Burkitt Lymphoma Cell Line Raji Research Articles

Tumor-Specific Reactive Oxygen Species Accelerators Improve Chimeric Antigen Receptor T Cell Therapy in B Cell Malignancies.

Chimeric antigen receptor T cell (CART) therapy is currently one of the most promising treatment approaches in cancer immunotherapy. However, the immunosuppressive nature of the tumor microenvironment, in particular increased reactive oxygen species (ROS) levels, provides considerable limitations. In this study, we aimed to exploit increased ROS levels in the tumor microenvironment with prodrugs of ROS accelerators, which are specifically activated in cancer cells. Upon activation, ROS accelerators induce further generation of ROS. This leads to an accumulation of ROS in tumor cells. We hypothesized that the latter cells will be more susceptible to CARTs. CD19-specific CARTs were generated with a CD19.CAR.CD28.CD137zeta third-generation retroviral vector. Cytotoxicity was determined by chromium-51 release assay. Influence of the ROS accelerators on viability and phenotype of CARTs was determined by flow cytometry. The combination of CARTs with the ROS accelerator PipFcB significantly increased their cytotoxicity in the Burkitt lymphoma cell lines Raji and Daudi, as well as primary chronic lymphocytic leukemia cells. Exposure of CARTs to PipFcB for 48 h did not influence T cell exhaustion, viability, or T cell subpopulations. In summary, the combination of CARTs with ROS accelerators may improve adoptive immunotherapy and help to overcome tumor microenvironment-mediated treatment resistance.

Inhibition of Pro-Survival Pathways in DLBCL Cells By Functional Lipoprotein-like Nanoparticles

Introduction: Diffuse large B cell lymphoma (DLBCL) cells have an increased demand for cholesterol and cholesteryl esters to maintain membrane-anchored pro-survival signaling pathways, such as B cell receptor (BCR) signaling. In addition to BCR-mediated stimulation of de novo cholesterol synthesis, another mechanism employed by lymphoma cells to maintain cholesterol and cholesteryl ester homeostasis is by binding cholesterol-rich HDLs via the high-affinity receptor, scavenger receptor type B-1 (SCARB1). We previously reported that, unlike normal B cells, DLBCL cells highly express SCARB1, and that synthetic, cholesterol-poor high-density lipoprotein (HDL)-like nanoparticles (HDL NPs) exquisitely target SCARB1, deplete cellular cholesterol, and induce lymphoma cell death in vitro and in vivo. Given the critical role of cholesterol in maintaining proper membrane organization for BCR-mediated intracellular signaling, we hypothesized that HDL NP binding to SCARB1 and subsequent cellular cholesterol depletion modulates the organization of the cell membrane, reduces intracellular signaling downstream of membrane-anchored pathways like the BCR, and modulates the expression of genes relevant to cholesterol synthesis. Collectively, these mechanisms potently induce lymphoma cell death.Methods: We focused on germinal center (GC) DLBCL and Burkitt's Lymphoma (BL), due to the previously reported sensitivity of the SUDHL4 (GC DLBCL) and Ramos (BL) cell lines to HDL NP-induced cell death. In addition to SUDHL4 and Ramos, we tested the GC DLBCL cell line SUDHL6 and the BL cell lines Raji, Daudi and Namalwa. HDL NPs were synthesized, purified and characterized using standard protocols. Cell viability was quantified using the MTS assay, and total cholesterol levels were measured using the Amplex Red cholesterol assay. A blocking antibody against SCARB1 was used to inhibit binding of HDL NPs to the receptor. Confocal microscopy was used to visualize changes in membrane organization of SUDHL4 and Ramos cells following HDL NP treatment. Changes in the phosphorylation of signaling kinases (e.g. AKT, ERK1/2) following HDL NP treatment was measured using phospho-kinase arrays, phos-flow analysis, and western blot assays. RNA was harvested from cells treated with the HDL NPs for 48 hours and analyzed using Illumina's HT-12 microarray and RT-qPCR. Protein expression changes were measured using western blot assays.Results: HDL NPs potently induced cell death in all of the GC DBLCL and BL cell lines, all of which expressed SCARB1, with HDL NP IC50 values between 1 ~ 5 nM. As expected, HDL NP-induced cell death correlated with reduced total cellular cholesterol levels. Inhibition of HDL NP binding to SCARB1 by antibody blockade protected cells from HDL NP-induced cell death. HDL NP treatment induced a reorganization of the plasma membrane in SUDHL4 and Ramos cells, resulting in clustering of SCARB1, a phenomenon not seen in cholesterol-rich human HDL or saline controls. HDL NP treatment led to a decrease in the phosphorylation of a number of kinases downstream of BCR signaling, including AKT, ERK1/2, LCK and LYN over time. HDL NPs demonstrated synergy with small molecule inhibitors of various signaling kinases, such as ATK (inhibitor = GDC-0068) and SYK (inhibitor = R406), in Ramos and SUDHL4 cells. By microarray analysis and RT-qPCR, HDL NPs up-regulated a number of cholesterol biosynthesis genes, as well as the cell cycle inhibitor p21 and the pro-apoptotic protein APOPT1.Conclusions: These data demonstrate that HDL NPs bind SCARB1, altering the organization of the plasma membrane and reducing cellular cholesterol levels. Collectively, this results in decreased membrane-anchored pro-survival signaling, changes in gene expression pathways, and, ultimately, lymphoma cell death. Enhancing cholesterol depletion strategies, such as the combination of the targeted cholesterol depletion agent HDL NP with small molecule inhibitors of signaling kinases (e.g. AKT and SYK inhibitors), represents a novel and targeted therapeutic strategy for DLBCL and may be broadly applicable to other malignancies dependent on cholesterol homeostasis and membrane-anchored signaling pathways. DisclosuresKarmali:Gilead: Speakers Bureau; AstraZeneca: Speakers Bureau. Thaxton:AuraSense: Other: Co-founder of the biotech company AuraSense.

Omipalisib (GSK458), a Novel Pan-PI3K/mTOR Inhibitor, Exhibits In Vitro Anti-Lymphoma Activity in Chemotherapy-Sensitive and -Resistant Models of Burkitt Lymphoma

Background: Despite significant gains achieved in the treatment of Burkitt lymphoma (BL), current multi-agent immunochemotherapeutic regimens lead to high rates of acute toxicity, and relapsed/refractory disease still represents a significant hurdle with survival expected in only about 20-30% of such patients. Novel targeted therapeutic approaches are necessary to reduce treatment related toxicity in the up-front setting and improve survival in the relapsed/refractory setting. Analyses of genomic abnormalities in BL have identified increased activation of the PI3K/Akt/mTOR pathway in BL, induced by tonic B-cell receptor signaling and increased expression of Myc induced microRNAs (miRs), as having a significant role in Burkitt lymphomagensis. Additionally, recent reports have implicated higher expression of PI3K activating, Myc induced miRs in pediatric patients with a higher risk of relapse. While focused targeting of PI3K with the PI3K-delta isoform specific inhibitor idelalisib has led to significant activity in indolent B-cell lymphomas, limited activity has been noted in the setting of more aggressive forms. A broader inhibition of both upstream and downstream components of the pathway may exhibit more significant anti-lymphoma activity. To this end, we investigated the in vitro effects of PI3K/Akt/mTOR pathway inhibition with the dual pan-PI3K/mTOR inhibitor Omipalisib (GSK458) in chemotherapy-sensitive and -resistant BL cell line models.Methods: The in vitro effect of omipalisib was investigated in the BL cell lines Raji, Raji 4RH (chemotherapy-rituximab resistant), Raji 8RH (rituximab resistant), Ramos, and Daudi. Cell viability following exposure to omipalisib alone and in combination with cytotoxic chemotherapeutic agents was analyzed using Cell-Titer Glo and Alamar Blue assays. Apoptosis was analyzed using western blotting for PARP and by flow cytometry with Annexin V-propidium iodide staining. Downstream targets in the PI3K/Akt/mTOR pathway were analyzed using western blotting. Cell cycle analysis was performed by flow cytometry using propidium iodide staining. Synergistic activity of combination exposures was determined by calculation of a combination index using CalcuSyn software.Results: In vitro exposure of BL cell lines to omipalisib in concentrations ranging from 0.05μM to 50μM for 24, 48 or 72 hours resulted in a dose and time dependent decrease in viable cells with significant activity noted at even low nM concentrations (48 hour IC50 values: Raji=1.2μM, Raji 4RH=0.02μM, Raji 8RH=1.9μM, Ramos=0.01μM, Daudi=0.01μM). Flow cytometry for Annexin V and propidium iodide, after 72 hours of single agent exposure to omipalisib, showed a marked induction of apoptosis in all cell lines. For example, at an omipalisib concentration of 200nM, the percentage of Annexin V positive cells were Raji=40.7%, Raji 4RH=4.4%, Raji 8RH=41.5%, Ramos=59.4% and Daudi=46.9%. Approximately ten-fold higher omipalisib concentrations were required to induce similar degrees of apoptosis in the chemotherapy resistant Raji 4RH cell line compared to chemotherapy sensitive cell lines. Western blotting for downstream targets of the PI3K/Akt/mTOR pathway, including S6 and GSK3Β, showed a reduction in phosphorylation after 30 minutes of exposure to omipalisib in all cell lines. Determination of cell cycle progression following exposure to omipalisib for 72 hours at concentrations ranging from 0.006μM to 25μM showed dose-dependent cell cycle arrest in G1 phase in all cell lines; however the chemotherapy resistant Raji 4RH cells arrested in G2/M at higher concentrations. When BL cells were exposed to omipalisib in combination with either doxorubicin or dexamethasone, synergistic anti-tumor activity was observed in all cell lines tested.Conclusion: Inhibition of PI3K and mTOR by the dual inhibitor omipalisib suppresses activation of the PI3K/Akt/mTOR pathway leading to impaired BL cell proliferation with G1 cell cycle arrest and induction of apoptosis in chemotherapy-sensitive and -resistant cell line models of BL. Inhibition of the PI3K/Akt/mTOR pathway with omipalisib also increases the in vitro response to cytotoxic chemotherapeutic agents. Our findings note the pre-clinical activity of PI3K/Akt/mTOR pathway inhibition in BL and highlight the relevance of pursuing PI3K/Akt/mTOR pathway inhibition as a potential therapeutic option in BL. DisclosuresNo relevant conflicts of interest to declare.

Targeting BET Bromodomains in Pre-Clinical Models of Burkitt Lymphoma

Background: Burkitt lymphoma (BL) is the most common NHL type in children. Although treatment for pediatric BL has improved significantly, there is an urgent need for novel therapies that reduce the toxicity of modern treatment regimens and improve on the dismal survival observed in the relapsed/refractory setting where only about 20-30% of patients survive their disease. Recent reports have implicated co-activation of c-Myc and PI3K in Burkitt lymphomagenesis. Genomic analysis of recurrent oncogenic mutations in BL have identified tonic B-cell receptor signaling and the over-expression of Myc induced microRNAs from the MIR17-92 family, e.g. mir17 and mir19, as possible mechanisms of PI3K activation in BL. Mir17 and mir19, have been implicated in Burkitt lymphomagenesis and their overexpression may be associated with a higher risk of relapse. The protein phosphatase and tensin homolog (PTEN) is a major regulator of PI3K pathway activation. MIR17-92 cluster members have been shown to target PTEN leading to increased PI3K activation. We have previously identified increased expression of mir17 and mir19 along with increased activation of AKT in cell line models of chemotherapy resistant BL suggesting a potential mechanism for increased resistance. BET bromodomains interact with chromatin and enhance transcriptional activation of numerous genes including c-Myc. Thus, BET bromodomains represent a promising target in BL. To investigate the effects of inhibition of c-Myc driven activation of the PI3K/AKT/mTOR pathway, we characterized the activity of the highly potent small molecule bromodomain inhibitor JQ1 in chemotherapy sensitive and resistant BL cell lines. Additionally, we analyzed the ability to enhance anti-lymphoma activity of PI3K/Akt/mTOR pathway inhibition in BL by the combination of BET bromodomain inhibition and targeted inhibition of the PI3K/AKT/mTOR pathway.Methods: The in vitro activity of JQ1 was investigated in the BL cell lines Raji, Raji 4RH (chemotherapy-rituximab resistant), Raji 8RH (rituximab resistant), Ramos, and Daudi. Cell Viability following exposure to JQ1 alone and in combination with the PI3K/mTOR inhibitor omipalisib (GSK458)) was analyzed using Cell-Titer Glo or Alamar Blue assays following 24, 48, and 72 hour exposure over a range of inhibitor concentrations. Induction of apoptosis was analyzed using western blotting for cleaved PARP. C-Myc expression following JQ1 exposure was determined by western blot following 48 hour JQ1 exposure. The effect of JQ1 exposure on the expression of c-Myc induced microRNA expression was determined by qRT-PCR in cells exposed to JQ1 for 48hours. Synergy of combination exposures was determined using CalcuSyn to generate combination index (CI) values.Results: In vitro exposure of BL cell lines to JQ1 for 24, 48, and 72 hours resulted in a significant dose and time dependent decrease in viable cells (72 hour IC50 values: Raji 0.12µM, Raji 4RH 1.7µM, Raji 8RH 0.7µM, Ramos 0.22µM and Daudi 4µM). There was an increase in cleaved PARP after 72 hour exposure indicating induction of apoptosis. While single agent effect was seen in the resistant Raji 4RH cell line, activity was noted to primarily occur at the higher end of the dosing. Western blot analysis demonstrated a reduction in c-Myc expression following exposure to JQ1 1µM for 24 hours (relative band intensity normalized to control: Raji=0.12, Raji 4RH=0.18, Raji 8RH=0.11). qPCR showed a reduction in Mir17 relative transcription levels after 48 hours of exposure to JQ1 2.5mM (relative expression compared to control: Raji=0.72, Raji 4RH=0.98, Raji 8RH=0.83, Ramos=0.57, Daudi=0.46). When combined with omipalisib, an increased effect on cell viability was noted. The combination effect was noted to be synergistic (CI<0.9) at multiple dose combinations while other combinations exhibited primarily additive effects.Conclusion: In vitro inhibition of BET bromodomains with JQ1 results in a decrease in c-Myc expression and a decrease in c-Myc dependent miR expression with impaired proliferation and induction of apoptosis in chemotherapy-sensitive and -resistant BL cell lines. Augmented, and in some cases synergistic, activity is also noted with dual inhibition of BET bromodomains and the PI3K/Akt/mTOR pathway in BL cell lines. DisclosuresNo relevant conflicts of interest to declare.

Identification and Characterization of a New Protein Isoform of Human 5-Lipoxygenase.

Leukotrienes (LTs) are inflammatory mediators that play a pivotal role in many diseases like asthma bronchiale, atherosclerosis and in various types of cancer. The key enzyme for generation of LTs is the 5-lipoxygenase (5-LO). Here, we present a novel putative protein isoform of human 5-LO that lacks exon 4, termed 5-LOΔ4, identified in cells of lymphoid origin, namely the Burkitt lymphoma cell lines Raji and BL41 as well as primary B and T cells. Deletion of exon 4 does not shift the reading frame and therefore the mRNA is not subjected to non-mediated mRNA decay (NMD). By eliminating exon 4, the amino acids Trp144 until Ala184 are omitted in the corresponding protein. Transfection of HEK293T cells with a 5-LOΔ4 expression plasmid led to expression of the corresponding protein which suggests that the 5-LOΔ4 isoform is a stable protein in eukaryotic cells. We were also able to obtain soluble protein after expression in E. coli and purification. The isoform itself lacks canonical enzymatic activity as it misses the non-heme iron but it still retains ATP-binding affinity. Differential scanning fluorimetric analysis shows two transitions, corresponding to the two domains of 5-LO. Whilst the catalytic domain of 5-LO WT is destabilized by calcium, addition of calcium has no influence on the catalytic domain of 5-LOΔ4. Furthermore, we investigated the influence of 5-LOΔ4 on the activity of 5-LO WT and proved that it stimulates 5-LO product formation at low protein concentrations. Therefore regulation of 5-LO by its isoform 5-LOΔ4 might represent a novel mechanism of controlling the biosynthesis of lipid mediators.

Abstract 3893: Obinutuzumab (GA101) versus rituximab against rituximab-sensitive and -resistant Burkitt lymphoma (BL) differentially phosphorylate BCR, Fc-gamma receptor, and natural killer cell-mediated cytotoxicity signaling pathways

Abstract Background: Burkitt Lymphoma (BL) is the most common NHL in children and adolescents and has an excellent prognosis (≥80% 5years, EFS, Cairo et al. Blood, 2007) and further improved with the addition of rituximab (Goldman/Cairo et al, Leukemia, 2013, Cairo et al. JCO, 2012). However, a subset of patients with chemoimmunotherapy resistant disease has a dismal prognosis (≤ 10% 5 years, EFS) (Miles/Cairo et al. BJH, 2012, Barth et al. BJH, 2013). Obinutuzumab, a novel glycoengineered type II CD20 Ab, mediates enhanced cell death &amp; ADCC against B-cell lymphoma vs. RTX (Awasthi/Cairo et al. BJH, 2015), and was recently FDA and EMA approved for first line treatment of CLL in combination with chlorambucil. Objective: To evaluate phosphorylation of signaling pathway altered differentially following obinutuzumab vs RTX against RTX-sensitive/resistant BL Methods: Raji (CD20+) and Raji-4RH) cells were cultured in RPMI with 10% FBS. Tumor cells were incubated with 100 μg/ml obinutuzumab, and/or RTX for 24 hrs. For phosphoproteomics analysis, we performed a mass spectrometry-based label-free quantitative phosphoproteomic profiling of the BL cell lines Raji /Raji4RH in the presence/absence of obinutuzumab or rituximab or isotype control. Silencing of PLCG2 (Dharmacon, USA) and MAPK1 (Sigma Aldrich, USA) in Raji/Raji4RH cell lines was carried out according to the manufacturer's instructions Results: In all 978 unique phosphorylated proteins were identified. Out of these 661 phosphoproteins were identified after obinutuzumab vs. 615 in RTX treatment, respectively. For the Raji4RH, 534 phosphoproteins were identified after obinutuzumab and 534 in RTX treatment, respectively (Fig.1). Functional annotation of proteins differentially phosphorylated in response to obinutuzumab vs. RTX (&amp;gt;1.5-fold) reveals the involvement of the BCR (PLCG2, BTK &amp; GSK3B), FC gamma phagocytosis (FCRG2B, MAPK1 &amp; RAF1), and Natural killer cell-mediated cytotoxicity (MAPK1, RAF1&amp; PLCG2) signaling pathways (Fig. 2). Differential phosphorylations of proteins involved in BCR or cytotoxicity pathways were validated by western blot after incubation with obinutuzumab vs. RTX in Raji/ Raji4RH cell lines, revealed up regulation of BTK, PLCY2 and ERK1/RAF1 after obinutuzumab vs. RTX treatment in Raji. Silencing, of PLCG2 and MAPK1 pathway, significantly increased cell proliferation and decreased cytotoxicity after obinutuzumab treatment in Raji (P = 0.0002 &amp; 0.000002) but no change in Raji4RH. Conclusions: Obinutuzumab and RTX differentially phosphorylate BCR, and cytotoxicity signaling pathways. Obinutuzumab function differentially in RTX resistant and sensitive BL cell lines which may provide insights into alternate therapeutic strategy in RTX resistant BL. Citation Format: Aradhana Awasthi Tiwari, Delphine C.m. Rolland, Mona Elmacken, Janet Ayello, Lisa Kurien, Carmella van de Ven, Venkatesha Basrur, Kevin Conlon, Damine Fermin, Matthew J. Barth, Christian Klein, Kojo S.j. Elenitoba-Johnson, Megan Lim, Mitchell S. Cairo. Obinutuzumab (GA101) versus rituximab against rituximab-sensitive and -resistant Burkitt lymphoma (BL) differentially phosphorylate BCR, Fc-gamma receptor, and natural killer cell-mediated cytotoxicity signaling pathways. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3893.

In Silico HLA Typing Using Standard RNA-Seq Sequence Reads.

Next-generation sequencing (NGS) enables high-throughput transcriptome profiling using the RNA-Seq assay, resulting in billions of short sequence reads. Worldwide adoption has been rapid: many laboratories worldwide generate transcriptome sequence reads daily. Here, we describe methods for obtaining a sample's human leukocyte antigen (HLA) class I and II types and HLA expression using standard NGS RNA-Seq sequence reads. We demonstrate the application using our algorithm, seq2HLA, and a publicly available RNA-Seq dataset from the Burkitt lymphoma cell line Raji.

A Comparative Global Phosphoproteomics Analysis of Obinutuzumab (GA101) Versus Rituximab (RTX) Against RTX Sensitive and Resistant Burkitt Lymphoma (BL) Demonstrates Differential Phosphorylation of Signaling Pathways Proteins

Background: Burkitt Lymphoma (BL) is the most common NHL in children and adolescents and has an excellent prognosis (≥80% 5years, EFS, Cairo et al. Blood, 2007). The prognosis has improved with the addition of targeted immunotherapy with rituximab (Goldman/Cairo et al, Leukemia, 2013, Cairo et al. JCO, 2012). However, a subset of patients with chemoimmunotherapy-resistant disease has a dismal prognosis (≤ 10% 5 years, EFS) (Miles/Cairo et al. BJH, 2012). Deregulation of signaling pathways controlled by protein phosphorylation underlies the pathogenesis of B-cell lymphomas, however, the extent to which they contribute to rituximab resistance is largely unknown (Barth et al. BJH, 2013). Obinutuzumab (GA101), a novel glycoengineered type II CD20 Ab, mediates enhanced cell death & ADCC against diffuse B-cell lymphoma vs. RTX (Mössner et al. Blood, 2010), and was recently approved by FDA and EMA for first line treatment of CLL in combination with chlorambucil.Objective: To evaluate phosphorylation of signaling pathway proteins altered differentially after obinutuzumab or RTX treatment against RTX sensitive/ resistant BL cell lines.Methods: Raji (CD20+, ATCC, Manhass, VA) and Raji-4RH (provided by M. Barth, Roswell Park Cancer Institute) were cultured in RPMI with 10% FBS. For in-vitro studies, tumor cells were incubated with 100 µg/ml obinutuzumab (supplied by Christian Klein, PhD, Roche Research & Early Development, Zurich), and/or RTX for 24 hrs . Cell death was evaluated by staining with AnnexinV/7AAD and analyzed by flow-cytometry. ADCC were performed with K562-IL-15-41BBL expanded NK cells at 20:1 effector: target ratio (E: T, n=3) using an europium release assay (Perkin-Elmer). For Phosphoproteomics analysis, we performed a mass spectrometry-based label-free quantitative phosphoproteomic profiling of the BL cell lines Raji, /Raji4RH in the presence/absence of obinutuzumab or rituximab (100µg/ml for 24h) or isotype control. Six milligrams of protein from each condition were digested by trypsin and peptides were subjected to phosphopeptide enrichment using metal oxide affinity chromatography (MOAC) and immunoprecipitation. An LTQ Orbitrap XL in-line with a Paradigm MS2 HPLC was employed for acquiring high-resolution MS and MS/MS data that were searched with the Swissprot Human taxonomic protein database.Results: Obinutuzumab, compared to RTX, significantly enhanced cell death in Raji 45.1±3.3% vs. 32.7±6.8%, (p=0.005) & Raji4RH 15.8±2.2% vs. 2.1±1.5% (p=0.001), respectively. Overall survival of mice receiving 30 mg/kg of obinutuzumab was significantly increased when compared to mice receiving 30 mg/kg of RTX in Raji (p=0.05) & Raji4RH (p=0.024), respectively.In Raji, 329 proteins were differentially phosphorylated (>1.5-fold change) between obinutuzumab vs. RTX. Of these proteins, 171 were expressed at higher levels in obinutuzumab than in RTX. Proteins differentially phosphorylated in response to obinutuzumab and RTX treatment were involved in the B-cell receptor (BCR) signaling pathway (LYN, BTK, CD19, PLCG2, INPP5D, NFATC1 and PIK3AP1), the spliceosome (TRA2A, DDX46 and PRPF31), and the cell cycle signaling pathway (WEE1, MMC3, GSK3B and CCNH). (Fig.1A)Obinutuzumab and RTX also resulted in a differential phosphorylation of 606 proteins in Raji4RH. These proteins were involved in the spliceosome (CDC42, TRA2A and DDX42), tight junction (HCLS1, PRKCD, EPB41 and MYH2) and nucleotide excision repair (POLD3, CCNH and LIG1) pathways. (Fig.1B)Differential phosphorylation of BCR signaling pathways proteins (BTK, PLCG2 and GSK3B) was validated by western blot studies after incubation with obinutuzumab vs. RTX in Raji/Raji4RH cell lines, reveled up regulation of BTK and PLCY2 after obinutuzumab treatment vs. RTX treatment in Raji BL cell line.Conclusions: These data suggest that obinutuzumab vs. rituximab treatment result in global changes in BL proteins involved in BCR, spliceosome, cell cycle, nucleotide excision repair & tight junction signaling pathway. Furthermore, BCR signaling pathways appear more affected by obinutuzumab compared to RTX in Raji cell lines as compared to Raji4RH. Further, these data revealed the utility of unbiased phosphoproteome interrogation of obinutuzumab vs. rituximab mediated signaling events as well as characterizing signaling networks that may provide insights into pathogenetic mechanisms of rituximab resistance. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Global Phosphoproteomics Analysis Of Burkitt Lymphoma (BL) Demonstrates Abnormal Protein Phosphorylation In BCR, Spliceosome, mTOR and Chemokine Signaling Pathways In Rituximab Induced Resistant BL

BackgroundBurkitt Lymphoma (BL) is the most common non-Hodgkin lymphoma (NHL) in children and adolescents and has an excellent prognosis (≥80% 5years, EFS) following short but intense multi-agent chemotherapy (Cairo et al JCO, 2012). The prognosis has improved following the addition of targeted immunotherapy with rituximab (Goldman/Cairo et al, Leukemia, 2013). However, a subset of patients with chemoimmunotherapy resistant disease have a dismal prognosis (≤ 10% 5 years, EFS) (Miles/Cairo et al. BJH, 2012 and Cairo et al Blood, 2007). Deregulation of signaling pathways controlled by protein phosphorylation underlies the pathogenesis of B-cell lymphomas, however the extent to which they contribute to rituximab resistance is largely unknown (Barth et al. BJH, 2013). Recently, Czuczman et al have generated a chemoimmunotherapy resistant BL cell line Raji4RH from the native Raji BL line after serial exposures to rituximab (Czuczman et al Clin Cancer Res, 2008). ObjectiveWe therefore sought to compare the in-vitro cytotoxicity of rituximab to Raji vs. Raji4RH and the resulting phosphoproteomics changes between untreated and rituximab treated Raji vs. Raji4RH by global phosphoproteomics. MethodsIn order to identify deregulated signaling proteins/pathways that play a role in BL rituximab resistance, we performed a mass spectrometry-based label-free quantitative phosphoproteomic profiling of the BL cell line Raji, (CD20+,ATCC, Manhassas, VA) and its rituximab resistant derivative Raji 4RH in the presence or absence of Rituximab (100µg/ml for 24 hr) or isotype control. Six milligrams of protein from each condition were digested by trypsin and peptides were subjected to phosphopeptide enrichment using metal oxide affinity chromatography (MOAC) and immunoprecipitation using a cocktail of 3 anti-phosphotyrosine antibodies. An LTQ Orbitrap XL in-line with a Paradigm MS2 HPLC was employed for acquiring high-resolution MS and MS/MS data which were searched with the Swissprot Human taxonomic protein database. Quantitation of identified phosphopeptides was based on spectral counts of phosphorylated peptides. The accuracy of the phospho-site localization was predicted using Luciphor program. Pathway analyses were performed using the Database for Annotation, Visualization and Integrated Discovery (DAVID) ResultsOur in-vitro results shows that rituximab induced significantly more cell death against Raji vs. Raji4RH (32.7±6.8% vs.5.5±2.2%, p=0.001) compared to isotype control (9.35±0.75% vs.8.9±1.1%, p=NS) at 24 hrs. Furthermore, 2272 unique phosphopeptides were identified and quantified with a probability P> 0.7 and a false localization rate (FLR) < 0.01. These unique phosphopeptides corresponded to 850 unique phosphorylated proteins. Six hundred and nine proteins were differentially phosphorylated (>1.5-fold change) between Raji and Raji 4RH (Figure 1A). Out of these proteins, 374 were more phosphorylated in Raji4RH than in Raji and 235 were more phosphorylated in Raji than in Raji 4RH. Proteins in the spliceosome and the BCR signaling pathway were significantly represented.Changes in the phosphorylation of 430 proteins were induced by Rituximab in Raji cells and involved those in the B-cell receptor (BCR) signaling pathway (LYN, LCK, SLAMF1, PAG1, CD20), the spliceosome (SF3A1, SF3B1, SRSF6), the mTOR pathway (RICTOR, PDPK1, RPS6KA1, BRAF) and the chemokine signaling pathway (CXCR5, PTK2B, CCL1, PRKCD).After rituximab treatment, 559 proteins were differentially phosphorylated between Raji and Raji 4RH (Figure 1B). Rituximab treatment increased the phosphorylation of 262 proteins in Raji 4R while it increased the phosphorylation of 299 proteins in Raji. ConclusionsThe data indicate that Rituximab results in global changes in proteins in BL involved in not just BCR signaling but also in the spliceosome, the mTOR and the chemokine signaling pathway. Furthermore, aberrant phosphorylation-induced signaling events in the spliceosome and the BCR signaling pathways may contribute to resistance to rituximab. These data revealed the utility of unbiased phosphoproteome interrogation of rituximab mediated signaling events as well as characterizing signaling networks that may provide insights into pathogenetic mechanisms of rituximab resistance. [Display omitted] AA and DR considered co-primary first authors, and ML and MC considered co-senior authors. Disclosures: Cairo:Roche/Genentech : advisory board Other.

Chromosomes 2q, 4q and 13q21 Are Preferential Targets of Epstein-Barr Virus (EBV) Integration In Burkitt's Lymphoma Cell Lines

AbstractAbstract 4452The Epstein-Barr virus (EBV) was first described in 1964 as a possible cause of Burkitt's lymphoma (BL), the most common childhood cancer in equatorial Africa. EBV is an oncogenic virus found in about 95% of the endemic BL. In latently infected cells, the EBV DNA can be maintained in episomal form, but integrated EBV could also be present. Data on the exact integration loci of EBV in BL are very rare, however, integration of EBV-DNA into the human genome has been pointed as an important mechanism in malignant cellular transformation. We have investigated the EBV integration loci (EBV-IL) in 20 BL cell lines using fluorescence in situ hybridization (FISH).FISH was performed using, as EBV-DNA probe, a biotinylated 3,000 bp fragment amplified by long distance-PCR from DNA of the EBV-positive BL cell line Raji. FISH on metaphase spreads of the BL cell line Namalwa was performed to validate the quality of the EBV-FISH probe. Negative controls were performed on EBV-negative BL cell lines CA-46, CW698, and Tanoue. Integration of EBV was defined by the presence of symmetrical doublet hybridization signals at the same chromosomal loci in both sister chromatids. A minimum of 15 well-conserved and complete metaphases was evaluated in each case.As expected, Namalwa showed symmetrical doublet hybridization signals of our EBV-FISH probe on chromosome 1p35. EBV-negative cell lines did not show hybridization signals of the probe. We detected 632 EBV-IL in the total of metaphases analyzed in 20 BL cell lines. Integration of EBV was seen in all chromosomal arms except Yp, 8p, 14p, 20q, 21p, and 22p. An analysis of the chromosomal distribution of all EBV-IL revealed a pattern of preferential insertion for EBV-IL on chromosomes 13q (14% of the metaphases), 2q and 4q (12%, respectively), 7q (10,5%), 3q (9%), and 17q (6,5%).Regarding the recurrency of EBV-IL, the BL cell lines were divided in 4 groups. The first group consisted of 8 cell lines with high-recurrent EBV-IL at a specific chromosomal band (75%-100% of the metaphases analyzed). This first group showed specific EBV-IL at a precise chromosomal location on homologous chromatids on 2p23 (Seraphine), 2q21~31 (Switzer), 22q12 (PA-682), 7p22 and 13q21 (AG876), 7q11 and 17q25 (Naliaka), and 7q21 and 13q21 (LY-67). Interestingly, in BL60 and Maku, the unique EBV-IL visualized was localized near the translocation breakpoint junction of a der(19)t(17;19) and a der(13)t(3;13), respectively. The second group contained 5 cell lines with medium-recurrent EBV-IL (27%-50%). In this group, we observed metaphases with 1 to 3 recurrent EBV-IL localized on 2p12 (Rael), 15q11~14 (JI), 15q13~15 (JBL2), 4q21 and 4q32 (BL16), and 3q21~26, 4p12, and 13q21 (Akuba). The third and fourth groups comprised 4 cell lines with low-recurrent EBV-IL (13%-20%) and 3 cell lines with absence of recurrent EBV-IL, respectively. Non-recurrently integrated EBV-DNA signals on additional chromosomes were observed in cell lines of all 4 groups, except in 4 of the high-recurrent group. We further investigated the distribution of the EBV-IL of groups 3 and 4, of those in groups 1 and 2 with a recurrency less than 27% of the metaphases, and of those that were non-recurrently integrated. We observed again that the viral integration targeted preferentially chromosomes 2q (21%), 4q (14%), and 3q (11%).Our results identify for the first time the integration loci of EBV in BL cell lines and that this integration succeed preferentially on chromosomes 2q, 3q, and 4q. Moreover, in 12 cell lines, this integration ocurred non-randomly at a specific chromosomal sites, targeting 13q21 recurrently. Whether the integration of EBV in these loci affects the expression of genes important for the pathogenesis of BL or whether the EBV integration contributes to an enhanced chromosomal instability remains to be elucidated. Disclosures:No relevant conflicts of interest to declare.

Speckled-like Pattern in the Germinal Center (SLIP-GC), a Nuclear GTPase Expressed in Activation-induced Deaminase-expressing Lymphomas and Germinal Center B Cells

We identified a novel GTPase, SLIP-GC, with expression limited to a few tissues, in particular germinal center B cells. It lacks homology to any known proteins, indicating that it may belong to a novel family of GTPases. SLIP-GC is expressed in germinal center B cells and in lymphomas derived from germinal center B cells such as large diffuse B cell lymphomas. In cell lines, SLIP-GC is expressed in lymphomas that express activation-induced deaminase (AID) and that likely undergo somatic hypermutation. SLIP-GC is a nuclear protein, and it localizes to replication factories. Reduction of SLIP-GC levels in the Burkitt lymphoma cell line Raji and in non-Hodgkin lymphoma cell lines resulted in an increase in DNA breaks and apoptosis that was AID-dependent, as simultaneous reduction of AID abrogated the deleterious effects of SLIP-GC reduction. These results strongly suggest that SLIP-GC is a replication-related protein in germinal center B cells whose reduction is toxic to cells through an AID-dependent mechanism.

Lysophosphatidic acid inhibits the cytotoxic activity of NK cells: involvement of Gs protein-mediated signaling

Lysophosphatidic acid (LPA) is an activator and chemoattractant of NK cells, which are critical members of the immunological tumor surveillance machinery. Here, we analyzed the influence of LPA on the interaction of human NK cells with tumor cells such as the Burkitt lymphoma cell line Raji and the human melanoma cell line A2058. Thereby we found that LPA inhibits the release of perforin and cytotoxic activity of NK cells. Analysis of signal transduction showed that LPA induces common signaling pathways of chemotaxins such as G(i) protein-dependent actin re-organization, activation of the mitogen-activated protein kinase p38 as well as phosphatidylinositol-3-kinase-dependent signal molecules [protein kinase B/Akt and glycogen synthase kinase-3beta (GSK-3beta)]. In contrast to most chemotaxins, LPA is also able to activate G(s)-dependent signaling molecules. This signaling cascade involves the LPA receptor type-2, increase cAMP levels and protein kinase A (PKA) activation, which in turn are responsible for the modulatory effect of LPA on NK cell-mediated cytotoxicity. Moreover, blocking the regulatory subunits of PKA I abrogates the inhibitory effect of LPA, whereas the catalytic subunits are not involved. Based on our data, one can assume that LPA contributes to the tumor escape from the immunological surveillance machinery.

Cross-linking of CD54 on Burkitt Lymphoma Cell Line Raji and Ramos Induces FasL Expression by Reactive Oxygen Species and Apoptosis of Adjacent Cells in Fas/FasL Interaction

CD54 is a cell surface adhesive glycoprotein, which is expressed in most cells. Interaction between CD54 and its ligands is involved in several cellular events including activation, proliferation, and cell death and also cell-to-cell adhesion. In the present study, we found that cross-linking of CD54 on Burkitt lymphoma cell lines, Raji and Ramos, induced apoptosis. We investigated that cross-linking of CD54 on Raji and Ramos using immobilized anti-CD54 mAb (clone 6.5B5) leads to apoptosis. CD54-induced apoptosis took place in association with an increase of intracellular reactive oxygen species (ROS) and a loss of the mitochondrial membrane potential and also the activation of caspases 3 and 9, resulting in the degradation of the proteolytic poly (ADP-ribose) polymerase. Pretreatment of each N-acetyl cystein and N-benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone (a broad caspase inhibitor) blocked apoptosis. Cross-linking of CD54 immediately induced expression of fasL, which was inhibited by pretreatment of N-acetyl cystein. NOK-1 (antagonistic anti-fasL), ZB4 (antagonistic anti-fas), and N-benzyloxycarbonyl-Ile-Glu-Thr-Asp-fluoromethylketon (caspase 8 inhibitor) effectively rescued cells from apoptosis via adjacent fas-fasL interaction but did not block ROS generation. Taken together, it is concluded that engagement of CD54 on B lymphoma cell lines by anti-CD54 mAb may trigger fasL expression through ROS generation and may subsequently induce apoptosis in adjacent fas-fasL interaction.

Episomal vectors to monitor and induce somatic hypermutation in human Burkitt-Lymphoma cell lines

Somatic hypermutation (SHM) occurs at a specific B-cell differentiation stage, during the germinal centre reaction, and provides a means to diversify and shape the antibody repertoire of the adaptive immune system. Burkitt-Lymphoma (BL) is a germinal centre derived B-cell malignancy. Presumably deregulation of the somatic hypermutation- and/or class switch recombination process causes a translocation between the myc-locus and one of the Ig-loci, which is characteristic for BL. We show here that we developed an episomal-based vector system to monitor and induce AID-dependent somatic hypermutation in human cell lines. Exemplarily, we utilized this system to investigate the hypermutation status of various BL cell lines. Analysis of the influence of Ig-specific cis-regulatory elements for the mutability of a green fluorescent protein (GFP) reporter transgene revealed, that the presence of Ig-enhancers is required for an efficient targeting of a constitutively transcribed GFP transgene. In contrast, a transcriptional inducible GFP transgene was hypermutated by overexpression of activation-induced cytidine deaminase (AID), even in the absence of Ig-specific sequences. This observation verifies that overexpression of AID in non hypermutating BL cell lines as well as the expression of endogenous AID in the hypermutating BL cell line Raji can overcome the target restriction of AID.

Reovirus therapy of lymphoid malignancies

Reoviruses infect cells that manifest an activated Ras-signaling pathway, and have been shown to effectively destroy many different types of neoplastic cells, including those derived from brain, breast, colon, ovaries, and prostate. In this study, we investigated the reovirus as a potential therapeutic agent against lymphoid malignancies. A total of 9 lymphoid cell lines and 27 primary human lymphoid malignancies, as well as normal lymphocytes and hematopoietic stem/progenitor cells, were tested for susceptibility to reovirus infection. For in vitro studies, the cells were challenged with reovirus (serotype 3 Dearing), and viral infection was assessed by cytopathic effects, viability, viral protein synthesis, and progeny virus production. We present evidence of efficient reovirus infection and cell lysis in the diffuse large B-cell lymphoma cell lines and Burkitt lymphoma cell lines Raji and CA46 but not Daudi, Ramos, or ST486. Moreover, when Raji and Daudi cell lines were grown subcutaneously in severe combined immunodeficient/nonobese diabetic (SCID/NOD) mice and subsequently injected with reovirus intratumorally or intravenously, significant regression was observed in the Raji-induced, but not the Daudi-induced, tumors, which is consistent with the in vitro results. Susceptibility to reovirus infection was also detected in 21 of the 27 primary lymphoid neoplasias tested but not in the normal lymphocytes or hematopoietic stem/progenitor cells. Our results suggest that reovirus may be an effective agent against several types of human lymphoid malignancies.

Origin and properties of soluble CD21 (CR2) in human blood.

By analysis with a panel of CD21 MoAbs it is shown that a large part of the soluble CD21 in human blood plasma is of the long isoform (CD21L), as judged by comparison with antigen produced by mouse L cells transfected with CD21L-cDNA and reactivity with the restricted CD21 MoAb R4/23. This is compatible with the hypothesis that soluble CD21 in the blood is mainly derived from follicular dendritic cells (FDC). Cells from a human keratinocyte cell line transfected with cDNA from the Burkitt lymphoma cell line Raji also produced soluble CD21L (sCD21L), whereas the short form of sCD21 (sCD21S) was the major component of sCD21 produced by the B lymphoblastoid cell line LICR-LON-HMy and the T cell line Jurkat. Confocal studies of FDC isolated from human tonsil revealed that CD21 was present in the cytoplasm. On gel filtration sCD21 from untreated serum has an apparent size considerably greater than the 130kD found by SDS-PAGE analysis. This may be partly accounted for by the non-globular shape of the molecule, but may also indicate, as reported by others, that in its native state sCD21 is complexed with other proteins. However, no evidence of complexing with sCD23 or C3d could be found.

Cloning and expression of the human single copy homologue of the mouse zinc finger protein zfr

A human homologue of the murine zinc finger protein zfr is transcriptionally induced in the Epstein–Barr virus-positive Burkitt lymphoma cell line Raji upon treatment with the granulocyte/macrophage lineage ganglioside IV 3NeuAc-nLcOse 4Cer. The gene was cloned by a rapid amplification of cDNA ends approach based on a cDNA clone. The resulting hzfr sequence is 3393 base pairs in length coding for a protein of 1057 amino acids. Sequence alignments between hzfr and zfr reveal an identity of 92% on the nucleotide level and an identity of 96.4% on the amino acid level, respectively. Based on Southern blot data hzfr can be addressed as a single copy gene. Tissue-specific expression was determined by semi-quantitative PCR of normalized cDNA populations from various human tissues with glyceraldehyde-3-phosphate dehydrogenase as an internal control. Highest levels of transcripts were found in brain. hzfr transcripts could not be detected in skeletal muscle.

Early steps in termination of the immortalization state in Burkitt lymphoma: induction of genes involved in signal transduction, transcription, and trafficking by the ganglioside IV 3NeuAc-nLcOse 4Cer

Stimulation by the ganglioside IV 3NeuAc-nLcOse 4Cer leads to growth arrest in the Burkitt lymphoma cell line Raji. In order to analyze the primary response of Raji cells to that stimulus, a cDNA array screen and a suppression subtractive hybridization-PCR approach were performed. Twenty-four genes with assigned functions were confirmed to be induced by the ganglioside in reverse Northern blot experiments covering e.g. protein kinase B, phospholipase C, the MAP-kinase ERK3, the transcription factors YY1, DR1 and NSEP, the membrane traffic protein TAP, and the nuclear export protein CRM1. Most of the genes identified are involved in signal transduction, transcription, and cell trafficking. For selected genes, the induction of expression was quantified by semiquantitative RT-PCR.

Role of the epstein-barr virus RTA protein in activation of distinct classes of viral lytic cycle genes.

Initiation of the Epstein-Barr virus (EBV) lytic cycle is controlled by two immediate-early genes, BZLF1 and BRLF1. In certain epithelial and B-cell lines, their protein products, ZEBRA and Rta, stimulate their own expression, reciprocally stimulate each other's expression, and activate downstream viral targets. It has been difficult to examine the individual roles of these two transactivators in EBV-infected lymphocytes, as they are expressed simultaneously upon induction of the lytic cycle. Here we show that the Burkitt lymphoma cell line Raji represents an experimental system that allows the study of Rta's role in the lytic cycle of EBV in the absence and presence of ZEBRA. When expressed in Raji cells, exogenous Rta does not activate endogenous BZLF1 expression, yet Rta remains competent to transactivate certain downstream viral targets. Some genes, such as BaRF1, BMLF1, and a late gene, BLRF2, are maximally activated by Rta itself in the absence of detectable ZEBRA. The use of the Z(S186A) mutant form of ZEBRA, whose transactivation function is manifest only by coexpression of Rta, allows identification of a second class of lytic cycle genes, such as BMRF1 and BHRF1, that are activated in synergy by Rta and ZEBRA. It has already been documented that of the two activators, only ZEBRA stimulates the BRLF1 gene in Raji cells. Thus, there is a third class of viral genes activated by ZEBRA but not Rta. Moreover, ZEBRA exhibits an inhibitory effect on Rta's capacity to stimulate the late gene, BLRF2. Consequently ZEBRA may function to repress Rta's potential to activate some late genes. Raji cells thus allow delineation of the combinatorial roles of Rta and ZEBRA in control of several distinct classes of lytic cycle genes.

HSV type 1 genome variants from persistently productive infections in Raji and BJAB cell lines.

We studied possible genomic changes occurring in herpes simplex virus type 1 (HSV-1) during long-term cell culture which served as a model system for persistence and latency studies as introduced earlier. Sixteen HSV-1 reisolates were isolated from persistently productive HSV-1 (strains F and AK)-infected Burkitt lymphoma cell lines Raji and BJAB at four different times. They were roughly characterized in plaque morphology, plaque size, and infectivity. The viral reisolate DNAs revealed deletions and insertions of up to 1,150 base pairs in fragments BamHI-B, -E, -F, -J, -V, -X, and in the L-terminal and junction fragments S and K. Results were confirmed by additional restriction enzyme analyses and DNA sequencing of selected genomic regions between map units 0.642-0.650, 0.763-0.778 and 0.887-0.934. There was a progressive increase in genomic variability over a three-year period. However, changes in DNA fragment size occurred at different rates, with some reisolates showing stability over several months. The selective pressure for HSV-1 (F and AK) genomic changes was stronger in Raji than in BJAB cells, and stronger for F than for AK strain.