A Fully Human Anti-CD40 Antagonistic Antibody, CHIR-12.12, Inhibit the Proliferation of Human B Cell Non-Hodgkin’s Lymphoma.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 3279-3279 ◽  
Author(s):  
Wen-Kai Weng ◽  
Xia Tong ◽  
Mohammad Luqman ◽  
Ronald Levy
Keyword(s):  
Follicular Lymphoma ◽  
B Cell ◽  
Cell Lymphoma ◽  
B Cell Lymphoma ◽  
Expression Level ◽  
Primary Lymphoma ◽  
Immunoglobulin Gene ◽  
Dependent Manner ◽  
Lymphoma Cells

Abstract Immunotherapy using anti-tumor antibodies has become a feasible alternative for treating patients with lymphoma. These anti-tumor antibodies may target a specific receptor to disrupt proliferative signaling or mediate their anti-tumor effect by antibody-dependent cellular cytotoxicity (ADCC) or complement-mediated killing. The CD40 antigen is a good target for such anti-tumor antibodies for several reasons: CD40 is expressed on the vast majority of the non-Hodgkin’s B cell lymphomas and it has been proposed that the CD40/CD40L interaction provides a critical survival or proliferative signal for B cell lymphoma, especially the low-grade follicular lymphoma. In addition, B lymphoma cell lines become less sensitive to chemotherapy-induced apoptosis after CD40 cross-linking in an in vitro study. Therefore, an anti-CD40 antagonist that disrupts the CD40/CD40L interaction and mediates effector mechanism could have a therapeutic advantage. In this report, we describe a fully human anti-CD40 antagonistic IgG1 monoclonal antibody, CHIR-12.12 that was generated from mice with a human immunoglobulin gene loci (XenoMouse®mice, Abgenix Inc.). We first compared the antigen expression level of CD40 to the level of CD20, the target for rituximab, on primary lymphoma cells. While the expression level of CD40 was similar between different samples of primary follicular lymphoma cells, it was 10 fold less than the level of CD20. The expression of CD40 and CD20 on chronic lymphocytic leukemia/small lymphocytic lymphoma cells (CLL/SLL) was more variable. However, the level of CD20 was still significantly higher than the level of CD40 in all samples tested (2.4 to 13 fold). While CHIR-12.12 binds to primary lymphoma cells similarly to several other anti-CD40 antibodies, CHIR-12.12 did not induce proliferation of these primary tumore cells. By contrast, an agonist anti-CD40 antibody induced proliferation of these lymphoma cells up to 6-fold over baseline. To study the ability of CHIR-12.12 to interrupt the CD40-CD40L interaction, we cultured lymphoma cells with CD40L-transfected feeder cells in the presence of control IgG1, CHIR-12.12 or rituximab. In this system, the lymphoma cells proliferate in response to CD40-CD40L interaction. The addition of rituximab did not influence the proliferation. However, CHIR-12.12 inhibited the proliferation of follicular lymphoma and of CLL/SLL cells in a dose-dependent manner. The inhibition was observed with antibody concentration at 1 μg/ml and reached maximum of 90% inhibition at 10 μg/ml. We then evaluated the ability of CHIR-12.12 to elicit complement-mediated killing or ADCC. In vitro, rituximab induced complement-mediated cytotoxicity, while CHIR-12.12 did not. However, both CHIR-12.12 and rituximab induced effective ADCC of primary follicular lymphoma cells using purified NK cells from a healthy donor. Even though the level of CD40 is 10-fold less than the level of CD20 on the cell surface of these tumor cells, CHIR-12.12 induced the same degree of ADCC killing as did rituximab. Thus, this novel antagonist CHIR-12.12 antibody both blocks tumor-stimulatory CD40/CD40L interaction and mediates ADCC in the presence of a low number of target antigen. Our results support further development of this antibody to treat patients with B cell lymphoma.

Discrepancy of CD20 Protein Expression In IHC and FCM Analyses In Primary B-Cell Lymphoma: Relationship Between FCM-Negative Phenotype and Rituximab Binding with Lymphoma Cells

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 5087-5087 ◽  
Author(s):  
Takashi Tokunaga ◽  
Akihiro Tomita ◽  
Kazuyuki Shimada ◽  
Junji Hiraga ◽  
Takumi Sugimoto ◽  
...  
Keyword(s):  
B Cell ◽  
Research Funding ◽  
Cell Lymphoma ◽  
B Cell Lymphoma ◽  
Primary Lymphoma ◽  
Newly Diagnosed ◽  
Rt Pcr ◽  
Lymphoma Cells ◽  
Anti Cd20

Abstract Abstract 5087 Background Rituximab is an anti-CD20 chimeric-monoclonal antibody, and its effectiveness for treatment of CD20-positive B-cell lymphomas has been proven over the past 10 years. Although rituximab is now a key molecular targeting drug for CD20-positive lymphomas, some patients with rituximab resistance have emerged. We previously reported that the CD20-protein-negative phenotypic change after using rituximab is one of the critical mechanisms in rituximab resistance (Hiraga J, Tomita A, et al., Blood, 2009., Sugimoto T, Tomita A, et al., Biochem Biophys Res Commun, 2009.). Recently, we have recognized that some newly-diagnosed B-cell lymphomas show CD20-protein-positive in immunohistochemistry (IHC) but -negative in flow cytometry (FCM) analyses. For these patients, so far, neither the molecular mechanisms of CD20 IHC(+)/FCM(−) phenotype, nor the relationship between this phenotype and rituximab resistance are clear. Thus, the clinical significance of introducing rituximab therapy for these patients must be elucidated. Aims Analyses of the molecular backgrounds of CD20 IHC(+)/FCM(−) phenotype in primary B-lymphoma cells, and confirmation of the effectiveness of rituximab therapy for the patients who show CD20 IHC(+)/FCM(−) phenotype. Results Primary B-cell lymphoma (diffuse large B-cell (DLBCL), follicular, MALT, mantle cell, and Burkitt) tissues and cells were analyzed by IHC and FCM. Four newly-diagnosed B-cell lymphoma patients showed IHC CD79(+)/CD20(+) and FCM CD19(+)/CD20(−) phenotype using anti-CD20 antibodies L26 for IHC and B1 for FCM, and all were diagnosed as DLBCL. Chromosomal analysis showed complex karyotypes in 3 out of 3 patients analyzed, and no shared abnormalities were confirmed. Primary lymphoma cells from 3 patients were available for further molecular analyses, and the genomic DNA, the total RNA, and the protein from whole cell lysate were obtained from these lymphoma cells. DNA sequencing analysis indicated no significant genetic mutations on the coding sequences (CDS) of MS4A1 (CD20) gene. Semi-quantitative and quantitative RT-PCR indicated that CD20 mRNA expression was almost normal in 2 patients and ≂~f10 times lower in 1 patient compared to the positive control B-lymphoma/leukemia cells. Almost the same expression tendency with RT-PCR was confirmed in immunoblot analysis using whole cell lysate and the two different anti-CD20 antibodies. The molecular weight of the CD20 protein in immunoblotting corresponded to the wild type in these patients. Rituximab binding assay in vitro was performed using primary lymphoma cells from a patient and the fluorescent-labeled rituximab (Alexa488-rituximab). Interestingly, rituximab binding on the surface of the CD19 positive lymphoma cells was confirmed in vitro. Rituximab containing combination chemotherapy was performed, resulting in complete response in all 4 cases after completing 4 to 8 courses. Conclusions and Discussion CD20 IHC(+)/FCM(−) phenotype was confirmed in newly-diagnosed DLBCL patients. Significant abnormalities in CD20 protein and mRNA expression in immunoblotting and RT-PCR were not confirmed, and genetic mutations on CDS of MS4A1 gene, resulting in the conformation change of CD20 protein, were not detected. The possibility of abnormal post-translational modification or aberrant localization of CD20 protein, leading to interference with antibody binding, can not be excluded. Rituximab binding with CD19-positive primary lymphoma cells was confirmed in a patient, suggesting that CD20 IHC(+)/FCM(-) phenotype does not directly indicate the ineffectiveness of rituximab for these cells. Further investigations, performing in vitro CDC and ADCC assay using primary lymphoma cells, are still warranted to show rituximab effectiveness and sensitivity to those cells. Disclosures: Kinoshita: Zenyaku Kogyo Co.: Research Funding; Chugai Pharmaceutical Co., Ltd.: Research Funding. Naoe:Zenyaku Kogyo Co.: Research Funding; Chugai Pharmaceutical Co., Ltd.: Research Funding.

The Green Tea Extract Epigallocatechin Induces In Vitro Cell Death in Primary Human Lymphoma Cells through an ROS Dependent Mechanism.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 234-234 ◽  
Author(s):  
Tait D. Shanafelt ◽  
Yean K. Lee ◽  
Susan M. Geyer ◽  
Deanna Grote ◽  
Mary Stenson ◽  
...  
Keyword(s):  
Cell Death ◽  
B Cell ◽  
Cell Lines ◽  
Green Tea ◽  
Cell Lymphoma ◽  
B Cell Lymphoma ◽  
Lymphoma Cell ◽  
Primary Lymphoma ◽  
Lymphoma Cells

Abstract BACKGROUND: We have demonstrated the green tea extract epigallocatechin-3-gallate (EGCG) has anticancer activity in primary CLL B-cells (Lee, Blood 2004). After dissemination of our in vitro findings by the lay press, many patients with CLL and other low grade non-Hodgkin lymphomas (NHL) began using over the counter green tea extracts as an alternative treatment strategy. We recently reported a case series of 3 patients with CLL and 1 patient with follicular lymphoma who appeared to derive objective clinical benefit from such treatment (Shanafelt, Leukemia Research 2006). Based on these findings EGCG has entered clinical testing for treatment of CLL at Mayo Clinic. Here we explore the in vitro antitumor activity of EGCG against other types of non-Hodgkin lymphoma. METHODS: Five established human B-cell lymphoma cell lines (HT, DOHH2, KARPAS, Ramos, RL) and primary lymphoma cells from 7 patients with various B-cell NHL sub-types [DLBC, FL, SMZ (2), MCL, SLL(2)] were used to evaluate the in vitro sensitivity of human lymphoma cells to EGCG. Freshly isolated primary lymphoma cells harvested in suspension from lymph nodes/spleen were obtained from patients with NHL who provided written informed consent. All patients were untreated at the time of biopsy. Lymphoma cell lines and primary lymphoma cells (n=7) were cultured with increasing doses of purified EGCG (3.12–50 ug/ml) for 24–72 hrs. Viability was assessed by using annexin/PI staining by FACS analysis. RESULTS: EGCG-induced dose dependent cell death in both established human B-cell lymphoma cell lines (average LD50 at 24 hrs between 25–50 ug/mL) and primary NHL cells (average LD50 at 24 hrs between 25–50ug/mL). In contrast, EGCG had minimal effect on purified normal B-cells (n=3) at the highest doses tested (50 ug/mL). By immunoblotting, EGCG-induced death in primary cells and cell lines was associated with PARP cleavage, suggesting the agent induced apoptotic cell death. Despite this finding, EGCG had no effect on levels of MCL-1, XIAP, or Bcl-1 by either immunoblot or FACS analysis. Based on reports that EGCG induces cell death in some cancer cell types through generation of oxidative stress (Furukawa, 2003; Nakazato, 2005), we explored this mechanism in lymphoma cells. To determine whether reactive oxygen species (ROS) generation was necessary for EGCG-induced cell death, lymphoma cell lines were cultured with or without catalase (which catalyzes the conversion of hydrogen peroxide to water and oxygen) for 30 min prior to subsequent 24 hr EGCG exposure (50 and 100 mg/ml). Pre-treatment with catalase (100 U) provided dramatic protection against cell death in both primary NHL cells and NHL cell lines suggesting that EGCG-induced cell death in lymphoma cells is dependent on ROS generation (Fig. 1 shows an example for a patient with mantle cell lymphoma and a patient with splenic marginal zone lymphoma). CONCLUSION: EGCG has in vitro anti-tumor activity against a variety of B-cell NHLs. Given its known favorable toxicity profile in vivo, EGCG is an attractive agent for clinical testing in patients with indolent NHL who otherwise are currently being managed with observation. Figure Figure

Pathologic Co-Expression and Physical Interaction of c-MYC and BCL6 in B-Cell Lymphomas.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 2-2 ◽  
Author(s):  
Masumichi Saito ◽  
Ryan T. Phan ◽  
Herbert C. Morse ◽  
Laura Pasqualucci ◽  
Riccardo Dalla-Favera
Keyword(s):  
B Cells ◽  
B Cell ◽  
Cell Lymphoma ◽  
Somatic Hypermutation ◽  
B Cell Lymphoma ◽  
Transgenic Animals ◽  
Primary Lymphoma ◽  
Physical Interaction

Abstract Deregulated expression of the proto-oncogenes BCL6 and c-MYC caused by chromosomal translocation or somatic hypermutation is common in non-Hodgkin B cell lymphoma derived from germinal center (GC) B cells, including diffuse large cell lymphoma (DLBCL) and Burkitt lymphoma (BL). Normal GC B cells express BCL6, whereas, surprisingly, they do not express c-MYC, suggesting that the expression of this oncogene in BL and DLBCL (20% of cases) is ectopic (Klein, U. et al. Proc Natl Acad Sci U S A100, 2639–2644, 2003). Here we report that c-MYC is absent in proliferating GC B cells because it is transcriptionally suppressed by BCL6, as demonstrated by the presence of specific BCL6 binding sites in the c-MYC promoter region and by chromatin immunoprecipitation experiments showing that BCL6 is bound to these sites in vivo. Thus, c-MYC escapes BCL6-mediated suppression in lymphoma leading to the co-expression of the two transcription factors, an event never observed in immunohistochemical and gene expression profile analysis of normal GC B cells. Surprisingly, co-immunoprecipitation experiments and in vitro binding experiments indicate that, when co-expressed, BCL6 and c-MYC are physically bound in a novel complex detectable in DLBCL and BL cell lines as well as in primary lymphoma cases. The formation of the BCL6/c-MYC complex has several significant functional consequences on the function of both c-MYC and BCL6: 1) a two fold, BCL6-binding dependent increase in c-MYC half-life, an event that has been shown to contribute to its oncogenic activation; 2) a synergistic increase in the ability of both BCL6 and c-MYC to suppress MIZ1-activated transcription of the p21CIP cell cycle arrest gene; 3) MYC-dependent inhibition of BCL6 acetylation by p300, an event that physiologically inactivates BCL6 via c-MYC-mediated recruitment of HDAC. Notably, the pathologic co-expression of c-MYC and BCL6 was shown to have pathologic consequences in vivo, since double transgenic BCL6/c-MYC mice display accelerated lymphoma development and the appearance of a novel GC-derived tumor phenotype not recognizable in single transgenic animals and containing the pathologic c-MYC/BCL6 complex. Thus, the pathologic co-expression and illegitimate physical interaction of BCL6 and c-MYC leads to an increase in the constitutive activity of both oncogenes. These results identify a novel mechanism of oncogenic function for BCL6 and c-MYC and a novel tumor-specific protein complex of potential therapeutic interest.

The Effect of Iodine-131-Rituximab on B Cell Lymphoma In Vitro and In Vivo.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 4512-4512
Author(s):  
Rongcheng Luo ◽  
Qiang Zuo ◽  
Li Wei ◽  
Wangjun Liang ◽  
Dayong Zhen ◽  
...  
Keyword(s):  
B Cell ◽  
Cell Lymphoma ◽  
Raji Cell ◽  
B Cell Lymphoma ◽  
Lethal Effect ◽  
Dependent Manner ◽  
Raji Cells ◽  
Iodine 131

Abstract The purpose of this study was to investigate the cell specific cytotoxic effect of Iodine-131 Rituximab on CD20-positive B cell lymphoma in vitro and on Raji cell tumors grown in vivo. Rituximab was labeled with Iodine -131 by the iodogen method. Cultured Raji cells or the nude mice bearing Raji tumors were treated with various concentrations of Iodine-131-Rituximab or Iodine-131 alone or Rituximab alone. The results showed that The lethal effect was found on Raji cells treated with Iodine-131-Rituximab in a dose-dependent manner; The proliferation rate of Raji cells was significantly lower in cells treated with Iodine-131-Rituximab, as compared to the cells treated with Iodine-131or Rituximab alone (P<0.05); Tumor inhibition was found to be greatest in the mice treated with Iodine-131-Rituximab through intratumor injection, as compared with Iodine-131-Rituximab i.p. injection or Rituximab alone (p<0.05). We conclude that Iodine-131-Rituximab specifically inhibits the growth of Raji tumor cells in vitro and in vivo. Iodine-131-Rituximab is a promising agent for radioimmunotherapy that targets CD20-positive B cell lymphoma.

Rituximab Activation of AKT and HIF Pathways Is Dependent on Membrane Lipid Raft Cholesterol Levels in Lymphoma Cells

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 1571-1571
Author(s):  
Yumi Nozaki ◽  
Toru Mitsumori ◽  
Norio Komatsu ◽  
Keita Kirito
Keyword(s):  
B Cell ◽  
Lipid Raft ◽  
Cell Lymphoma ◽  
B Cell Lymphoma ◽  
Membrane Cholesterol ◽  
Dependent Manner ◽  
Lymphoma Cells ◽  
Akt Activation ◽  
Cholesterol Levels ◽  
Induced Apoptosis

Abstract Abstract 1571 Rituximab, which is a monoclonal antibody directed against CD20 proteins, has significantly improved the treatment outcome of B-cell lymphoma patients. Recent studies have revealed that the lipid components of the membrane microdomain, also known as the lipid raft, determine the biological function and efficiency of the antibody. The raft-associated sphingolipid GM1 level also affects the susceptibility of lymphoma cells to rituximab. Clinical observations have suggested that the use of statins may affect the efficiency of rituximab by modulating lipid raft cholesterol levels. In the present study, we investigated whether differences in lipid raft components affected rituximab-induced intracellular signaling pathways and the biological activity of the antibody. Initially, we analyzed the membrane cholesterol and GM1 levels in several B-cell lymphoma cells (Raji, RL,Namalwa and Ramos cells). We found that two cell lines (Raji and RL cells) have higher cholesterol levels compared with Namalwa and Ramos cells; however, Namalwa and Ramos cells have higher GM1 expression compared with Raji and RL cells. Interestingly, rituximab clearly activated the PI3K/AKT pathway in the cholesterol-rich cells (Raji and RL cells). Conversely, treatment with rituximab suppressed the basal activity of AKT in the GM1-rich cells (Namalwa and Ramos cells). We also investigated whether cholesterol levels or the GM1 level affected rituximab-induced PI3K/AKT activation. We treated the cholesterol-rich cells with methyl-β-cyclodextrin (MβCD) to deplete cholesterol from the lipid rafts. Treatment with MβCD clearly disrupted rituximab-induced AKT activation. Importantly, cholesterol replacement restored rituximab-induced AKT activation. In contrast, treatment with D-threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol (PDMP), which inhibits the synthesis of GM1, did not reverse rituximab-induced AKT suppression in the GM1-rich cells. These results suggest that lipid raft cholesterol levels, but not the GM1 level, determine rituximab-induced AKT activation. We also examined the biological significance of rituximab-induced AKT activation in lymphoma cells. Although AKT activates a variety of downstream molecules, we focused our attention on hypoxia-inducible factor (HIF) because recent studies have revealed that abnormal expression of the alpha subunit of HIF-1 (HIF-1α) is frequently found in lymphoma cells. In agreement with the finding that rituximab induced AKT activation, treatment with rituximab markedly increased the expression of HIF-1α in the cholesterol-rich cells. In contrast, rituximab reduced the basal HIF-1α level in the GM1-rich cells. Interestingly, rituximab enhanced the expression of the anti-apoptotic protein survivin in a HIF-1-dependent manner in Raji and RL cells. In addition, rituximab suppressed the chemotherapeutic reagent-induced apoptosis of Raji and RL cells. Interestingly, depletion of membrane cholesterol by MβCD completely blocked all these processes. In conclusion, rituximab exerts different effects on lymphoma cells that are dependent on lipid raft cholesterol levels. Our observations suggest that a high level of membrane cholesterol may diminish rituximab-induced apoptosis through AKT activation and subsequent induction of HIF-1α. Importantly, a reduction of membrane cholesterol may enhance the efficiency of rituximab. Disclosures: No relevant conflicts of interest to declare.

Apoptotic and Non-Apoptotic Cellular Pathways Executed by Bortezomib (BTZ) in Rituximab-Resistant Lymphomas

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 4967-4967
Author(s):  
Juan Gu ◽  
Francisco J. Hernandez-Ilizaliturri ◽  
Cory Mavis ◽  
Natalie M Czuczman ◽  
Karen E Thudium ◽  
...  
Keyword(s):  
Cell Cycle ◽  
B Cell ◽  
Tumor Cells ◽  
Research Funding ◽  
Cell Lymphoma ◽  
B Cell Lymphoma ◽  
Mitotic Catastrophe ◽  
M Cell ◽  
Lymphoma Cells

Abstract Abstract 4967 Rituximab-chemotherapy relapsed/refractory (r/r) B-cell lymphomas represent an emerging clinical challenge that underlies the need to develop alternative therapeutic strategies. A better understanding of the mechanism(s)-of-action of BTZ and other proteasome inhibitors (PI) is likely to aid in the identification of biomarkers that can be used to determine clinical responsiveness and/or help in the rational development of novel PI-based therapeutic combinations (e.g. incorporating biologics, small molecules and/or chemotherapy) in r/r B-cell lymphoma. Previously we demonstrated that rituximab resistance was associated with increased proteasome activity leading to a de-regulation in the apoptotic threshold of lymphoma cells to multiple chemotherapy agents. Pharmacological and genetic (e.g. siRNA silencing of BAK/BAX) inhibition of apoptosis partially affected BTZ activity in rituximab-resistant (RSCL) but not in rituximab-sensitive cell lines (RSCL) suggesting the existence of alternative pathways of cell death associated with PI exposure. To this end we evaluated the contribution of cellular senescence, cell cycle inhibition, or mitotic catastrophe to the anti-tumor activity of BTZ as a single agent or in combination with chemotherapeutic agents in RSCL, RRCL and in primary tumor cells. Lymphoma cells were exposed to BTZ (10-25nM) for 24–48 hrs. Cell senescence was determined by SA-β-gal staining using a senescence assay kit and inverted phase-contrast microscopy was performed. Changes in cell cycle were analyzed by the FACScan DNA method and changes in cell cycle regulatory proteins (i.e. cdc2, cyclinA/B, p21, CDK2/4/6) were analyzed by Western blotting. Mitotic index was determined by Wright-Giemsa stain and positive cells were counted under a Nikon microscope. Mitotic catastrophe was determined by confocal microscopy by staining with α-tubulin antibody. Finally, changes in ATP content was determined by the Cell Titer Glo assay. Baseline differences were observed between RSCL and RRCL in terms of cell morphology, proliferation rate and senescence. RRCL (Raji2R and Raji4RH) were considerably larger in size, had a slower proliferation rate and an exhibited a 3-fold increase the number of cells in senescence than RSCL. In vitro exposure of RSCL and RRCL to BTZ attenuated the number of cells in senescence by 50–75%. Cell cycle analysis demonstrated that RRCL had more cells in S phase when compared to RSCL. In vitro exposure to BTZ-induced G2/M arrest in RRCL, but not in RSCL. Overexpression of G2/M cell cycle regulatory proteins cyclin B and cdc2 were observed in RRCL and in tumor cells isolated from r/r B-cell lymphoma patients. Mitotic catastrophe with multi-nucleated cells were only detected in RRCLs exposed to BTZ. In vitro and ex vivo exposure of RSCL and RRCL to BTZ potentiated the cytotoxic effects of paclitaxel and overcame the acquired resistance to chemotherapy drugs in RRCL and primary tumor cells isolated from r/r lymphoma patients in a dose-dependent manner. Our results suggested that BTZ activates several death pathways in B-cell lymphoma pre-clinical models. In addition to apoptosis, BTZ is capable in triggering mitotic catastrophe in rituximab-chemotherapy lymphoma cells with decreased levels of pro-apoptotic proteins. Moreover, sensitization of RRCL to drug therapy involves interplay between cellular senescence attenuation, G2/M cell cycle regulation, and mitotic catastrophe. Hence, proteasome inhibition may provide a novel therapeutic approach for treating apoptosis-resistant B-cell lymphoma. Research, supported in part as a subproject of NIH grant R01 CA136907-01A1 awarded to Roswell Park Cancer Institute. Disclosures: Hernandez-Ilizaliturri: Genmab: Research Funding; Amgen: Research Funding; Celgene: Consultancy. Czuczman:Millennium: Honoraria, Research Funding.

Cross-Species Investigations Reveal Role, Mechanism and Predictive Power of Paracrine, Secondary Senescence in B-Cell Lymphoma Therapy

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 3715-3715
Author(s):  
Jan R. Dörr ◽  
Maja Milanovic ◽  
Yong Yu ◽  
Julia Kase ◽  
Dido Lenze ◽  
...  
Keyword(s):  
B Cell ◽  
Predictive Power ◽  
Conflicts Of Interest ◽  
Cell Lymphoma ◽  
B Cell Lymphoma ◽  
Brdu Incorporation ◽  
Lymphoma Cells ◽  
Long Term Outcome

Abstract Abstract 3715 Apoptosis and cellular senescence operate as anti-tumor safeguard mechanisms. Unlike apoptotic cells, senescent cells remain viable, and, hence, may crosstalk to other cells in their vicinity over extended periods of time. In fact, cells that entered oncogene-induced senescence or anticancer therapy-induced senescence (TIS) present with a senescence-associated secretory phenotype (SASP), a massive production of secretable factors, which reportedly reinforces senescence through an intracellular mechanism. Utilizing the Eμ-myc transgenic mouse lymphoma model, we provide evidence for an outcome-relevant paracrine, DNA damage-independent secondary senescence program (SecS) in vitro and in vivo. Apoptosis-blocked (bcl2-infected) lymphoma cells from different genetic backgrounds were treated with the DNA-damaging anticancer agent adriamycin in vitro or the alkylating agent cyclophosphamide upon lymphoma formation in mice in vivo. TIS and SecS was detected based on senescence-associated b-galactosidase activity (SA-b-gal), Ki67 staining and BrdU incorporation. The secretome of senescent cells was analyzed by proteomics, gene expression and protein arrays. Overall and progression free survival in mice and patients was assessed by Kaplan-Meier analysis. Transcriptome and secretome analyses followed by functional studies found extracellular matrix proteins, especially small leucine-rich proteoglycans (SLRP), but not NF-kB-dependent cytokines and chemokines, to induce SecS in proliferating lymphoma cells in a paracrine fashion, and linked a “high secretor” status to stronger SecS induction. Dissecting senescence-mediating pathways in recipient cells by biochemical, genetic and pharmacological means unveiled an essential role for the LDL receptor-related protein 1 (LRP1), a receptor for SLRP and other SASP components, through the cell-cycle inhibitor p21CIP1 in SecS. Accordingly, mice harboring TIS-capable but genetically SecS-defective lymphomas (e.g. lacking LRP1 or p21CIP1 expression) experienced inferior long-term outcome to therapy. Not only the recipient cell-based LRP1 status but also the genetically and biologically distinct donor cell-based secretor gene signature stratified outcome in mice. Strikingly, humanized versions of both classifiers were predictive in a large cohort of diffuse large B-cell lymphoma (DLBCL) patients, where they identified – although composed of different gene sets – largely overlapping patient subgroups with superior prognosis, again suggesting SecS as the critical underlying treatment effector principle. Our study highlights the predictive power of senescence for treatment outcome in DLBCL, and provides functional examples (which will be discussed at the meeting) for SASP-related non-genotoxic pro-senescent therapies. Disclosures: No relevant conflicts of interest to declare.

Bufalin inhibits human diffuse large B-cell lymphoma tumorigenesis by inducing cell death through the Ca2+/NFATC1/cMYC pathway

2020 ◽  
Author(s):  
Jincheng Song ◽  
Dan Zou ◽  
Xiaoxuan Zhao ◽  
Yang Chen ◽  
Fei Lv ◽  
...  
Keyword(s):  
Antitumor Activity ◽  
B Cell ◽  
Cell Lymphoma ◽  
B Cell Lymphoma ◽  
Dependent Manner ◽  
Multiple Cancer ◽  
Large B Cell Lymphoma ◽  
Large B Cell

Abstract The 5-year survival rate of diffuse large B-cell lymphoma (DLBCL) can reach 60%. However, nearly half of patients undergo relapse/refractory issues with a survival period of less than 2 years. New therapeutic approaches are therefore needed to improve chemotherapy efficacy and patient survival. Bufalin (BF), isolated from the traditional Chinese medicine Chansu, has been reported to play an anticancer role in multiple cancer cell types. However, there are few reports of the effects of BF on the growth of DLBCL. In the present study, we demonstrated that BF exerts antitumor activity in DLBCL cells, both in vitro and in vivo. Treatment of DLBCL cells with BF resulted in increased proliferation and apoptosis in a dose- and time-dependent manner. Daily intraperitoneal injection of 1.5 mg/kg BF significantly delayed DLBCL xenograft growth in NOD/SCID mice without affecting body weight. Bioinformatics analysis showed that BF may regulate NFATC1 protein and affect expression of its downstream gene, cMYC. Our results suggest that BF can attenuate NFATC1 translocation by reducing the intracellular calcium concentration; BF may also have a low synergistic effect with cyclosporin A. In conclusion, we demonstrated that BF exerts antitumor activity that is mediated at least in part by the Ca2+/NFATC1/cMYC pathway. Our findings suggest that BF can be effectively applied as a novel potential therapeutic agent for DLBCL.

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