Burkitt's Lymphoma Cell Lines Research Articles

CX-4945, an Orally Bioavailable Selective Inhibitor of Casein Kinase 2 (CK2), Exhibits Anti-Tumor Activity in Chronic Lymphocytic Leukemia and Exhibits Synergy When Combined with the PI3K-Δ Inhibitor GS-1101 or Fludarabine.

Abstract 2471 Background:Casein kinase 2 (CK2) is a highly conserved and constitutively active serine-threonine protein kinase that plays a fundamental role in maintaining cell survival through pro-proliferative, anti-apoptotic and pro-angiogenic signaling. CK2 promotes malignant cell viability by regulating critical downstream signaling pathways including PI3K/AKT, and overexpression and hyperactivation of this kinase has been well described in B-cell malignancies including chronic lymphocytic leukemia (CLL) and multiple myeloma (MM).In B-cell malignancies, the B-cell receptor (BCR) represents another key survival pathway. BCR activation results in downstream signaling of multiple pro-survival kinases including PI3K and AKT. Drugs that inhibit the transmission of signals through the BCR have been shown to reduce cell survival. Given that BCR-mediated kinase signaling relies on the convergence of many regulatory inputs, we hypothesize that inhibiting this pathway at more than one site would be most effective at killing cells.CX-4945 is the first orally bioavailable small molecule inhibitor of CK2 to progress to human clinical trials. CX-4945 has been found to be highly selective for CK2 and has been shown to inhibit AKT phosphorylation. We have previously reported pre-clinical single agent activity of CX-4945 across a broad range of primary hematologic malignancy samples including CLL. Many of the samples sensitive to CX-4945 were also sensitive to other inhibitors of the BCR pathway. Here we present our findings combining CX-4945 with GS-1101, the isoform selective inhibitor of PI3KΔ, or fludarabine in CLL. Methods:Fresh peripheral blood mononuclear cells from CLL patients were purified using a Ficoll-Paque gradient. Purified cells were then added to wells (5 × 104 per well) containing HS-5 stromal cell conditioned media and serial dilutions (ranging from 4.5 nM to 10 μM) of CX-4945 alone and in combination with serial dilutions of GS-1101 and fludarabine (both ranging from 4.5 nM to 10 μM). Cells were cultured for 72 hours and a tetrazolium-based MTS assay was performed to measure cell viability. Synergy analysis was performed using R or CalcuSyn.Ramos, a Burkitt's lymphoma cell line, and primary patient samples were treated with increasing concentrations of CX-4945 alone and in combination with GS-1101, and phosphorylation of the BCR pathway including AKT and ERK were evaluated by immunoblotting. Results:Synergy was observed between CX-4945 and GS-1101 in all samples tested (9 primary CLL patient samples). The median combination index (CI) for all samples was < 1, which indicates overall synergy between CX-4945 and GS-1101. The mean IC50 of CX-4945 alone was 4.3 μM, and the mean IC50 for GS-1101 alone was 4.2 μM suggesting limited single agent activity of each individual drug in the samples tested. However, in combination, the mean IC50 was reduced to 321 nM. Synergy was also observed in 3 of 4 CLL samples treated with a combination of CX-4945 and fludarabine.Downstream targets of CK2 and the BCR were examined. Decreased levels of phospho-AKT and phospho-ERK were seen in response to treatment with CX-4945 alone and in combination with GS-1101. Conclusions:Our data confirms that CX-4945 demonstrates significant pre-clinical activity in CLL when combined with GS-1101 or fludarabine. As such, CX-4945 may be an attractive compound for clinical development in lymphoid malignancies in combination with other agents, especially those that target other kinases. Additional studies focused on evaluating the efficacy of CX-4945 in combination with other inhibitors of the BCR signaling pathway and in combination with other cytotoxic chemotherapies are underway and will be presented. Disclosures:Druker:Gilead: Consultancy, Membership on an entity's Board of Directors or advisory committees; Cylene: Consultancy, Membership on an entity's Board of Directors or advisory committees. Spurgeon:Gilead: Research Funding.

BTK inhibitor PCI-32765 targets endogenous and microenvironment-mediated B-cell receptor signaling to suppress lymphoma cell growth and is highly synergistic with Bortezmib against non-Hodgkin B-cell lymphomas

Abstract 4721 Background:B-cell receptor (BCR) signaling is a critical pathway in the pathogenesis of B-cell malignancies and Bruton tyrosine kinase(Btk) is essential for BCR signaling and function. PCI-32765, a specific and irreversible small-molecule Btk inhibitor, has recently been reported to display a significant clinical activity against non-Hodgkin B-cell lymphomas (NHL) especially chronic lymphocytic leukemia and small lymphocytic lymphoma (CLL/SLL). In this study we set to explore 1) the role of Btk in NHL cell apoptosis and proliferation, 2) the role of BtK in bone marrow strom-mediated lymphoma cell survival and 3) to test if PCI-32765 as a therapeutic agent in single or in combination with Bortezomib for NHL therapy. Methods:B-cell lymphoma cell lines including mantle cell lymphoma lines (Jeko-1 and HBL-2), Burkitt lymphoma cell line (Raji) and transformed large cell lymphoma cell line (SUDHL-10) as well as primary lymphoma cells from various NHL samples were used for the experiments. These cells were cultured in the presence or absence of bone marrow mesenchymal stromal cells (MSC). The endogenous and MSC-induced Btk and its signaling activation such as BtK, ERK1/2 and AKT expression and phosphorylation status as well as its inhibition by were examined PCI-32765 by Western blot. The effects of PCI-32765 on lymphoma cell growth and appotosis were analyzed by using MTT, DAPI stain and flow cytometric annexin V/PI staining. Furthermore, the combined effect of PCI-32765 and Bortezomib on lymphoma cell growth and apoptosis was analyzed using the CalcuSyn software program in search for a synergistic or additive effect. Results:We found constitutive expression and activation of Btk and its downstream signaling in most of these cell lines and primary lymphoma cells. Furthermore, co-culture with MSC cells further enhanced the phosphoration of Btk and AKT in these cells. Incubation of Jeko-1, Raji, HBL-2 and SUDHL-10 cell lines with PCI-32765 induced cell growth inhibitory effects. We found that PCI-32765 exhibited a significant dose-dependent induction of cytotoxicity in these cells at various time points as measured by MTT. We also found significant apoptosis in these cells treated with PCI-32765. In addition, PCI-32765 significantly inhibited phpsphorylation of AKT and Btk, confirming the block of BCK signal pathways in these cells. Finally, MTT assays indicated that combined PCI-32765 with Bortezomib induced a synergistic cytotoxicity against these NHL cells (CI<1). Discussion:Our studies therefore highlight the biological significance of Btk in B-cell lymphoma cell growth and survival. PCI-32765 effectively antagonizes B-cell survival provided by bone marrow stromal cells and synergistically in combination with Bortezomib eliminates lymphoma cells. This study provide rational for targeting BCR and Btk as a novel therapeutic approach for NHL. Disclosures:No relevant conflicts of interest to declare.

The PI3K Inhibitor GS-1101 (CAL-101) Synergistically Potentiates HDAC-Induced Proliferation Inhibition and Apoptosis Through the Activation of JNK in Lymphoma Cells

AbstractAbstract 3714Pre-clinical studies demonstrated the potential therapeutic value of targeting the PI3K Delta (PI3Kδ) pathway in lymphoma. A selective PI3Kδ inhibitor, GS-1101, shows promising single agent response rates in patients with a variety of lymphoid malignancies. Previously, we showed that inhibition of PKCβ/AKT pathways (pathways that augments survival and proliferation signals) augments histone deacetylase (HDAC) inhibitor-induced stress response, and the irreversible engagement of the apoptotic cascade in lymphoma cells. In the present study, we investigated the cytotoxicity and mechanisms of cell death induced by GS-1101 in combination with HDAC inhibitors panobinostat (LBH589) and suberoylanilide hydroxamic acid (SAHA).Cytotoxicity was determined by WST-1 proliferation assay. Apoptosis was measured by flow cytometry, caspase activation and PARP cleavage. Cell signaling transduction pathways were analyzed by Western blot and by phosphoproteome array.Six DLBCL, two MCL and two Burkitt lymphoma cell lines were treated with different concentration of GS-1101. To determine the molecular mechanisms by which GS-1101 decreases the viability in various cell lines differently, three cell models (SU-DHL-6 as the most sensitive cells, SU-DHL-16 with moderate sensitivity, and Raji the most resistant cells) were selected for phosphoproteome profiling. SU-DHL-6 cells with IC50 only 0.2 μM had constitutively active AKT pathway and highly active ERK pathway. GS-1101 treatment significantly decreased the level p-AKT in this cell line, but more interestingly it also resulted in a decrease in the level of p-ERK. Significant dephosphorylation of ERK was observed also in SU-DHL-16 cells, however with a minimal effect on p-AKT levels. In contrast, the analysis of Raji cells highlighted a very low activity of AKT pathway and no changes in p-ERK levels after GS-1101 treatment.To enhance the effect of GS-1101, SU-DHL-6/16 cells and NHL primary cells were simultaneously treated with the HDAC inhibitor LBH589. An interaction of the LBH589/GS-1101 combination was formally examined by using various concentrations of LBH589 and GS-1101. Combined treatment resulted in a synergistic inhibition of proliferation in all tested cells and induced a significant increase of caspase-dependent apoptosis in SU-DHL-6/16 cells. For example, in SU-DHL-6 cells co-treatment of 0.1 μM GS-1101 with 2.5 nM LBH589 induced 62% increase of apoptotic population in comparison to the untreated sample, while GS-1101 alone induced 13% increase and LBH589 induced 11% increase. Moreover, substituting SAHA for LBH589 suggests that the synergy between GS-1101 and LBH589 is a class effect between HDAC inhibitors and GS-1101, rather than specific to LBH589.Mechanistically, in SU-DHL-6 cells, LBH589 single treatment increased the level of many phosphoproteins; such as p-ERK, p-AKT, p-CREB, p-AMPKα1/2, p-GSK3α/β, p-HSP27, p-MEK1/2, p-MSK1/2, p-P38a, and p-STAT2. Phosphoproteome analysis of cells treated simultaneously with both agents mostly recapitulated the profile of cells treated with GS-1101 only. Exceptions are p-Chk2, p-c-Jun, p-JNK, and p-STAT5, where levels were significantly higher than in either GS-1101 or LBH589 single treatments. This suggests that GS-1101 and LBH589 may enhance stress-related responses through JNK pathway.These data suggest that PI3Kδ inhibition in combination with HDAC inhibitors might have potential therapeutic value in lymphoma treatment, and provide pre-clinical rationale for combination therapy in patients with lymphoid malignancies. [Display omitted] Disclosures:Lannutti:Gilead Sciences Inc: Employment.

Evaluation of the Anti-Tumor Activity of MLN4924, A Novel NEDD8 Activating Enzyme Inhibitor, in Pre-Clinical Models of Rituximab Chemotherapy-Sensitive or -Resistant B-Cell Lymphoma.

AbstractAbstract 2761Clinical outcome of patients with relapsed/refractory diffuse large B-cell lymphoma (DLBCL) and mantle cell lymphoma (MCL) remains poor with currently available therapies. Recently, the ubiquitin-proteasome system (UPS) appears to play an important role in the development of resistance in MCL and some subtypes of DLBCL. Targeting UPS represents a rational approach in an attempt to eradicate drug-resistant lymphoma clones. MLN4924 is a novel, potent and selective inhibitor of the NEDD8-activating enzyme (NAE) that is necessary for the modification of cullin-RING ubiquitin ligases. We evaluated the anti-tumor activity of MLN4924 against a panel of rituximab-sensitive (RSCL) or rituximab/chemotherapy–resistant (RCRCL) DLBCL and Burkitt lymphoma cell lines, cytarabine-sensitive or -resistant (AraCR) MCL cell lines, and primary tumor cells freshly isolated from lymphoma patients (n=13). Lymphoma cells were exposed to escalating doses of MLN4924 alone or in combination with selected chemotherapy agents for up to 72 hrs. Changes in the cell viability or ATP content were determined by alamar Blue reduction or CellTiterGlo assays, respectively. Induction of apoptosis and changes in the levels of NFkB and UPS regulatory proteins were analyzed by Western blotting. Cell cycle alterations were determined by propidium iodide staining and NFkB activity was quantified by flow cytometry using the Imagestream technology. MLN4924 demonstrated time- and dose-dependent anti-lymphoma activity in all cell lines tested. The IC50 in RSCLs were Raji=400nM, RL=1uM and U2932=>3uM. All RCRCLs were less responsive to MLN4924 as a single agent with IC50 concentrations 4–10× those of their respective sensitive parental cell lines. The MCL cell lines Mino, MinoAraCR, Z-138, HBL-2 and HBL-2AraCR were most sensitive to MLN4924 anti-tumor effects (IC50=250nM) with no significant difference between cytarabine-sensitive and -resistant cell lines; while the MCL cell lines Rec-1, Rec-1AraCR, Jeko-1 and Jeko-1AraCR were less sensitive (IC50=500–1000nM). A variable degree of anti-tumor activity was also observed in primary lymphoma cells. In addition to single-agent activity, MLN4924 plus selected anti-lymphoma chemotherapy agents (bortezomib, bendamustine and cytarabine) demonstrated synergy in cytarabine-sensitive and (to a lesser degree) cytarabine-resistant MCL cell lines. Combinations with additional chemotherapeutic agents (doxorubicin and vincristine) resulted in additive effects. Exposure of MCL cells to MLN4924 resulted in G1 cell cycle arrest. In vitro exposure of the more sensitive MCL cell lines Mino and MinoAraCR to MLN4924 resulted in an increase in p-IkBα and down-regulation of both total and nuclear NFkB. The less sensitive cell lines Rec-1 and Rec-1AraCR demonstrated little to no change in NFkB activation following exposure to MLN4924. Additional studies are ongoing to further define the molecular mechanisms of the anti-tumor activity observed following NAE inhibition by MLN4924 in these pre-clinical models and to further evaluate the activity of MLN4924 in in vivo SCID mouse models of B-cell lymphoma. Our data suggests that MLN4924, a novel NAE inhibitor, is active against B-cell lymphomas, particularly MCL, and is a promising agent warranting further investigation in relapsed/refractory aggressive B-cell lymphomas. Disclosures:No relevant conflicts of interest to declare.

Vitamin D Triggers Killing of Burkitt Lymphoma Cells by Human Macrophages

Abstract 1035 Introduction:Distinct macrophage subsets have been linked with either protective or pathogenic roles in cancer. M2 macrophages have been shown to suppress adaptive tumour-specific immune responses and promote tumour growth, while M1 macrophages eliminate neoplastic cells and activate tumour-killing mechanisms. Macrophages isolated from solid tumours (tumor-associated macrophages, TAM) reveal a suppressive and tumor promoting M2-like phenotype. However, it remains unclear which tumoricidal effector mechanisms are compromised in theses immune cells. While infilatration of macrophages is a characteristic morphological hallmark in Burkitts' lymphoma (BL), the phenotype and functional properties of TAM as part of the stroma are poorly understood. In this study we investigated the phenotype of lymphoma associated macrophages (LAM) and the mechanisms by which macrophages are able to modulate the growth of tumor cells. Methods:We included 19 patients diagnosed with BL and 20 patients with benign reactive lymphadenopathy as a control group. Phenotypic characterizations of LAM were evaluated by immunohistochemistry and by qPCR in paraffin embedded tissues. To investigate anti lymphoma cytotoxicity of distinct macrophage subsets we generated macrophages from PBMC either by GM-CSF to create the M1 phenotype, or by M-CSF to obtain the M2 phenotype. M1 and M2 macrophages were coincubated them with several BL cell lines and cytotoxicity was analyzed by flow cytometry, qPCR and immunofluorescence. Results:First, we could demonstrate that BL infiltrating macrophages displayed an anti-inflammatory M2-phenotype characterized by expression of surface markers such as CD68 and CD163. Second, we identified impaired vitamin D metabolism in LAM and M2 macrophages as demonstrated by low expression of vitamin D receptor and Cyp27B1, and increased expression of Cyp24A1. Third, macrophages revealed a reduced cytotoxic potential towards lymphoma cells which was dependent on the expression of the vitamin D dependent peptide cathelicidin. Finally, we could demonstrate that excess supplementation of calcitriol led to increased cytotoxicity of M1- and M2 macrophages towards BL cells. Conclusions:These results suggest a mechanism in which vitamin D is required for innate immunity to overcome the ability of lymphoma cells to evade macrophage-mediated antitumoral responses. The present findings underscore the importance of vitamin D for sustaining innate immunity and imply that the therapeutic activation of the vitamin D pathway may even result in triggering tumoricidal effector mechanisms of LAM. Disclosures:No relevant conflicts of interest to declare.

Target-Directed Development of a Proposed Biosimilar Rituximab (GP2013): Comparability of Antibody-Dependent Cellular Cytotoxicity Activity and Pre-Clinical Pharmacokinetics and Pharmacodynamics with Originator Rituximab

AbstractAbstract 4867 Background:Biosimilars are biologics approved by highly-regulated markets to be similar to existing agents that aim to offer more affordable treatment, thereby increasing patient access. Development of a biosimilar involves extensive characterization of the originator product over several years and a target-directed iterative development process to ensure a product that is highly comparable to the originator with similar clinical efficacy, safety and quality. Using antibody-dependent cellular cytotoxicity (ADCC), a main mode of action of rituximab, we illustrate how functional/structural relationship can be engineered into a biosimilar to ensure comparability at the in vitro level. Here we also present pre-clinical data confirming in vivo comparability for the proposed biosimilar rituximab GP2013, in terms of pharmacokinetics (PK), pharmacodynamics (PD) and efficacy. Methods:By employing a highly sensitive glycan quantitation method, relevant post-translational glycosylation patterns were assessed for their impact on in vitro ADCC relative potency data using the Raji and NK3.3 cell lines as target- and effector cells, respectively. Subsequently, bioactivity of GP2013 and originator rituximab were evaluated in a dose-response manner across a wide concentration range against SU-DHL-4 (diffuse large B-cell lymphoma) and Daudi (Burkitt's lymphoma) cell lines using freshly purified human NK cells. In vivo anti-tumor activity was assessed in two xenograft SCID mouse models of non-Hodgkin's lymphoma (SU-DHL-4 and Jeko-1 cell lines). Comparative PK and PD were assessed in single (5 mg/kg, n=14) and multiple (20 or 100 mg/kg, n=8) dose studies in cynomolgus monkeys, the pharmacologically most relevant species. Results:GP2013 and originator rituximab showed similar ADCC potency against both SU-DHL-4 and Daudi cells, with ADCC being reflective of engineered glycosylation patterns and structure-function relationships. In both xenograft mouse models, GP2013 and originator rituximab inhibited tumor growth to a similar extent, including at the more sensitive sub-optimal dose levels that are most likely to identify any potential differences. In primates, PK analysis confirmed bioequivalence between GP2013 and originator rituximab with nearly identical AUC values and 90% CIs entirely within the standard acceptance range of 0.8–1.25. Bioequivalence of PD response (B-cell depletion) was also shown, with 95% CIs of areas under the effect-time curves (AUEC) ratios for relative change from baseline in B-cell populations within the 0.8–1.25 acceptance range. The use of different doses indicated that comparable exposure and PD response can be expected for GP2013 and originator rituximab using indication-specific dosing regimens. Conclusions:This pre-clinical comparability exercise confirms that GP2013 and originator rituximab are pharmacologically similar with regard to ADCC potency, anti-tumor activity, PK exposure (AUC) and B-cell depletion. As such, it is hypothesized that GP2013 will show similar efficacy and safety as the originator product in ongoing clinical trials across different clinical indications. Disclosures:da Silva:Sandoz Biopharmaceuticals/HEXAL AG: Employment. Kronthaler:Sandoz Biopharmaceuticals/HEXAL AG: Employment. Meyer:Sandoz Biopharmaceuticals/HEXAL AG: Employment. Papandrikopoulou:Sandoz Biopharmaceuticals/HEXAL AG: Employment. Stangler:Sandoz Biopharmaceuticals/HEXAL AG: Employment. Visser:Sandoz Biopharmaceuticals/HEXAL AG: Employment.

New Therapeutic Approach for the Treatment of B-Cell Disorders Using Chlorambucil/Hydroxychloroquine-Loaded AntiCD20 Nanoparticles

AbstractAbstract 158B-cell disorders show highly variable clinical courses, ranging between indolent diseases like the chronic lymphocytic leukemia (CLL) and highly aggressive lymphoproliferative disorders like Burkitt Lymphoma. The treatments of these disorders have been characterized by the development of new approaches, including dose-intensive chemotherapy regimens and immunotherapy via monoclonal antibodies (Ab). Despite the promising survival rates, these multi-agent treatments are flawed by a high degree of toxicity and a significant fraction of patients do not respond. The use of core shell nanoparticles design with specific Ab-coating represents a new strategy to target only tumor cells with high-dose chemotherapy. We characterized, both in vitro and in vivo, the effects of a new kind of biodegradable nanoparticles (BNP) coated with an anti-CD20 antibody and loaded with Chlorambucil (CLB) and Hydroxychloroquine (HCQ).BJAB and MEC1 are two well characterized Burkitt lymphoma and CLL cell line, respectively; both are mutated in P53, express different amount of CD20 and result quite resistant to CLB, but more susceptible to HCQ. HCQ/CLB-loaded antiCD20 BNP can release enough amounts of drugs inside the cancer cells and overcome multi-drug resistance mechanisms, which are over-expressed in many B-cell disorders. The presence of antiCD20 antibody targets BNP on cells expressing this antigen, inducing a selective penetration in these cells, as demonstrated by electron microscopy studies; the cytotoxic effect was evident analyzing apoptosis and after 24–48 hours all the cancer B-cells resulted destroyed. Data indicate that BNP were able to kill also cells expressing very low amount of CD20, where Rituximab was almost ineffective.These results were confirmed using primary cells isolated from more than 40 patients with untreated chronic lymphocytic leukemia. These data were obtained incubating fresh isolated blood with HCQ/CLB-loaded antiCD20 BNP and analyzing residual viable or apoptotic cells.Endovenous or intraperitoneal injection of HCQ/CLB-loaded antiCD20 BNP in mice did not induced tissue damages, weight loss or death, while the same amount of free cytotoxic agent cause death of all the animals.Biodistribution analysis demonstrated a selective targeting of the antiCD20 BNP to tumor cells, with a peak 48 hours after injection; BNP elimination was mainly documented in liver and intestine but not by spleen, kidney, lungs or other organs.The intraperitoneal injection of BJAB cells in SCID mice determined the formation of a tumor mass visible at the site of injection followed by early multi-organ involvement and death of all animals. Eight injections of antiCD20 BNP containing CLB/HCQ (400 micrograms each) in 17 days were able to increase survival in 100% of mice and 90% of animals were cured. The same amount of CLB+HCQ cannot be used as free drugs because of their toxicity able to kill all the animals in about a week.The intravenous injection of MEC1 cells in SCID mice allowed the development a new human/mouse model of leukemia. The treatment with antiCD20 BNP containing CLB/HCQ showed comparable therapeutic efficacy, while free drugs were almost uneffective.In conclusion, the results of the present study demonstrate that antiCD20 BNP containing HCQ/CLB can be effective as a single agent in controlling both aggressive and indolent models of disseminated B-cell disorders and provide a rationale for adopting this new therapeutic approach in clinical trials. Furthermore, the results demonstrate that nanoparticles are effective via different administration methods. Disclosures:No relevant conflicts of interest to declare.

Ofatumumab (OFA) Is a Novel Anti-CD20 Monoclonal Antibody (mAb) with Improved Anti-Tumor Activity in Vitro and in Vivo in Mantle Cell Lymphoma (MCL) Pre-Clinical Models.

AbstractAbstract 2757MCL is typically characterized by an aggressive clinical course and inevitable development of refractory disease despite early intervention that often includes: immunotherapy (e.g., rituximab), multi-agent induction chemotherapy and consolidation with high dose chemotherapy and autologous stem cell transplant in first remission. Residual disease at the time of stem cell collection is an important cause for treatment failure. There is a need to evaluate more potent anti-CD20 mAbs capable to kill lymphoma cells with low CD20 surface levels. In Burkitt’s lymphoma (BL) and diffuse large B-cell lymphoma (DLBCL) pre-clinical models we previously demonstrated that OFA was more potent than rituximab (RIT) in vitro and in vivo. In order to characterize the activity of OFA against MCL, we evaluated the activity of OFA against cytarabine (Ara-C)-sensitive (eg. Mino, Jeko-1, Rec-1, HBL-2, Granta and Z-138); –resistant MCL cell lines (eg. MinoAraCR, Jeko-1AraCR, Rec-1AraCR, HBL-2AraCR and GrantaAraCR); and primary tumor cells derived from MCL patients (n=2). Antibody-dependent cellular cytotoxicity (ADCC) and complement dependent cytotoxicity (CDC) were measured by standard 51Cr release assays in MCL exposed to OFA, RIT or isotype control. OFA vs. RIT direct anti-proliferative effects were measured in by alamar blue reduction assay. Apoptosis following in vitro exposure to OFA or RIT was detected by caspase 3/PARP cleavage. Patient tumor cells were isolated from biopsy specimens by negative selection using magnetic beads and incubated with OFA or RIT +/− human serum as a complement source. Cell viability was determined at 48 hours by CellTiterGlo assay. Surface CD20 and the complement inhibitory proteins (CIPs) (CD55 and CD59) density in MCL cell lines was determined by flow cytometry (Image stream) and compared to BL or DLBCL cell lines. For in vivo experiments 6–8 week-old SID mice were inoculated subcutaneously with 5×106 matrigel suspended Z-138 cells. Upon tumor engraftment, mice were assigned to RIT (10mg/kg), OFA (10mg/kg) or control groups. Tumor growth curves were calculated for each group. Mice were sacrificed if tumor size reached >2cm in any dimension. After 6 months, survival was analyzed by Kaplan-Meier analysis and compared by log-rank test. OFA induced significantly higher levels of CDC associated cell lysis compared to RIT in almost all MCL cell lines tested (10/11) (Mino: 53.2% vs 0.2%; MinoAraCR: 72.6% vs. 0.6%; Jeko-1: 33.4% vs. 9.8%; Jeko-1AraCR: 38.3% vs. 2.8%; REC-1: 17% vs 3%; Rec-1AraCR: 7.8% vs. 0.2%; HBL-2: 27.1% vs. 19.2%; HBL-2AraCR: 86.6% vs. 72.2%; GrantaAraCR: 17% vs 0.9%; Z-138: 56.4% vs. 0.65%; all p-values <0.05). No differences in RIT or OFA mediated ADCC or direct signaling was observed. As previously noted in BL and DLBCL models, OFA was capable of inducing a higher degree of CMC even at low CD20 levels in contrast to RIT. In vivo, OFA slowed tumor growth, and prolonged survival in Z-138 bearing SCID mice compared to RIT (median survival for RIT was 127 days vs. not reached for OFA treated animals; p<0.05). Our data suggest that, OFA is more potent than RIT against Ara-C-sensitive and –resistant MCL cells in vitro, delays tumor growth and prolongs survival compared to RIT in an in vivo MCL SCID mouse model, and retains CDC activity despite low CD20 and high CIP surface expression levels. OFA appears to be a promising mAb targeting CD20 in MCL and is undergoing clinical testing in the front-line setting (NCT01527149). Disclosures:Czuczman:Genmab: Unrestricted Research Grant Other.

The SMAC Mimetic Lcl-161 Demonstrates Anti-Tumor Activity As a Single Agent and Decreases the Apoptotic Threshold of Chemotherapy Agents in Rituximab-Chemotherapy Sensitive and Resistant Lymphoma Pre-Clinical Models

Abstract 61The loss of response to apoptotic stimulus in lymphoma is a major obstacle in the treatment of primary and refractory B-cell malignancies. While the role of the anti-apoptotic Bcl-2 family proteins in the pathogenesis, maintenance, and progression of many sub-types of B-cell lymphoma is well characterized, the impact of the expression level(s) of other key regulatory proteins of cell death pathways (i.e. inhibitor of apoptosis [IAP] proteins) is less defined. The role of IAP proteins in the acquirement of resistance to rituximab or chemotherapy in B-cell lymphoma is unclear. Overexpression of IAP proteins and loss of expression of its antagonist, the second mitochondria-derived activator of caspases (SMAC) correlates with inferior clinical outcomes in a range of malignancies. Perhaps related to the acquisition of resistance, we found that rituximab-resistant cell lines (RRCL) have a deregulation of pro-apoptotic (Bak/Bax) and anti-apoptotic (Mcl-1, Bcl-XL) Bcl-2 family protein expression along with increased expression of the IAP protein survivin. Small molecule SMAC mimetics like LCL-161 are promising agents for lowering the threshold of tumor cell apoptosis, and represent a potential new avenue of therapy for de novo and refractory drug-resistant lymphoma. To this end, we evaluated the anti-tumor activity of LCL-161 in a range of rituximab-sensitive (RSCL), RRCL, and primary lymphoma cells. Cells were exposed to escalating doses of LCL-161 alone or in combination with various chemotherapy agents (i.e. etoposide, doxorubicin, vincristine, gemcitabine, carboplatin, oxaliplatin, bortezomib and cytarabine) for 48 and 72 hrs. Changes in cell viability and ATP content were determined by the CellTiter-Glo viability assay. Protein lysates were obtained from RSCL and RRCL to determine baseline levels of IAP protein family members. LCL-161 displayed significant anti-tumor activity against Burkitt's lymphoma (BL), diffuse large B-cell (DLBCL) and mantle cell lymphoma (MCL) cell lines. Activity was observed in both RSCL and RRCL cell lines. IC50 values for LCL-161 alone were between 35uM and 45uM for the DLBCL lines. Responses were slightly lower in BL and MCL compared to DLBCL cell lines. Synergistic activity between LCL-161 and several chemotherapy agents (e.g. gemcitabine, cytarabine, carboplatin, vincristine, etoposide and bortezomib) commonly used in the management of aggressive lymphoma was seen at physiologically-relevant doses. In vitro exposure of lymphoma cells to LCL-161 decreased the cytotoxic threshold of chemotherapy by 50%, while ex vivo studies with primary patient lymphoma samples showed a decrease of nearly 60%. In vivo studies using a xenograft SCID murine model are planned. In summary, LCL-161 has shown activity both as a single agent, and when combined with several chemotherapy agents in BL, MCL, and DLBCL cell lines as well as primary patient samples. Additionally, LCL-161 exhibits significant cytotoxic activity against RRCLs suggesting an ability to antagonize IAP proteins. This data supports the continued investigation of LCL-161 as a novel and effective targeted agent for the treatment of de novo and refractory aggressive B-cell lymphomas. Disclosures:No relevant conflicts of interest to declare.

Daratumumab, a Novel Human Anti-CD38 Monoclonal Antibody for the Treatment of Chronic Lymphocytic Leukemia and B-Cell Non–Hodgkin Lymphoma

AbstractAbstract 3935Daratumumab (DARA) is a human CD38 antibody with broad-spectrum killing activity. DARA induces killing of tumor cells, mainly via complement-dependent cytotoxicity (CDC) and antibody-dependent cell-mediated cytotoxicity (ADCC) (de Weers M, J Immunol 2011). DARA is currently being evaluated in phase I/II clinical trials in patients with multiple myeloma. In these clinical studies the adverse events have been manageable and marked reductions in paraprotein and bone marrow plasma cells have been observed. In the present study, we have analyzed the potential of targeting CD38 using DARA in two types of B-cell non-Hodgkin lymphoma (B-NHL) (follicular lymphoma (FL) and mantle cell lymphoma (MCL)), and in chronic lymphocytic leukemia (CLL). Flow cytometry analysis demonstrated that MCL and CLL tumor cells show heterogeneous expression of CD38, while FL cells showed invariable high CD38 levels. CD38 has attracted special attention in CLL where high CD38 expression is a marker of bad prognosis (Hamblin et al, Blood 1999 and 2002) and is expressed preferentially in the proliferating fraction of the tumor (Damle RN, Blood 2007). In addition, we have recently shown that high CD38 expression in MCL was associated with resistance to the proteasome inhibitor bortezomib (Pérez-Galán P, Blood 2011).Here, we tested the cytotoxic activity of DARA in tumor cell lines and in fresh tumor cells obtained from patients. DARA did not induce CDC in MCL cell lines (MINO, REC, HBL2, JEKO), irrespective of CD38 expression levels. Also, FL cell lines (WSU-FSCCL, RL) expressing relatively high CD38 levels were insensitive to DARA-induced CDC. This low CDC was associated with high expression of the complement inhibitors CD55 and CD59. In addition, the number of CD38 molecules per cell in these MCL and FL cell lines was lower than that found on the CDC-sensitive Daudi Burkitt lymphoma cell line, suggesting a threshold for CD38-targeted CDC lysis.In the presence of PBMC effector cells obtained from healthy donors, DARA showed significant levels of ADCC in cells from MCL, FL and CLL. In CLL primary cases (n=8) tested, DARA (14–43% lysis) was generally superior or at least equally effective (mean+/−SD=28,78 +/− 9,78) in inducing ADCC as compared to the anti-CD20 antibodies ofatumumab (mean+/−SD=21,35 +/− 15,71) and rituximab (mean+/−SD=29,30 +/− 15,90).The immunomodulatory agent lenalidomide shows considerable single agent activity in MCL, FL and CLL. Interestingly, it has been shown that lenalidomide may be able to increase ADCC, probably via activation of NK cells. We therefore tested whether the combination of lenalidomide and DARA could enhance ADCC. Noteworthy, DARA-induced ADCC in MCL, FL cell lines and primary CLL cells was significantly (p<0,05) enhanced when effector cells were pretreated with the immunomodulatory agent lenalidomide (3 μM, 72 h) with DARA doses ranging from 0,01–1 μg/ml.Finally, CD38 is important for cell migration and adhesion, especially for CXCR4-CXCL12-induced migration of tumor cells (Vaisitti et al, Leukemia 2010). Our preliminary results suggest that in the CLL subtype with high CD38 and more migratory capacity DARA (10–30 μg/ml) inhibits CXCL12/SDF1α mediated migration up to 70%. These results are of high importance because inhibition of tumor cell trafficking to tissue sites as bone marrow and lymph nodes, may be clinically relevant, as increasing evidence indicates that tumor–microenvironment interactions may play an important role in drug resistance and contribute to clinical failures. We are currently validating this in vitro results in a CLL mouse model.Taken together, these results suggest that DARA may be a promising therapeutic agent both for MCL, FL and CLL, in which DARA exerts its effects mainly via ADCC and for CLL also via inhibition of migration of tumor cells. Interestingly, the cytotoxic activity of DARA by ADCC could be further augmented by addition of lenalidomide in these three models. Disclosures:Lammerts van Bueren:Genmab: Employment. Bakker:genmab: Employment. Parren:genmab: Employment. Perez-Galan:Genmab: Research Funding.

Chronic Active B-Cell Receptor Signaling in Lymphoma

Abstract SCI-26We have developed loss-of-function, RNA interference-based screens to reveal genes essential for cancer cell proliferation and survival. In parallel, we are using high-throughput RNA resequencing (RNA-seq) to identify somatic mutations and other structural abnormalities in cancer. The intersection of these two data sets has helped us to discover novel pathogenetic pathways in lymphoma that are amenable to therapeutic attack. The activated B-cell-like (ABC) subtype of diffuse large B-cell lymphoma (DLBCL) has constitutive activation of the NF-κB pathway, which we traced to the signaling adapter CARD11. In some ABC DLBCL biopsies (∼10%), somatic mutations produce CARD11 isoforms that spontaneously activate NF-κB signaling. In ABC DLBCL tumors with wild-type CARD11, we defined a “chronic active” form of B-cell receptor (BCR) signaling that activates NF-κB. Such ABC DLBCLs are killed by knockdown of BCR signaling components, such as Bruton’s tyrosine kinase (BTK), or components of the BCR itself. Over one-fifth of ABC DLBCLs have mutations in the CD79B or CD79A subunits of the BCR. In 18 percent of cases, mutations occur in a single tyrosine residue in the critical “ITAM” signaling motif, generating BCRs that avoid negative autoregulation by the LYN tyrosine kinase. Based on these findings, we are conducting clinical trials of ibrutinib in relapsed/refractory DLBCL. Ibrutinib is an irreversible and highly selective small-molecule inhibitor of BTK. Thus far, ibrutinib monotherapy has induced many complete and partial responses in patients with ABC DLBCL, including those with “primary refractory” tumors that had never responded to any prior therapy. One patient has been in a sustained complete response for over 19 months, taking ibrutinib daily with no discernible side effects. Of note, ABC DLBCL tumors with and without CD79B mutations have responded, suggesting that BCR pathway addiction may be a prevalent feature in this lymphoma subtype. More recently, we have uncovered a “tonic” form of BCR signaling in Burkitt lymphoma that engages the prosurvival PI(3) kinase pathway. Two-thirds of Burkitt lymphoma cell lines die upon knockdown of BCR subunits or the proximal kinase SYK, due to loss of PI(3) kinase signaling. Moreover, a gene expression signature of PI(3) kinase activity is more highly expressed in Burkitt lymphoma biopsies than in biopsies of other aggressive lymphomas. Tonic BCR signaling in Burkitt lymphoma is mechanistically distinct from chronic active BCR signaling in ABC DLBCL, since it does not engage BTK, CARD11, or NF-κB. RNA-seq revealed that 70 percent of sporadic Burkitt lymphoma cases harbor somatic mutations that potentiate the action of the transcription factor TCF3 by preventing its inhibitory heterodimerization with the DNA-binding inhibitor ID3. TCF3 promotes tonic BCR signaling and PI(3) kinase activity in Burkitt lymphoma by transactivating the immunoglobulin heavy- and light-chain genes, thereby increasing surface BCR expression, and by repressing the phosphatase SHP-1, a potent negative regulator of BCR signaling. Hence, inhibitors of proximal BCR signaling and the PI(3) kinase pathway should be evaluated in Burkitt lymphoma, especially in patients for whom high-dose chemotherapy is infeasible, such as older individuals and those with the endemic form of this lymphoma. Disclosures:Off Label Use: I will be discussing clinical trials of ibrutinib (PCI-32765) in lymphoma. Ibrutinib is an investigational drug that has not yet received FDA approval for any indication.

MiR-221 and MiR-222 Patterns Characterize Burkitt Lymphoma in Human and Mouse Model

AbstractAbstract 1304Recently, a class of noncoding RNAs called microRNA (miRNAs) has emerged as critical gene regulators in cell growth, differentiation, disease and development. MiRNAs are 18–24 nucleotide long noncoding RNAs, which regulate gene expression by pairing with 3′ untranslated region (UTR) of target mRNA and inhibiting protein translation and/or inducing mRNA degradation. Deregulated miRNA expression is reported in various human diseases including lymphomas, suggesting an important role in their pathogenesis.According to WHO classification, Burkitt lymphoma (BL) is a rare, highly aggressive NHL composed of monomorphic medium-sized B cells with multiple nucleoli and numerous mitotic figures and is more common in children than in adults. The molecular feature of BL is the translocation that places MYC under the control of immunoglobulin gene regulatory elements. High levels of c-MYC have been clearly shown to have a tumour-promoting effect. However, there is recent evidence that infrequent cases may lack an identifiable MYC translocation, the explanation for which is still uncertain, though suggesting the existence of pathogenetic mechanisms alternative to genetic alterations.Over the past years miRNA signatures have been described to characterize and classify different types of BL or to investigate the expression of miRNAs possibly regulated by c-Myc in BL cases positive or negative for Myc translocation. However, it remained unclear the functional role of differentially expressed miRNAs and no further studies have been conducted. We performed miRNA expression profile to gain further insights into the molecular pathology of BL. We conducted array analysis on a set of 5 sporadic BL patients, 3 endemic BL patients, 9 reactive tissues and 11 cases of mononucleosis. Our profile is the first one that shows the different expression between BL cases and normal B cells whereas recent miRNA profiles have been conducted in BL compared to other B-NHL (B-CLL, MCL & FL). A common trend of miRNAs altered expression was also observed by NanoString analysis in 10 BL cell lines compared to 5 normal CD-19+ B cells. Among several miRNAs previously described be deregulated in BL we identified a severe down-regulation of miR-221, miR-222 in all classes of comparisons we analyzed. The down-regulation of miR-221 and miR-222 associated to BL has been also confirmed by q-RT-PCR method in a different cohort of BL patients (20) compared to the healthy controls (6). We found that interesting considering the up-regulation of miR-221 and miR-222 previously confirmed in a lot of solid tumors by multiple studies. We are investigating a different role of the cluster miR-222 and miR-221 in lymphomas that have a different process in carcinogenesis than solid tumors.In vivo models to study the lymphomagenesis of BL have been created but until now no one studied the importance of the miRNAs in vivo. We analyzed the expression of miR-221 and miR-222 in a Myc transgenic mouse model. The transgene construct consists of the Myc oncogene (c-myc) in association with the Emu immunoglobulin heavy chain enhancer and Myc promoter. Expression of the mouse Myc transgene is restricted to the B cell lineage. Previously it has been shown an increase of pre-B cells in the bone marrow throughout life of hemizygotes and a transient increase in large pre-B cells in the blood at 3–4 weeks of age; moreover spontaneous pre-B and B cell lymphomas reach an incidence of 50% at 15–20 weeks in hemizygous progeny of a wildtype female mated with a hemizygous male. We observed the development of Burkitt lymphoma within 10 weeks of birth in 14 out of 25 Eu-Myc transgenic mice and a premature death in 5 out or 25 transgenic mice within 6–8 weeks of birth without showing any enlarged lymph nodes. Transgenic mice with masses showed the same phenotype characterized by enlarged spleen (3 fold), lymphosarcomas associated with BL and enlarged lymph nodes around the neck area. B-cells have been negatively selected from enlarged lymph nodes and enlarged spleen. A qRT-PCR has been conducted to evaluate the miR-221 and miR-222 expression. The miRNA levels showed a down-regulation in B cells collected from the masses when compared to normal B cells derived from the spleen of WT mice.In conclusion, our study reveals new insights into the functional significance in loss of miR-221 and miR-222 expression in BL pathogenesis. Disclosures:No relevant conflicts of interest to declare.

A new target in Burkitt's lymphoma

This study identified a new pathway involved in the pathogenesis of Burkitt's lymphoma that is amenable to therapeutic targeting. Using RNA sequencing of patient biopsy samples and Burkitt's lymphoma cell lines, together with re-analysis of published sequence data, the authors identified recurrent mutations in the transcription factor TCF3 and its negative regulator ID3. TCF3 activated the pro-survival phosphatidylinositol 3-kinase pathway in Burkitt's lymphoma cells, in part by augmenting tonic B cell receptor signalling. Inhibitors of the TCF3 pathway inhibited growth of Burkitt's lymphoma cells and tumour xenografts.

Epstein-Barr nuclear antigen 1 induces expression of the cellular microRNA hsa-miR-127 and impairing B-cell differentiation in EBV-infected memory B cells. New insights into the pathogenesis of Burkitt lymphoma

Epstein-Barr Virus (EBV) is a γ-herpesvirus that infects >90% of the human population. Although EBV persists in its latent form in healthy carriers, the virus is also associated with several human cancers. EBV is strongly associated with Burkitt lymphoma (BL), even though there is still no satisfactory explanation of how EBV participates in BL pathogenesis. However, new insights into the interplay between viruses and microRNAs (miRNAs) have recently been proposed. In particular, it has been shown that B-cell differentiation in EBV-positive BL is impaired at the post-transcriptional level by altered expression of hsa-miR-127. Here, we show that the overexpression of hsa-miR-127 is due to the presence of the EBV-encoded nuclear antigen 1 (EBNA1) and give evidence of a novel mechanism of direct regulation of the human miRNA by this viral product. Finally, we show that the combinatorial expression of EBNA1 and hsa-miR-127 affects the expression of master B-cell regulators in human memory B cells, confirming the scenario previously observed in EBV-positive BL primary tumors and cell lines. A good understanding of these mechanisms will help to clarify the complex regulatory networks between host and pathogen, and favor the design of more specific treatments for EBV-associated malignancies.

B7-H1, Which Represses EBV-Immortalized B Cell Killing by Autologous T and NK Cells, Is Oppositely Regulated by c-Myc and EBV Latency III Program at Both mRNA and Secretory Lysosome Levels

EBV-immortalized B cells induce a complex immune response such that the virus persists as a clinically silent infection for the lifetime of the infected host. B7-H1, also called PD-L1, is a cosignaling molecule of the B7 family that can inhibit activated T cell effectors by interaction with its receptor PD-1. In this work, we have studied the dependence of B7-H1 on NF-κB and c-Myc, the two main transcription factors in EBV latency III proliferating B cells, on various lymphoblastoid and Burkitt lymphoma cell lines, some of them being inducible or not for the EBV latency III program and/or for c-Myc. We found that B7-H1 repressed killing of EBV-immortalized B cells by their autologous T and NK cells. At the mRNA level, NF-κB was a weak inducer whereas c-Myc was a strong repressor of B7-H1 expression, an effect mediated by STAT1 inhibition. At the protein level, B7-H1 molecules were stored in both degradative and unconventional secretory lysosomes. Surface membrane B7-H1 molecules were constitutively internalized and proteolyzed in lysosomes. The EBV latency III program increased the amounts of B7-H1-containing secretory lysosomes and their export to the surface membrane. By repressing actin polymerization, c-Myc blocked secretory lysosome migration and B7-H1 surface membrane export. In addition to B7-H1, various immunoregulatory molecules participating in the immunological synapse are stored in secretory lysosomes. By playing on actin polymerization, c-Myc could thus globally regulate the immunogenicity of transformed B cells, acting on export of secretory lysosomes to plasma membrane.

Anticancer Activity and Cellular Repression of c-MYC by the G-Quadruplex-Stabilizing 11-Piperazinylquindoline Is Not Dependent on Direct Targeting of the G-Quadruplex in the c-MYC Promoter

This G-rich region of the c-MYC promoter has been shown to form a G-quadruplex structure that acts as a silencer element for c-MYC transcriptional control. In the present work, we have synthesized a series of 11-substituted quindoline analogues as c-MYC G-quadruplex-stabilizing compounds, and the cell-free and in vitro activity of these compounds were evaluated. Two lead compounds (4 and 12) demonstrated good cell-free profiles, and compound 4 (2-(4-(10H-indolo[3,2-b]quinolin-11-yl)piperazin-1-yl)-N,N-dimethylethanamine) significantly down-regulated c-MYC expression. However, despite the good cell-free activity and the effect of these compounds on c-MYC gene expression, we have demonstrated, using a cellular assay in a Burkitt's lymphoma cell line (CA46-specific), that these effects were not mediated through targeting of the c-MYC G-quadruplex. Thus, caution should be used in assigning the effects of G-quadruplex-interactive compounds that lower c-MYC to direct targeting of these promoter elements unless this assay, or similar ones, demonstrates direct targeting of the G-quadruplex in cells.

Parthenolide induces apoptosis and lytic cytotoxicity in Epstein-Barr virus-positive Burkitt lymphoma

Burkitt lymphoma (BL) has been reported to be strongly associated with Epstein-Barr virus (EBV) infection. The fact that EBV is generally present in cancer cells but rarely found in healthy cells represents an opportunity for targeted cancer therapy. One approach is to activate the lytic replication cycle of the latent EBV. Nuclear factor (NF)-κB is thought to play an essential role in EBV lytic infection. Elevated NF-κB levels inhibit EBV lytic replication. Parthenolide (PN) is a sesquiterpene lactone found in medicinal plants, particularly in feverfew (Tanacetum parthenium). The aim of the present study was to analyze the effect of PN on the survival of Raji EBV-positive lymphoma cells. Raji cells were treated with 0, 4 or 6 μmol/l PN for 48 h. MTT assay and western blot analysis were performed to evaluate the findings. Results showd that PN suppressed the growth of the EBV-positive BL cell line, Raji, and activated the transcription of BZLF1 and BRLF1 by inhibiting NF-κB activity. Most notably, when PN was used in combination with ganciclovir (GCV), the cytotoxic effect of PN was amplified. These data suggest that the induction of lytic EBV infection with PN in combination with GCV may be a viral-targeted therapy for EBV-associated BL.

Roles of Cell Signaling Pathways in Cell-to-Cell Contact-Mediated Epstein-Barr Virus Transmission

Epstein-Barr virus (EBV), a human gamma herpesvirus, establishes a life-long latent infection in B lymphocytes and epithelial cells following primary infection. Several lines of evidence indicate that the efficiency of EBV infection in epithelial cells is accelerated up to 10(4)-fold by coculturing with EBV-infected Burkitt's lymphoma (BL) cells compared to infection with cell-free virions, indicating that EBV infection into epithelial cells is mainly mediated via cell-to-cell contact. However, the molecular mechanisms involved in this pathway are poorly understood. Here, we establish a novel assay to assess cell-to-cell contact-mediated EBV transmission by coculturing an EBV-infected BL cell line with an EBV-negative epithelial cell line under stimulation for lytic cycle induction. By using this assay, we confirmed that EBV was transmitted from BL cells to epithelial cells via cell-to-cell contact but not via cell-to-cell fusion. The inhibitor treatments of extracellular signal-regulated kinase (ERK) and nuclear factor (NF)-κB pathways blocked EBV transmission in addition to lytic induction. The blockage of the phosphoinositide 3-kinase (PI3K) pathway impaired EBV transmission coupled with the inhibition of lytic induction. Knockdown of the RelA/p65 subunit of NF-κB reduced viral transmission. Moreover, these signaling pathways were activated in cocultured BL cells and in epithelial cells. Finally, we observed that viral replication was induced in cocultured BL cells. Taken together, our data suggest that cell-to-cell contact induces multiple cell signaling pathways in BL cells and epithelial cells, contributing to the induction of the viral lytic cycle in BL cells and the enhancement of viral transmission to epithelial cells.

TLR9 agonists induced cell death in Burkitt's lymphoma cells is variable and influenced by TLR9 polymorphism

Toll-like receptor 9 (TLR9) triggering is a promising novel strategy to combat cancer as it induces innate and adaptive immunity responses. B-cell lymphoma is unique in this context as tumor cells express TLR9 and may harbor latent Epstein-Barr virus (EBV), a gamma-herpesvirus with remarkable oncogenic potential when latent. Latent EBV may be promoted by TLR9 triggering via suppression of lytic EBV. Here, we elaborated an initial assessment of the impact of TLR9 triggering on EBV-positive and EBV-negative B-cell lymphoma using Burkitt's lymphoma (BL) cell lines as an in vitro model. We show that, independent of the presence of EBV, the TLR9 ligand oligodeoxynucleotide (ODN) CpG-2006 may or may not induce caspase-dependent cell death in BL cells. Moreover, ODN CpG-2006-induced cell death responses of BL cells were associated with TLR9 single-nucleotide polymorphisms (SNPs) rs5743836 or rs352140, which we detected in primary BL tumors and in peripheral blood from healthy individuals at similar frequencies. Thus, our findings suggest that the effect of TLR9 agonists on BL cells should be tested in vitro before installment of therapy and TLR9 SNPs in BL patients should be determined as potential biological markers for the therapeutic response to treatment targeting innate immunity.

Abstract 1128: The role of peptide deformylase in the apoptosis of c-Myc overexpressing cancers

Abstract In prokaryotes, the removal of an N-terminal formyl group by the peptide deformylase is required for post-translational protein processing. Our identification of human mitochondrial PDF (HsPDF) and deformylated peptides in the mitochondria shows that a similar post-translational processing of the 13 human mitochondrial DNA-encoded, and formylated, proteins can occur. Inhibition of HsPDF reduced proliferation of many human cancer lines. We developed a monoclonal antibody to hsPDF; profiling of cancer and normal cell lines by three immunoassays, flow cytometry, immunoprecipitation and in-cell western, showed over-expression of HsPDF in cancer cell lines. This data was confirmed at the mRNA level by RT-PCR. As the c-myc oncogene regulates mitochondria and metabolism in cancer, we investigated its role in regulating HsPDF. In a tet-off c-myc cell line, the expression of HsPDF was regulated by c-myc; inhibiting this protein resulted in the selective inhibition of mitochondrial protein translation, and eventual death of myc-positive, but not myc-negative cells. Furthmore, knocking down c-myc in a Burkitt's Lymphoma cell line significantly reduced the expression of both the mRNA and the protein levels of HsPDF relative to beta-Actin. The mechanism of death by actinonin was apoptosis in two c-myc over-expressing cell lines. Death was initiated with mitochondrial membrane depolarization, followed by inhibition of mitochondrial translation, and eventually caspase-3 activation and apoptosis. To confirm the specificity of PDF in cell death, we used two other hydroxamic acid peptidomimetics that do not target HsPDF. These compounds did not result in apoptotic death. Furthermore, inhibition of translation of the mitochondrial DNA by chloramphenicol and tetracycline, two structurally different inhibitors of the mitochondrial ribosome, which is upstream of deformylation, followed by treatment with actinonin resulted in a delay and marked reduction in apoptosis by actinonin, but not by staurosporine and the HDAC inhibitors SAHA and Trichostatin A. Thus, death was not a result of ATP depletion, but a unique PDF-mediated mechanism. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1128. doi:1538-7445.AM2012-1128