel. These scaffolds act as direct c-Rel inhibitors by modifying the conformation of the c-Rel protein, thus preventing DNA binding. We previously reported that in vitro treatment of T cells with the thiohydantoin IT-603 induces c-Rel deficiency, resulting in suppression of T cell alloactivation without compromising T cell activation triggered by recognition of tumor-associated or viral antigens (Shono et al., Cancer Discovery, 2014). Here, we for the first time demonstrate in vivo efficacy of a c-Rel inhibitor treatment regimen in mouse models of graft-versus-host disease (GVHD) and graft-versus-lymphoma (GVL), as well as xenograft models of human B cell lymphomas, revealing that inhibition of c-Rel activity allows not only for suppression of GVHD while retaining GVL activity, but it also mediates promising anti-lymphoma effects. We first show that the novel small molecule IT-901 is a more potent c-Rel inhibitor than IT-603 and has a superior pharmacokinetic profile. IT-901 displayed significantly improved in vivo efficacy, ameliorating GVHD while preserving the anti-lymphoma activity of T cells (Figure 1a,b). Recent genetic evidence has established a pathogenetic role for NF-kB signaling in lymphoid malignancies. We therefore sought to explore the potential of IT-901 for targeted therapy of human lymphomas. We analyzed six representative diffuse large B cell lymphoma (DLBCL) cell lines including activated B-like (ABC; HBL1, TMD8, U2932) and germinal center B-like (GCB; Ly19, SU-DHL4, SU-DHL8) cell lines for nuclear translocation of c-Rel and found that c-Rel was constitutively active in all cell lines. To examine if c-Rel inhibition with IT-901 alters cytokine production by DLBCL cells, we analyzed cytokine levels in the supernatant after in vitro incubation with IT-901. IT-901 treatment resulted in decreased levels of a wide range of cytokines in TMD8 cells, with the notable exceptions of interleukin 8 (IL-8), tumor necrosis factor (TNF)-α, and TNF-β (P<0.05, Figure 2a). We next investigated if IT-901 treatment affected growth of DLBCL cells in vitro. We found that IT-901 dose-dependently inhibited cell growth of both ABC and GCB cell lines with IC50 values between 3µM to 4µM. Interestingly, IT-901 at a concentration of 3µM did not have an anti-proliferative effect on TMD8 cells, suggesting that cytokines such as IL-8 and TNF-α may be upregulated as a mechanism of resistance to c-Rel inhibition by activating alternative survival pathways. Indeed, in vitro treatment of TMD8 cells with a TNF-α neutralizing antibody inhibited cell growth, and this effect was enhanced when combining TNF-α blockade with c-Rel inhibition (P<0.01, Figure 2b). Furthermore, we detected high HMOX1 protein levels in DLBCL cells treated with IT-901, suggesting that HMOX1 expression was induced, which is a hallmark of oxidative stress. Indeed IT-901 induced production of high levels of reactive oxygen species in lymphoma cells. This suggests that induction of oxidative stress may be a second mechanism contributing to the anti-lymphoma activity of IT-901. We next analyzed primary lymphoma cells and found that the c-Rel gene is widely expressed in human B cell malignancies and frequently amplified in DLBCL and EBV-transformed B cells. Importantly, intranuclear analysis of the c-Rel protein demonstrated that this transcription factor can be constitutively active in a wide range of human lymphomas. IT-901 efficiently inhibited growth of EBV-transformed B cells in vitro, and mediated significant anti-lymphoma activity in a xenograft model of EBV-induced lymphoma (P<0.01, Figure 2c). In summary, our findings underscore multiple therapeutic benefits and great potential for clinical translation of a novel c-Rel inhibitor. Figure 1 Figure 1. Figure 2 Figure 2. Disclosures No relevant conflicts of interest to declare.

scholarly journals Inhibition of human B-cell lymphoma growth by CD40 stimulation

Blood ◽  
1994 ◽  
Vol 83 (10) ◽  
pp. 2787-2794 ◽  
Author(s):  
S Funakoshi ◽  
DL Longo ◽  
M Beckwith ◽  
DK Conley ◽  
G Tsarfaty ◽  
...  
Keyword(s):  
B Cell ◽  
Cell Lymphoma ◽  
B Cell Lymphoma ◽  
Cell Surface Expression ◽  
B Cell Differentiation ◽  
Severe Combined Immune Deficiency ◽  
B Cell Lymphomas ◽  
Inhibition Of Proliferation ◽  
Cd40 Stimulation ◽  
Human B Cell

Abstract CD40 is a molecule present on B lymphocyte lineage cells that is important in B-cell differentiation and activation. Signaling through CD40 has been shown to exert costimulatory signals on normal B cells resulting in proliferative and differentiation responses. Examination of several B-cell lymphomas showed cell-surface expression of the CD40 molecule. Incubation of these lymphomas with anti-CD40 antibodies resulted in significant growth inhibition in vitro. Cross-linking of the CD40 antibodies resulted in even greater inhibition of proliferation. A recombinant soluble human CD40 ligand was also shown to inhibit lymphoma proliferation. When various human B-cell lymphomas were transferred into mice with severe combined immune deficiency, the treatment of the mice with anti-CD40 antibodies resulted in significant increases in survival showing that anti-CD40 is efficacious after in vivo administration. Thus, CD40 stimulation by either the antibody or soluble ligand directly inhibits human B-cell lymphoma growth and therefore, may be of significant clinical use in their treatment.

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